SCREENING, ASSAY DEVELOPMENT, AND TRAIT SEGREGATION FOR ANTHRACNOSE SUSCEPTIBILITY IN SOUTHERN HIGHBUSH BLUEBERRY By DOUGLAS A. PHILLIPS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIRE MENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017
2 2017 Douglas A. Phillips
3 To my wonderful wife, children, family and friends, for all of their patience and suppor t throughout my graduate career
4 ACKNOWLEDGEMENTS I would like to thank my committee members: James Olmstead, Patricio Munoz, Natalia Peres, Philip Harmon, and Vance Whitaker for all of their guidance and support during my graduate school education and research. In addition, I would like to thank the lab members and personnel of my committee members labs for their assistance and support.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................. 4 LIST OF TABLES ........................................................................................................ 7 LIST OF FIGURES ...................................................................................................... 8 ABSTRACT ................................................................................................................. 9 CHAPTER 1 LITERATURE REVIEW .................................................................................. 11 Blueberry Background and Production .......................................................... 1 1 Florida Blueberry Production .......................................................................... 1 1 Blueberry Cultivar Development for Florida .................................................... 1 2 University of Florida Blueberry Breeding Program .......................................... 13 Highbush Blueberry Diseases ........................................................................ 14 Anthracnose ................................................................................................... 14 Infection Process.. ........ 17 Temperature Effects ................................................................................ 19 Chemical Cont rols ................................................................................... 20 Recent Stem Lesion Infec tions in Florida ................................................. 2 1 Disease Resistance and Susc eptibility ........................................................... 22 Inoculation Methodology for Fungal Pathogens ............................................. 25 Trait Segregation in Autotetraploids ............................................................... 27 Research Objectives by Chapter .................................................................... 29 2 SCREENING FOR ANTHRACNOSE SUSCEPTIBILITY IN SOUTHERN HIGHBUSH BLUEBERRY CULTIVARS ......................................................... 31 Materials and Methods ................................................................................... 31 Plant Material ........................................................................................... 31 Experimental De sign ................................................................................ 31 Source of Isolate and Inoculum Preparation ............................................ 32 Inoculation and Collection of Data ........................................................... 33 Statistical Analys is ................................................................................... 34 Results and Discussion .................................................................................. 34 3 DEVELOPMENT OF DETACHED STEM SCREENING ASSAY FOR ANTHRACNOSE SUSCEPTIBILITY IN SOUTHERN HIGHBUSH BLUEBERRY .................................................................................................. 4 2
6 Materials and Methods ................................................................................... 4 2 Plant Material ........................................................................................... 4 2 Experimental Design ................................................................................ 4 2 Source of Isolate and Inoculum Preparation ............................................ 4 3 Inoculation and Collection of Data ........................................................... 43 Statistical Analys is ................................................................................... 4 5 Results and Discussion .................................................................................. 4 5 4 TRAIT SEGREGATION OF ANTHRACNOSE STEM LESION SUSCEPTBILITY IN SOUTHERN HIGHBUSH BLUEBERRY 5 6 Materials and Methods ................................................................................... 5 6 Biparental Populations ............................................................................. 5 6 Inoculation and Incubation Conditions ..................................................... 5 7 Isolate Source and Inoculum Preparation ................................................ 5 8 Inoculation Protocol and Collection of Data ............................................. 59 Results and Discussion .................................................................................. 6 0 5 CONCLUSIONS ............................................................................................. 75 REFERENCES .......................................................................................................... 78 BIOGRAPHICAL SKETCH ........................................................................................ 89
7 LIST OF TABLES Table p age 1 1 Possible genotypes in autotetraploids at one locus with two alleles 29 2 1 Cultivars inoculated and their nursery source 39 3 1 Cultivars inoculated and their nursery source 5 2 4 1 Parents used in controlled crosses 7 0 4 2 Expected Segregation Ratios Assuming Monogenic Control Fully Recessive Susceptible Parent and No Double Reduction 7 1 4 3 Expected Segregation Ratios Assuming Two Fully Recessive Genes Control Susceptibility Trait and No Double Reduction 7 1 4 4 Chi Square Results Based on Cumulative Infected Plants in Experiments 1 4 7 1 4 5 Experiment 2 Mean and Range of Anthracnose Lesion Lengths 7 2 4 6 Experiment 3 Mean and Range of Anthracnose Lesion Lengths 7 2 4 7 Experimental Conditions 7 2
8 LIST OF FIGURES Figure p age 2 1 Mean number of anthracnose lesions per plant Experiment 1 40 2 2 Mean AUDPC per cultivar Experiment 1 4 0 2 3 Mean number of anthracnose lesions per plant Experiment 2 4 1 2 4 Mean AUDPC per cultivar Experiment 2 4 1 3 1 Image of detached stems in glass culture tubes 52 3 2 Protocol Adjustment Test 1 Mean number of anthracnose lesions per stem 5 3 3 3 Protocol Adjustment Test 2 Mean number of anthracnose lesions per stem 53 3 4 Protocol Adjustment Test 3 Mean number of anthracnose lesions per stem 5 4 3 5 Experiment 1 Mean number of anthracnose lesions per stem 54 3 6 Experiment 2 Mean number of anthracnose les ions per stem 5 5 4 1 Experiments 14 Individual Experiment Results 7 3 4 2 Experiments 14 Cumulative Results 7 4 4 3 Effects of Temperature on Growth of C. gloeosporioides isolate 74
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Require ments for the Degree of Master of Science SCREENING, ASSAY DEVELOPMENT, AND TRAIT SEGREGATION FOR ANTHRACNOSE SUSCEPTIBILITY IN SOUTHERN HIGHBUSH BLUEBERRY By Douglas A. Phillips December 2017 Chair: Patricio R. Munoz Major: Horticultural Sciences Southern highbush blueberry (SHB) is an important early season crop in Florida, able to capture high market prices from March through midMay. Anthracnose, a disease caused by Colletotrichum spp. affects stems, leaves, and fruit in many crops, including blueberries. In recent years, a fungicideresistant stem lesion form of anthracnose has been reported in central Florida on two SHB cultivars, Flicker and Scintilla, a direct progeny of Flicker. A whole plant screening assay was performed on Flicker and nine other SHB cultivars commercially grown in Florida. Flicker had a significantly higher mean number of anthracnose lesions and area under the disease progress curve (AUDPC) than all other tested cultivars. Additional screening tests are required on other SHB cultivars grown in Florida. A detached stem inoculation as say was tested to determine whether this protocol provided comparable results to the whole plant screening assay. If the results were comparable, it would provide a fast, more cost effective method of screening field and
10 breeding selections for anthracnose. Although the results of the detached stem assay were not comparable to those in the whole plant screening, this protocol may still be useful as an initial screening tool on large volumes of plant material, to identify potentially susceptible cultivars or selections for additional testing. Since susceptibility has been observed in Scintilla, a progeny of Flicker, it appears that the susceptibility trait is heritable to some degree. Flicker has been used in the University of Florida blueberry breeding program, raising concerns that there may be susceptible plants present in the programs germplasm. Bi parental populations derived from crosses including either Flicker or Scintilla as a susceptible parent, and an assumed resistant advanced selection as the other parent, were inoculated with C. gloeosporioides to determine the segregation ratio of the susceptibility trait. The initial hypothesis was that the susceptibility trait was controlled by a single gene and was fully recessive. In two populations, the percentages of infected plants on a cumulative basis across experiments were consistent with a single gene trait using a chi square analysis and three additional populations were close to being good fits. Further research is needed to genotype thes e populations and identify single nucleotide polymorphism (SNP) markers to be used to screen for susceptibility in the UF breeding program early selection stages
11 CHAPTER 1 LITERATURE REVIEW Blueberry Background and Production Blueberry ( Vaccinium L. section Cyanococcus) is native to eastern North America, and includes diploid (2n = 2x = 24), tetraploid (2n = 4x = 48) and hexaploid (2n = 6x = 72) species ( Sharpe, 1959, Vander Kloet, 1988). The principal cultivated species are highbush blueberry ( Va ccinium corymbosum ), lowbush blueberry ( Vaccinium angustifolium ), and rabbiteye blueberry ( Vaccinium virgatum Aiton), all of which are commercially grown in the United States ( Chavez and Lyrene, 2009b ). The US is the worlds largest blueberry producer, w ith total production of over 254 million kilograms (5 60 million pounds) in 201 5 The top five US states in terms of utilized production in 2015 were Washington, Oregon, Georgia, Michigan, and California ( USDA, 201 5 ). Although the US is the largest global exporter of blueberries (primarily to Canada and Japan), it is also a net importer, primarily sourcing imports from Chile ( Evans and Ballen 2014, Ag Marketing Resource Center, 2015). Florida Blueberry Production The cultivars used in Florida blueberry production are primarily southern highbush blueberries (SHB), which are interspecific hybrids between V. virgatum, V. corymbosum, and V. darrowii (a small number of rabbiteye cultivars are grown in northern Florida). Major production areas are north central F lorida (Alachua, Marion, Putnam, Sumter, and Lake counties), accounting for approximately 40% of blueberry acreage in the state, central Florida (Polk, Orange, Pasco, Hernando, and Hillsborough counties), approximately 35% of total acreage, and south centr al Florida (Highlands, Hardee, Desoto, Manatee, and Sarasota counties), with approximately 20% of total
12 acreage ( Williamson, 2015). Total acres under production in Florida increased from 405 hectares (1,000 acres) to over 1,820 hectares (4,500 acres) between 1982 and 2012, and there was an increase in blueberry production between 2000 and 2011 of 637.93%, with over 10 million kilograms (23 million pounds) produced in 2011 ( Evans and Ballen 2014, Williamson et al. 2014). Florida had the highest price per pound realized in the US in 201 5 at $ 3.35, due to the ability of growers to utilize early ripening cultivars and go to market between April and early May, before other US states can harvest and ship their fruit. Almost all of Floridas blueberry production is for the fresh fruit market ( Williamson et al. 2015, USDA, 201 5 Blueberry Statistics ). Blueberry Cultivar Development for Florida Blueberries require a certain number of chilling hours (length of time in dormancy between 0C and 7C) for proper reproductive and vegetative development, and this varies by species and cultivar ( Chandler et al. 1937, Olmstead et al. 2015). This is an adaptation of woody species in temperate zones, to keep plants dormant during the season of winter freezes, and is highly heritable ( Lyrene, 2005). Florida has a subtropical climate, with lower available chilling hours than in temperate climates. Historical accumulated chilling hours per year have on average been approximately 300 to 500 hours in north central Florida, 200 to 300 hours in central Florida, and 100 to 200 hours in south central Florida ( Andersen et al. 2016). In addition, Floridas hot and humid climate is favorable for the development of several fungal diseases on blueberry plants that can result in premature d efoliation and dieback ( Lyrene, 2005). Therefore, it was necessary to develop blueberry cultivars requiring a lower number of chilling hours and possessing improved disease resistance for commercial production in Florida.
