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1 FUNGI IN THE BOTRYOSPHAERIACEAE C AUSING STEM BLIGHT O N VACCINIUM SPP. IN THE SOUTHEAS TERN UNITED STATES A ND STEM BLIGHT DISEASE MANAGEMENT ON SOUTHE RN HIGHBUSH BLUEBERR IES IN FLORIDA By AMANDA FAITH WRIGHT A DISSERTATION P RESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011
2 2011 Amanda Faith Wright
3 To my family, friends, and wonderful husband f or all of their kindness, patience and support throughout my graduate car eer
4 ACKNOWLEDGMENTS I would like to thank my family and friends for all of their help and support throughout my time at UF. I would most especially like to thank my husband Jon for his love encouragement, and hugs when I began to feel down. Lastly, I would like to thank my committee members : Aaron Palmateer, Ariena VanBruggen, James Olmstead, and Philip Harmon, for all of their guida nce and patience with me as well
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 L ITERATURE REVIEW ................................ ................................ .......................... 11 Blueberry Industry in the Southeastern United Stat es ................................ ............ 11 Vaccinium ................................ ................................ ................................ ............... 11 Breeding Southern Highbush Blueberry in Florida ................................ .................. 12 Stem Blight Resistance Breeding ................................ ................................ ..... 13 Vaccinium Plant Propagation ................................ ................................ .................. 14 Classification of Botryosphaeria dothidea ................................ ............................... 15 Anamorphs of Botryosphaeriaceae ................................ ................................ ......... 16 Higher Classification of Botryosphaeriaceae ................................ .......................... 17 Stem Blight and Dieback of Blueberries ................................ ................................ .. 20 Host Range ................................ ................................ ................................ ............. 20 Botryosphaeria Latent Phase ................................ ................................ .................. 21 Disease Cycle of Fungi in the Botryosphaeriaceae ................................ ................ 22 Plant Health and Disease Transmission ................................ ................................ 24 Manag ement of Botryosphaeria Stem Blight ................................ ........................... 25 Dissertation Thesis ................................ ................................ ................................ 27 Research Objectives By Chapter ................................ ................................ ............ 27 2 T AXONOMY, PHYLOGENY, AND IDENTIFICATION OF FUNGI IN THE BOTRYOSPHAERIACEAE CAUSING STEM BLIGHT AND DIEBACK OF VACCINIUM SPP. IN THE SOUTHEASTERN UNITED STATES ......................... 30 Overview of Botryosphaeriaceae Taxonomy ................................ .......................... 30 Materials and Methods ................................ ................................ ............................ 33 Sample Collection and Pathogen Isolation ................................ ....................... 33 Molecular Methods ................................ ................................ ........................... 33 rDNA Restriction Analysis ................................ ................................ ................ 34 Sequence and Phylogenetic Analysis ................................ .............................. 35 Results ................................ ................................ ................................ .................... 36 Molecular and Morphological Identification of Botryosphaeria spp. .................. 36 Sequence Analysis ................................ ................................ ........................... 37 Conidia Morphology ................................ ................................ ......................... 38 Discussion of Stem Blight and Dieback in the Southeaster n United States ............ 39
6 3 S USCEPTIBILITY OF SOUTHERN HIGHBUSH BLUEBERRY GENEOTYPES TO BOTRYOSPHAERIACEAE (BOT.) PATHOGENS ................................ ............ 52 Overview of Stem Blight Resistance Breeding ................................ ........................ 52 Materials and Methods ................................ ................................ ............................ 54 Inoculum preparation for accession screening ................................ ................. 54 Differential Response to Pathogen Isolates ................................ ...................... 55 Un rooted Softwood Cutting Inoculations ................................ ......................... 55 Micro cutting Inoculations ................................ ................................ ................. 56 Clonally Propagated Seedling Inoculations ................................ ...................... 57 Results ................................ ................................ ................................ .................... 58 Differential Response to Pathogen Isolates ................................ ...................... 58 Un rooted Softwood Cutting Inoculations ................................ ......................... 58 Micro cu tting Inoculations ................................ ................................ ................. 59 Clonally Propagated Seedling Inoculations ................................ ...................... 59 Discussion of Stem Blight Resistance Screening in Florida ................................ .... 59 4 EVALUATION OF SOUTHERN HIGHBUSH CULTIVER AND PROPAGATION METHODS FOR STEM BLIGHT MORTALITY DURING THE FIRST TWO YEARS OF GROWTH IN FLORIDA ................................ ................................ ....... 69 Overview of Stem Blight Disease Managment ................................ ........................ 69 Materials and Methods ................................ ................................ ............................ 70 Plot Installation and Maintenance ................................ ................................ ..... 70 Pathogen Isolation and Identification ................................ ................................ 72 Latent Isolations and Identification ................................ ................................ ... 72 Pathogenicity ................................ ................................ ................................ .... 75 Results ................................ ................................ ................................ .................... 75 Plot Ratings and Pathogen Incidence ................................ .............................. 75 Latent Isolations ................................ ................................ ............................... 76 Pathogenicity ................................ ................................ ................................ .... 77 Discussion of Stem Blight Mortality in Florida ................................ ......................... 77 LIST OF REFERENCES ................................ ................................ ............................... 91 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 103
7 LIST OF TABLES Table page 2 1 Isolates of Bot. fungi from Vaccinium spp. in the s outheastern US .................... 44 2 2 Botryosphaeriaceae i solates from GenBa nk ................................ ...................... 45 2 3 Isolation f requency of Bot. fungi in the southeastern Unit ed States .................... 46 2 4 C onidia l dimensions of Bot. fungi in southeastern US ................................ ........ 51 3 1 Differentia l response to L. theobromae and N. ribis ................................ ............ 64 3 2 Combined results of the un rooted so ftwood cutting analysis ............................. 64 3 3 Un rooted micro propagation analysis ................................ ................................ 65 3 4 Combined results from the seedling sc reen ................................ ....................... 67 4 1 Bot. fungi recov e red from apparently healthy softwood cuttings ........................ 81 4 2 Bot. isolates from GenBank used in phylogenetic analysis ................................ 81 4 3 Plant mortali ty and Bot. colony identification ................................ ...................... 82 4 4 ANOVA of disease incidence and Bot. severity. ................................ ................. 83 4 5 Bot. colonies isolated from the li y s ate of s oft w ood cuttings ................................ 88 4 6 Bot. colonies isolated from surface sterilized softwood cuttings ......................... 88
8 LIST OF FIGURES Figure page 2 1 Map of the southeastern United States ................................ .............................. 43 2 2 A schematic diagram of the restriction sites in the ITS rDNA region .................. 47 2 3 Example of restriction digest patterns ................................ ................................ 47 2 4 Phylogentic tree of all Bot. species recovered in survey ................................ .... 48 2 5 Phylogenetic Neofusicoccum spp.tree ................................ ............................... 49 2 6 Phylogenetic Lasiodiplodia spp. tree ................................ ................................ .. 50 3 1 Corre lat ion graph for u n rooted softwood cuttings.. ................................ ............ 65 3 2 Corr elation graph for un rooted microcuttings ................................ ................... 66 3 3 Correlation graph for the cloned seedlin g inoculations. ................................ .... 68 4 1 Average number of dead plants per plot at Alachua Co.. ................................ ... 85 4 2 Average number of dead plants per plot at D eSoto Co .. ................................ ... 86 4 3 Average numbe r of dead plants per plot at Polk Co.. ................................ ......... 87 4 4 AUDPC of latent isolates ................................ ................................ .................... 89 4 5 Phylogenetic tree of latent isolates ................................ ................................ .... 90
9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requi rements for the Degree of Doctor of Philosophy FUNGI IN THE BOTRYOSPHAERIACEAE C AUSING STEM BLIGHT O N VACCINIUM SPP. IN THE SOUTHEAS TERN UNITED STATES A ND STEM BLIGHT DISEASE MANAGEMENT ON SOUTHERN HIGHBUSH BLUEBERRIES IN FLORI DA By Amanda Faith Wright December 2011 Chair: Philip F. Harmon Major: Plant Pathology Stem blight of southern highbush blueberry (SHB ; hybrid of Vaccinium corymbosum L. hybrid V. darrowi Camp ) and rabbiteye blueberry (REB; Vaccinium virgratum Aiton ) blueberries h as been c ite d by growers in the S outheast as the most important disease s for the industry. In 2010, 365 stem blight samples were collected from SHB and REB cultivars from 28 sites in the southeastern United States (AL, FL, GA, NC, and SC); 86% of the samples were initial ly identified as Botryosphaeria ( Bot .) species S equence analysis of fungal DNA PCR restriction fragment length polymorphism (PCR RFLP) assays and morphology of conidia were used to discriminate among Bot species. Neofusicoccum ribis and L. theobromae w ere identified as the two predominate species causing stem blight in the southeastern US; Botryosphaeria corticis B. dothidea, and Diplodia seriata were found infrequently. In an additional survey completed in 2010 p ropagative material collected monthly from May to Oct ober and subsequent fungal isolations identified latent Bot pathogens on apparently healthy softwood cuttings (sw ) Bot fungi also were isolated from l y sate washed from healthy sw Using PCR RFLP, L. theobromae and N. ribis were identified
10 and pathogenicity of select isolates was confirmed. Experimental plots plant ed at three separate locations in Florida tested the effec ts of natural Bot. infection Plants installed at these locations were locally propagated from a single sw distributor and propagated in the Pacific Northwest from a single tissue culture (tc) shipment Over a two year period, a greater number of plant s propagated from sw died than plant s propagated from tc ; Bot. was isolated from the majority of sw samples. Additional research is needed to determine the impact of these latent infections on blueberry production and pathogen distribution. Also plants propagated from sw should be collected from multiple distributors in Florida and stock should be evaluated over multiple y ears to determine potential and frequency of Bot. contamination A technique was devised to screen for stem blight resistant progeny Inoculation of un rooted micro cuttings produced repeatable results. However, i nconsistent results were obtained for the clonally propagated seedling and un rooted sw experiments. Lack of repeatability could be due to small sample size, differences in plant stress at the time of rooting or the presence of latent infections. Further research efforts should focus on the natu re of blueberry response to controlled stresses in relation to the severity of Bot. infection.
11 CHAPTER 1 LITERATURE REVIEW Blueberry Industry in the Southeastern United States Southern highbush blueberry (SHB; interspecific Vaccinium corymbosum L.and V. darrowi Camp hybrid section Cyanococcus ) and rabbiteye blueberry (REB; V. virgratum Aiton section Cyanococcs ) cultivars are grown in the southeastern US I n 2009 the blueberry industry in Alabama, Florida, Georgia, and North Carolina was valued to be $18 0,322,200 for approximately 19,016 harvested acres ( Table 3, USDA, National Agriculture Statistics Service, http://usda.mannlib.cornell.edu ) Blueberry production in Florida is characterized as an early season high dollar niche market ( 67 ). Vaccinium The genus Vaccinium (Ericaceae) contains about 400 species native to all continents except Australia and Antarctica (69) Vaccinium consists of five sub categories that comprise three major crops : Cyanococcus (blueberry), Myrtillus Oxycoccus (cranberry), Vac cinium, and Vitis idaea (lingonberry). Cyanococcus, Myrtillus, and Oxycoccus have a polyploidy series (2n = 24, 4n = 48, 6n = 72) ( 69 ). Most Vaccinium spp. exist as woody shrubs and vines which produce self incompatible flowers that are cross pollinated by insects. The habitat of Vaccinium spp. typically consists of areas with acidic soils that are well drained. Ecosystems that recently have been disturbed including coastal sand dunes, lakes and rivers banks, abandoned fields, mountainous areas with sha llow soils, and areas with frequent fires are sites where stands of Vaccinium spp. typically establish ( 136 ). Small fruits containing many seeds that are dispersed by birds and mammals are produced by Vaccinium spp. which promotes colonization of newly di sturbed sites ( 59,66,77 ). Other
12 methods of adaptation include clonal propagation via rhizomes; in areas with frequent forest fires V. vacillans is able to regrow quickly and recolonize the disturbed location ( 77 ). Breeding Southern Highbush Blueberry in Florida Professor Ralph Sharp began the SHB breeding program at the University of Florida in order to develop low chill early ripening cultivars ( 67 ). Highbush blueberries from Michigan and New Jersey provided initial breeding stock, but cultivars were poorly to produce cultivars with better adaptation ( 68 ). T etraploid highbush ( V. corymbosum ) and hexaploid rabbiteye ( V. virgratum ) blueberries have been bred at the University of Florida (66,67). B reeding programs have not merged because the tetraploid x hexaploid crosses produce pentaploids that have reduced male fertility and produce fruit which is too dark in color ( 68,69 ). Species incorporated into the southern highbush breeding program have included V. corymbosum from the peninsula, and north central Florida, V. elliottii, V. darrowi, and most recently V. arboreum (67 ) The r esulting crosses have produced cultivars with low chilling requirements ripen ing a mon th ahead of the earliest rabbiteye blueberries ( 66 69 ). The University of Florida (UF) breeding program is based on phenotypic recurrent selection which is used to manipulate traits controlled by hundreds of genes. Recurrent selection is based on two p rinciples The first being, heterozygous parents have variable progeny. The second is if progeny that are extreme in the expression of certain characteristics are crossed; the second generation progeny will be variable, and some will be more extreme in t he selected character than their parents (66).
13 Cultivar selection proceeds through four stages: in stage I, up to 20,000 seedlings are planted in high density plots. After one year, plants are evaluated, and the best 2,000 seedling plants are selected and advanced to stage II. Plants are rated for three years, and the best 200 plants are numbered and marked for asexual propagation. The 200 selections are planted in 15 plant plots, and are rated for three years for fruit and vegetative characteristics. The superior 20 stage III plants each year are asexually propagated and planted on multiple farms as stage IV plants. Stage IV plants are evaluated for three to six years. On average, one or two plants are selected for cultivar release from the original 20,000 seedling establishment ( Olmstead personal communication ). Stem Blight Resistance B reeding Stem blight of SHB is caused by a complex of fungi in the Botryosph ae riaceae ( 140 ). Fungicide applications (24,119) optimizing irrigation practices (81) an d aggressive pruning (137) for the control of stem blight do no t adequately manage the disease Cultivars produced through breeding efforts could offer stem blight control for growers with a minimum of additional inputs ( 25 ). Varying levels of susceptibil ity to diseases caused by Botryosph ae ria (Bot.) pathogens have been reported in Cornus florida (86) M alus domestica (13), Mangifera indica (107) Prunus persica ( 16,32 ) and ), Vaccinium corymbosum cultivars(22,103,120) Differences in cultivar resistan ce have been attributed to genetics (18,45) stress (74,105) differences in inoculation procedures (103,120) and isolate virulence ( 18 ). The use of disease ind ices succulent stem tissue, and virulent isolates have helped to standardize inoculation proc edures ( 25,120 ).