13 Ralph Sharpe began work on breeding highbush blueberries for the Florida climate in 1949 at the University of Florida. He collaborated on this work with George Darrow, the USDA blueberry breeder at the time in Beltsville, Maryland. Their efforts focused on crossing northern highbush cult ivars from Michigan and New Jersey with native Florida blueberry species, from which they hoped to obtain genes for a lower chilling hour requirement, longer leaf retention to support early bloom set, and improved adaptation to heat and disease ( Lyrene, 2002). The primary Florida species used was Vaccinium darrowii Camp, a diploid evergreen species with no chilling requirement, found extensively in peninsular Florida on poorly drained flatwoods soils ( Camp, 1945, Sharpe, 1953, Lyrene, 1997). In particular, a selection know as 4B, collected from the wild in central Florida by Sharpe and Darrow, was instrumental in reducing the northern highbush chilling requirement ( Draper and Scott 2003). V. darrowii genes are present in all southern highbush cultivars ( Lyrene, 2002 ). In addition, V. myrsinites and V. virgatum have also been used in attempts to reduce the highbush chilling hour requirement ( Sharpe, 1953, Sharpe, 1959, Chavez and Lyrene, 2009a ). University of Florida Blueberry Breeding Program The Universi ty of Florida has produced and released many cultivars that are used extensively by growers throughout the southeastern US. These cultivars include Star Emerald and Jewel released in the late 1990s, which still comprise most of the planted blueber ry acreage in Florida. Each meets the requirement for a lower number of chilling hours, and Jewel is often planted in combination with Emerald for pollination purposes. However, recent problems with necrotic ring blotch virus and bacterial leaf scorch (caused by Xylella fastidiosa) have led to a decrease in new plantings of Star
14 Additional cultivars widely grown in Florida are Springhigh and Primadonna both of which bloom and ripen early. More recently, Flicker was released in 2009. It has a l ow chilling requirement and can be grown in an evergreen management system (foliage maintained for longer periods to help support the following years flowers and fruit) in central and south Florida where winter freezes are rare, allowing for earlier harvest ( Williamson et al. 2014 ). The most recently released cultivars in 2015 were Avanti Arcadia and Endura all of which are well suited for the evergreen production system ( Buck, 2015). Highbush Blueberry Diseases Highbush blueberries grown in the southeastern US can be susceptible to several different diseases, caused by fungal, bacterial, and viral pathogens including Phytophthora root rot, botrytis blossom and fruit rot, Botrysphaeria stem blight, bacterial leaf scorch, bacterial wilt, red rings pot virus, necrotic ring blotch, blueberry mosaic virus, leaf rust, and anthracnose (Chang et al. 2009; Harmon et al. 2016; Polashock et al. 2017; QuitoAvila et al. 2013; ThekkeVeetil et al. 2014) Anthracnose Anthracnose is a group of diseases ca used by Colletotrichum a genus of fungal pathogens with a primarily tropical and subtropical distribution ( Cannon et al. 2012, Hyde et al. 2009). Colletotrichum is part of the Ascomycota phylum ( Perfect et al. 1999) and its life cycle primarily depends on asexual reproduction. Its taxonomy remains somewhat fluid, with the number of species ranging from 29 to in excess of 700 depending on the criteria used. Plant pathologists recently selected Colletotrichum as the eighth most important genus of plant pathogens on the basis of scientific or
15 economic importance, and almost every crop grown throughout the world is susceptible to one or more species ( Dean et al. 2012). Anthracnose typically results in dark, sunken necrotic lesions on the leaves, stems, flow ers, or frui t, with orangeto pink conidia being discharged from the lesions in later stages ( Freeman et al. 1998, Jeffries et al. 1990). Two species with wide host ranges for fruit infections are Colletotrichum gloeosporioides and Colletotrichum acutatum These species can be similar in morphology and cultural characteristics, and C. acutatum was often misclassified as C. gloeosporioides before molecular methods for taxonomy became available (e.g., PCR amplification with species specific primers) (Adaskaveg and Hartin, 1997, Peres et al. 2005). Anthracnose is typically seen as fruit rots, leaf spots, or stem lesions. Post harvest fruit rots can be a significant problem, including on blueberries, and are generally caused by infection of C. acutatum Infections typically remain latent until the fruit ripens, and symptoms on the fruit may not appear until after it is harvested ( Jeffries et al. 1990). Pre harvest fruit rots on immature fruit are also possible. Colletotrichum can also cause anthracnose on leaves. In an orchard in Korea, anthracnose was observed on the leaves of three highbush blueberry cultivars, Duke Darrow and Coville Symptoms appeared as brown circular or irregularly shaped spots on the leaves. All but one of the isolates from infected leaves were identified as C. gloeosporioides (the remaining one was C. acutatum ) (Kim et al. 2009). The third form of anthracnose is infection of the stem, characterized by dark, sunken lesions and tip dieback. C. gloeosporioides and C. acuta tum were found to have infected leaves, stems, and fruit on highbush blueberry in Korea. The infected stems
16 turned dark brown, then became gray and died. Of five stem isolates taken, all were identified as C. gloeosporioides based on morphological and cult ural characteristics. The pathogenicity of the isolates was tested by inoculating wounded and nonwounded highbush blueberry leaves of three cultivars with 20(2~3 x 106 conidia/ml). The inoculation resulted in anthracnose symptoms on all of the wounded leaves, but only weakly or not at all on the nonwounded leaves ( Kim et al. 2009). Also stem blight symptoms were observed on highbush blueberry in Japan, with the previous years shoots turning brown and then blighted, along with adjacent floral bud death. Also, small red or brown leaf spots were observed near the blighted stems. Isolates were identified as C. acutatum based solely on morphological and cultural characteristics. Pathogenicity was tested by inoculating stem s, leaves and fruit, both wounded and nonwounded, with a conidial suspension (2 x 106 conidia/ml). Necrotic lesions and spots formed on the wounded stems and leaves, but either did not appear or were less severe on the nonwounded tissue ( Yoshida and Tsukiboshi 2002). In addition, there were two reports of stem lesions on blueberries, one in the US and one in China. In 2010, there was a report of lesions on green blueberry canes in Michigan. The lesions were described as dark brown to black, circular or oval, with light brown to gray centers, and salmonpink masses of spores. The pathogen was identified as C. acutatum This report also noted that anthracnose stem lesions had been observed in Ontario, Canada and Michigan in 2003 and 2004 ( Schilder, 2010). In 2013, there was a first report of stem lesions and leaf spots on highbush blueberry caused by anthracnose in China. Symptoms were described as beginning
17 with yellow to red irregularly shaped lesions on stems and leaves, which expanded and turned dark br own, surrounded by a red halo. Isolates from stem tissue were initially identified as C. gloeosporioides based solely on morphological and cultural characteristics, which was then confirmed by amplifying genomic DNA with ITS1 and ITS4 primer pairs and perf orming a BLAST search. Pathogenicity was tested by inoculating wounded highbush blueberry plants with a conidial suspension (1 x 105 conidia/ml), resulting in lesions on stems and leaves from which the pathogen was reisolated and confirmed to be C. gloeos porioides ( Xu et al. 2013). C. gloeosporioides has also been found to cause stem lesion anthracnose on cassava ( Fokunang et al. ), mango ( Gupta et al. 2015), and dragon fruit ( Vijaya et al. 2015) Infection Process Although various species of Colletotrichum utilize different strategies to invade host tissue, the early stages of fungal development are effectively the same for all species. The steps for these early developmental stages are (1) conidia formation in acervuli, followed by dispersal with rain or irrigation splash, (2) adhesion of conidia to the plant cuticle, (3) germination and production of germ tubes, (4) differentiation of appressoria, and (5) cuticle penetration by a penetration hyphae. Penetration is accomplished through a combi nation of mechanical force and tissue degradation by enzymes ( OConnell et al. 2000 ). Mechanical force is applied through increased turgor pressure, a result of melanization of the appressori um (thought to make the cell wall selectively permeable) and the synthesis of osmotically active compounds ( Munch et al. 2008). Previous studies have shown a correlation between temperature, wetness,
18 and infection. A study on blueberry fruit infection by C. acutatum found that 25C was the optimum temperature for appr essorium formation and mycelial growth (with little to no mycelial growth at either 5C or 35C), and that interruption of wetness helped to inhibit the infection process ( Miles et al. 2013 ). This was consistent with earlier studies on C. o r biculare infec tion in watermelon ( Monroe et al. 1997) and C. coccodes in tomato ( Sanogo et al. 1997). Following penetration, the infection strategies are intracellular hemibiotrophy and subcuticular/intramural necrotrophy. Intracellular hemibiotrophy has a short biotr ophic phase (typically one to three days) associated with intracellular primary hyphae. Initially, the penetrated cells remain alive, with an interface being deposited between the plasma membrane and the cell wall of the pathogen. This interface allows the transfer of nutrients from the host to the pathogen. Within 24 hours the host plasma membrane begins to degenerate and host cells start to die. C ell death is gradual and confined to infected cells, and biotrophic relationships continue to be established w ith newly infected cells. Ther e are no symptoms at this point and no apparent host recognition of the pathogen or defense response. The biotrophic phase is followed by a necrotrophic phase associated with more narrow secondary hyphae, which spread both int ercellularly and intracellularly throughout the host tissue. This is characterized by extensive enzymatic degradation and death of host cells, releasing sugars available to the pathogen. Symptoms appear with cell necrosis and the host begins to produce def ensive compounds, but the cells synthesizing these compounds often die before concentrations sufficient to inhibit fungal growth can accumulate.