14 SHB stem blight resistance is h eritable; however, the amount of progress that can be made in a SHB breeding program is difficult to estimate ( 18,45,142 ). In 1989, Gupton and Smith (45) concluded only moderate progress could be made in re sistance breeding using mass selection because of equal general and specific combining abilities (GCA and SCA). However, in 1990 Buckely (18) concluded GCA and SCA were significant using a highly virulent isolate and recommended progeny screening for iden tification of resistant parents R esistant seedlings were recovered from all cross combinations (18) SHB cultivar variation to stem blight has been identified by in vitro and by cut stem inoculations ( 103,120 ). Cultivar susceptibility has been shown to vary between inoculated greenhouse trials and field observations ( 120 ). Vaccinium Plant Propagation In Florida and throughout much of the southeastern United States SHB and REB blueberries are propagated from softwood cuttings. Cuttings are collected fro m the beginning of May until the end of September and planted in a 50/50 peat perlite mix. Plants are rooted under intermittent mist for approximately 2 months. O nce cuttings have rooted, plants are hardened off for another 1 to 2 months before field ins tallation ( Lyrene personal communication ). Another system for the propagation of Vaccinium species is the use of in vitro micropropagation techniques ( 23,43,61 ) Micropropag a ted blueberry, blackberry, and raspberry plants were free from bacterial, funga l, and viral diseases which were previously detected in mother plants (43). I n vitro blueberry shoots also have been shown to root better under intermittent mist than plants propagated from softwood cuttings ( 23 ).
15 ower, produced shorter shoots, and individuals were more variable when propagated from softwood cuttings than compared to micropropagated plants ( 61 ). Classification of Botryosphaeria dothidea In 1863, Botryosphaeria was described by Cesat i and De Notaris, and originally included twelve species which lacked detailed morphological descriptions ( 116 ) Despite obvious similarities researchers described new speci es occurring on different hosts. I n 1954 Von Arx and Mller synonymized many of t hese species and created the species complexes, B. dothidea and B. quercuum (35) However, the taxonomic revisions of Von Arx and Mller were not accepted widely ; differences in anamorph morphology prohibited many researchers from accepting the synonymizat ion of B. ribis with B. dothidea ( 35,107,116 ) In 1985 Pennycook and Samuels (96) accepted Von Arx and Mllers synonyms and described Botryosphaeria parva In 2001 Smith and Stanoz (122) indicated paraphyl y within the B. dothidea species complex ; B ribis was phylogenetically separate from B. dothidea Cluster analysis and conidial morphology reinforced the separation of B. ribis from B. dothidea and B. parva ( 51). In 2004 multi gene phylogenetic analysis separated, B. ribis (anamorph Neofusicocc um ribis ) from B. dothidea (anamorph Fusicoccum aesculi ) and B. parva (anamorph N. parvum ) ( 116 ). N eofusicoccum parvum and N. ribis form a fungal complex and are difficult to distinguish molecularly and morphologically (35,51,72,144,145) Single gene p hylogenetic analysis and restriction fragment length polymorphisms (RFLP) are unable to differentiate these species ( 3,117,144,145 ). Conidia of N. parvum are slightly smaller than those of N. ribis and turn light brown and multi septate with age ( 116 ). P artial
16 sequences of elongation factor one alpha (EF 1 (RPB2) are used to distinguish these two species ( 88,93,94 ). Genetic regions such as tubulin, EF1 ITS, and RPB2 have been used to identify and describe cryptic species within N. parvum/N. ribis complex which i nclude: N. cordaticola N. kwambonambiense and N. umdonicola ( 93 ). Anamorphs of Botryosphaeriaceae More than 18 genera have been associated with Botryosphaeria and have included Diplodia, Dothiorella, Fusicoccum, Lasiodiplodia, Phylosticta, and Sphaeropsi s ( 31 ). In 2002 Denman (34) combined the anamorphs of Botryosphaeria into two lineages: Diplodia dark pigmented conidia, and Fusicoccum, hyaline conidia. The taxonomic subdivision was supported by subsequent studies which included a larger array of ta xa and DNA based markers ( 31 ). However, the distinction was questioned by Saccharata proteae, which was separate from the Diplodia like and Fusicoccum like groups ( 34,35 ). In 2006, Crous (31) redefined Botryosphaeriaceae, and limited Botryosphaeria to th e type specimen B. dothidea and B. corticis. The name Botryosphaeria was no longer available for species with Fusicoccum like or Diplodia like anamorphs. N ew genera such as Neofusicoccum were described to accommodate the reevaluation or previously defin ed taxa are referred to by the anamorph name only, and examples include Diplodia, Lasiodiplodia, and Macrophomina Within Botryosphaeriaceae 10 lineages were recognized including an unresolved clade ( Diplodia / Lasiodiplodia / Tiarosporella ). Phylogenetical ly resolved genera within Botryosphaeriaceae are Botryosphaeria str ( Fusicoccum anamorphs), Macrophomina Neoscytalidium Dothidotthia ( Dothiorella anamorphs), Neofusicoccum ( Botryosphaeria like teleomorphs, Dichomera like
17 synanamorphs), Pseudofusicoccum Saccharata ( Fusicoccum and Diplodia like Botryosphaeria quercuum ( Diplodia like anamorph), and Guignardia ( Phyllosticta anamorphs) ( 31 ). In 2008, Phillips (98) resolved the dark spored teleomorph genera in the Botryosphaeriaceae which i ncluded the Diplodia / Lasiodiplodia / Tiarosporella clade. Three lineages and at least six genera were identified and included Neodeightonia, Phaeobotryon, Phaeobotryosphaeria (anamorph Sphaeropsis ), Barriopsis, Spencermartinsia, and Dothiorella Dothidotth ia was moved to the newly created family Dothidotthiaceae and was placed in the Pleosporales instead of Botryosphaeriaceae where it originally was grouped by Crous (98) Higher Classification of Botryosphaeriaceae In the late 19 th century two systems of classifications emerged; Saccardo grouped species based on morphological characteristics of conidia and Lindua proposed a second system which attempted to place fungi in natural or phylogenetic groups. ant classification and is the system on which current Ascomycete taxonomy is based ( 34 ). In 1897 Lindua placed Botryosphaeria in the Melogrammatacaeae and was moved to the Sphaeriales which were defined by fungi lacking as costroma. T he Dothideales were characterized by the formation of asci in locules embedded in stroma. In 1907 Von Hhnel relocated Botryosphaeria into Pseudosphaeriaceae defined by single locule, multiascal ascostromata which was moved to Dothideales. In 1915 Thessen and Sydow created the sub family Botryosphaerieae, and in 1918 created Dothideineae and assigned Pseudosphaeriales, Botryosphaeriaceae, and Botryosphaeria into the new grouping ( 34 ).
18 Due to confusion over the morphology of ascostromata, interthecial tissues, and true peri thecia, Botryosphaeria was moved into the Pleosporaceae. In 1928 Miller demonstrated the fundamental difference between tissues forming the perithecium and the boundary of the locules which were defined as centrum features. Sphaeriales was defined b y true peritheciea and paraphyse s and Dothideales was characterized as having ascostromat ic ascomata and lacked paraphyse s; Botryosphaeria lacked a true perithecial wall and was moved to Dothideales ( 34 ) In 1932 Nannfeldt proposed the group Ascoloculares whic h built on earlier descriptions of ascolocular development; in 1955 Ascoloculares formerly was proposed as class Loculoascomycetes by Lutrell ( 65 ). All other filamentous ascomycetes were segregated to the Euascomycetes. Separation from the unitunicate as comycetes was widely accepted; however the placement and number of orders within the groups was disputed ( 31,114 ). Within the Loculoascomycetes, the centrum was further expanded by Lutrell; three different ascostromatal development types were described; D othidea, Pleospora, and Elsino are characters which define the modern orders Dothideales, Pleosporeales, and Myriangiales ( 115 ). Lutrell placed Botryosphaeria in the Pleosporales; vonArx and Mller did not support the placement of Guignardia and Botryos phaeria, two closely related genera into separate orders (Dothideales and Pleosporales) (31,35). Instead one order, the Dothideales, was delimited containing two sub orders and 24 families (31,35). Botryosphaeria remained in Botryosphaeriaceae and was re located to the Dothideales (31,35). Barr agreeing with Luttrel, disagreed with the consolidation, created ten orders
19 based on dicaryon and ascus type (7). By the end of the 1980s two systems of classification existed that of Barr & Luttrel, and vonArx & Mller In 1995 Berbee, Spatafora, and Tehler questioned the class validity of Loculoascomycota ( 9,10,62,63,123 ). Subsequent studies using ribosomal RNA and lichenized Verrucariales existed within the Eurotiomycetes as subclass Chaetothyriomycetidae ( 62,63 ). Further results retained the sister group status of the Dothideales and Pleoporales, and in 1997 the class Dothideomycetes was formally described by Eriksson and W inka which replaced the remaining part of the long recognized Loculoascomycetes ( 113,114 ). Disagreement concerning subdivision within the Dothideomycetes was unresolved; the weight and importance of taxonomic characters was heavily disputed and included: centrum development, the presence of p seudothecia, and pseudoparaphyse s ( 114 ). In 2006 Schoch defined two Dothideomycete lineages the pseudoparaphyste Pleosporomycetidae (Pleosporales) and aparaphysate Dothideomycetidae (Dothideales, Capnodiales, and Myri angiales). Currently, 10 orders resided within the Dothideomycetes: Pleosporales, Hysteriales,Mytilinidiales, Patellariales, Botryosphaeriales, Jahnulales, Dothideales, Capnodiales, Myriangiales and Trypetheliales ( 113 ). In an analysis using conserved pr otein regions, fungi in the Botryosphaeriaceae did not group phylogenetically with any previously described orders. Schoch proposed a new order in 2006, Botryosphaeriales, to accommodate fungi in Botryosphaeria and Guignardia. Higher taxonomic classificat ion for fungi in the Botryosphaeriaceae has been enigmatic because of intermediate morphology: pseudoparaphyses are present in
20 immature and absent in mature fruiting bodies ( 113 ). Currently, Botryosphaeriales has one family, Botryosphaeriaceae; however, recent studies have called for a more narrow interpretation of Botryosphaeriaceae, Guignardia (Phyllosticta anamorphs) reside on a separate clade from fungi traditionally identified as Botryosphaeria (114). Stem Blight and Dieback of Blueberries A survey c ompleted in 2007 in Florida found stem blight and dieback of SHB is caused by a complex of fungi in the Botryosphaeriaceae and includes: Botryosphaeria dothidea ((Moug.:Fr.) Ces. & DeNot. anamorph is Fusicoccum aesculi ) Lasiodiplodia theobromae ( ((Pat.) Griffon & Maubl), formerly B. rhodina ) and Neofusicoccum ribis ( ((Slippers.:Crous.:Wingf.) Slippers & Phillips formerly B. ribis ) ( 140 ) Stem blight typically infects current season growth and can rapidly colonize the xylem ( 84 ). Partial or complete oc clusion of the vasculature results in reddening or drying of leaves on affected shoots and brown discoloration on one side of the infected branch ( 84,139 ). If the pathogen s progress into and colonizes the crown of a plant systemic branch dieback occurs a nd eventually kills the plant ( 29,84 ) Host Range Fungi in the Botryosph aeriaceae are non host specific (15,121) I n 1934 Smith (121) reported, Botryosphaeria dothidea was pathogenic on 20 plant familie s, 34 genera, and 50 species. More recently, B. doth idea was isolated from active lesions of 14 plant families, 22 genera, and 50 species in California alone ( 15 ). These fungi also infect many economically important crops which include almonds (49) apples (13,16) blueberries (35,134) eucalyptus (18) gra pes (88,132 135) mangos (107) peaches (32), and pistachios ( 72,80 ). Botryosphaeria also can affect plant species diversity in
21 tropical ecosystems, and in years of drought cause severe dieback on chaparral vegetation ( 15,131 ). Botryosphaeria Latent P hase The majority of fungi the Botryosphaeriaceae have been characterized as fungal endophytes and include Botryosphaeria Diplodia, Dothidotthia, Guignardia, Lasiodiplodia, Neofusicoccum and Pseudofusicoccum ( 118 ). Individual hosts can be infected by a diverse community of endophytes; eight s pecies in the Botryosphaeriaceae have been found co inhabiting Syzgium cordatum in South Africa ( 95 ). P athogens have been known to be op portunistic endophytes; Neofusicoccum australe has been isolated from twigs of Eucalyptus gl o bulus in Western Australia However, in its native ranges in Australia and Tasmania N. eucalyptorum and N. eucalpticola were isolated in the highest frequency ( 19 ). Latent infections begin w hen fungi in the Botryosphaeriaceae penetrate the host through natural openings such as lenticels, stomata, or other openings on healthy plants ( 22,106,118 ). The formation of appresoria on the surface of apple fruits also has been observed ( 56 ). Almost all plant organs have been reported to host late nt species in the Botryosphaeriaceae from the bark and vascular tissue of stems, to fruits, flowers, and seed capsules ( 38,46,56,84 ). Lasiodiplodia theobromae and Diplodia pinea have been reported to infect pine and Podocarpus seed (46) Likewise, Neofusico ccum batangarum is a seed born pathogen and has been reported as a potential biocontrol agent of Brazilian pepper (115). H owever, little evidence is present that systemic infection occurs as plants mature ( 39,115,118,124 ). Conidia and ascospores are so urces of inoculum for dispersal, dissemination, and latent infection for fungi in the Botryos phaeriaceae ( 1,5,80,91,126 ). In nursery settings
22 the number of latent infections on seedlings increased when seedlings were placed under mature plants ( 118 ). In fection frequency typically favors increased periods of wetness; in California Botryosphaeria dothidea infects the panicles and shoots of pistachios during the fall and winter (rainy season) (72) Pistachio i nfections typically remain latent until conditi ons are favorable (temperatures above 27C) ( 1,82,83,89 ). Latent infection also occurs during the early developmental sta g es of fruit (55,56,92) For example Botryosphaeria infection of apples can occur within 7 weeks of petal fall (92). P hytoalexins an d benzoic acid inhibit fungal growth on immature fruit, as the fruit matures sugar levels increase and macrosco pic symptoms begin to appear ( 54 57,92 ). Disease Cycle of Fungi in the Botryosphaeriaceae When Botryosphaeria dothidea enters through natural o penings on blueberries the fungus is restricted to the outer portion of the lesion by a thickened periderm layer ; a s a result the fungus is unable to move through the vasculature (84) In contrast, Botryosphaeria corticis penetrates blueberry stems throu gh natural openings; cankers result from increased cell numbers due to the disorganization of the cortex and phloem (85) Invasion of wounded or succulent stems by fungi in the Botryosphaeriaceae results in the rapid breakdown of phloem and cortical tiss ues ( 12,17,108 ). After pathogen entry, mycelium progresses rapidly down the vasculature while lateral movement through pits and intercellular spaces occurs more slowly ( 12 ). All cell types are colonized and include: callus parenchyma, cortical parenchym a, xylem ray parenchyma, trachieds, and vessels ( 17,108 ). Host defenses such as callus tissue and lignified cells do not restr ict fungi in the Botryosphaeriaceae ; mortality is the result of partial or complete occlusion of the vasculature ( 17,108 ).
23 Pis tachios infected with B. dothidea are retained longer on the tree than healthy plant anatomy; partially submerged pycnidia provide a source of inoculum ( 17,83 ). Pycnidia maturation occurs after 12 days at temperatures ranging for 10 36C and peak productio n occurs at 30C ( 17,81,82 ). Conidial production occurs 4 6 weeks after inoculation at temperatures between 10 30C for isolates of B. dothidea, D. seriata, and L. theobromae (27,81,132) Optimum reported temperatures for sporulation are 24C for B. doth idea, 18C and 24C for D. seriata, and 12C, 18C, and 24C for L. theobromae ( 22,132 ). Conidia germinate between 4 6 hours after inoculation at temperatures ranging from 5 40C; germ tubes consistently grew toward the wounded area suggesting a chemotac tic response (12,137). Germination is favored between 98 100% relative humidity; percent germination decline s severely when humidity levels are less than 100% (91). Continuous moisture is necessary for 12 h for B. dothidea to penetrate pistachio lenticels stomata, fruit, and wounds ( 82,83 ). Interrupted wetness periods of one hour or more irreversibly stop infection and significantly reduce disease incidence ( 5 ). Inoculum is produced on current and prior season growth; on pine cones, conidia continuousl y are produced in three year old pycnidia of Diplodia pin e a ( 38,87 ). Conidia have been detected from February to November in blueberries (28) peach (137) and pistachio orchards ( 80,89 ). The highest levels of conidial inoculum have been recorded during t he months which receive the highest amounts of rainfall: May to July for blueberries in North Carolina (28) July to mid August for peaches in Georgia (137) and December to February for grapevines in California ( 132 ). Conidia are spread
24 mostly by rain; however insects, birds, and water from irrigation systems have been reported to spread conidia ( 52,81 ). Light rain is more conducive for spore deposition than heavy rain because splashed dispersed conidia of B. dothidea have a relatively short dispersal distance ( 1,28 ). The number of continuous rainy days and increased su mmer temperatures are correlated positively with disease severity ( 82,83,89 ). Plant Health and Disease Transmission Stresses such as drought (86,105) defoliation (90) and nutrient def iciencies (14) predispose a plant to infection by fungi in the Botryosphaeriaceae. Drought limits photosynthetic production and the accumulation of carbohydrates which aid the host in defense ( 36 ). Stress related diseases typically are ca used by facultat ive saprophytes and latent pathogens ( 117,128 ). Susceptibility to B. dothidea increases as plant water 12 to 13 MPa ( 30 ). Once tu r gor pressure is restored resistance to B. dothidea is res tored within 3 5 days (30) If a plant is exposed to prolonged periods of drought an d extremely low susceptibility to B. dothidea can be irreversible ( 112 ). Defense responses are affe cted in drought stressed plants; protein and enzyme synthesis as w ell as carbohydrate production decrease ( 36 ). Hyphal growth is large and round in stressed birch stems, as compared to hyphal growth inside healthy stems which is irregular, contorted, and restricted to the xylem vesicles (78 ). In stressed hosts defense responses such as lytic activity also are suppressed ( 14 ) Stored carbohydrates are utilized due to reduced photosynthetic production; callus formation is limited which compromises the ability of the host to wall off invading hypha e or close wounds ( 90 ). Catechol and salicin, compounds inhibitory to Hypox y lon mammatum, are inhibited when aspen are drought stressed ( 60 ).
25 74 ). Spore ge rmination, germ tube elongation, and mycelia growth of B. dothidea 2.0 MPa ( 74 ). Mycelial growth increases as water potentials decrease for other fungi including: Botrytis squamos a, Fusarium oxysporum, Monilinia fructicola and Macrophomina phaseolina (1 4 ). Management of Botryosphaeria Stem Blight Cultural and chemical management options for control of Botryosphaeria diseases are similar in many cropping systems including apple (57) blueberry (24) grape (11) peach (8,1 05,137) and pistachio ( 48) Benzimidazoles q uinone outside inhibitors ( QoI ) and sterol biosynthesis inhibitors ( DMI ) reduced external symptoms of Botryosphaeria blight in apple (16) cut flower (33,122) grape (11) and pistachio cropping systems ( 73,7 5 ). Root dip treatments for container grown blueberries limit the development of B. dothidea ( 24 ). Applications of captan, ziram, and tebuconazole on inoculated blueberries produced stem lesions as long as the untreated control while pyraclostrobin reduce d stem lesions but did not prevent disease ( 119 ). Applications of select fungicides such as thiophanate methyl and carbendazim reduced canker on cork oak after cork was removed from trees ( 64 ). Captan and Captifol improved peach tree fruit yield and trunk diameter; however, Botryosphaeria infection was not prevented ( 8 ). Paclobutrazol (PB Z ), a gibberellin inhibitor reduced mycelial growth and spore germination for a broad range of woody pathogens including: Armillaria gallica, Botryosphaeria dothidea, and Fusarium roseum ( 50 ). PBZ enhanced tolerance to environmental stresses and has reduced foliar diseases such as dollar spot ( 20 ). Pre inoculation of arbuscular mycorrhizal fungi improved survival and growth rates for
26 apples when infected with N. ribis ( 59 ) The use of Lippia scaberrima essential oils have displayed anti fungal activity on N. parvum; and plant extracts of Macleaya cordata, Polygonum cuspidatum and Scutellaria have been used to control pop lar stem canker caused by B. dothidea ( 109,146 ) Fungicides have been effective controls for fruit rots caused by fungi in the Botryosphaeriaceae Partial pressure infiltration of prochloraz and pyrimethanil controlled mango stem end rot ( 101 ). Fungicidal applications approximately 10wks after bud brea k have reduced fungal foliar and fruit diseases of cranberry ( 53 ). Late season applications of tebuconazole reduced latent apple white rot infections ( 57 ). The herbicide paraquat has been shown to enhance white rot, and has been used to predict latent inf ections ( 13 ). Botryosphaeria isolates resistant to tebuconazole and iprodione were produced in vitro and retained high le vels of virulence on pistachios (73, 75). T ebuconazole retained efficacy while iprodione could not control mycelia growth of resistant isolates ( 73,75 ). Integrated pest management (IPM) programs including orchard sanitation and irrigation management have offered promising methods for the control of Botryosphaeria diseases. Altering the trajectory angle of sprinklers, drip irrigation, and a reduction in the length of overhead irrigation time has reduced spore release, dispersal, and germination of Botryosphaeria dothidea in pistachio orchards ( 81,83 ). The use of empirical models incorporating wetness durat ion, rain amounts, and temperatur e to predict panicle and shoot blight infection events reduced disease incidence ( 1,82,83,89 ). In apple orchards 24 h irrigation periods are not recommended due to increased disease incidence ( 92 ).
27 Removal of inoculum sources such as blighted shoots re duced the amount of available inoculum for current and prior seasons in pistachio orchards ( 1 ). Infected stems are pruned during peach dormancy and chipped to increase decomposition ( 137 ). In blueberry orchards removal of cold damaged shoots, and the sub sequent treatment with fungicides helped to reduce disease incidence ( 24 ). Delayed pruning of grapevines in California is recommended; current timing coincides with highest periods of spore dispersal by fungi in the Botryosphaeriaceae ( 133 ) Dissertation T hesis S pecies distribution of fungi in the Botryosphaeriaceae cause blueberry stem blight and dieback in the southeastern United States could be dependent on geographic region and Vaccinium spp Effective disease management practices for stem blight coul d include the development of stem blight resistant cultivars through the creation of a n effective Bot. screening protocol. Bot. could be a latent pathogen in propagation material in Florida, w hich could contribute to the early onset of plant morality R esearch O bjectives By Chap t er In Florida, Lasiodiplodia theobromae and Neofusicoccum ribis were identified as the primary pathogens causing stem blight and dieback on SHB (140). However, f ungi in the Botryosphaeriaceae have a wide host range and geograph ical distribution; species allocation is dependent on climate cultivar and geographic location (15,31,49,116). Also, classification and identification of fungi in the Botryo sphaeriaceae has been enigmatic. At least 18 anamorph lineages have been asso ci ated with Botryosphaeriaceae (31). Difficulties exist using the anamorph for species identification: conidia can take up to five weeks to be produced (116,134), production is inconsistent for some species such as Neofusicoccum parvum (102), and morphology
28 can vary with culture age (51). Amplification a nd sequencing of phylogenetically informative regions have aided in rapid identification and phylogenetic elucidation of these fungi (31,72). The refore, an obj ective in chapter two w as to develop a molecula r diagnostic technique which would differentiate fungi in the Botryosphaeriaceae causing stem blight and dieback in Florida. A second objective was to determine if species distribution w as dependent on climate, geographic location, and Vaccinium spp. R esearch on blueberries in the United States, during the late 1980s and early 1990s primarily focused on the development of resistance screening techniques of stem blight caused by B otryosphaeria dothidea (18,45) However, screening for resistance to B. do thidea has been difficult due to ambiguities surrounding how much progress could be made within the breeding stock. In 1989, Gupton and Smith (45) concluded only moderate progress could be made when using mass selection because of equal general and specif ic combining abilities (GCA and SCA). However, in 1990 Buckely (18) concluded GCA and SCA were significant using a highly virulent isolate and recommended progeny screening for identification of resistant parents R esistant seedlings were recovered from all cross combinations (18) Resistance to stem blight is heritable (141); however, the ability to screen for Bot. resistance during the earliest stages of the breeding processes is unclear. The refore, the objective of chapter three was to develop a protoc ol which could screen SHB seedlings for resistance to fungi in the Botryosphaeriaceae. The life cycles of fungi in the Botryosphaeriaceae can consist of a saprophytic or an endophytic phas e ; entry typically is gained through wounds ( 34,118,128). However, fungi in the Botryosphaeriaceae also can colonize a healthy plant through natural
29 openings inclu ding lenticels and stomata. C olonization can result in a latent stage in the disea se cycle (118 ). Disease development typically is triggered when the host un dergoes a period of stress induced by physiological and environmental factors includi ng cold damage (30), defoliation (90 ), and drought stress ( 112 ). SHB are propagated from softwood cuttings (sw) during the summer months or rainy season (May 1 to Oct 1) in Florida During the months of sw propagation, environmental conditions are within the optimum ranges for Bot. spore production (28,132), dissemination (28 ), an d germination (12,27). The objective of chapter four was to determine t he frequency and path ogenicity of Bot. fungi latently infecting SHB propagation material in Florida. As well as to determine if s tem blight disease incidence and severity var y between plants propagated from tissue culture at a single location in Lowell, OR and softwood cuttin gs propagated from a single site in Hawthorne, FL at three commercial locations in Florida.
30 CHAPTER 2 TAXONOMY, PHYLOGENY, AND IDENTIFICATION O F FUNGI IN BOTRYOSPHAERIACEAE CAUSING STEM BLIGHT AND DIEBACK OF VACCINIUM SPP. IN THE SOUTHEAS TERN UNITED ST ATES Overview of Botryosphaeriaceae Taxonomy The blueberry industry in the s outheastern United States has capitalized on an early season high dollar niche market which is due to the development of low chill early ripening cultivars (72). Southern highbus h blueberry (SHB; interspecific Vaccinium corymbosum L.and V. darrowi Camp hybrid section Cyanococcus ) and rabbiteye blueberry (REB; V. virgratum Aiton section Cyanococc u s ) cultivars are grown in the southeastern US; in 2009 the blueberry industry in Alaba ma, Florida, Georgia, and North Carolina was valued to be $180,322,200 for approximately 1 9,016 harvested acres ( Table 3, USDA, National Agriculture Statistics Service, http://usda.mannlib.cornell.edu ). However, fungal vascular diseases are an ongoing pro blem for the southeastern blueberry industry (6). Stem blight and dieback result in premature plant mortality and have been cited by growers as the most economically important diseases that they face. In Florida stem blight and dieback of blueberry are c aused by a complex of fungi in the Botryosphaeriaceae (Bot.), which includes many opportunistic plant pathogenic genera (140). Bot. pathogens have been found parasitizing many agricultural crops including apples (13), grapes (88,133 135), mangos (107), pea ches (12,137), and pistachios (80). These pathogens enter through lenticels, stomata, wounds, a nd colonize the vasculature; life cycles can consist of a saprophytic or an endophytic phase (12,28,89,118,128). Disease development typically begins when a plan t sustains a
31 wound or is stressed by environmental or physiological conditions such as defoliation (90), drought (30), or winter freeze damage (112). Specifically, infection of blueberries by fungi in the Botryosphaeriaceae results in partial or complete occlusion of the vasculature, causing the rapid breakdown of xylem tissues (84). Characteristic stem blight symptoms include reddening and drying of leaves on affected shoots and brown discoloration typically is present in cross section on one side of a n infected branch (29,84). Systemic branch dieback occurs when the pathogen progresses into and colonizes the crown of a plant which results in mortality (84). Stem blight reduces yield, and dieback reduces plant longevity, which can result in substantial replant costs. Management recommendations include, aggressive pruning (136), fungicide applications (24), str ess management (74,111), and ro g ue ing of dead plants and infected tissue (1,13 17). However ; cultural practices do not consistently control the diseases (6). Botryosphaeria dothidea ((Moug.:Fr.) Ces. & DeNot.) was first described in 1963 by Witcher and Clayton (139) as the causal agent of stem blight on blueberries in North Carolina. In latter half of the 20th century, B. ribis and 23 other desc ribed fungal species were considered as synonyms of B. dothidea (116,139). Currently, Neofusicoccum ribis ((Slippers.:Crous.:Wingf.) Slippers & Phillips formerly B. ribis ) and N. parvum (Pennycook.:Samuels) Slippers & Phillips formerly B. parva ) are consi dered distinct species from B. dothidea (31,116). Recently, the genus was reclassified to a single monophyletic group including the type species B. dothidea (anamorph = Fusicoccum aesculi ) and B. corticis ( ( Demaree.:M.S. Wilcox) Arx and Mll ) (31). Taxa e xcluded from the monophyletic clade either were transferred into newly described
32 genera such as Neofusicoccum or now are referred to by anamorph names such as Diplodia De. Not. Lasiodiplodia ((Pat.) Griffon & Maubl), and Macrophomina ( (Tassi) Goid., Ann ali Sper) (31). Thus far, no teleomorph names have been proposed for species with Fusicoccum like and Diplodia like anamorphs (31,49). At least 18 anamorph lineages have been associated with Botryosphaeriaceae and are commonly discriminated by conidia m orphology (31). Difficulties exist using the anamorph for species identification: conidia can take up to five weeks to be produced (116,134), production is inconsistent for some species such as N. parvum (102), and morphology can vary with culture age (51 ). Amplification a nd sequencing of phylogenetically informative regions have aided in rapid identification and phylogenetic elucidation of these fungi (31,72). Molecular techniques such as amplified ribosomal DNA restriction analysis (ARDRA) (4), PCR fin ger printing (3), and PCR restriction fragment length polymorphisms (PCR RFLP) have been used to discriminate Botryosphaeriaceae species (95,117). In Florida a complex of fungi in the Botryosphaeriaceae, L. theobromae and N. ribis were identified as the primary pathogens causing stem blight and dieback on SHB (140). Botryosphaeria dothidea was only recovered from two samples outside the survey area. Isolation was independent of the type of infection, sample location and time of year (140). Fungi in the Botryosphaeriaceae have a wide host range and geographical distribution; species allocation is dependent on climate cultivar and geographic location (15,31,49,116). The refore yearly shifts in the pathogen population of fungi in the Botryosphaeriaceae are possible. The objectives of the study were to
33 identify fungi in the Botryosphaeriaceae causing stem blight and dieback on Vaccinium spp. in the southeastern United States using morphological and molecular techniques. Materials and Methods Sample Colle ction and Pathogen Isolation Stem blight and dieback samples were collected from SHB and REB c ultivars from Alabama, Florida, Georgia, North Carolina and South Carolina between 2009 and 2010 (Figure 2 1). In total 346 samples were collected; 246 samples w ere collected from SHB, and 100 samples were collected from REB cultivars. Stem blight samples consisted of partially hardened succulent stems from current season growth. Dieback samples consisted of a major cane approximately 5 to 15 cm in diameter which was taken no further than 12 cm above the crown of a plant. Pathogen isolation and storage followed the methods of Wright and Harmon (140). Botryosphaeria like colonies were characterized as having white aerial mycelium which became light to oliva cious gr ay within 5 to 7 days. Isolates were grown on V8 agar [ (1 liter is 15.0g agar, 200ml V8 juice, 2.0g CaCO 3 ) amended with 0.01mg of rifampicin (rif) and 0.25 g of ampici llin sodium salt (amp) ] The morphology of conidia was evaluated following the procedur es described by Wright and Harmon (140). Molecular Methods Fungal samples were prepared by collecting 0.1 mg of a e r i al mycelium from 7 day old colonies. Genomic DNA was extracted using a Qiagen Dneasy Kit (Qiagen Santa Clarita, CA). As previously describ ed by Wright and Harmon 2010, the ITS rDNA and partial EF1 s were amplified using the ITS1 F/ITS4 and EF1 728/EF1 tubulin were amplified using the primer pairs Bt2a/ Bt2b (40).