19 With subcuticular/intramural necrotrophy, the pathogen does not immediately penetrate the cell, but develops beneath the cuticle. Cell wall swelling and dissolution occur, although symptoms do not develop for 24 hours. This is followed by a rapid spread of the pathogen throughout the host tissue, with host cell dissolution and death. In this strategy there is no di stinction between primary and secondary hyphae, as there is with intracellular hemibiotrophy. C. gloeosporioides on papaya is known to use this strategy, although C. gloeosporioides on avocado, citrus and rubber combine both infection strategies ( Munch et al. 2008, OConnell et al. 2000, Perfect et al. 1999). In some cases, the post penetration infection strategy depends on the species of host, the susceptibility of the host, or on the type of tissue being colonized. For example, C. acutatum the pat hogen responsible for fruit rot in blueberry, utilized an intracellular hemibiotrophic strategy on a susceptible blueberry cultivar, whereas a subcuticular intramural strategy was used on a resistant cultivar, possibly due to an attempt to overcome the hos ts defense response ( Wharton and Schilder 2008). Colletotrichum infections may also be quiescent, where the appressori um go es through an extended dormancy phase before forming a penetration hyphae. This is often the case with post harvest fruit rots where symptoms appear only after harvest ( Jeffries et al. 1990). Temperature Effects Appressorial formation and conidia germination in Colletotrichum is affected by both temperature and incubation time (Dodd et al. 1991, King et al. 1997). Several studies have been conducted to determine the optimal temperatures for coni dia germination and appressorial formation in both C. gloeosporioides and C. acutatum
20 The studies were performed by cutting agar disks of Colletotrichum mycelia from the growing edge of an ac tively growing culture and incubating them on nutrient agar or wet filter paper, generally at temperatures between 5 and 35C (at 5 C increments) and measuring radial growth after some defined period of days (Hartung et al. 1981, Lee, 1993, King et al. 1997). In some cases conidia were applied to the surface of fruit or leaves prior to incubation (Dodd et al. 1991, Leandro et al. 2002). For C. gloeosporioides the optimal temperature for mycelial growth was 20C with almost no growth at 30C (Hartung et al. 1981); for conidial production was 15 to 25C, with maximum sporulation at 25C, decreasing at or above 30 C (King et al. 1997); for conidial germination and development of appressoria was between 20 and 30C (Dodd et al. 1991); for conidial germination was 28C (Denner et al. 1986); and for germination of conidia and germ tube elongation was between 20 and 30C, with no appressorial formation at or above 30C (Lee, 1993). Taken together, t hese studies found that the optimal temperature fo r growth, conidial formation and appressorial formation in C. gloeosporioides is between 20 and 28 C, with a decrease at or above 30C Chemical Controls Anthracnose diseases in blueberry can generally be controlled by a range of fungicides Azoxystrobin (Abound, fungicide class strobilurins or QoI) is frequently used in Florida. In addition, pyraclostrobin + boscalid (Pristine), cyprodinil + fludioxonil (Switch), and Captan are used to treat anthracnose ripe rot and leaf spot ( Williamson et al. 201 6 )
21 Recent Stem Lesion Infections in Florida Flicker is a SHB cultivar that had been frequently selected by growers in central and south Florida. Characteristics favoring production of Flicker in this region are that it has very low chilling requirements, can be grown in an evergreen management system, and tends to ripen early. In 2014, a stem lesion form of anthracnose was observed on certain SHB cultivars (Flicker and, to a lesser degree, Scintilla) on four farms in central Florida (Harmon, pers. com mun.) One grower stated that he had 15 acres of Flicker, and between 80 and 100% of those plants became infected with this form of anthracnose when they w ere approximately two years old. In 2015 he stopped planting Flicker, and by the end of harvest i n 2016 he had removed all of the Flicker plants from his fields. He indicated that the financial impact to him was in the hundreds of thousands of dollars due to loss of yield, lost years of production from the young plants, and the need to replant that acreage with other cultivars (Morris, pers. commun.). One central Florida nursery saw a large increase in orders for Flicker about 2012 due to that cultivars early success as a low chill plant that could be grown in an evergreen management system. The nursery had significant advance orders for Flicker in 2014 and propagated plants based on those orders, but ultimately did not ship any Flicker plants due to the anthracnose outbreak and its impact on growers (Sheffield, pers. commun.). Using molecular methods, the causal pathogen was identified as C. gloeosporioides (Harmon, pers. commun.). Certain isolates collected from infected Flicker plants in central Florida appear to be resistant to fungicides in the QoI class, including Abound, Cabrio, and Pristine. It has been suggested that heavy QoI fungicide
22 use may have minimized competition from other pathogens and allowed C. gloeosporioides to thrive. This is based on reports that fungicide insensitivity has been observed in areas where QoI fungicides were widely used, but not yet in areas without widespread QoI use (Harmon, pers. commun.). Recommendations for control include rotations of DMI fungicides (Demethylation Inhibitors) with compatible contact fungicides, not applying more than the label rate of any one active ingredient for each season, changing between products with different active ingredients, and good cultural practices ( Harmon, 2014). D ue to this outbreak of anthracnose, there has been a sharp reduct ion in new plantings of this cultivar and many growers have removed all Flicker plantings (Harmon, pers. commun.) By 2017, it was very difficult to find commercial nurseries producing Flicker plants In addition, Flicker has been used as a parent in the UF breeding program, raising concerns regarding potential susceptibility of offspring from these crosses. This is the primary concern that led to the development of this thesis research project. The desired outcome of this research, which will extend beyond the experiments covered in this thesis, is to develop a SNP marker based screening assay to identify susceptible germplasm in the UF breeding program, to assist in select ing parents that do not have the anthracnose susceptibility trait. Disease Resi stance and Susceptibility Disease resistance has been defined as the ability of an organism to exclude or overcome, completely or in some degree, the effect of a pathogen or other damaging factor, and is characterized by limited symptoms, which is a result of the inability of a pathogen to grow or spread throughout the organism ( van Loon, 1997). Host resistance is pathogen specific, and nonhost resistance is resistance to all races of a pathogen. A
23 plants defense system against infection by pathogens is comprised of both preformed and induced responses. Preformed responses include both structural barriers and antimicrobial compounds that provide nonspecific protection against a broad range of pathogens. Induced responses rely upon recognition of the pat hogen by the host, which triggers signaling pathways that activate other defenses, including an oxidative burst, expression of pathogenesis related (PR) proteins, structural barriers (e.g., cell wall lignification), and a hypersensitive response ( Thatcher et al. 2005). With induced resistance, the general level of resistance to pathogens is boosted, which has been termed systemic acquired resistance (SAR) ( van Loon, 1997). Plant disease resistance is characterized by complete (qualitative) resistance, often controlled by a single gene (monogenic) or incomplete (quantitative) resistance, often controlled by multiple genes (polygenic) with each gene having a partial effect. It is important to note that it is possible for a single gene to result in only part ial disease resistance. Monogenic (q ualitative ) disease resistanc e typically follows four phases and involves twoway communication between the plant and the pathogen whereby the pathogen is recognized by the plant. This communication takes the form of an allele specific interaction between a plant host R gene and a pathogen avirulence ( Avr ) gene, which triggers a plant defense response, and is known as genefor gene resistance ( Belkhadir et al. 2004). The genefor gene concept states that for each gene co nditioning a reaction in the host ( R ), there is a corresponding gene conditioning pathogenicity in the pathogen ( Avr ) (Flor, 1971 ). This appears to involve an indirect recognition of an Avr gene, referred to as the guard hypothesis, where the R gene
24 interacts with another plant protein that is modified by the pathogen, and the R gene recognizes a product of the pathogen attack on that plant protein ( Gururani et al. 2012). A great deal of uncertainty continues to surround the concept of partial resistance. Many researchers have suggested that the loci controlling qualitative and partial resistance are not distinct, and that in fact the same genetic mechanisms are involved. There could be partial resistance variation to the four phases of qualita tive resistance, resulting in modifications to the extremes of complete resistance. Other hypotheses regarding partial resistance include mechanisms that regulate developmental and morphological phenotypes, different alleles involved in basal defense, and the detoxification of pathogen phytotoxins. While the effectiveness of qualitative R genes can have a short lifespan due to selection pressures, the effectiveness of genes producing partial resistance can be more durable, since their partial effects can result in lower selection pressure, so mutated Avr genes only provide the pathogen with a marginal advantage ( Poland et al. 2008). In contrast, polygenic disease resistance is not associated with a specific mechanism, but instead only refers to the number of genes involved in resistance. The location of polygenic genes controlling a trait is referred to as quantitative trait loci (QTL ), with each QTL having an additive effect on disease resistance. The effect of each gene is typically small and is often influenced by environmental factors, or by interaction with other genes (i.e., epistasis). This type of resistance is characterized by a quantitative distribution of differenc es in the level of resistance (Lindhout, 2002).
25 Inoculation Methodology for Fungal Pathogens The concept of a disease triangle was developed by George McNew to study the interrelationships of factors impacting a plant disease epidemic. The three factor s represented by the legs of the disease triangle are (1) the susceptibility of the host to the pathogen, (2) the inoculation potential of the pathogen (i.e., the presence of a virulent pathogen), and (3) the extent to which environmental conditions are favorable for infection ( McNew, 1960). In developing an inoculation protocol to screen for susceptibility to a specific disease, the second and third factors must be fully considered. T o design an objective disease screening experiment that can be replicated the pathogen from which the inoculum is to be developed must be virulent, and the environmental conditions at and following the time of inoculation must be favorable for infection by the pathogen. If environmental conditions are not sufficiently favorabl e for infection by the specific pathogen, there could be escapes (i.e., susceptible plants showing no symptoms) or disease symptoms may be less severe than would be observed under more favorable conditions A commonly used method of inoculating whole plants ( as well as detached stems or leaf tissue) with a fungal pathogen is to spray a suspension of the pathogen directly onto the tissue, followed by some method of keeping the inoculated tissue in a warm, humid environment favorable for infection, such as in a sealed plastic bag or container. The suspension is typically sprayed onto the tissue surface until runoff using either a hand atomizer or a pressurized spraying device ( Lewers et al. 2007 Rahman et al. 2015, Yun et al. 2006).
26 Several methods of inoculation have been used involving wounding stem or leaf tissue prior to or as part of the inoculation, including stem incision (creating a flap), needle puncture, and leaf tearing followed by placement of mycelium over the wound and sealing with Kimwipes and parafilm ( Baker et al. 1995); stem puncture with a needle coated with inoculum ( Talg and Stensvand, 2013); forcing a severed stem or leaf petiole into an agar plug of PDA and mycelium ( Twizeyimana et al. 2012 Zhao, 2004) ; the placement of drops o f conidial suspension on the stem surface followed by stem incision through the drops ( Baayen and Schrama, 1990); injection of inoculum into the stem ( Baayen and Schrama, 1990); abrasion of stem tissue followed by inoculum application with a brush and seal ing with cheesecloth and parafilm ( Tooley et al. 2014); and stem incision followed by insertion of an agar inoculum disk into the incision ( Denman and Sadie 2001, Stewart et al. 2005). In general the level of resistance in plant tissues tends to decrease following detachment, as the tissue begins to senesce and deteriorate (Dhingra and Sinclair 1995; Pettit et al. 2011). T o keep a detached stem or leaf alive during the observation period following inoculation, various methods have been used incl uding placing the detached stem into a glass jar containing water ( Denman and Sadie 2001); placement of the detached stems on a platform of moist towels and filter paper inside a sealed container ( Mei et al. 2012); placing cuttings on racks in containers suspended above 2 cm of water and misting the inside of the container once per day ( Stewart et al. 2005); lining the table under a plastic cover with wet paper towels to maintain an humid environment ( Hopkins and Harris 2000); and placement of the petiole of a cut leaf in water agar ( Cowley et al. 2012).
27 Trait Segregation in Autotetraploids Ploidy level refers to the number of genomes that comprise a cell nucleus. For diploid organisms, there are two genomes, or sets of chromosomes, in the cell nucleus. Polyploids have more than two genomes, and are separated into allopolyploids and autopolyploids. Allopolyploids arise through interspecific hybridization followed by chromosome doubling, resulting in the association of different, nonhomologous genomes. Pairing during meiosis is similar to the pairing observed in diploid organisms, where bivalents are formed between homologous pairs of chromosomes (i.e., the nonhomologous chromosomes arising from different ancestral genomes do not pair or recombine). Thi s inheritance structure is referred to as disomic ( Gallais, 2003). Autopolyploids originate from chromosome doubling of a withinspecies diploid genome, possibly through unreduced gametes. They have more than two homologous chromosomes, each having an equal opportunity to pair during meiosis. Pairing can take place between (1) random pairs of homologous chromosomes (bivalents), or (2) more than two homologous chromosomes (multivalents), where chromosomes pair, synapse, and recombine with multiple chromosomes simultaneously ( Gallais, 2003, Lloyd and Bomblies 2016). Inheritance under autopolyploid structures is polysomic, arising through (1) random assortment of multiple homologous chromosomes, (2) dosage allelic combinations and (3) m ore than two alleles at a locus ( Grandont and Lloyd, 2013). Diploid organisms have two partitions regarding variation at a single locus, homozygous (e.g ., AA and aa) and heterozygous (e.g. Aa) In contrast tetraploids have
28 four partitions for variation at a single locus monogenic, digenic simplex, digenic duplex, and digenic triplex, allowing for five different genotypes at a locus (Table 1 1 ). The terms simplex, duplex and triplex refer to t he number of dominant (i.e., reference) alleles (dosage) in the genotype ( Fisher, 1947). Under tetrasomic inheritance ratios, the four homologous chromosomes randomly associate, with each chromosome having an equal capability of pairing and recombining wit h any of the other four homologous chromosomes ( Lloyd and Bomblies 2016 ). This complex segregation results in a higher number of possible genotypes than occurs under diploid segregation. For example, the genotypes created by selfing the tetraploid digenic duplex AAaa will result in 1/36 AAAA: 2/9 AAAa: 1/2 AAaa: 2/9 Aaaa: 1/36 aaaa. In contrast, selfing the diploid Aa results in 1/4 AA: 1/2 Aa: 1/4 aa (Gallais, 2003). In addition, the phenomenon referred to as double reduction can further complicate the segregation patterns of autopolyploids (Gallais, 2003, Luo et al. 2004). Although segregation of genetic variability occurs in autotetraploids, it is at a much slower rate than takes place for diploid organisms ( Qu and Hancock 1995). Therefore, recessive d etrimental alleles take longer to be remove d from a tetraploid population than it would for diploid organisms ( Gallais, 2003). Highbush blueberry ( V. corymbosum ) has tetrasomic inheritance ratios ( Draper and Scott 1971, Krebs and Hancock 1989 Qu and Hancock 1995). This includes the interspecific hybrid US 75, which had a key role in the development of SHB ( Qu and Hancock 1995). In a more recent study, most of the traits studied in SHB (yield, weight, fruit diameter, and stem scar) were found to have tetrasomic inheritance with no double
29 reduction, but fruit firmness appeared to have some level of double reduction ( Amadeu et al. 2016) Table 1 1 Possible genotypes in autotetraploids at one locus with two alleles (Gallais, 2003) Genotype Term AAAA monogenic quadriplex AAAa digenic triplex AAaa digenic du plex Aaaa digenic sim plex aaaa monogenic nulliplex Research Objectives by Chapter Flicker and Scintilla cultiv ars were frequently planted by c entral Florida commercial blueberry growers. However, they were found to be susceptible to a stem lesion form of anthracnose that is resistant to commonly used fungicides. As a result, many growers did not continue to plant these cul tivars, and many removed c urrent plantings (Harmon, 2014, pers. commun.). Also, Flicker has been frequently used as a parent in the University of Florida blueberry breeding program, and Scintilla is a progeny of Flicker. This raises concerns that the genetic trait for anthracnose susceptibility may have been passed on to other selections or cultivars in the UF blueberry breeding program, which could have detrimental impacts for both growers and the breeding program. The objective of Chapter 2 was to screen blueberry cultivars commercially grown in Florida for susceptibility and communicate the findings to growers for their use in making decisions on selection of cultivars. Although significant economically important symptoms of this disease have onl y been observed in Flicker and Scintilla to date, it is
30 important to determine whether other cultivars are susceptible so that if this is the case growers can be notified and treatment plans considered. Screening of multiple replicates of blueberry plants for disease can be costly. Field inoculations can risk spread of the inoculum outside of the test area and may render the inoculated plants unsuitable for further evaluation or research. Greenhouse inoculations mitigate the risk of inoculum spread, but can be costly in terms of space and the cost of purchasing a sufficient number of plants for multiple replicates. The objective of Chapter 3 was to develop a cos t efficient, smallscale assay for screening field selections and cultivars for susceptibili ty to anthracnose stem lesions. The effectiveness of this protocol in screening for susceptibility will be tested by comparing the results with those obtained in the whole plant screening in Chapter 2. The risks of using disease susceptible germplasm in a breeding program can be significant, for both the breeding program and end users of released cultivars. The likelihood of the heritability of the trait for anthracnose stem lesion susceptibility has been demonstrated in the susceptibility of Scintilla, a direct progeny of Flicker. T o limit use of susceptible germplasm in the UF breeding program, a molecular screening assay must be developed. The first step in developing that assay is determining the segregation of the susceptibility trait in bi parental populations with a susceptible and an assumed resistant parent. The objective for Chapter 4 was to determine the segregation ratio of this trait, and select a population or populations for sequencing and SNP marker identification, leading to a marker based screening tool for identifying susceptible germplasm. The sequencing and marker identification work will be performed subsequent to the research covered by this thesis.