34 PC R reaction mixtures, as well as the PCR conditions for the amplification of ITS rDNA and EF1 Harmon 2010. tubulin PCR conditions were selected as follows: initial denaturation at 94C for 3 min; 40 cycles at 94C for 60 s, 60C for 45 s, and 72C for 60 s; final e xtension at 72C for 10 min; then amplification products were held at 4C (116). Five (Fisher Sci Borate EDTA buffer (TBE) (Promega Corporation, Madison, WI) and were visualized with UV light. PCR products were purified using the MinElute PCR Purificatio n Kit (Qiagen Inc. ). Sel ect EF1 tubulin, and ITS rDNA sequences were cloned into Escherichia coli JM109 high efficiency competent cells (Promega) using pGEM T Easy vector were sent for bidi rectional sequencing to the Interdisciplinary Center for Biotechnology Research (ICBR, University of Florida, Gainesville, FL). rDNA Restriction A nalysis Forty Botryosphaeria isolates were used to identify restriction fragment length polymorphisms (RFLP) in the ITS rDNA amplicon using the web based REPK program (26). The ITS rDNA was digested with the restriction enzymes Kas I, Sma I, or Sty I (New England Biolab s (NEB), Ipswich, MA), all digests contained a final volume of 15L The restriction digest of Ka s I consisted of 1.5L of NEB Buffer 4, 1.5uL of 10x b ovine serum albumin (BSA), 1.0L of en zyme, 2L of PCR product and 9L of sterile water. The digest of Sma I consisted of 1.5L of NEB buffer 4, 1.0L of enzyme 2l L of PCR product, and 11.5L of sterile water. Likewise the Sty I digest consisted of 1.5L of NEB
35 buffer 3, 1.0L of enzyme 2L of PCR product and 11.5L of sterile water. Digests were incubated for 1 h at 25C for Sma I and 37C for Kas I and Sty I. Restriction fragments were separated on a 1 .8% agarose gel amended with 0.5g/1mL of ethidium bromide then visualized with UV light. Sequence and Phylogenetic Analysis Sequence data for each gene was aligned locally using CLUSTAL W (130); alignment accuracy was checked manually using McClade 4.08 OSX (77). Phylogenetic analysis was completed using 24 isolates selected at random from the southeastern US (Table 2 1) Isolate sequences were compared to related sequences published in GenBank (Table 2 2). CMW7063, a Bionectria isolate, and SEGA49, a G ui g n ardia isolate, were used as outgroups in this study. Single locus datasets were analyzed using maximum parsimony in PAUP 4.0b10 (127). A partition homogeneity test in PAUP was used to verify statistical congruence for the multiple data sets. The comb ined data sets were analyzed using two algorithms: maximum parsimony in PAUP and Bayesian posterior probabilities in Mr. Bayes vs 3.1 (110). For the combined data set, maximum parsimony was analyzed using the heuristic search option (TBR branch swapping) with an additional 100 random sequence additions. Alignment gaps were treated as missing data; all characters were considered unordered and of equal weight. To estimate branch support, bootstrap values were determined using 1000 replicates and 100 random sequence additions. The best nucleotide substitution model was determined using jModelTest, using the Akaike and Bayesian information criteria (AIC and BIC, respectively) (104). In Mr. Bayes th e general time reversible model with gamma distribution (GTR + G) was used for all loci, and different rates of evolutio n were applied to each locus. Metropolis
36 couple Markov chain Monte Carlo (MCMC) consisted of two simultaneous runs for 10 6 generations. Bayesian probability distributions were obtained from the 50% majority rule consensus tree by sampling trees every 10 2 generations. After the analysis the first 200 trees for each run were discarded. The consensus tree was based on the remaining 800 trees for each run. Results Molecular and Morphological Ident ification of Botryosphaeria spp. Two hundred ninety nine colonies exhibiting Botryosphaeria like growth were identified from a total of 346 SHB and REB samples. Botryosphaeria like colonies were recovered from 38 out of 47 samples in AL, 59 out of 68 in FL 88 out of 102 in GA, 104 out of 119 in NC, and 10 out of 10 samples in SC (Table 2 3). Either Alternaria spp. Pestalotia spp. or Phomopsis spp. were isolated from the remaining 48 samples. Restriction digests of the ITS1F/ITS4 amplicon for 299 sample s resulted in 5 distinct patterns. Using Sma I, a two fragment pattern of 417 and 99 bp was produced for 7 isolates which were identified as B. dothidea Kas I produced two distinct banding pattern s consisting of 365 and 178 bp or 418 and 198 bp, for 241 an d 11 isolates, respectively which corresponded to isolates of N. ribis and D. seriata. Lastly, Sty I produced two distinct pattern s consisting of 265 and 216 bp or 308 and 208 bp, for 241 and 38 isolates, respectively and corresponded to isolates of N. rib is and L. theobromae ( Figure s 2 1 and 2 2, and Table 2 3). N eofusicoccum ribis was isolated from 241 samples throughout the survey area, whereas L. theobromae was isolated from 38 samp les and was only found in AL, FL, and GA Botryosphaeria corticis was isolated from one SHB sample in NC; B. dothidea was isolated from 7 samples and was recovered from all states with the exception of
37 SC. Likewise, D. seriata was isolated fro m 7 samples, and was found in AL GA, and SC Sequence Analysis tubulin, EF1 rDNA sequences were amplified f rom a subset of 24 Botryosphaeria isolates which were representative for the observed PCR RFLP patterns Each PCR product consisted of 550 bp for ITS rDNA, 410 bp for tubulin and 280 bp for EF1 (data n o t shown) PCR products were cloned sequenced and deposite d in GenBank accession No. JN60783 to JN607151 ( Table 2 1 ) The initial consensus tree in Figure 2 4 was composed of 36 taxa including 24 taxa obtained in the current study and 12 taxa c ollected from GenBank (Tables 2 1 and 2 2). A partition homogeneity test indicated datasets were combinable ( p > 0.17). Of the 1256 characters analyzed, 231 characters were parsimony informative ; maximum parsimony analysis yielded 66 most parsimonious tre es (Length = 684 CI = 0.88 RI = 0.96) (Figure 2 4). Bayesian analysis produced trees with topology identical to the parsimony analysis. Isolates considered in the phylogenetic analysis formed 8 clades, and two main phylogenetic groups. The two phylogeneti c groups corresponded to species in the Botryosphaeriaceae with Diplodia like and Fusicoccum like anamorphs; branches were strongly supported with bootstrap and posterior probabilities of 100% and 1.00, respectively. Five groups comprised the Fusicoccum cl ade and included N. parvum, N. ribis, the southeastern United States Neofusicoccum clade, B. corticis and B. dothidea. The Diplodia clade consisted of a D. seriata clade and two Lasiodiplodia clades (Figure. 2 4) Two additional trees were constructed u sing maximum parsimony and Bayesian algorithms. The tree shown in Figure 2 5 included samples from an additional 9
38 Neofusicoccum species (Table 2 2). Of the 1347 characters analyzed 244 were parsimony informative and a partition homogeneity test ( p > 0. 4) indicated that the ITS, tubulin, and EF1 the combined data set yield ed 6 most parsimonious trees (length = 816 CI= 0.874 RI= 0.845) (Figure 2 5) Bayesian and the maximum parsimony trees of the combine d data sets had identical tree topology Six distinct clades were formed; clade one included isolates of N. mediterraneum, N. luteum, and N. australe C lade two was comprised of isolates of N. nonquaesitum, N. arbuti, and N. andium Clade 3 only was compo sed of isolates of N. cordaticola ; clade 4 included isolates of N. kwambonambinse and the southeastern Neofusicoccum isolates. W hile clade 5 included the type isolates of N. ribis the southeastern N. ribis isolates, and isolates of N. umdonicola Finally clade 6 was composed of N. parvum isolates. The tree shown in Figure 2 6 consisted of 7 isolates of L. theobromae identified in this study and 11 isolates represen t ing 7 different Lasiodiplodia spp ., and two isolates of D. seriata (Table s 2 1 and 2 2) The ITS and EF1 analysis and a partition homogeneity test ( p > 0.14) indicated that data set s could be combined. Maximum parsimony analysis for the combined data set yield ed 12 most pa rsimonious trees (length = 309 CI= 0. 841 RI= 0.751). Three clades were formed: clade one included isolates of L. theobromae, L. parva, and L. psuedotheobromae ; clade two was composed of isolates of L. crassisporea, L. gonubienses, L. rubropurpurea, and L. venezuelensis; and clade three cons isted of isolates of D. seriata Conidia Morphology All 24 isolates listed in Table 2 1 could be separated into two groups based on conidial morphology. The first group was characterized by the production of thick
39 walled hyaline conidia which became dar k brown or honey colored with age and corresponded to isolates of D. seriata and L. psuedotheobromae and L. theobromae Conidia of L. theobromae and L. psuedotheobromae became septate w ith age ; longitudinal striations extend ed the length of the conidia However, conidia of L. psuedotheobromae were large r than conidia of L. theobromae. The second group was characterized by production of thin walled hyaline conidia and included B corticis, B. dothidea, and N ribis. Conidial dimensions for these fungi are shown in Table 2 4. Discussion of Stem Blight and Dieback in the Southeastern United States Initial identification of fungi in the Botryosphaeriaceae recovered from stem bight and dieback samples in the southeastern United States was based on PCR RFLP ana lysis of ITS rDNA sequence data. Fungi isolated included: B. dot hidea, B. corticis, D. seriata, L. theobromae and N. ribis Neofusicoccum ribis was isolated in the highest frequen c y; 241 isolates were identified throu ghout the survey area. Lasiodiplodia theobromae was isolated from 38 samples and was not recovered in SC or NC, the northern most areas included in the survey. Likewise Botryosphaeria corticis, B. dothidea and D. seriata were recovered in low numbers. Based on results from the present su rvey N. ribis should be considered the primary pathogen causing stem blight and dieback of Vaccinium spp. in the southeastern United States. PCR RFLP was unable to differentiate the cryptic Neofusicoccum species residing within the N. parvum / N. ribis complex and between a cryptic species of L. theobromae (Figure s 2 4 and 2 5). Findings from the current study are consistent with previous studies which were unable to distinguish cryptic species within Botryosphaeriaceae (94,95,117). Genealogical concord ance phylogenetic species recognition (GCPSR ) was needed to distinguish closely related species (94,95,117). Results from the current
40 study are similar to the 2007 survey completed in Florida; N. ribis was isolated in the highest frequency from sample mat erial. I solates identified as N. ribis in the previous study grouped into a clade separate from the type N. ribis isolates with low phylogenetic resolution (140). T he analysis only included partial sequen ces of EF1 (140). T he additions of tubulin and the newly described cryptic Neofusicoccum spp. provided greater resolution and helped resolve the status of the southeastern Neofusicoccum clade. The southeastern Neofusicoccum clade did not assemble within the traditional N. ribis or N. pa rvum groups ; and instead clustered with isolates of N. kwambonambiense a cryptic species of N. ribis (Figure s 2 4 and 2 5) (93). Further studies should include a fo u rth gene to analyze the cryptic species in the southeastern United States in order to det ermine the distribution and the predominance of the fungi in the N. ribis and N. kwambonambiense groups. Conidia in the N. ribis / N. parvum complex remained hyaline, and did not turn colors with age. Many of the isolates tested did not produce conidia ( data not shown). Results were congruent with previous morphological studies of N. parvum ; the majority of the isolates tested were sterile (102). When conidial production occurred, conidia of N. ribis and southeastern Neofusicoccum group could not be diff erentiated based on morphological characteristics (Table 2 4). In contrast morphological differences between the L. theobromae groups was more apparent, conidia of L. theobromae were smaller than the conidia of L. psuedotheobromae (Table 2 4). The reco very of L. psuedotheobromae and L. theobromae in the southern most sampling locations and its absence in the northern regions of South Carolina and the coastal plains of North Carolina could be due to climatic differences. Similar findings
41 have been repor ted in studies on grapevines in California and Western Australia; L. theobromae was isolated from the warmest areas surveyed (129,135). Other environmental factors such as humidity and precipitation may also influence the distribution of L. psuedotheobrom ae and L. theobromae in the Southeastern United States. Fungi in the Botryosphaeriaceae which were isolated infrequently from the present study included: Botryosphaeria corticis B. dothidea and D. seriata Botryosphaeria corticis was isolated o nce from a SHB sample in NC. In 1942, Damaree and Wilcox described Botryosphaeria corticis as the causal agent of blueberry cane canker; and at the time it was considered the most devastating pathogen for blueberries in the region (6,85). In 1970 stem bl ight caused by B. dothidea became more of limiting factor in production rather than cane canker (25). Decline in B. corticis predominance was caused by the release of resistant cultivars (6). Also histopathological differences between B. corticis and B. d othidea could have contributed to the decline of B. corticis as a major pathogen of blueberries (84,85). Botryosphaeria corticis proliferates the cortical parenchy ma of the xylem causing cankers; in contrast B. dothidea rapidly moves down the xylem and br eaks down all plant tissues (84,85). Despite the ability of Botryosphaeria dothidea to more efficiently colonize blueberry plant tissue relative to B. corticis the pathogen only was isolated from seven samples in the southeastern United States. With the exception of South Carolina, B. dothidea was isolated from all states in the survey area; results could be due to a relatively small sample size collected from South Carolina. Infrequency of B. dothidea also could be a ttributed to the recent taxonomic cha nges ; Neofusicoccum ribis and N.
42 parvum now are considered separate species (116 ). In a previous study completed in Florida, B. dothidea wa s shown to be less virulent than N. ribis or L. theobromae (141). Therefore the ability of B. dothidea to infect a host also could affect isolation frequency. R ecovery of Diplodia seriata was sporadic; eleven isolates were identified from samples in Alabama, Georgia, and South Carolina. The majority of D. seriata isolates also were identified from REB samples. D. se riata is an important pathogen of pome and stone fruits causin g cankers, and black rot on fruits (13,99). On grapes reports of the pathogenicity of D. seriata range from weakly pathogenic to a primary cause of canker and vascular streaking (133). In Mex ico virulence of D. seriata is reported to be dependent on the type of host tissue used for inoculation (134). In the southeastern United States, pathogenicity and impact of D. seriata on blueberries has yet to be determined. This study inclu des only one year of data from the southeastern United States, pathogen population shifts from year to year are a possibility. However samplings from mult iple years in Florida, indicate a relatively stable pathogen population: B. dothidea, L. theobromae and N. ribis were recovered in relatively the same frequency (140). Further sampling of the pathogen population over multipl e years in other states in the s outheastern US and from wild Vaccinium spp. could help to further define the potential for populat ion shifts. The i mpact of individual fungi in the Botryosphaeriaceae on the blueberry industry is unclear; species have been isolated independently from stem blight and dieback symptoms and from SHB and REB cultivars. Currently, management of stem blight and dieback on blueberries does not target specific fungi in the
43 Botryosphaeriaceae Further research such as fungicide sensitivity assays, and differences in cultivar resistance between the species should be evaluated. Figure 2 1 Map of the south eastern United States indicating counties with commercial blueber ry production where stem blight and dieback were collected. Alabama = Chilton, Clay, Coosa, Henry, Huston Co.; Georgia = Atkinson, Bacon, Camden, Clinch, Irwin, and McIntosh Co.; Florida = A lachua, Citrus, Desoto, Marion, Polk, and Sumter Co.; North Carolina = Bladen, Craven Duplin, New Hanover, and Pender Co.; and South Carolina = Pickens Co.
44 T able 2 1. Representative isolates of fungi in the Botryosphaeriaceae from Vaccinium spp. in the southeastern United States used in phylogenetic analysis GenBank Accession No. Isolate a Species Host b Season Origin tubulin EF1 c ITS d SENC96b Botryosphaeria corticis SHB Summer New Hanover Co, NC JN607129 JN607106 JN60 783 SEAL20b B. dothidea R EB Summer Huston Co, AL JN607130 JN607107 JN60784 SEFL62 B. dothidea SHB Summer Marion Co, FL JN607131 JN607108 JN60785 SENC65 B. dothidea SHB Summer Bladen Co, NC JN607132 JN607109 JN60786 SEAL5 Diplodia seriata REB Summer Henry Co, AL JN607133 SEAL 34 D. seriata REB Summer Coosa Co, AL JN607134 JN607110 JN60787 SEGA6d D. seriata REB Summer Bacon Co, GA JN607135 JN607111 JN60788 UF0628 D. seriata Ilex spp. Alachua Co, FL JN607136 JN607112 JN60789 SEAL9a Lasiodiplodia theobromae REB Summer Henry Co AL JN607137 JN607113 JN60790 SEFL3 L. theobromae SHB Summer Alachua Co, FL JN607138 JN607114 JN60791 SEFL28b L. theobromae SHB Summer Polk Co, FL JN607139 JN607115 JN60792 SEGA21 L. psuedotheobromae SHB Summer Mackintosh Co, GA JN607140 JN607116 JN607 93 SEFL61a L. psuedotheobromae SHB Summer Erwin Co, GA JN607141 JN607117 JN60794 SEGA70 L. psuedotheobromae REB Summer Marion Co, FL JN607142 JN607118 JN60795 SEFL49 L. psuedotheobromae SHB Summer Desoto Co, FL JN607143 JN607119 JN60796 SEAL2 Neofusico ccum ribis REB Summer Henry Co, AL JN607144 JN607120 JN60797 SEGA5 N. ribis REB Summer Bacon Co, FL JN607145 JN607121 JN60798 SEGA8 N. ribis REB Summer Bacon Co, FL JN607146 JN607122 JN60799 SENC29 N. ribis SHB Summer Craven Co, NC JN607147 JN607123 JN 607100 SENC34a N. ribis SHB Summer Craven Co, NC JN607148 JN607124 JN607101 SEFL27 Neofusicoccum spp. SHB Summer Polk Co, FL JN607149 JN607125 JN607102 SENC7 Neofusicoccum spp. SHB Summer Duplin Co, NC JN607150 JN607126 JN607103 SENC105b Neofusicoccum spp. SHB Summer Bladen Co, NC JN607151 JN607127 JN607104 SEGA49 Guig n ardia spp. REB Summer Mackintosh Co., GA JN607152 JN607128 JN607105 a Fungi identified based on morphology, PCR RFLP, and phylogenetic analyses b SHB = Southern highbush blueberry; RE B = Vaccinium virg r atum c Elongation factor 1 alpha d Internal transcribed spacer region
45 Table 2 2 Botryosphaeriaceae isolates from GenBank used in phylogenetic analysis Isolate a Species Host Origin GenBank Accession No tubulin EF1 b ITS c ATCC 22927 Botryosphaeria corticis Vaccinium spp USA EU673108 EU673291 DQ299247 CBS 119047 B. corticis V. co r ymbosum USA EU673107 EU017539 DQ299245 CMW991 B. dothidea P. nigra New Zealand AY236924 AY236895 AF027751 CMW8230 Diplodia seriata Picea glauca Canada AY972119 DQ280418 AY972104 CMW110492 Lasiodiplodia crassispora Unknown Unknown N/A EF622066 EF622086 CMW13488 L. crassispora Eucalyptus urophylla Venezuela N/A DQ103559 DQ103552 CMW14077 L. gonubiensis Syzygium corda tum South Africa N/A DQ103566 AY639595 CBS456.78 L. parva Cassava field Colombia N/A EF622063 EF622083 CBS494.78 L. parva Cassava field Colombia N/A EF622064 EF622084 CBS 116459 L. psue dotheobromae Gmelina arborea Suriname N/A EF622059 EF622077 CBS 44 7.62 L. psue dotheobromae Citrus aurantium Costa Rica N/A EF622060 EF622081 WAC12535 L. rubropurpurea Eucalyptus grandis Australia N/A DQ103571 DQ103553 CMW10130 L. theobromae Vitex doniana Uganda AY236929 AY236900 AY236951 PD161 L. theobromae Pistacia vera AZ, USA GU251782 GU251254 GU251122 CMW13513 L. venzuelensis Acacia mangium Venezuela N/A DQ103570 DQ103549 PD252 Neofusicoccum andinum Eucalyptus spp. Venezuela GU251815 GU251287 GU251155 PD284 N. arbuti Arbutus menziesii CA, USA GU251813 GU251285 GU251153 PD283 N. arbuti A. menziesii CA, USA GU251814 GU251286 GU251154 PD253 N. australe Acacia spp. Australia GU251879 GU251351 GU251219 CMW13992 N. cordaticola Syzygium cordatum South Africa EU821838 EU821868 EU821898 CMW14056 N. cordaticola S. cor datum South Africa EU821843 EU821873 EU821903 CMW14023 N. kwambonambiense S. cordatum South Africa EU821840 EU821870 EU821900 CMW14140 N. kwambonambiense S. cordatum South Africa EU821859 EU821889 EU821919 PD285 N. luteum Vitis vinifera Portugal GU25188 1 GU251353 GU251221 PD147 N. mediterraneum P. dulcis CA, USA GU251870 GU251342 GU251210 PD311 N. mediterraneum Olea europea Italy GU251835 GU251307 GU251175 PD301 N. nonquaesitum V. corymbosum Chile GU251824 GU251296 GU251164 PD302 N. nonquaestum V. co rymbosum Chile GU251825 GU251297 GU251165 CMW1130 N. parvum Sequoia gigantean South Africa AY236919 AY236890 AY236945 CBS110301 N. parvum Vitus vinifera Portugal EU673095 AY573221 AY259098 CMW7054 N. ribis Ribis rubrum NY, USA AY236908 AY236879 AF 241177
46 Table 2 2 Continued CMW7773 N. ribis Ribis sp. NY, USA AY236907 AY236878 AY236936 PD289 N. ribis Eucalyptus Australia GU251788 GU251260 GU251128 PD300 N. ribis V. corymbosum Chile GU251808 GU251280 GU251148 CMW14079 N umdonicola S. cordatum South Africa EU821855 EU821885 EU821915 CMW14127 N. umdonicola S. cordatum South Africa EU821866 EU821896 EU821926 a Acronyms of culture collections: ATCC = CMW = Culture Collection Forestry and Agricultural Biotechnology Institu te, University of Pretoria, South Africa CBS = Contraalbureau Schimmelcultures, Utrecht, Netherlands PD = University of California Davi s, USA Asterisks represents isolates published in GenBank which were used in initial phylogen e tic analysis b Elongatio n factor 1 alpha c Internal transcribed spacer region N/ A not available Table 2 3 Incidence and isolation frequency of fungi the Botryosphaeriaceae occurring the in the southeastern United States identified by PCR RFLP s State No. Samples Sites SHB/RE B Bot. samples % of Bot Sma I Sty I Kas I B. dothidea N. ribis L. theobromae N. ribis D. seriata Alabama 47 5 18/29 38 81 1 25 5 25 7 Georgia 102 7 38/64 88 86 2 68 17 68 1 Florida 68 6 68/ 59 87 1 42 16 42 North Carolina 119 9 119/ 104 87 3 99 99 South Carolina 10 1 3/7 10 100 0 7 7 3 Total 346 28 246/100 299 86 7 241 38 241 11
47 Figure 2 2 A schematic diagram of the nuclear ribosomal DNA gene cluster and includes the small subunit (SSU), 5.8s, the large subunit, as well as the two internal transcr ibed spacers (ITS1 and ITS 2) Restrict ion sites found in the ITS1F and ITS4 amplification product for B. dothidea, D. seriata, L. theobromae, and N. ribis Figure 2 3 Example of restriction digest patterns produced from the ITS1, 5.8s, and ITS2 region of rDNA for fungi in the Botryosphaeriaceae that occur in the southeastern United States. 1 = Botryosphaeria dothidea 2 = Lasiodiplodia theobromae 3 = Diplodia seriata and 4 = Neofusicoccum ribis 100bp makers separate rest riction enzymes used for the digest.
48 Fig ure 2 4 One of 66 most parsimonious trees generated by heuristic searches (Length = 684 CI = 0.88 RI = 0.96). Bootstrapping percentages and Bayesian posterior probabilities by branches are displayed in order; b ranches without bootstrap support are labeled with Branches with maximal support for both analyses, and isolates obtained from the current study are marked with asterisks.
49 F igure 2 5 One of 6 most parsimonious trees generated by heuristic searches ( L ength = 816 CI= 0.874 RI= 0.845). Bootstrapping percentages and Bayesian posterior probabilities by branches are displayed in order. Branches with maximal support for both analyses and isolates obtained from the current study are marked with asterisks.
50 Fig ure 2 6. One of 12 most parsimonious trees generated by heuristic searches ( L ength = 309 CI= 0.841 RI= 0.751) Bootstrapping percentages and Bayesian posterior probabilities by branches are displayed in order. Branches with maximal support for both analyses and isolates obtained from the current study are marked with asterisks.
51 Table 2 4. Dimensions of conidia of fungi in Botryosphaeriaceae from the Southeastern US in comparison with those reported from previous studies Isolates from this study Fungi from previous studies Species, isolate a b References B. corticis SENC96b 28.91 3.11 7.51 0.98 23.5 32.5 5.5 7 100 B. dothidea SEAL20b 20.03 1.65 5.96 0.52 (15 )20 26( 32) (4 )5 6( 9) 96 SEFL51b 23.07 2.18 6.67 0.89 (20 )23 27( 30) 4 6( 6) 116 SENC65 23.33 1.83 5.29 0.55 26.22.1 5.80.6 139 D. seriata SEAL34 22.82 2.27 8.46 0.73 (18 )21 26( 28) (10 )11 14( 16) 99 SEGA6d 24.63 2.64 8.82 0.86 (29.2 )23 16( 12) (12)10.7 8.4( 7) 135 SESC5 24.57 1 .64 10.7 1.95 L. theobromae SEAL9a 20.9 1.3 10.2 0.5 ( 20)23 27( 30) ( 10)11 13( 14) 129 SEFL3 20.2 1.3 11.9 0.83 (18 )20 25 (9)11 14 135 L. psuedotheobromae SEGA21 25.86 1.91 16.03 1.32 23.5 32 14 18 2 SEGA60 24.57 2.2 14.91 1.91 N. ribis SEAL6 16.71 1.88 5.59 0.52 (16 ) 19 23( 24) 5 6( 7) 116 SEGA8 17.37 1.41 6.23 0.58 16 22.9 4.9 7.8 139 SENC34a 16.67 1.85 5.43 0.6 Neofusicoccum spp UF0440 20.3 1.8 6.8 0.7 16 28 5 8 95 SENC7 20.8 1.6 6.2 1.1 a b Minimum size, most common values, and maximum size for length and width of conidia c Conidia were produced directly on PDA
52 CHAPTER 3 SUSCEPTIBILITY OF SO UTHERN HIGHBUSH BLUEBERRY G ENOTYPES TO BOTRYOSPHAERIACEAE ( BOT.) PATHOGENS Overview of Stem Blight Resistance Breeding The southern highbush blueberry [ SHB; Vaccinium corymbosum L. hybrid V. darrowi Reade ] industry in Florida is an early season high value nich e market; in 2009 the average farm gate value for blueberry production was $72,900,000 for approximately 3 ,000 harvested acres ( Table 3, USDA, National Agriculture Statistics Service, http://usda.mannlib.cornell.edu ). The most economically damaging funga l disease in Florida blueberry production is stem blight, which results in yield reduction and premature plant mortality. Stem blight in blueberry is caused by a complex of fungi in the Botryosphaeriaceae (Bot) and includes: Botryosphaeria dothidea, Lasio diplodia theobromae and Neofusicoccum ribis (140 ). These fungi are opportunistic pathogens, and primarily live as endophytes or saprophytes I nfection often occur s when plants become stress ed ( 118 ). Bot. f ungi infect current season blueberry growth, a nd basipetal movement of the pathogen in the vasculature can progress rapidly ( 139 ). Stem blight symptoms typically include rapid witling and reddening of leaves on affected branches (84 ). Partial or complete occlusion of the vasculature results in br own discoloration typically observed in cross section on one side of the infected branch ( 84,139 ). In severe cases, infection progresses into the crown of the blueberry plant and results in systemic branch dieback which eventually kills the plant. Mortality a ssociated with dieback reduces production and results in substantial replant costs. Fungicide applications ( 24, 119) optimizing irrigation practices ( 26,81) and aggressive pruning (137) for the control of stem blight increase s production costs and d oes not adequately manage the disease (25 ).