31 CHAPTER 2 SCREENING FOR ANTHRACNOSE SUSCEPTIBILITY IN SOUTHERN HIGHBUSH BLUEBERRY CULTIVARS Materials and Methods Plant Material Plant material used in the evaluation of anthracnose susceptibility consisted of ten cultivars of Vaccinium corymbosum (southern highbush blueberry), all of which are commercially grown in Florida. Plants were purchased from Fall Creek Nursery in Lowell, Oregon and AgriSt arts in Apopka, Florida (Table 2 1 ) both of which use tissue culture to propagate their nursery plants, to avoid issues with known plant pathogens Flicker was not available from Fall Creek Nursery. The plants were approximatel y 10 cm tall, in 38 cell trays. All of the cultivars with the exception of Rebel were developed by the University of Florida blueberry breeding program. The plants were transplanted into Kord 15 cm plastic pots (HC Companies, Middlefield, OH) filled with 100% Fafard peat (Sun Gro Horticulture, Agawam, MA) and allowed to grow for four months before inoculation. Plants were placed 11.5 cm apart within rows 11.5 cm apart on greenhouse benches. The plants were watered regularly, and fertilized with a Peters P rofessional 202020 fertilizer (Scotts, Marysville, OH) every two weeks. The experiments were conducted in a temperaturecontrolled greenhouse without supplemental lighting at the University of Florida, Gainesville, Florida. Experimental Design The experimental design was a randomized complete block design with ten replicates, one plant per cultivar per replicate. The block design was used to account for variation in temperature and light in the greenhouse. The experiment was conducted
32 twice, both during May 2016. The treatment was composed of ten different cultivars of V. corymbosum (Table 2 1 ) each of which was inoculated with a single isolate of Colletotrichum gloeosporioides (isolate 15646) F ifty Flicker plants (known to be susceptible to anthracnose) were used as negative standards, instead of an equal number of control replicates for each cultivar allowing for more cultivar replicates to be inoculated with the pathogen. The negative standard results were then compared with the pathogeninoculated Flicker plants to determine whether any additional control procedures were required. Source of Isolate and Inoculum Preparation The pathogen used for inoculation was a singleconidium isolate (15 646) of C. gloeosporioides originally isolated in 2015 from naturally infected stems of Flicker on a commercial blueberry farm in central Florida ( Harmon, pers. commun.). The isolate was incubated on autoclaved potato dextrose agar (PDA) under continuo us fluorescent lighting at 25C for five days, at which point sufficient sporulation had occurred. The petri plate containing the pathogen was flooded with deionized water, the conidia dislodged into a suspension with a sterilized Lshaped glass rod, and t he suspension filtered through two layers of cheesecloth to remove dislodged mycelia. The conidial concentration was determined using a hem a cytometer (Bright Line Hem a cytometer, Hausser Scientific), and then diluted to obtain a conidial suspension of 1x105 conidia mL1. The control Flicker plants were sprayed with deionized water.
33 Inoculation and Collection of Data Inoculation was performed on 12 May 2016 and 26 May 2016. The conidial suspension was sprayed until run off onto each plant using a Crown Spra Tool aerosol spray gun (Aervoe, Gardnerville, NV), coating all stem surfaces and upper surfaces of leaves. Each plant was imm ediately sealed inside a 30.5 x 61 cm 4 mil polyethylene bag (Uline, Pleasant Prairie, WI) along with a moistened Kimwipe (Ki mberly Clark, Irving, TX), for 16 hours to maintain a moist environment conducive for infection by Colletotrichum After removal of the polyethylene bags, the plants were watered daily. Greenhouse temperature ranged between 16 C and 36 C, measured using a HOBO temperature logger (HOBO U23002, Onset Computer Corp.). Each plant was examined daily to identify any disease symptoms. Two criteria were used to measure the disease phenotype (i.e., necrotic stem lesions). First, the number of lesions on each infected plant was recorded to evaluate disease incidence. Second, one representative lesion per infected plant was selected, marked, and measured with a digital caliper daily for seven days following first observation of disease symptoms to evaluate disease s everity using area under the disease progress curve (AUDPC). For the AUDPC calculations, the trapezoidal method (Madden et al 2007) was i n 1[(yi + yi+1)/2](ti+1 ti), where n = number of assessments y = disease severity score for each plant at assessment, and t = time at each assessment. Following the end of the observation period, infection by C. gloeosporioides was confirmed through isolations from necrotic lesion boundaries to fulfill Kochs post ulates
34 The isolated tissue was surface disinfested with a 10% bleach (0.6%) ( sodium hypochlorite) solution for one minute, followed by three water rinses for one minute each. The isolated tissue was then placed on PDA and incubated under continuous fluore scent lighting at 25C until conidia covered most of the plate. Confirmation was made by comparing the morphological characteristics of the isolate with those of the original isolate. Statistical Analysis The response variables (mean lesion number and AU DPC) were analyzed utilizing a one way analysis of variance (ANOVA) to assess the effect of treatment (cultivar), using the following linear model: e re fixed effects, and e is the random residual effect ~ N 2). Tukeys honestly significant difference test was utilized when the ANOVA test showed significant difference among treatments, to separate treatment means at P 0.05. All analyses were conducted using the R statistical software (R Development Core Team, 2016). Results and Discussion Anthracnose stem lesions were observed on young green stems of certain cultivars in both experiments; they were primarily subcircular, sunken, and enlarging over time. Where multiple lesions were located in close proximity, they usually coalesced into a large necrotic area, often causing dieback of the stem tip. On certain lesions orange sporulation was observed erupting from the necrotic area.
35 In both experimen ts, Flicker consistently had the highest level of infection among tested cultivars. In Experiment 1, the mean number of lesions on Flicker was significantly higher using Tukeys Honestly Significant Difference (HSD) test than for all tested cultivars except for Emerald, Farthing, and Kestrel. Flicker had a mean of 0.6 lesions per plant, whereas Farthing and Kestrel had mean s of 0.2 lesions, and Em erald had a mean of 0.1 lesion (Figure 2 1) In the second experiment, Flicker had a signif icantly higher mean number of lesions per plant (1.5) than all other tested cultivars (0.1 or 0.2) ( Figure 22 ). Disease severity was measured by calculating the area under the disease progress curve (AUDPC). In the first experiment, the mean AUDPC value for Flicker was significantly higher than for all tested cultivars other than Emerald and Kestrel (Figure 2 3) In the second experiment, the mean AUDPC value for Flicker was significantly higher than all other tested cultivars ( Figure 24 ). No di sease was observ ed on any of the noninoculated Flicker plants in either experiment. Isolated stem tissue taken from each infected plant confirmed that in all cases, the stem lesions were attributable to C. gloeosporioides based on comparing the morphol ogical characteristics of the isolate from the infected plants with those of the original inoculum isolate. The lesions observed in these experiments were consistent with descriptions in published reports on anthracnose. They were subcircular (Jeffries et al. 1990), sunken (Freeman et al. 1998), exhibited orange sporulation (Freeman et al. 1998), coalesced into larger necrotic areas (Jeffries et al. 1990), and resulted in stem dieback (Kim et al. 2009).
36 The results of this study regarding cultivar specific susceptibility were consistent with reports from commercial blueberry growers in central Florida, who reported anthracnose stem lesions only on Flicker, and to a lesser extent Scintilla, which is a progeny of Flicker (Harmon, pers. commun.). In the second experiment in this study, where no wounding occurred (discussed below), only Flicker showed statistically significant symptoms of this disease following inoculation with a C. gloeosporioides isolate collected from central Florida farms W e wer e unable to obtain Scintilla plants in time for inclusion in this experiment; however, it will be included in future screening studies. The formation of anthracnose stem lesions in these experiments was similar in some respects to those reported on nort hern highbush blueberries in Asia and the US. In a case of anthracnose observed on blueberry in northeastern China, the lesions were described as yellow to reddish, irregularly shaped, becoming dark brown in the center and expanding (Xu et al. 2013). A report from Japan described anthracnose lesions on blueberry stems as the shoot tips turning brown, then blighted within 20 cm of the tips. Most of these lesions remained constant in size (Yoshida and Tsukiboshi 2002). Also, K im et al. (2009) reported ant hracnose on blueberry stems in Korea that turned brown to dark brown, became gray, followed by stem death. Finally, a report from Michigan indicated that anthracnose lesions on green blueberry canes were dark brown to black, circular or oval, with light br own to gray centers and salmonpink spore masses (Schilder, 2010). The stem lesions observed in the experiments in the present research were dark brown to black, subcircular to oval, with orange or salmon colored conidia erupting from some of the lesions, and stem blight at the tips, which are all similar
37 characteristic s to published reports. However, there were differences with some of the reported characteristics, including color of the lesion (yellow to reddish according to Xu et al. light brown to gray centers per Schilder, stems turning gray per Kim et al. ) and lesions remaining constant in size per Yoshida and Tsukiboshi The cause of these morphological differences is unknown, but it may have been due to cultivar characteristics (northern highbush vs SHB), pathogen species differences ( C. gloeosporioides vs. C. acutatum ), or environment al conditions As discussed above, there were some differences between the two experiments in this study in the development of lesions on certain cultivars. In Experi ment 1 the mean number of lesions and AUDPC on Flicker were not significantly different from certain other cultivars, but in Experiment 2 the results for Flicker were significantly different from all other cultivars. In addition, Flicker had a high er mean number of lesions in the second experiment (1.5 per plant) than in the first experiment (0.6 per plant). In Experiment 1, some of the young stems had heat damage due to high temperatures in the greenhouse while the plants were covered with polyethylene bags, which may have affected the results. The greenhouse temperature during inoculation was approximately 29 C (and up to 36C thereafter), and would have been higher in close proximity to the plants while sealed inside the bags. Inoculation and cov ering of the plants with polyethylene bags was performed later in the afternoon for Experiment 2, minimizing the amount of time the plants were exposed to higher temperatures while covered (the temperature during inoculation was between 22 and 25C), and no heat damage was observed upon removing the bags.
38 It is possible that the heat inflicted wounding allowed Colletotrichum to directly infect senescent tissue of some of the plants or opportunistically as a saprophyte, where infection otherwise may not h ave occurred. In particular, Emerald and Kestrel, which both had heat damage on some young tips, developed stem lesions in the first experiment, whereas none of the ten replicates of these two cultivars developed lesions in the second experiment where there was no wounding. Vijaya et al (2015) suggested that wounds might increase a plants susceptibility to infection through direct introduction of the pathogen into plant tissues, whereas on nonwounded stems the cuticle may protect the plant against infection. In one study where redfleshed dragon fruit was inoculated with C. truncatum they observed no anthracnose symptoms on nonwounded stems, whereas wounded stems were symptomatic (Vij ay a et al. 2015). In addition, in two reports of anthracnose stem lesions on northern highbush blueberry inoculations were performed on wounded and nonwounded tissue (stems and leaves) W hile the wounded tissue became infected, the nonwounded tissue showed symptoms either weakly or not at all (Kim et al. 2009; Yoshida and Tsukiboshi 2002). It is also possible that the high temperatures had an inhibiting effect on growth of the Colletotrichum isolate (15 646) resulting in fewer mean lesions per plant on Flicker in Experiment 1. Appressoria l formation ( an infection mechanism used by Colletotrichum ) and conidial germination are affected by temperature (Dodd et al. 1991, King et al. 1997). Several studies have found that that the optimal temperature for conidial growth and appressorial formation in C. gloeosporioides was between 20 and 28C, with a decrease at or above 30C (Dodd et al. 1991; Hartung et al. 1981; Lee, 1993; King et al. 1997). Temperatures in excess of 30C in the first experiment,
39 especially during the immediate post inoculation period, may have created an environment that was not favorable for growth of the pathogen, resulting in lower rates of infection on Flicker, whereas the heat inflicted wounding may have resulted in infect ion on Emerald, Farthing, and Kestrel Due in part to the lower infection rate on Flicker in Experiment 1, the mean number of lesions was not significantly greater than the other cultivars. In Experiment 2 the mean number of lesions on cultivars other than Flicker was similar to that in Experiment 1, but the higher mean number of lesions on Flicker resulted in a significant difference from the other cultivars. Therefore, the number of lesions in Experiment 2 may represent more accurately what w ould be observed in these cultivars under ideal infection conditions. Table 2 1 Cultivars inoculated and their nursery source Cultivar Name Source Chickadee Fall Creek Nursery Emerald Fall Creek Nursery Farthing Fall Creek Nursery Flicker AgriStarts Jewel Fall Creek Nursery Kestrel Fall Creek Nursery Rebel Fall Creek Nursery San Joaquin Fall Creek Nursery Springhigh Fall Creek Nursery Star Fall Creek Nursery
40 Figure 21 Mean number of anthracnose lesions per plant Experiment 1. Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). Figure 22 Mean AUDPC per cultivar Experiment 1. Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). b ab ab a b ab b b b b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean Lesions per Plant b ab b a b ab b b b b 0 5 10 15 20 25 30 35 40 45 Mean AUDPC
41 Figure 23 Mean number of anthracnose lesions per plant Experiment 2. Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). Figure 24 Mean AUDPC per cultivar Experiment 2. Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). b b b a b b b b b b 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Mean Lesions per Plant b b b a b b b b b b 0 5 10 15 20 25 30 35 40 45 Mean AUDPC
42 CHAPTER 3 DEVELOPMENT OF DETACHED STEM SCREENING ASSAY FOR ANTHRACNOSE SUSCEPTIBILITY IN SOUTHERN HIGHBUSH BLUEBERRY Materials and Methods Plant Material Ten different cultivars of Vaccinium corymbosum (southern highbush blueberry) grown commercially in Florida were evaluated for susceptibility to the stem lesion form of anthracnose (nine cultivars were used in Experiment 2 as San Joaquin plants could not be obtained for the final experiment) The plants were obtained from Island Grove Nursery and Lochloosa Lake F arm in Hawthorne, Florida, and Cornelius Farms in Manor, Georgia (Table 3 1 ). The plants were grown out in 100% peat until there were sufficient stems with approximately 10 cm of new, green gr owth Plants were watered and fertilized (Petersons 2020 20) on a regular basis. Three protocol adjustment tests were conducted prior to Experiments 1 and 2 to fine tune the specific protocol to be tested Protocol adjustment tests 1 and 2 were conducted in a temperaturecontrolled laboratory on the Univer sity of Florida camp us in Gainesville, Florida (temperature approximately 2 4 C ) Protocol adjustment test 3 and Experiment s 1 and 2 were conducted in a temperaturecontrolled growth room at the University of Florida Gulf Coast Research and Education Center in Wimauma Florida (temperature maintained at 27 C ) Experimental Design A randomized complete block design with ten replicates was utilized, with one stem per cultivar per replicate, and the experiment was conducted twice during September 2017. The treatment was ten different V. corymbosum cultivars (Table 3 1 )
43 (nine for Experiment 2) each of which was inoculated with an isolate of C. gloeosporioides (15 646) Ten Flicker stems (kn own to be susceptible to anthracnose stem lesions) were used as negative controls instead of using control replicates for each cultivar. This allowed an increase in the number of replicates that could be inoculated with the pathogen. Source of Isolate an d Inoculum Preparation A single conidium isolate of C. gloeosporioides (15 646) was prepared and used as the pathogen in this experiment. The isolate was originally collected from infected Flicker stems i n a blueberry orchard in central Florida in 2015. The isolate was incubated in a petri plate on autoclaved potato dextrose agar (PDA) under continuous fluorescent lighting at 25C, until sufficient sporulation had occurred (five days). The plate was then flooded with sterile deionized water (SDW) a sterilized plastic rod with a triangular tip was used to dislodge the conidia into suspension, and two layers of cheesecloth were used to remove mycelial particles. Conidia l concentration was determined using a hemacytometer (Bright Line Hemacytometer, Hausser Scientific), and then diluted to 1x105 conidia mL1. Inoculation and Collection of Data Prior to inoculation, ten stems were harvested from whole plants for each cultivar (each stem comprised of ap proximately 10 cm of new growth). These stems were pl aced into a plastic bag, from which one stem per block was arbitrarily selected. The conidial suspension was applied to each stem using a Smith Professional 2gallon sprayer (The Fountainhead Group, New York Mills, NY) coating the entire stem surface and upper surfaces of leaves. The control stems were sprayed with SDW Each
44 inoculated and control stem was placed into a 25 x 150 mm borosilicate glass culture tube (Kimble Chase, Vineland, NJ ) into which 10 mL of SDW had been added, c overed with a plastic cap, and placed into racks separated by block (Figure 3 1) For Experiments 1 and 2, t he rack s were placed into a temperaturecontrolled growth room at 27 C under fluorescent lighting (1 4 hours light, 1 0 hours dark) A room humidifier ( Vicks 1.5 Gallon Wa rm Steam Vaporizer Model V150SGNL, Proctor & Gamble) was used to increase relative humidity in the growth rooms, which was maintained between 60 and 80%. The plastic caps were removed from the tubes after 24 hours in Experiment 1, and after 48 hours in Ex periment 2. Each stem was examined daily to identify any disease symptoms. The number of lesions on each infected stem was recorded to determine disease incidence. After the observation period, infection by C. gloeosporioides was confirmed by isolating f rom necrotic lesion boundaries to fulfill Kochs postulates The isolated tissue was surface disinfested with a 10% bleach (0.6%) ( sodium hypochlorite) solution for one minute, followed by three water rinses for one minute each. The isolated tissue was then placed on PDA media and incubated under continuous fluorescent lighting at 25C. For Experiment 2, since tissue isolations for all previous tests of protocol and Experiment 1 had showed C. gloeosporioides as the cause of the disease symptoms on all infected stems, three infected stems per cultivar were selected arbitrarily to perform tissue isolations instead of is olating from all ten infected stems.
45 Statistical Analysis The response variable (mean lesion number) was analyzed utilizing a oneway analysis of variance (ANOVA) to assess the effect of treatment (cultivar), using the following linear model: e where Y is the effects, and e is the random residual effect ~ N 2). Tukeys honestly significant difference test was utilized when the ANOVA test showed significant differences among treatments. T his test was used to separate treatment means at P 0.05. All analyses were conducted using the R statistical software (R Development Core Team, 2016) Results and Discussion Three protocol adjustment test s were performed to help determine the optimal procedures and conditions to use in the research experiments. In the first multiple stem cuttings were taken from a single stem, so that some stems had a cut at the top of the cutting. The tube caps were kept in place for the entire test (approximately two weeks) to help keep the stems from becoming desiccated. In that test, anthracnose infection was observed across all inoculated cultivars, but not in the controls The mean number of lesions for Kestrel, San Joaquin, Springhigh and Star were not significantly different than the mean number of lesions for Flicker at P 0.05 (Figure 3 2 ) There was no significant effect of the blocking structure in any of the protocol adjustment tests. Tissue isolations were not taken for the protocol adjustment tests, but anthracnose was identified solely through morphological symptoms (dark, sunken lesions, expanding over time, orange sporulation erupting from necrotic areas).