53 With the lack of effective chemical and cultural control methods, resistance breeding for management of stem blight in SHB is the most promising method for control. Stem blight resistance in SHB is heritable b ut the degree of additive genetic var iation contributing to resistance to the disease ha s not often been estimated ( 18 ,45 ,140 ). Difficulties in assigning a heritability estimate have been attributed to differences in isolate virulence ( 18 ), and plant stres s ( 120 ). Cultivar variation for stem blight susceptibility has been identified by in vitr o and cut stem inoculations ( 98 ,120 ). However, cultivar susceptibility has been shown to vary between inoculated greenhouse trials and field observations, indicating a high degree of non genetic variation for the trait ( 120 ). The University of Florida (UF) breeding program is based on phenotypic recurrent selection. Cultivar selection proceeds through four stages: in stage I, up to 20,000 seedlings are plan ted in high density plots. After one year plants are evaluated, and the best 2,000 seedling plants are selected and advanced to stage II. Plants are rated for three years, and the best 200 plants are numbered and marked for asexual propagation. The 200 s election s are planted in 15 plant plots and are rated for three years for fruit and vegetative characteristics. The superior 20 stage III plants each year are asexually propagated and planted at multiple locations as stage IV plants. Stage IV plants are evaluated for three to six years. On average, one or two plants are selected for cultivar release from the original 20,000 seedling establishment ( Olmstead personal communication ). Progeny in the UF breeding program are phenotypically diverse and have va rying levels of stem blight susceptibility as observed in the early stages of cultivar
54 development ( 142 ). Stem blight resistance in the UF germplasm is heritable; however, the variation in genotype susceptibility previously reported could be due to varyin g levels of resistance to the two predominate pathogens found in Florida, Lasiodiplodia theobromae and Neofusicoccum ribis ( 142 ) Previous studies have examined inoculated stem cuttings (18,27,105,122 ) H owever, a seedling screening protocol would be mos t usef ul for breeding selection. Therefore, the objective of this experiment was to develop a screening protocol for the earliest stage of plant development that would consistently identify tolerance to stem blight in cultivars and selections of SHB from the UF blueberry breeding program. Materials and Methods Inoculum preparation for accession screening T wo isolates were used : Lasiodiplodia theobromae isolate MixFC6 and Neofusicoccum ribis isolate UF0440. Isolates had been collected and stored on filte r paper at 4C as part of a previous study. Isolates were revived by placing infested filter paper strips onto plates of V8 agar (15.0g agar, 200ml V8 juice (CSC Brands LP ), 2.0g CaCO 3 0.01mg of rifampicin and 0.25g of ampicillin sodium salt in 1 L) at 2 5 C I noculum was prepared from five, three day old cultures of N. ribis, isolate UF0440, grown on V8 agar Cultures were ground i n a blender for 30 s with 250 mL of sterile distilled water (sdw). Mock inoculum consisted of three sterile V8 agar plates blended with 150 mL of sdw. Cuttings were sprayed with inoculum until the leaves were dripping. A sterile paper towel moistened with sdw was placed on top of the inoculated cuttings. P lants were bagged and placed in a 25C incubator (Percival Scientific, Perry IA) receiving 12 h of light (GE Chroma 50, 20W lights) per day; environmental conditions remained unchanged for the duration of the experiments.
55 Differential Response to Pathogen Isolates In the summer of 2009, the cultivars Emerald, Jewel, Mis ty, Primadonna, Snowchaser, and Springhigh were obtained from a commercial blueberry nursery (Fall Creek Farm & Nursery, Inc. Lowell, OR). Plants were maintained in a greenhouse at UF before and after inoculation at temperatures ranging between 20C to 30 C. Five plants of each cultivar were inoculated with L theobromae isolate MixFC6 N ribis isolate UF0440 and a sterile V8 agar plug was used as the negative control. Isolates were grown for three days at 25C on V8 agar as described previously The e xperiment was a randomized complete block design (RCBD); each plant was considered a block. Eight millimeter plugs were excised from the colony margin of each pathogen species, and 8mm control plugs were also taken from sterile V8 agar and placed on the cu t end of a separat ely pruned stem. Inoculum was secured with Parafilm (Pechiney Plastic Packaging Co. Chicago, IL), and the developing lesions were measured twice weekly for three weeks Area under the disease progress curve ( AUDPC ) was calculated The e xperiments were repeated twice sequentially To test if data from the experiments could be combined, a one way Wilcoxon rank sum test was analyzed in SAS (SAS institute Cary, NC). Statistical s ignificance was calculated using analysis of variance with a and inoculum effects. Un rooted Softwood Cutting Inoculations Four cultivars (Snowchaser, Sweetcrisp, Springhigh, and Windsor) were used along with five selections from the UF blueberry breeding program (FL98 325, FL06 372, FL06 382, FL06 483, and FL06 559). Clonal softwood cuttings, between 9 and 16 cm in length were taken from a location mid way down a non woody cane The cuttings
56 ranged from approximately 0.5 to 0.8 cm in diameter. Shears were sterilized with 95% ethanol between each genot ype and after every 4 to 5 cuts Approximately 15 cuttings were taken per genotype. The cuttings were moistened with tap water and were placed on ice for 1 h. After 1 h, the cuttings w ere washed with 10% bleach for 1 min prior to planting in disinfested gallon pots (10% bleach for 1 min). The un rooted cuttings were planted in a RCBD in 100% sphagnum moss moistened with tap water. The cutting was trimmed so that a section of the stem w ith the top two to three leaves was removed. Cuttings then were inoculated the same day as collected from the field using the methods described for the accession screen After one week, the sterile paper towel was removed and lesion lengths were measured twice a week for two weeks. After the paper towel was removed all pots remained bagged and cuttings remained in a moist environment. Lesion lengths after two weeks were used for analysis in SAS. The experiment was repeated twice. The Wilcoxon rank sum test was used to test the experimental effect, and the data were analyzed using a mixed model. Micro cutting Inoculations Un rooted clonal micro cuttings of the cultivars Abundance, Emerald, Jewel, Primadon na, and Snowchaser propagated by tissue culture (Agri Starts Apopka, FL) were obtained. Upon receipt, the plant material was washed with sdw and 20 clonal replicates of each cultivar were transplanted into 30 x 30 cm Petri dishes ( Corning Inc. Corning, NY ) containing moist sphagnum moss. The cuttings were trimmed so that a section of the stem with the top two to three leaves was removed with scissors sterilized with 95% ethanol. Petri plates were sealed with Parafilm and inoculation proce dures followed t he protocol developed for the accession screen
57 After 24 h, the paper towel was removed and plants were rated on an ordinal scale of 8,6,4,2, and 0 corresponding to t he number of cuttings that died; had stem blights extending 3/4, 1/2, or 1/4 of the to tal length of a plant, and cuttings with no symptoms The mean disease score was calculated for micro cuttings of the same cultivar. Ratings were taken in 24 h increments for ten days and AUDPC values were calculated. The experiment was repeated twice. Data were analyzed in SAS using a general linear model and cultivar effects were separated using the Waller Duncan k ratio t test ( k =100). Clonally Propagated Seedling Inoculations Seedlings were selected at random from numbered crosses in th e UF blueberry breeding program and grown in 100% sphagnum moss until four single node cuttings could be made. Cuttings were transferred to a 50:50 w/w peat perlite mix and were rooted under intermittent mist for two months. Mother plants were re grown, and the cloni ng process was repeated. Once cuttings were rooted, the mist application decreased in frequency for approximately one month. The plants were transplanted using a RCBD design into 48 cell trays that were cut in half so 24 cells were used for each block (i ndividual cells were 5.1 x 5.1 cm ). A 50:50 w/w peat perlite mix was used as the transplant medium The inoculation prot ocol followed the accession screening methods as described above. A fter 48 h the moistened paper towel was removed, and lesion length s were measured twice a week for two weeks, and AUDPC was calculated. The Wilcoxon rank sum test was used to test the experimental effect. Data were analyzed using a mixed model, and genotype means we LSD
58 Results Differential R esponse to Pathogen Isolates The SHB cultivars Emerald, Jewel, Misty, Primadonna, Springhigh and Snowchaser were inoculated with a single virulent isolate of either L theobromae or N ribis Cultivar was the only significant factor ( p < 0.05), and AUDPC d id not vary depending on the inoculum source (Table 3 1). The cultivar with the lowest AUDPC was Jewel followed by Snowchaser, Misty, Emerald, Primadonna, and Springhigh, respectively (Table 3 1). Un rooted Softwood Cutting Inoculations Softwood cuttings w ere taken from the cultivars Snowchaser, Springhigh, Sweetcrisp, and Windsor, and the breeding selections FL06 372, FL06 382, FL06 438, FL06 559, and FL98 325. Fifteen replicates of each genotype were inoculated with an isolate of N ribis The experiment was repeated twice within a 4 week period of time. Data from both experiments could be combined after replicates which produced no visible lesions and were removed from the analysis. In total 13 replicates were removed from the analysis. Blighting wa s apparent on some of the un inoculated softwood cuttings; however most un inoculated cuttings were asymptomatic and appeared healthy after two weeks with no visible signs of wi lting or drying Snowchaser had the shortest average lesion length followe d by Windsor Springhigh FL06 382, FL06 483, FL98 325, FL06 372, Sweetcrisp and FL06 559 respectively (Table 3 2). The additional removal of FL06 372 which had the largest discrepancy in results between the two trials improved the R square value in correlation analysis from 0.01 (data not shown) to 0.501 between both trials (Figure 3 1).
59 Micro cutting Inoculations Un rooted micro from tissue culture were taken directly from a sterile environment and inoculated with an isolate of N ribis The experiment was repeated twice. Data were combined based on the Wilcoxon rank sum test. The cultivar effect was significant ( p < 0.05) for the un rooted micro Snowchaser and Abundance respectively (Table 3 3). The correlation analysis between both trials was 0.68 (Figure 3 2). Clonally Propagated Seedling Inoculations Seedlings were taken from the initial SHB bre eding stock were clonally propagated, inoculated with N. ribis isolate UF0440, and were measured for three weeks to calculate AUDPC. Inoculation was repeated after six weeks; the first inoculation was in September and the second inoculation was in Novemb er. For the first experiment cuttings were rooted under intermittent mist beginning in July, mother plants were regrown until the four node stage then cuttings for the second inoculation were rooted under intermittent mist beginning in August. The over a ll model was not significant ( p > 0.05) (Table 3 4); no statistical differences were apparent between different seedling clones. When data from both experiments w ere combined the experimental factor was significant ( p < 0.05). The correlation coefficient for average lesion length was 0.183 between the two trials (Figure 3 3 ). Discussion of Stem Blight Resistance Screening in Florida Lasiodiplodia theobromae, and Neofusicoccum ribis are the most economically damaging fungal pest s of blueberry in Florida. A long term goal of the University of Florida blueberry breeding program is to develop cultivars resistant to Bot. pathogens.
60 Differing levels of susceptibility to diseases caused by Bot. pathogens have been reported in blueberry (103 ) C o rnus spp (86 ) ., Malus domestica (13 ) and Mangifera indica cultivars (107 ) In a previous study, heritability for resistance to Bot. pathogens was demonstrated in the UF blueberry breeding germplasm ( 142 ). However, the germplasm used for calculating heritability had al ready gone through two rounds of selection. A more efficient method would be able to screen for Bot. resistance at the seedling stage of the selection process. The object ive of the current study was to develop a screening assay which would reliably and repeatedly screen blueberry genotypes for Bot. resistance. Additionally, differential cultivar re sponse to Bot. pathogens was ass essed to determine if one pathogen could be used to screen for Bot. resistance. In a preliminary study, isolates of the Bo t. species Botryosphaeria dothidea Lasiodiplodia theobromae and Neofusicoccum ribis were inoculated onto a single blueberry cultivar; B. dothidea was the least virulent of the three pathogens ( 141 ). Therefore, isolates of L. theobromae and N. ribis were evaluated on a subset of genotypes to determine whether susceptibility to the two isolates differed. Results from the present experiments were similar to previous studies; cultivars could be ranked for susceptibility to Bot. pathogens based on lesion leng th ( 25,27,103,120 ) However, no differences were identified when plants were inoculated with different Bot. species (Table 2 1) Based on these results a single virulent isolate of N. ribis was used for all subsequent studies. Neofusicoccum ribis is th e most common species of Bot. in Florida production areas T herefore, resistance to this species should be the primary focus of the University of Florida breeding program (140 ) However, inconsistent results
61 observed during the seedling inoculation experim ent could indicate an inadequate sampling of SHB genotypes or the use of only a single virulent isolate from each Bot species Future studies should include more cultivars and advanced accessions to test for differential levels of susceptibility to fung i in the Botryosphaeriaceae. Additionally, different isolates of L. theobromae and N. ribis should be tested in future experiments. Neofusicoccum ribis is an opportunistic pathogen that invades wounds, and latently colonizes healthy tissue such as petio les, stems, and seed (34,118,128) W hen a host becomes stressed due to abiotic or biotic factors the infecti on cycle is activated (90 112,118 ) Fungi in the Botryosphaeriaceae were previously recovered from un inoculated genotypes used in a SHB resistanc e screening study (142 ). To determine the degree that latent infection can impact lesion length scoring, softwood cuttings from nine genotypes were collected and washed with 10% bleach prior to inoculation. Lesion length variation was sufficient to deter mine genotype differences in susceptibility (Table 2 2) However, the correlation between repeated experiments was low (Figure 2 1). After two weeks, stem lesions were visible on control treatments for both experiments; however the controls used in the s econd experiment were more severely blighted. The general trend was for increased disease severity (as measured by lesion length) in the second experiment. Softwood cuttings for the second experiment were taken 4 weeks later in November The additional d elay in collection could have increased the possibility of exposure to additional biotic or abiotic factors prior to inoculation In Florida conidia production of L. theobromae was observed most frequently during late October and early November in 2007 (1 42 ) The presence of Bot. prior to inoculation likely contributed to the discrepancies between repeated experiments.
62 To address the potential for latent contamination of stem cuttings un rooted micro cuttings propagated from tissue culture were used in the subsequent experiment. Again, genotypic differences in susceptibility to Bot. were apparent based on lesion length scores (Table 2 3) However, disease was not apparent on the un inoculated controls. The correlation between repeated experiments was much higher (Figure 3 3), indicating the screening protocol was repeatable in the absence of latent contamination. The seedling stage in the breeding process is the most attractive stage for Bot. resistance selection because a large number of individuals can be culled from the initial breeding population. For adequate statistical power, the seedling material used was replicated prior to inoculation This was done by softwood cuttings. Based on the previous results, the longer plant material is exposed to the endemic Bot. pathogens in open field and greenhouse settings, the more likely latent Bot. infection may be present (118,142 ) However, blighting symptoms were not observed on the experimental controls, suggesting Bot. was not latently present on cu ttings or present in blueberry seed. Screening seedlings for Bot. resistance produced inconsistent results which varied significantly between trials. Significant differences between experimental results could potentially be attributed to differences in p lant stress at the time of rooting. In July the greenhouse was significantly hotter than when plants were placed under intermittent mist in August. Additionally, plant height could have a role in the variable results between repeated experiments. Plants ranged from 3 to 20 cm in height and generally plants in trial 1 were taller than plants in trial 2. Lesion lengths caused by Bot. could have varied naturally depending on the length of the plant stem (Table 2 4). Non repeatable results could also be du e to a low number of replicates per seedling F urther
63 research should include additional replicates per selection rooting during the same time of the year and selecting plants which are roughly the same height Environmental factors play a key role in initial Bot. colonization and the subsequent onset of the disease ( 74,82,117 ). In healthy plants Bot. is able to colonize the host through natural openings such as lenticels and stomata O nce inside the host the fungus ability to colonize the plant is dependent on the host defense response and stress level (112,118 ). The ability of the host to prevent latent colonization and the severity in which a pathogen is able to latently colonize the plant could be a heritable factor which affects cultivar r esistance. Also the ability of cultivars to recover from stress could be additional heritable factors which contribute to the severity of infection of some SHB cultivars. Further research efforts should focus on the nature of blueberry response to control led stresses in relation to severity of Bot. infection.
64 Table 3 1 Differential response of SHB to inoculation with Lasiodiplodia theobromae (MixFC6) and Neofusicoccum ribis (UF0440) DF Value Pr
65 Figure 3 1 Correlation graph for trials 1 and 2 of un rooted softwood cuttings. Average lesion lengths after two weeks were measured in cm and nine inoculated replicates were measured for each accession in both trials. The correlation coefficient between both trials was 0.51. Table 3 3 Un rooted micro propagated cuttings response Neofusicoccum ribis isolate UF0440 DF F value Pr < F Experiment a 1 1.93 0.18 Cultivar 4 3.75 0.02 Mean b Waller Grouping Primadonna 18.24 A Emerald 23.99 A B Jewel 24.48 ABC Snowchaser 28.88 B C Abundance 33.36 C a The combined results included eight inoculated petri dishes and a total of 160 micro cuttings were rated per cultivar. For each separate experiment one petri dish per cultivar as used as an un inoculated control (data not shown). b. Mean separation for the micro cutting experiments using a Waller Duncan k ratio t test (k=100) ; the experiment wide Minimum Significant Difference (MSE) was 9.04.
66 Figure 3 2 Correlation graph for trials 1 and 2 of un rooted microcuttings. AUDPC was calculated aft er 10 days calculation was based on an ordinal scale (see materials and methods). For each trial a total of four plates were inoculated which contained 20 clones; a total of 80 clones were rated per cultivar. An additional petri plate was designated as th e negative control (data not shown).
67 Table 3 4. Combined res ults from the seedling screen using Neofusicoccum ribis isolate UF0440 DF F value Pr < F Cultivar 19 1.37 0.13 Experiment 1 12.45 0.05 Cultivar*Experiment 19 0.7 0.86 Average Plant Height d. Seedling Trial 1 Trial 2 Means 189s1 6.47 9.7 3.88 230s1 7.33 7.47 5.20 245s1 9.2 6.87 5.26 275s1 2.07 9.13 5.44 251s1 8.83 6.5 5.83 187s1 10.6 7.5 8.53 279s1 13.93 10.87 10.47 160s1 9.93 7.8 11.28 265s1 3.93 8.82 11.67 200s1 10.1 10.47 16. 63 219s1 11.9 6.1 19.46 251s2 14.46 8.5 19.56 172s1 13.4 8.6 19.59 146s1 11.3 10.9 21.97 224s1 11.13 8.9 24.12 194s1 10.67 7.37 25.41 236s1 15.5 7.23 25.56 164s1 13.4 7.21 30.99 286s1 6.2 2.87 31.22 200s2 11.86 5.1 41.77 a. Main effects table of the combined results of the accessions from the seedling screening trial which included 6 inoculated seedlings. One un inoculated seedling of each accession was included with each experiment (data not shown) b. Mean AUDPC values calculated from both e xperiments and the minimum significant difference (MSD) was 9.34. d. Mean plant height in both trials (cm), inoculated replicates were used in each experiment
68 Figure 3 3. Correlation graph between trials 1 and 2 for the cloned seedling inoculations. AUDPC values were calculated after measuring lesion lengths in cm after two weeks. In each trial averages were based on three seedling replicates The correlation coefficient between both inoculation trials was 0.183.
69 CHAPTER 4 EVALU ATION OF SOUTHERN HI GHBUSH CULTIVAR AND PROPAGATION METHODS FOR STEM BLI GHT MORTALITY DURING THE FIRST TWO YEARS OF GROWTH IN FLORIDA Overview of Stem Blight Disease Managment Since the 1980s, blueberry production in the southeastern United States has seen considerable growth due to the development of low chill early ripening cultivars, which has enabled the industry to capitalize on an early season high dollar niche market (66) In 2009, Florida ranked 7 th for commercial blueberry acreage and 2 nd in the United Sta tes for ave rage farm gate value (USDA Economic research service (ERS) ). However, diseases are prevalent in the southeastern production region; f ungal vascular diseases such as stem blight and dieback are an ongoing problem for the blueberr y industry ( 67 ) Stem blight affects yield, while dieback affects plant longevity (142) During the first two years of establishment, mortality associated with dieback results in substantial replant costs (141) As a result stem blight has been cited by Florida blueberry growers a s the most economically important disease problem affecting the industry Stem blight typically af fects current season growth and can rapidly progress down the vasculature ( 85,139 ). Partial or complete occlusion of the vasculature results in reddening or drying of leaves on affected shoots, and brown discoloration is visible on one side o f an infected branch (85 139 ). If infection progresses into the crown of a plant, systemic branch dieback occurs and eventually kills the plant ( 85). Aggressive pruning (137 ), f ungicide applications (24), stress management (112 ), and ro g ue ing of dead plants and infe cted tissue (80,137 ) commonly are recommended for stem blight and dieback management. However, cultural practices do not consistentl y control the diseases (6 ).
70 In Florida, stem blight and dieback are predominantly caused by a complex of fungi in the Botryosphaeriaceae (Bot.) and include: Botryosphaeria dothidea, Lasiodiplodia theobromae, and Neofusicoccum ribis (140 ). The life cycles of Bot. fungi can con sist of a saprophytic or an endophytic phas e ; entry typically is gained through wounds ( 34,118,128 ). However, Bot. pathogens also can colonize a healthy plant through natural openings inclu ding lenticels and stomata. C olonization can result in a latent s tage in the disea se cycle (118 ). Disease development typically is triggered when the host undergoes a period of stress induced by physiological and environmental factors includi ng cold damage (30), defoliation (90 ), and drought stress ( 112 ). SHB are pro pagated from softwood cuttings (sw) during the summer months or rainy season (May 1 to Oct 1) in Florida During the months of sw propagation, environmental conditions are within the optimum ranges for Bot. spore production (28,132), dissemination (28 ), a n d germination (12,27 ). P lants propagated from tissue culture (tc) or produced in regions where stem blight disease pressure is low could have fewer latent Bot. infections and subsequent stem blight symptoms after planting than plants conventionally propa gated in Florida. The objective of this study was to determine t he frequency and pathogenicity of Bot. fungi latently infecting SHB propagation material in Florida and to determine s tem blight disease incidence and severity on plants propagated from tc an d sw at three locations in Florida. Materials and Methods Plot Installation and Maintenance Three commercial sites were located in Alachua, Desoto, and Polk, Co. FL. The Alachua site is 25 km northeast the DeSoto site is 338 km south and the Polk, Co site is 208 km southeast from the University of Florida in Gainesville. All sites were located
71 in centers of SHB production Plant material was purchased once from a softwood cutting propagator in Hawthorne, FL (Island Grove Ag. Products) and a tissue c ulture Jewel at the Desoto and Polk, Co. sites. The Alachua county experiment was incorp orated into a commercial field with raised beds consisting of a mix of soil and pine bark in rows with a single drip irrigation tube. Overhead irrigation sprinklers were installed in every third row. Pine bark mulch was applied as a top dressing to rows prior to planting. Additionally, some beds also were covered with black plastic mulch. Experimental plots were arranged in a randomized complete block design (RCBD) consisting of 8 rows (blocks) each at 0.80 m spacing, with 25 plants per plot. T he Deso to county site was incor porated into a commercial field. The experiment was a RCBD consisting of 7 rows planted at 0.76 m spacing with 29 plants per plot. Plants were installed in raised 100% pine bark beds. Overhead irrigation was placed every third row After one year, single drip irrigation tubs were instal led and placed down each row. T he Polk Co. site also was incorporated into a commercial field. The plot was installed in a RCBD initially consisting of 14 rows planted at 0.76 m spacing with 14 pla nts per plot. After one year two of the end rows were removed. Plants were installed in raised 100% pine bark be ds, do u ble rows of drip irrigation placed d own the center of each row, and b lack plastic mulch covered all rows. Overhead irrigation was not installed at this location. Plots were maintained by growers according to individual
72 management strategies. For all of the field locations, b eds were not fumigated and blueberries had not previou sly been planted Plots were rated for stem blight inciden ce and severity on the Horsfall Barrett scale every two to three weeks starting February 2009 and ending February 2011. A mixed model in SAS (Statistical Analysis Software Cary, NC) and LSD with Tukey adjusted p values tested for differences between treatm ents at each evaluation date. Pathogen Isolation and Identification Dead plants in the plots were removed and cause of mortality was determined. For Bot. isolation, bark of blueberry samples was removed to ex pose the discolored vasculature, and margins w ere excised and cut into pieces between 0.3 and 0.5 cm in length. Four pieces of each sample were surface disinfested in 10% household bleach (0.615% sodium hypochlorite) for 1 minute and rinsed with distilled water. Tissue samples were dried with a pape r towel and plated on 85 mm Petri dishes containing V8 juice agar (1 liter is 15.0g agar, 200mL V8 juice [ CSC Brands LP) 2.0g CaCO3 amended with 0.01mg of rifampicin (rif) and 0.25g of ampicillin sodium salt (amp) ] ). Cultures were incubated at 25C for 5 days. Fungal colonies were identified by growth habit and by spore morphology when present. After two years, plant mortality was compared by propagation method; at each separate location using a mixed model in SAS. In addition a t test also compared r esults between the propagation treatments at each location. Latent Isolations and Identification Apparently he althy softwood cuttings (sw = 3 to 4 node cuttings of newly flushed growth from the upper portion of a plants canopy) of the cultivar Star were c ollected randomly once a month from May to September at the commercial site in Alachua Co.,
73 FL. Pruning shears were washed with 95% EtOH every 10 th cutting. Thirty cuttings were placed in a plastic zip top bag, were moistened with sterile distilled water (sdw), and placed on ice for approximately 1 h. Between 4 and 6 bags of cuttings were collected for a sampling period. F or each experiment 20 cuttings rando ml y were selected. T he leaves of sw were removed, and stems were sliced into 8 10 pieces. Piec es were disinfes ted with 10% household bleach for 90 s, rinsed with sdw, and dried with a sterile paper towel. SW were plated onto potato dextrose agar (PDA)( 1 liter is 39g of PDA amended with 0.01mg of rif and 0.25g of amp). Cultures were incubated at 2 5C for 5 days. Fungal colonies were identifi ed by growth habit and by spore morphology. The margins of colonies consistent with Bot. growth habit were transferred onto PDA. Cultures were sto red following the methods of Wright and Harmon 2010 (140) The leaves and stems of 20 cuttings were placed into individual sterilized 250 mL Erlenmeyer flasks with 50 mL of sdw and shaken at 120 rpm for 2 h. The l y sate was suctioned onto sterile filter paper disks with pores between 2 8m in diameter and placed onto PDA. Incubation, identification, and isolation followed above methods. All Bot. isolates were grown on PDA. Cultures were incubated for 7 days at 25C before DNA extraction. Genomic DNA from all isolates was extracted using a Qiagen Dneasy Kit (Qiagen Santa Clarita, CA). After extraction, oligonucleotide primers ITS1F/ITS4 were used to amplify the internal transcribed spacer regions (ITS1 and ITS2) and 5.8s region of rDNA ( 42,138 ). PCR restriction fragment length polymorphisms (PCR RFLP) were used to id entify Bot. colonies. Reaction mixtures, the thermocycler program, and gel electrophoresi s followed the methods of Wright and Harmon 2010.
74 Purification Kit (Qiagen). ITS rDNA restriction ana lysis was performed on the ITS1 F/ITS4 amplicon as follows: 2 l of purified PCR product were digested with Kas I, S ma I, and Sty I; (New England Biolabs, Ipswich, MA) (New England Biolabs Inc., Beverly, MA) according to ltra violet (UV) light. Select sequences were cloned into Escherichia coli JM109 high efficiency competent cells (Promega) using pGEM T Easy vector systems II Kit (Promega) following the bidi rectional sequencing to the Interdisciplinary Center for Biotechnology Research (ICBR, University of Florida, Gainesville, FL). The ITS rDNA of select isolates was compared using phylogenetic analysis to related sequences of isolates recovered from Vaccin ium spp. in the s outheastern US and with other isolates previously published in GenBank (Table s 4 1 and 4 2). Data sets were aligned using Clustal X 2.06 macosx (Conway Institute UCD., Dublin, Ireland) (130) The Guignardia isolate, SEGA49 was used as the outgroup for phylogenetic analysis. Alignment gaps were treated as missing data, and all characters were considered unordered and of equal weight. Maximum parsimony was performed in PAUP 4.0b10 using the heuristic search option (TBR branch swapping) with an additional 100 random sequence additions (127) To estimate branch support, bootstrap values were determined using 1,000 replications and 100 random sequence additions.
75 Pathogenicity Bot. isolates were selected at random and used for pathogenicit y trials. The experiment was a RCBD each plant was considered a block. Four plants of the SHB cultivar Abundance were inoculated with three isolates each of L. theobromae and N. ribis A sterile agar plug was used as the negative control. Isolates were g rown for three days on V8 agar at 25C. Eight millimeter plugs were excised from the colony margin of each species and were placed on a freshly pruned stem. Inoculum was secured with Parafilm ( Pechiney Plastic Packaging Co. Chicago, IL) and lesions were measured once a week for three weeks. The experiment was repeated twice using different plant material from the same nursery AUDPC values were calculated, and data were analyzed in SAS first using a t test to determine if experimental data could be com bined. ANOVA tested model parameters; Waller Duncan k ratio t test ( k =100) was used to evaluate treatment effects. Results Plot Ratings and Pathogen Incidence The Alachua Co. site had 32 and 31 plant mortalities from plants propagated from sw and tc; Bot. was identified from 65% and 54% of the samples, respectively. Likewise, the Desoto Co. site had 242 and 43 plant mortalities; Bot. was isolated from 96% and 81% of the sw and tc samples. Fifty and 6 mortalities were recorded for plants propagated from sw and tc at the Polk Co. site, Bot. was isolated from 74% and 33% of the sw and tc samples (Table 4 3). Cultivar was the only potential factor affecting plant mortality and Bot. severity at the Alachua Co. site throughout the two year period (Figure 4 1 a nd Table 4 4 ).