46 Area under the disease progress curve (AUDPC) was used for the w hole plant inoculations in Chapter 2 as a measure of disease severity. However, it was decided not to use this measurement for the detached stem inoculations since the observed disease symptoms in the first protocol adjustment test (as well as the following tests) were much more severe (i.e., longer lesions lengths, with the entire stem becoming necrotic in several cases) than those seen in the whole plant inoculations, possibly due to the extended period of leaf wetness (discussed below). It was hypothesiz ed that the wounding at the tops of many stems might have resulted in infection that would not otherwise have occurred. Wounding, including pruning and insect damage, provides a direct path for pathogen infection (Cheong et al. 2002) where there may not otherwise have been infection. In studies where highbush blueberry was inoculated with C. gloeosporioides infection was observed on all wounded inoculated leaves or stems, but symptoms were only weakly expressed, or not at all, with nonwounded t issue (Kim et al. 2009; Yoshida and Tsukiboshi 2002). In experiments addressing stem anthracnose on redfleshed dragon fruit, wounded stems predisposed the plants to infection by C. truncatum It was suggested that protective wax and cuticle layers on non wounded stems might have protected plants from the pathogen to some degree (Vijaya et al. 2015). In the second protocol adjustment test only a single cutting was taken from any given plant stem to avoid the mechanical wounding present in the first test However, there was blueberry gall midge damage on the tips of several stems, and infection was again observed across all tested cultivars except for Emerald and Kestrel. The mean number of lesions for Flicker was significantly different from all ot her tested cultivars
47 except for San Joaquin (Figure 3 3 ) It was hypothesized that th e insect inflicted wounding might have resulted in disease symptoms on non susceptible cultivars which might not have been observed absent the wounding. In the third protocol adjustment test healthy stems without any wounding were inoculated. However, a high rate of infection across all tested cultivars except for Emerald was observed (Figure 3 4 ) with higher mean numbers of lesions for several cultivars than were observed in the first two tests of protocol At this point it was hypothesized that keeping the tube caps in place for the entire observation period was creating an unnatural environment where relative humidity in the tubes remained close to 100%. Condensation was observed on the inside of all tubes for the entire observation period. Since tissue wetness is a key environment al factor affecting rates of disease incidence and severity (Miles et al. 2003; Moraes et al. 2014; Moral, 2012; Schilder, 2010) this environmental stress may have resulted in infection where, absent this factor disease may not have occurred. It was decided that for Experiment 1 the tube caps would remain in place for 24 hours following inoculation, then removed, which is approximately the same as the time period the plastic bags were kept in place for the whole plant inoculation experiments (16 hours) discussed in Chapter 2. In addition, th e third protocol adjustment test was the first one conducted in a temperature controlled growth room, where the temperature was maintained at 27C, an optimal temperature for Colletotrichum growth ( Dodd et al. 1991 ; Denner et al., 1986; Hartung et al. 1981; King et al. 1997; Lee, 1993). Acting t ogether with the very humid environment in the tubes, the optimal temperature may have contributed to the higher mean number of
48 lesions on several tested cultivars although the temperature in the laboratory for protocol adjustment tests 1 and 2 was only slightly lower Once the prot ocol to be tested in the experi ments had been adjusted, Experiment 1 was conducted. A nthracnose stem lesions were observed on young green stems of certain tested cultivars; they were primarily subcircular, sunken, and enlarging over ti me. Where multiple lesions were located in close proximity, they often coalesced into a large necrotic area, at times causing dieback of the stem tip or necrosis of the entire stem On some lesions orange sporulation was observed erupting from the necrotic area. These symptoms are consistent with those observed in the whole plant screening experiments discussed in Chapter 2 as well as in published reports ( F reeman et al., 1998; Jeffries et al. 199 0; Kim and Tsukiboshi 2009) T he mean number of lesions on Flicker was significantly different from all cultivars except Jewel, Emerald, and Star (Figure 3 5) There was no significant effect of the blocking structure. An analysis of disease incidence (i.e., the presence or absence of anthracnose symptoms on each stem ) was identical to the analysis of mean number of lesions. In Experiment 2, the time during which the tube caps remained in place was increased from 24 to 48 hours to determine whether this would result in a higher number of lesions on Flicker and separate its means from those of the other cultivars. A nthracnose stem lesions were observed on young green stems of some cultivars and were similar in appearance to those observed in Exper iment 1. T he mean number of lesions on Flicker was twice the number observed in Experiment 1 (1.0 vs. 0.5) and was significantly different from all other cultivars except Jewel and Rebel (Figure 3 6 ) An analysis of disease incidence was very similar to the analysis of mean number of
49 lesions. Flicker and Rebel had slightly lower means using disease incidence, but the significance of the significant differences in means remained the same. The objecti ve of these experiments was to determine whether results comparable to whole plant inoculation results could be obtained using detached stems indicating it would be an effective screening assay (Cowley et al. 2012) The results of the whole plant inocula tion in Chapter 2, Experiment 2 showed a statistically significant difference between the susceptibility of Flicker and all other tested cultivars (Figures 23 and 24), although the results of Experiment 1 were less clear, possibly due to heat damage su ffered in the greenhouse. The results of the detached stem inoculations did not provide a statistically significant difference between Flicker and other tested cultivars (Figures 3 5 and 36 ). In Experiment 1, the mean numbers of lesions for Emerald, Jewel, and Star were not significantly different from Flicker, although the P values for Emerald and Star (0.063) were only slightly above the significance level of 0.05. In Experiment 2, the mean numbers of lesions for Emerald, Jewel, and Rebel were not significantly different from Flicker. The P value for Rebel was 0.917, and its mean number of lesions (0.7) was close to the results for Flicker (1.0) the known susceptible cultivar It does not appear that the detached stem protocol used in these experiments will provide results comparable to whole plant inoculations. Based on the outcomes of the tests of protocol and Experiments 1 and 2, there are a few reasons why the detached stem protocol may not be as precise as whole plant inoculations in screening blueberry plants for susceptibility to anthracnose. One limitation on the use of detached stem assays is the possibility of changes in susceptibility/resistance reactions following removal of the stem from the plant, at which
50 point it begins to senesce and deteriorate. In general the level of resistance in plant tissues tends to decrease following detachment (Dhingra and Sinclair 1995; Pettit et al. 2011). The stems used in the present experiments were young tender tips, which senesce more quickly than older tissue. This may partially explain the increased level of disease observed on detached stems in the present experiments. In addition, the environment inside the glass tubes remained very humid, even when the tube caps were removed after 24 and 48 hours. Condensation was observed on the inside of several tubes even during the second week following inoculation. In addition, the narrow width of the tubes resulted in leaves curling around the stems, in many c ases remaining in contact with the stems. There are several published articles on the impact of leaf wetness duration on conidial growth and appressorial development in Colletotrichum A study on anthracnose in blueberry canes in Michigan noted that the in fections may have been related to a late season increase in precipitation, providing sufficient wetness to allow infection of young green canes at a time when spores were abundant (Schilder, 2010). A report on C. acutatum and anthracnose fruit rot in blueb erry found higher percentages of appressoria formation and infected fruit with increasing periods of wetness. The average percentage of melanized appressoria (a penetration structure formed by Colletotrichum ) was 6, 30, and 63% following 12, 24, and 48 hours of wetness (Miles et al. 2013). The extended period of leaf and stem wetness in this present research possibly resulted in an increase in the rate of anthracnose infection, above that observed in whole plant inoculations where leaf and stem wetness persisted only during the time when the plants were covered with bags (16 hours).
51 Another reason for the increased levels of infection could be anthracnose infections on leaves which provided a secondary source of inoculum to infect the stems. Various published reports have identified secondary sources of inoculum as important in anthracnose infections where secondary inoculum was produced on leaves in corn, banana, and mangoes ( Berg strom and Nicholson, 1999; de Bellaire and Mourichon, 1997; Fitzell and Peak 1984). Bergstrom and Nicholson (1999) noted that as individual leaves become senescent they become more susceptible to anthracnose. As discussed above, in many cases in this pres ent research the leaves remained in contact with the stems inside the tubes. Anthracnose leaf spots, many with orange sporulation (i.e., conidia) erupting from these necrotic areas, were observed for several stems. It is possible that this additional sourc e of inoculum increased the likelihood of anthracnose infection above the levels in the whole plant inoculations, where minimal leaf spotting was observed and the leaves were generall y not in contact with the stems. One additional variable in the level of infection is the different stages of growth of the plant stems. Although the plants were acquired at the same time and efforts were made to have sufficient uniform growth, there remained some variation in the stages of growth, due to the genetic variation between cultivars. I n some cases the re were also an insufficient number of relatively identical stems for the ten replicates and slightly older stems were utilized in addition to the younger stems It is possible that this pathogen requires a very specif ic stage of growth for infection, and the lack of uniformity affected the results. Although the detached stem protocol tested in the present experiments does not appear to be as accurate as whole plant inoculations in screening for anthracnose
52 susceptibili ty in blueberries, and likely would not be an effective replacement for whole plant screening, it may have some value. The usefulness of this protocol may be in using it as an initial test in screening large volumes of plant material to identify potentiall y susceptible cultivars or selections for further testing (Denman and Sadie, 2001) Table 31 Cultivars inoculated and their nursery source Cultivar Name Source Chickadee a Island Grove Nursery Emerald a Island Grove Nursery Farthing a Island Grove Nursery Flicker a Lochloosa Lake Farm Jewel a Island Grove Nursery Kestrel a Island Grove Nursery Rebel Cornelius Farms Springhigh a Island Grove Nursery Star a Island Grove Nursery a Developed by the University of Florida blueberry breeding program Figure 31. Image of detached stems in glass culture tubes. Photo courtesy of author.
53 Figure 32 Protocol Adjustment Test 1 Mean number of anthracnose lesions per stem Columns with the same letter are not significantl y different according to Tukeys HSD test ( 0.05). Figure 33. Protocol Adjustment Test 2 Mean number of anthracnose lesions per stem Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). b b b a b ab b ab ab ab 0 0.5 1 1.5 2 2.5 Mean Lesions per Stem b b b a b b b ab b b 0 0.5 1 1.5 2 2.5 Mean Lesions per Stem
54 Figure 34. Protocol Adjustment Test 3 Mean number of anthracnose lesions per stem Figure 35 Experiment 1 Mean number of anthracnose lesions per stem Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). 0 0.5 1 1.5 2 2.5 Mean Lesions per Stem b ab b a ab b b b ab 0 0.5 1 1.5 2 2.5 Mean Lesions per Stem
55 Figure 36 Experiment 2 Mean number of anthracnose lesions per stem Columns with the same letter are not significantly different according to Tukeys HSD test ( 0.05). b ab b a ab b ab b b 0 0.5 1 1.5 2 2.5 Mean Lesions per Stem
56 CHAPTER 4 TRAIT SEGREGATION OF ANTHRACNOSE STEM LESION SUSCEPTBILITY IN SOUTHERN HIGHBUSH BLUEBERRY Materials and Methods Biparental Populations Six F1 segregating (or pseudo F2) populations (i.e. parents are presumed to be heterozygous at many loci ) were created from controlled crosses between Flicker or Scintilla (cultivars known to be susceptible to anthracnose stem lesions) and stage four parental clones ( not known to be susceptible) Flicker and Scintilla were used as both male and female parent in different populations In addition, three F1 segregating populations were obtained from controlled crosses between elite clones not known to be susceptible and assumed to be resistant to the stem lesion form of anthracnose. A search was performed through the University of Florida blueberry breeding database to confirm that none of the se elite clones had either Flicker or Scintilla in their pedigree lineage. All plant material comes from the University of Florida blu eberry breeding program (Table 4 1 ). Seeds from the resulti ng offspring were sown into one gallon pots in 100% peat, and following germination, 200 seedlings from each cross were arbitrarily sel ected and transferred into 46 x 61 cm flats containing 100% Fafard peat (Sun Gro Horticulture, Agawam, MA) at 50 seedlings per flat. About six m onths later, these seedlings were transplanted into the same size flats, filled with 100% peat, at 24 plants per flat. P lant s were provided water as needed and Peters Professional 2020 20 fertilizer (Scotts, Marysville, OH) applied every ten days.