76 ratings throughout the evaluation period (Table 4 4 ). Cultivar affected both plant mortality and Bot. severity at the Desoto Co. site during the two yea r period (Figure 4 2 and Table 4 4 ). Propagation also affected plant mortality throughout the rating period; however after two years propagation did not contribut e to Bot. severity (Table 4 4 incidence and 4 2 and Table 4 4 ). Likewise, cultivar, propagation, and the interaction between the two main effects affected disease incidence and severity ratings at the Po lk Co. site (Figure 4 3 and Table 4 4 (Figure 4 3 and T able 4 4). Latent Isolations In total 31 Bot. samples were re covered from the l y sate and sterilized sw. Five Bot. colonies were recovered from the l y sate; colonies were not recovered on 5/28/2010 and 6/28/2010 (Table 4 5). Three l y sate samples were identified as L. theobromae one sample was recovered on 7/28/2010 and two samples were recovered on 9/28/2010. Likewise, three samples were identified as N. ribis one was recovered on 8/30/2010 and tw o samples were recovered on 9/28 /2010. In total 25 Bot. isolates were recovered from apparently healthy sw from May to September (Table 4 6). One isolate of Diplodia seriata was the recovered on 5/28/2010; likewise L. theobromae was recovered once on 6/28/2010. Neofusicoccum
77 ribis was recovered from all isolation periods except 5/28/2010; 23 N. ribis isolates were re covered. Of the 545 characters analyzed, 91 characters were parsimony informative, maximum parsimony analysis yielded 78 most parsimonious trees (Length = 234 CI = 0.86 RI = 0.9 ), which differed by the arrangement of short branches within the B. dothidea D. seriata, L. theobromae, L. psuedotheobromae and N. ribis clades and were supported by bootstrap values between and 95% and 100% (Figure 4 4). Isolates preliminarily identified as either N. ribis or L. theobromae by PCR RFLP grouped within respective c lades. Pathogenicity Select isolates chosen for pathogenicity included N. ribis isolates swc4, swc7b, and swc10b and were collected on 8/30/2010. Three L. theobromae isolates were selected and included swc6b collected on 6/30/2010, as well as wash3 and wa sh7 recovered on 9/28/2010. Both the L. theobromae and N. ribis isolates were pathogenic on the cultivar Abundance (Figure 4 5). AUDPC values after three weeks were significantly different (p < 0.05); isolates of N. ribis had AUDPC values of 106.8, 117.6 6, and 104 for swc4, swec7, and swc10b, respectively. Likewise, isolates of L. theobromae had AUDPC values which were 51.7, 65.3, and 52.5 for swc6b, wash3 and wash7, respectively. Fungi were re isolated from inoculated tissue and were identified by morp hological characteristics. Discussion of Stem Blight Mortality in Florida P lants were received once from a single sw propagator in Florida, and a single tc pr opagator in Oregon. N o s tatistical degrees of freedom exist for the initial planting stock. Ther efore, r esearch inferences, implications and conclusions which can be
78 drawn from the field portio n of this project are limited P la nts propagated from sw died more frequently than plants propagated from tc. Bot. was consistently isolated in higher perce ntages from samples of plant s propagated from sw at all field locations (Table 4 3). Results could indicate that plants were infected with Bot. prior to planting. However the potential for Bot. contamination could vary based on environmental conditions, age of the planting source, and geographic location, and slight variation between individual propagation standards between SHB distributors in Florida. Propagation stock from different sw distributors should be evaluated over multiple years to determine the degree and severity of initial Bot. contamination in the propagation stock in Florida. T he severity of Bot. epidemics varied by locations more plants died at the DeSoto Co. site followed by the Alachua and Polk Co. sites, respectively ( Figures 4 1 thr ough 4 3 and Table 4 2 ) Also plants propagated from tc had more sever e stem blighting symptoms after two years at the DeSoto and Alachua Co. sites (Table 4 4). However, stem blight severity did not increase for plants propagated from tissue culture at t he Polk Co. site T he cultivars Snowchaser and Primadonna which are known to be susceptible to stem blight and dieback were the most severely blighted (Figure 4 1 through 4 3) Dissimilar disease incidence and severity ratings could be due to individual management practices and well as slight differences in environmental conditions between the locations such as chilling hours, rain fall, photo period, and soil type. C lean propagation material could have delay ed infection and symptom development for appr oximately one year for highly susceptible cultivars such as Snowchaser Further research should address the factors contributing to Bot. infection in the field as well as
79 stresses which activate t he infectious cycle. Also tc stock should be either collec ted at different times from the same provider, or received from multip le tc propagators results from these studies could determine how well tc plan ts grow and survive in Florida Bot. isolates were recovered in higher numbers from surface sterilized sw tha n from the l y sate (Table s 4 3 and 4 4). Select isolates collected from the l y sate and sterilized sw were pathogenic, indicating Bot. species recovered from asymptomatic sw could cause disease. Bot. spore dissemination has been reported to be dependent on the amount and duration of rainfall (28 ). When humidity drops below 100%, germination dec lines rapidly ; interrupted wetness periods can irreversibly stop infection (5 ). Therefore, Bot. spores on the outer surface of a stem or leaf may die after a period of drought. As the season progressed, the number of Bot. isolates recovered from sw increased and were the highest during the September sampling period (Table 2 6 ). The highest levels of conidial inoculum have been reported to be between June and July for blueberries in North Carolina and between July and August for peaches in Georgia ( 29,137 ). In Florida observations of conidia production of L. theobromae primarily was observed between October and Nov ember in 2007 (140 ). A greater number of Bot. spores c ould have been produced during the late summer in correspondence with the observed conidial production of L. theobromae Increased recovery also could be due to continuous latent infection events over the previous summer months. Neofusicoccum ribis and La siodiplodia theobromae were the two species predominantly recovered from apparently healthy sw (Table s 4 5 and 4 6 ). In two surveys of stem blight and dieback completed in 2007 and 2010 in Florida and in the s outheastern United States; N. ribis was found to be the predomi nate pathogen (140 ).
80 In 2010, N. ribis was recovered as the pre dominate latent pathogen infecting apparently healthy sw (Table 4 6 ). Select isolates also were more virulent than isolates of L. theobromae (Figure 4 5). Therefore, N. ribis could be propagated in tandem with SHB and rabbiteye cultivars, and could be a potential explanation of the homogeneous distribution of N. ribis in the s outheastern United States The latent Bot. pathogen population should be surveyed over multiple ye ars to assess Bot. ability to latently colonize different cultivars and r esults could aide in breeding efforts to develop resistan ce Further research should include determining if the number and severity of latent infections varies by the amount and dur ation of precipitation. Studies also should address how Bot. is spread through the propagation system and would allow the SHB industry to adopt a set of standards which could help reduce the amount, spread, and movement of pathogens within the propagation system
81 Table 4 1 Representative isolates of Bot. fungi recovered from apparently healthy SHB propagation material in Florida Isolate a Species Host b Origin Genbank Accession No c swc10b col: 8/30/2010 Neofusicoccum ribis SHB Alachua Co., FL JN39874 swc15 col: 9/28/2010 N. ribis SHB Alachua Co., FL JN39873 swc6b col: 6/30/2010 Lasiodiplodia theobromae SHB Alachua Co., FL JN39876 wash7 col: 9/28/2010 L. theobromae SHB Alachua Co., FL JN39875 a Fungi identified based on morphology, PCR RFLP, and ph ylogenetic analyses b SHB = Southern highbush blueberry c Internal transcribed spacer region Table 4 2. Bot. isolates from GenBank used in phylogenetic analysis Isolate a Species Host Origin GenBank Accession No. b SENC96b Botryosphaeria corticis V accinium spp NC, USA JN067083 UF0730 B. dothidea Vaccinium spp FL, USA GU595170 CMW7054 B. ribis Ribis rubrum NY, USA AF241177 CMW8230 Diplodia seriata Picea glauca Canada AY972104 UF0628 D. seriata Ilex spp. FL, USA JN06 0 789 CMW10130 Lasiodiplodia th eobromae Vitex doniana Uganda AY236951 SEAL9a L. theobromae Vaccinium spp AL, USA JN067090 SEGA70 L. psuedotheobromae Vaccinium spp GA, USA JN0607095 SEFL61a L. psuedotheobromae Vaccinium spp FL,USA JN0607094 SEFL28b L. psuedotheobromae Vaccinium spp F L, USA JN607092 UF0440 Neofusicoccum ribis Vaccinium spp FL, USA FJ877139 SENC7 N. ribis Vaccinium spp NC, USA JN607103 SEGA49 Guignardia spp. Vaccinium spp GA, USA JN607105 a Acronyms of culture collections: CMW = Culture Collection Forestry and Agric ultural Biotechnology University of Pretoria, South Africa b Internal transcribed spacer region
82 Table 4 3. Number of dead plants and Bot. colony identification separated by propagation at each location over two years of growth Total a No. of Bot. Samples b Percent c Alachua Co. SW 32 21 65% TC 31 17 54% Total 63 38 60% Desoto Co SW 242* 234 96% TC 43 35 81% Total 285 268 94% Polk Co. SW 50* 37 74% TC 6 1 33% Total 56 38 68% a. Number of dead plants after two years in the fiel d. Asterisks denote significant differences (p < 0.05) between propagation methods for each location. Statistics were tested using a mixed model in SAS. b. Number of samples having colonies consistent with Bot. growth habit c. Percentage of Bot. samp les isolated from dead plants for each propagation method
83 Table 4 4. ANOVA table of disease incidence and Bot. severity rating s for the main effects one and two years after pla nting, as well as treatment means for mortality and severity. Average No. o f Dead Plants Per Plot Average Stem Blight Severity Per Plot One Year Two Years One Year Two Years Site a Effect F value p value F value p value F value p value F value p value Alachua Co. Cultivar 1.5 0.23 12.02 0.05 0.71 0.55 52.4 0.05 Propagati on 1.01 0.32 0.14 0.71 0 1 0.32 0.58 C*P c 1.4 0.25 0.51 0.67 2.13 0.12 0.13 0.94 Cultivar Propagation Mean MSD b Mean MSD b Mean MSD b Mean MSD b Emerald SW 0.5 0.56 0.75 0.83 1 0.24 1.25 0.33 TC 0.75 1.0 1.25 1.25 Primadonna SW 0.63 0.63 1 1 TC 0.63 0.63 1.25 1.125 Snowchaser SW 1.5 3.75 1.5 3.75 TC .25 2.75 1 3.62 Star SW .125 .125 1 1 TC 0 0 1 1 Site Effect F value p value F value p value F value p value F value p value DeSoto Co. Cultivar 21.78 0.05 28.18 0.05 30.25 0.05 12.92 0.05 Propagation 17.45 0.05 21.01 0.05 39.09 0.05 2.23 0.14 C*P c 3.83 0.02 3.9 0.012 6.12 0.05 0.8 0.5 Cultivar Propagation Mean MSD b Mean MSD b Mean MSD b Mean MSD b Emerald SW 7.85 2.15 8.14 2.0 3.28 0 .66 3.45 1.01 TC 3.0 3.0 1.42 2 Jewel SW 4.43 4.57 2.57 3 TC 6.0 5.71 2.71 3.57 Primadonna SW 17.0 17.28 7.28 7 TC 9.43 9.71 3.71 4.57 Snowchaser SW 18.29 18.71 7.28 7.56 TC 11.14 12.0 4.28 6 Site Effect F value p value F value p value F value p value F value p value Polk Co. Cultivar 6.28 0.05 8.01 0.05 6 0.05 6 0.05 Propagation 26.11 0.05 37.3 0.05 32.77 0.05 25.49 0.05 C*P c 7.65 0.05 9.17 0.05 6.4 0.05 5.93 0.05
84 Table 4 4 C ontinued Cultivar Propagation Mean MSD b Mean MSD b Mean MSD b Mean MSD b Emerald SW 0.79 0.32 1.14 0.35 1.57 0.2 1.57 0.31 TC 0.29 0.36 1 1 Jewel SW 0.43 0.5 1.21 1.07 TC 0.0 0.0 1 1 Primadonna SW 0.5 0.64 1.21 1.85 TC 0.28 0.28 1 1.21 Snowchaser SW 2.21 2.71 2.28 2.85 TC 0.07 0.07 1 1 a T he experiment was a randomized complete block design at all locations ; a row was a blo ck. The Alachua Co. site had 8 rows, the De Soto Co. site had 7 rows and the Polk Co. site had twelve rows respectively. Each row had 8 treatments ; each cultiva r was replicated twice and was either propagated by softwood cuttings or tissue culture b Mixed model was complet ed in SAS and Minimum signif icant difference(MSD ) were calculated based on Tukey adjustments. The asterisk means the same MSD for an individual date. c C*P cultivar and propagation interaction
85 Figure 4 1. Average number of dead plants per plot at the Alachua Co. site over a two year period beginning 2/09/2009 and ending 2/24/2011; the experiment was a RCBD estimates for each treatment consisted of 8 replicates with 25 plants per plot. A mixed model in SA S separated treatment effects using Fi LSD with Tukey adjusted p value s. The error bars for each date are the experiment wide min imum significant difference (MSD ).
86 Figure 4 2. Average number of dead plants per plot at the DeSoto Co. site over a two year period beginning 2/06/09 and ending 2/23/11; the experiment was a RC BD estimates for each treatment consisted of 7 replicates having 29 plants per plot. A mixed model in SA S LSD with Tukey adj usted p values. The error bars for each date are the experiment wide min imum significan t difference (MSD ).
87 Figure 4 3. Average number of dead plants per plot at the Polk Co. site over a two year period beginning 2/06/09 and ending 2/23/11; the experiment was a RCBD estimates for each treatment consisted of 14 replicates with 14 plants per plot. A mixed model in SA S separated treatment effects using Fi with Tukey adjusted p values. The error bars for each date are the experiment wide min imum significant difference (MSD ).
88 Table 4 5 Total number of Bot. colonies isolated from the l y sate of 20 sw at each isolation period; identification was based on PCR RFLPs Date No. colonies Diplodia seriata Lasiodiplodia theobromae Neofusicoccum ribis 5/28/2010 0 6/28/2010 0 7/28/2010 1 1 8/30/2010 1 1 9/28/2010 4 2 2 Table 4 6 Total number of Bot. colonies isolated from 20 surface sterilized sw at each isolation period; identification was based on PCR RFLPs Date No. colonies Diplodia seriata Lasiodiplodia theobromae Neofusicoccum ribis 5/28/2010 1 1 6/28/ 2010 4 1 3 7/28/2010 4 4 8/30/2010 7 7 9/28/2010 9 9
89 Figure 4 4 AUDPC for isolates collected from the li y sate or apparently healthy sw. According to the Waller Duncan k ratio t test ( k =100), same letters are not consider ed sig nifica ntly different and minimum significant difference (M SD) was 39.46 The control inoculum developed no symptoms and was not included in the analysis. Results from both experiments were combined in total 10 plants were inoculated with all isolates plus a ne gative control.
90 Figure 4 5 One of 78 most parsimonious trees (Length = 234 CI = 0.86 RI = 0.9) generated by heuristic searches, 1000 bootstrap and 100 random addition sequence replicat es were completed in PAUP. The ITS region of rDNA was used to compare sequences in present study with those previously published in GenBank. Isolates highlighted in bold are from the present study.
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103 BIOGRAPHICAL SKETCH Amanda Faith Wright earned a Bachelor of S cience in biol ogical sciences from Clemson University in May 2006. Wh ile at Clemson she completed an undergraduate research project with Dr. Ste ven Jeffers there she fulfilled on foliage blight of hostas caused by Phytophthora nicotianae. After gradu ation Amanda went to the Plant Pathology Department at the University of Florida to compl ete a Master of Science and Doctor of Philosophy While at the University of Florida she worked with Dr. Philip Harmon and studied stem blight of blueberries caused by fungi in the Botryosphaeriaceae. After graduation she moved to Aiken, South Carolina with her husb and and cat named Spiller Currently, she is working at the Savannah River Nat ional Laboratory