57 Stem cu ttings of each of the populations were prepared the second week of January 2017 to propagate a clonal set of these populations, to be used in a second iteration of inoculations. Terminal 7 to 8 cm long multinodal stem cuttings (firm but green) were prepared, with the lowest leaves removed from the base of each cutting. The cuttings were placed into forty cell trays with a 50:50 mixture of peat (Sun Gro Horticulture, Agawam, MA) and perlite (Harborlite Corp., Lompoc, CA), and maintained on mist benches in a temperaturecontrolled greenhouse. Overhead mist was applied for six seconds every ten minutes during daylight hours. After the cuttings were sufficiently rooted, the mist was turned off and the cuttings were hand watered as needed. The cuttings were then transplanted into 46 cm x 61 cm flats containing 100% peat (Sun Gro Horticulture, Agawam, MA) at the same plant density used for the original seedling populations (i.e., 24 plants per flat) Flicker plants were acquired from Lochloosa Lake Farm nursery and grown out in onegallon pots to be used as positive standards. Experiments 1, 2, and 3 were performed on the first set of nine bi parental populations, and Experiment 4 was performed on the second, clonally propagated set of nine populations. Inoculation and Incubation Conditions A summary of the inoculation locations and conditions is presented in Table 45 Experiments 1 and 2 were conducted in a temperaturecontrolled greenhouse (17 C 3 6 C ) without supplemental lighting at the University of Florida Gulf Coast Research and Education Center (GCREC) in Wimauma Florida. Experiments 3 and 4 were conducted in temperaturecontrolled growth room s at the GCREC, where the
58 temperature was maintained at 27C (Experiment 12) A room humidifier ( Vicks 1.5 Gallon Warm Steam Vaporizer Model V150SGNL, Proctor & Gamble) was used to increase relative humidity in the growth rooms which were maintained between 60 and 8 0% To determine the optimal temperature range for the C. gloeosporioides isolate (15 646) used in these experiments ( following the initial inoculations in the greenhouse), the isolate was grown out in incubators under continuous fluorescent lighting at 10C to 35C, in 5 C increments. The isolate was transferred from filter paper storage onto potato dextrose agar ( PDA ) media, and 5 mm agar cubes from the growing edges of the colonies were subsequently cut and transferred to new PDA plates. The diameter of each isolate at its widest point was measured each day from the second to the sixt h day after placing in incubation. Isolate Source and Inoculum Preparation Each of the populations was inoculated with a singleconidium isolate of C. gloeosporioides (15 646) This isolate was originally collected in 2015 from naturally infected Flicker stems on a commercial blueberry farm in central Florida ( Harmon, pers. commun.). The isolate was incubated on PDA under continuous fluorescent lighting at 25C until sufficient sporulation had occurred (approximately five days). The plate containing the i solate was flooded with sterile deionized water (SDW), and the conidia were dislodged into suspension using a sterilized plastic rod with a triangular tip. This suspension was then filtered through two layers of cheesecloth to remove mycelial particles. C onidium concentration was determined using a hem a cytometer (Bright Line Hem a cytometer, Hausser Scientific), and the suspension was diluted to 1x105 conidia
59 mL1 for Experiment 1 and 1x106 conidia mL1 for all subsequent experiments. The conidial concentrat ion was increased following Experiment 1 to increase levels of infection. Inoculation Protocol and Collection of Data B i parental populations were inoculated on March 31, 2017 (Experiment 1), May 3 2017 (Experiment 2) July 7 2017 (Experiment 3) and September 23, 2017 (Experiment 4) In each experiment, t he conidial suspension was sprayed to run off onto each tray of plants using a Smith Professional 2gallon sprayer (The Fountainhead Group, New York Mills, NY), coating all stem surfaces and upper sur faces of leaves. The Flicker control plants were also inoculated with the conidial suspension in each experiment Each tray of plants was immediately sealed inside a 61 cm x 51 cm x 122 cm 2 mil polyethylene bag (Uline, Pleasant Prairie, WI), the interior surface of which had been moistened with SDW, for 16 hours for Experiment 1, 24 hours for Experiment s 2 and 3, and 72 hours for Experiment 4. The Flicker control plants were immediately sealed in moistened 30.5 x 61 cm 4mil polyethylene bags (Uline, P leasant Prairie, WI) Following removal of the polyethylene bags, the plants were hand watered as needed. Each plant for each population, as well as the control plants, w ere examined daily to identify any anthracnose symptoms (i.e., necrotic stem lesions). The number of plants per population with anthracnose stem lesions was used to determine segregation ratios of the susceptibility trait in the segregating populations. These ratios were compared to expected ratios in tetraploid species, initially assuming for purposes of comparison that susceptibility was determined monogenically by a fully recessive allele at a single locus
60 After the observation period was completed, tissue was isolated from the necrotic lesion boundaries of each infected plant to confirm infection by C. gloeosporioides The isolated tissue was surface disinfested using a 10% bleach (0.6%) ( sodium hypochlorit e ) solution, followed by three sterile deionized water rinses for one minute each. The tissue was then placed in a petri plate on PDA and incubated under continuous fluorescent lighting at 25C. For Experiment 2, since tissue isolations for all previous e x periments showed C. gloeosporioides as the cause of the disease symptoms on all infected plants, three infected plants per population were arbitrarily selected to perform tissue isolations instead of m aking isolations on all infected plants. Results and Di scussion The initial hypothesis was that the susceptibility trait is determined monogenically by a recessive allele at a single locus (i.e., a single dominant allele controls resistance to anthracnose). This provided a set of expected segregation ratios ( and percentages of susceptible plants) used to compare to the observed percentages of susceptible plants following inoculation with C. gloeosporioides (Table 4 2 ) Populations with Flicker and Scintilla as a parent developed anthracnose stem lesion symptoms, suggesting that the susceptibility trait was inherited from each of these susceptible parents. I solations were made from stem tissue to confirm that the observed lesions were caused by C. gloeosporioides the pathogen used as the inoculum. However, low percentages of infected plants were observed in each of the four experiments (Figure 4 1 ) Since the same individual plants did not necessarily show symptoms in each of the four experiments, the results were accumulated to record the individual s infected across all experiments (Figure 42 ). S ymptoms were observed in 18
61 of the same individual plants in two of the four experiments, and in 7 of the same individuals in three of the four experiments. For the remaining individuals, symptoms (if any) were observed in one of the four experiments. The cumulative infection percentages (and segregation ratios) for 08 18 x Flicker and 0843 x Flicker fit well with the expected ratios for singlegene control of susceptibility in a chi square goodness of fit test assuming a fully recessive susceptible parent (aaaa) and a heterozygous (AAaa) assumed resistant parent (Table 42) Chi square test P values for these populations of .267407 and .250592, respectively, were greater than the significance level of P > 0.05 with one degree of freedom (Table 44) In addition, the chi square tests for the three control populations had good fits between the observed and expected results, assuming two heterozygous (AAaa) parents (expected percentage of susceptible plants is 2.77%) In the case of the remaining populations, i t is possible that environmental conditions or plant stage of growth might have affected the number of infect ed plants This may have result ed in lower chi square P values (i.e., the number of observed infected plants did not fit well with the expected number of infected plants) There are a few possible reasons for lower than expected infection rates for the susceptibility trait It is possible that environmental factors (e.g., temperature, leaf wetness duration, relative humidity, etc.) or factors involving the stage of development of the plant affected the observed infection rates. As discussed above, researchers found that observed anthracnose resistance in white lupin was 29% controlled by environmental factors and environment x genetic interactions (Yang et al. 2010). A study of anthracnose in cassava determined that the phenotypic evaluation of anthracnose symptoms (stem lesions) might be significantly affected by environment, in
62 particular by temperature (Boonchanawiwat et al. 2016). Also, a study of Fusarium head blight in wheat found that possible quantitative trait loci ( QTLs ) for disease resistance behaved differently in different environments (i.e., the same locus may express varying degrees of resistance in different environments). Researchers determined that 49% of phenotypic variat ion was due to QTLenvironment interactions. A trait associated with disease severity was measured as 55% under field conditions, but 100% under greenhouse conditions (Ma et al. 2006). Similarly, it is possible that the incidence of anthracnose stem lesions in these experiments was lower than the disease incidence reported in grower fields (or expected with a monogenic recessive trait) due to different environmental conditions in both th e greenhouse and growth rooms, and the resulting interaction with the gene(s) controlling susceptibility/resistance. It was initially thought that the low incidence of anthracnose lesions in Experiments 1 and 2 might have been due to high afternoon temperatures in the greenhouse. Afternoon temperatures reached 36C and would have been higher inside the plastic bags covering the trays of plants. Various studies have found that the optimal temperature for C. gloeosporioides conidial germination and ap pressorial formation is between 20 and 28C with decreasing conidial germination and no appressorial formation above 30C ( Denner et al., 1986; Dodd et al., 1991; Hartung et al. 1981; King et al. 1997; Lee, 1993) R esults obtained from a test of C. gloeosporioides growth at different temperatures indicated that the optimal temperature range for the growth of th e isolate used in these experiments (15 646) is 25 C, with somewhat less growth at 20C and 30C, and little growth at 10 C, 15 C, and 35 C ( F igure 47 ). This was an indication that
63 high temperature might have been a factor in low infection rates in the first two inoculations (Experiments 1 and 2) To determine whether temperatures higher than the optimal range were a causal factor in the low infection rates, Experiment 3 was performed with the plants placed in growth room s where the temperature was maintained at 27 C. At that temperature in the growth rooms, the relative humidity (RH) was about 35 to 45%. To raise RH, room humidifiers were used, and the RH was maintained between 60 and 80% The materials and methods were otherwise the same as for the initial inoculation. However, the infection rates remained low ( Figure 4 3 ) and were similar to those observed in the prior inoculations ( Figure 4 6 ). Another possibility is that this C. gloeosporioides pathogen requires very specific periods of leaf wetness for a high rate of infection, which was not properly replicated for the inoculations in Experiments 1, 2 and 3. If this was the case, there may have been a number of escapes (i.e., no infection on susceptible plants because the factors required for disease did not occur at the proper time or for a sufficient period) (Agrios, 2005) There are many published articles addressing the impact of temperature and leaf wetness ( as well as RH in some cases ) on conidia growth and appressorial development in Colletotrichum A report on anthracnose in blueberry canes in Michigan noted that the infections might have been related to rainy weather later in the season, which could have provided sufficient wetness to allow infection of young green canes at a time when spores were abundant (Schilder, 2010). A study on C. acutatum and anthracnose fruit rot in blueberr y found that in general, higher percentages of appressorial formation and infected fruit were found with increasing periods of wetness.
64 The average percentage of melanized appressoria was 6, 30, and 63% following 12, 24, and 48 hours of wetness Also, the percentage of melanized appressoria on parafilm covered slides at 25C was 0, 1, 5, and 44% after 3 days where RH was 54, 84, 95, and 100%. The percentage of infected fruit at the same RH levels was 3, 17, 50, and 67% (Miles et al. 2013). A n investigation of anthracnose on olives found that in a group with wetness periods of 0 to 24 hours the average disease severity was 22%, whereas in a group containing a 48hour wetness period the average disease severity was 58% (Moral, 2012). A study on guava reported an increase in conidia l germination and appressorial melanization as wetness durat ion increased. At 25C, conidial germination was 33, 66, and 77% and appressorial development was 26, 59, and 68% at 6, 24, and 48 hours of wetness (Moraes et al. 2014). Si milar relationships between wetness duration and conidial growth, appressorial development and infection were found in studies on strawberry ( Wilson et al. 1990, Leandro, 2003), almond ( Dieguez Uribeondo et al. 2011), watermelon (Monroe et al. 1997), and evergreen azalea ( Bertetti et al. 2009) It is also possible that this pathogen requires a very specific stage of plant development for a high level of infection, which could not be uniformly replicated in the first three experiments Ontogeny in plants involves many aspects of various organs, including morphological characteristics of leaves, internode length, probability of branching on a shoot, etc. (Costes et al. 2012). For example, the development of a shoot is typically divided into a juveni le vegetative stage, an adult vegetative stage, and a reproductive stage (Poethig, 2003). In addition, changes in a plants morphology can
65 be related to environmental, nutritional, and seasonal changes (known as plant plasticity) (Costes et al. 2012). On togenic resistance is a change in a plants resistance to a pathogen with age (Agrios, 2005). A similar concept known as developmental resistance refers to a change in resistance to pathogens that is correlated with the developmental stage of the host plant or its organs Although in many cases ontogenic or development resistance has been observed as an increase in resistance as a plant matures (Whalen, 2005) there are also cases where a plant has demonstrated changing susceptibility and resistance at various stages of development. A study of resistance to anthracnose in Andean lupin found that resistance was not equally expressed at all developmental stages. Genotypes in this study were susceptible in the early 2 to 3 leaf and 4to 5 leaf stages, resistant at the 6to 7 leaf stage and susceptible at the 8to 9 leaf and 10 to 11leaf stages. The authors suggested that this could be due to stagespecific resistance genes (Falconi et al. 2015). A study of anthracnose in corn found susceptibility at the seedling stage, followed by resistance as leaves expand and mature, and then a second phase of susceptibility around the time of anthesis (likely associated with the onset of senescence) (Jamil and Nicholson 1987). Where anthracnose stem lesions were observed in central Florida blueberry plantings the plants were mature, with young green growth at the top due to post harvest pruning (topping). The lesions were observed in the young growth at the top of the plant. In contrast, t he plants inoculated in these experiments were seedlings approximately oneyear old, grown out in flats of peat which kept the plants small so
66 the stage of development was very different than the plants where field infections were observed. Abiotic stress (e.g., wounding) could also have an impact on a plants susceptibility to a pathogen through alteration of a hosts physiology, possibly in combination with stage of development. Wounding, including pruning and insect damage, provides a direct path for pathogen infection (Cheong et al. 2002) where there may not otherwise have been infection. In studies where highbush blueberry was inoculated with C. gloeosporioides infection was observed on all wounded inoculated leaves or stems but symptoms were only weakly expressed, or not at all with nonwounded tissues (Kim et al. 2009; Yoshida and Tsukiboshi 2002). In experiments addressing stem anthracnose on redfleshed dragon fruit, wounded stems predisposed the plants to infection by C. truncatum It was suggested that protective wax and cuticle layers on nonwounded stems might have protected plants from the pathogen to some degree (Vijaya et al. 2015). As discussed above, where anthracnose stem lesions were observed in central Florida blueberry plantings the plants were mature, with young green growth at the top due to post harvest topping Although this wounding may have had an impact on the level of infection observed in the field, since it could have provided a direct path for infec tion by the C. gloeosporioides pathogen it is not likely since disease symptoms in the field were observed in late July and August, two to three months after post harvest topping A nthracnose stem lesions have not been reported in other cultivars in the s ame production fields The plant populations inoculated in these experiments were not wounded.
67 For Experiment 4, efforts were made to replicate late summer environmental conditions when field infections were observed (appropriate temperatures, longer perio d of leaf wetness, and higher relative humidity). Growth rooms were again utilized to control temperature, which was maintained at 27C A lthough this is somewhat cooler than central Florida temperatures in August, it is the temperature at which the inoculum isolate grew best. Also, room humidifiers were used in the growth rooms to increase RH to between 60 and 80 % T here is frequent precipitation in centr al Florida during late summer months and in many cases overhead irrigation (including on the farms with the most severe anthracnose symptoms) (Harmon, pers. commun.). This may have increas ed leaf wetness duration beyond the 24 hours that the plants in the experiment were kept in moistened plastic bags when incubating the first set of populations. The decision was made to extend the length of time the plants were covered by plastic bags from 24 hours to at least 48 hours for the inoculation of the second set of populations to increase the duration of leaf wetness. The appearance of stem lesion symptoms on the Flicker control plants was used to indicate when the bags should be removed on the bi parental populations. This occurred 72 hours post inoculation, at which time the bags were removed. In addition, efforts were made to ensure that the plants each had young, green growth and were relatively uniform in their stage of development by providing uniform applications of water and fertilizer, and not pruning back any of the stems However, it is not possible to attain completely uniform growth due to genetic variability both between and within the segregating populations Despite the efforts to provide favorable environmental conditions for infection, the per centage of infected plants per population in Experiment 4 remained low (Figure 4 1 ).
68 However, the extended period of leaf wetness did result in higher rates of infection than in each of the previous experiments. In the 0854 x Scintilla population, the percentage of plants infected by anthracnose increased from 5.8%, 7.9% and 5.9% in Experiments 1, 2 and 3, respectively, to 14% ( Figure 41 ). The percentages in the other populations generally increased, but not as much The larger increase in the 0854 x Scintilla population may have been due to the interaction of the increased leaf wetness duration with the genetic component controlling susceptibility (gene x environment interaction), since the increases in the other popul ations were not as large. It is also possible that the susceptibility (or resistance) trait is polygenic. Lyrene (1993) indicated that most genetic variation in southern highbush blueberries is polygenic ally controlled. If the anthracnose susceptibility t rait in blueberry was, for example, controlled by two genes, for a fully recessive susceptible parent (aaaabbbb) and a heterozygous assumedresistant parent ( AaaaBbbb) the segregation ratio for the susceptibility trait would be 1: 4 ( 25% susceptible) (Table 4 3). This level of susceptibility is nearly identical to the percentage of infected plants observed in 08 54 x Scintilla on a cumulative basis although none of the other populations had infection percentages close to this level. Several published studies have identified polygenic systems responsible for susceptibility or resistance to anthracnose in various crops. Two studies in white lupin ( Lupinus albus ) found that anthracnose resistance was polygenic, with multiple genes working additively to confer resistance (Adhikari et al. 2009; Yang et al. 2009 ). However, 29% of phenotypic resistance was attributed to environmental factors and environment x genetic interactions (Yang et al. 20 09). Research of anthracnose resistance in pepper ( Capsicum annuum and Capsicum chinense) determined that
69 resistance was inherited quantitatively, with four quantitative trait loci (QTLs) identified as impacting the trait (Voorrips et al. 2004). A study on anthracnose resistance in water yam ( Dioscorea alata) found that resistance is dominantly but quantitatively inherited (Mignouna et al. 2002). An article on anthracnose resistance in lentil reported a polygenic effect of minor genes along with a major gene effect (Tullu et al. 2003). A study on common bean identified seven resistance genes for anthracnose (Kelly and Young 1996). Finally, research on anthracnose in cassava found that the disease phenotype (stem lesions) had a continuous distribution ranging from 12.5 to 27.0 mm, which the authors suggested indicated th at resistance was controlled by a complex of genes (Boonchanawiwat et al. 2016). With polygenic or quantitative resistance, the effect of each gene is typically small, and is often influenced by environmental factors, or by interaction with other genes (i .e., epistasis). This type of resistance is characterized by a quantitative distribution of differences in the level of resistance (Lindhout, 2002). The range of anthracnose stem l esions observed in Experiment 2 at seven days after inoculation was from 1.8 to 1 3 .9 mm (means per population between 3.6 and 12.1 mm) in individual plants with disease symptoms (Table 45) In Experiment 3 the range of lesions observed was from 1.8 to 28.7 mm (means between 3.5 and 28.7 mm) in individual plants with disease symptoms (Table 46 ). This continuous distribution could indicate control of the susceptibility/resistance trait by more than one gene. However, the mean lesion lengths among populations are quite close together, so this seems unlikely. The top end of the rang e in lesion lengths could have been due to a different stage of growth for those individual plants, since there is genetic variability within the populations.
70 In addition, although double reduction in autotetraploids can also impact segregation ratios, one recent study found that many traits in SHB (an autotetraploid) are inherited with no double reduction ( Amadeu et al. 2016). Further work needs to be performed to determine the specific gene structure controlling the anthracnose susceptibility trait. H owever, based on the present research a defensible argument can be made that this trait is controlled monogenically by a recessive allele at a single locus The good or nearly good, fit of the observed percentages of infected plants for multiple populatio ns with the expected percentages for a monogenic trait assuming a fully recessive susceptible parent (aaaa) and a heterozygous (AAaa) assumedresistant parent, is a strong point in favor of monogenic control Table 4 1 Par ents used in controlled crosses Female Parent Male Parent Type of Cross Scintilla 02 117 susceptible x resistant a 08 54 Scintilla r esistant a x susceptible Flicker 02 22 Crisp susceptible x resistant a 08 18 Flicker r esistant a x susceptible 08 43 Flicker r esistant a x susceptible Flicker Cor. 99 42 susceptible x resistant a 12 62 10 06 r esistant a x resistant a 04 245 11 80 r esistant a x resistant a 12 78 06 498 r esistant a x resistant a a assumed resistant Plant source: UF Blueberry Breeding Program
71 Table 4 2 Expected Segregation Ratios Assuming Monogenic Control Fully Recessive Susceptible Parent and No Double Reduction Fully Recessive Susceptible Parent Parent With Varying Recessive Alleles % Susceptible Plants % Resistant Plants aaaa AAAA 0 .0 % 100 .0 % aaaa AAAa 0 .0 % 100 .0 % aaaa AAaa 1 6. 7% 83 .3 % aaaa Aaaa 50 .0 % 50 .0 % Table 4 3 Expected Segregation Ratios Assuming Two Fully Recessive Genes Control Susceptibility Trait and No Double Reduction Fully Recessive Susceptible Parent Heterozygous Parent % Susceptible Plants % Resistant Plants aaaabbbb AAaaBBbb 2.8% 97.2% aaaabbbb AaaaBbbb 25 .0 % 75 .0 % Table 44. Chi Square Results Based on Cumulative Infected Plants in Experiments 14 Population Observed Infected Plants Expected Infected Plants P Value Scintilla x 02 117 16 29 (16.7%) .009641 08 54 x Scintilla 47 32 (16.7%) .003289 Flicker x 02 22 Cr 10 31 (16.7%) .000036 08 18 x Flicker 24 30 (16.7%) .267407 08 43 x Flicker 24 30 (16.7%) .250592 Flicker x Cor 99 42 16 30 (16.7%) .005553 12 62 x 10 06 4 5 (2.8%) .646767 04 245 x 11 80 5 5 (2.8%) 1.000000 12 78 x 06 498 6 5 (2.8%) .646767 Significance level: P = 0.05, one degree of freedom
72 Table 45 Experiment 2 Mean and Range of Anthracnose Lesion Lengths Bi Parental Population Infected Plants Mean Lesion Lengths (mm) Infected Plants Range of Lesion Lengths (mm) Scintilla x 02 117 3 4. 9 1.8 9.4 08 54 x Scintilla 15 6.7 3.3 1 3 .9 Flicker x 02 22 Crisp 1 5.7 5.7 08 18 x Flicker 9 4. 9 3.1 7.6 08 43 x Flicker 9 4.8 3.0 7.7 Flicker x Cor. 99 42 1 3.6 3.6 12 62 x 10 06 3 4.1 3.1 5.6 04 245 x 11 80 0 N/A N/A 12 78 x 06 498 1 12.1 12.1 Table 46 Experiment 3 Mean and Range of Anthracnose Lesion Lengths Bi Parental Population Infected Plants Mean Lesion Lengths (mm) Infected Plants Range of Lesion Lengths (mm) Scintilla x 02 117 7 8.5 3.5 15.0 08 54 x Scintilla 11 6.7 1.8 15.9 Flicker x 02 22 Crisp 1 3.5 3.5 08 18 x Flicker 11 7.1 2.9 17.9 08 43 x Flicker 7 6.6 4.2 8.9 Flicker x Cor. 99 42 5 4.2 2.2 5.1 12 62 x 10 06 1 28.7 28.7 04 245 x 11 80 0 N/A N/A 12 78 x 06 498 0 N/A N/A Table 4 7 Experimental Conditions Experimental Conditions Experiment 1 Experiment 2 Experiment 3 Experiment 4 Location Greenhouse Greenhouse Growth Room Growth Room High Temp ( C ) > 36 > 36 27 27 Time in Bags 16 h 24 h 24 h 72 h Rel. Humidity not measured not measured 60 8 0% 60 80% Inocul. Date 3/31/17 5/3/17 7/7/17 9/23/17
Figure 41. Experiments 14 Individual Experiment Results 0.00 0.05 0.10 0.15 0.20 0.25 % Infected PlantsExperiment 1 0.00 0.05 0.10 0.15 0.20 0.25 % Infected PlantsExperiment 2 0.00 0.05 0.10 0.15 0.20 0.25 % Infected PlantsExperiment 3 0.00 0.05 0.10 0.15 0.20 0.25 % Infected PlantsExperiment 4
Figure 42. Experiments 1 4 Cumulative Results Figure 43. Effects of Temperature on Growth of C. gloeosporioides isolate 0.00 0.05 0.10 0.15 0.20 0.25 Scintilla x 02117 0854 x Scintilla Flicker x 0222 Crisp 0818 x Flicker 0843 x Flicker Flicker x Cor. 99 42 12-62 x 10-06 04-245 x 11-80 12-78 x 06-498% Infected PlantsCumulative Results 0 10 20 30 40 50 60 70 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6Colony Mean Diameter in mm 10 15 20 25 30 35
75 CHAPTER 5 CONCLUSIONS Chapter 2 addresses the screening of commercially available SHB cultivars for susceptibility to a fungicide resistant, stem lesion form of anthracnose that ha s been found in grower fields on Flicker and Scintilla Given the significant financial impact to growers of this disease, there is a concern regarding whether other SHB cultivars could be sus ceptible. The results of the screening, in particular the results in Experiment 2, found that Flicker had a significantly greater mean number of lesions and AUDPC than other tested cultivars. As noted previously, Scintilla plants could not be located a t the time the screening experiments were conducted. While the results of Experiment 1 also showed Flicker as having a higher mean number of lesions and AUDPC, they were not significantly different from three other tested cultivars. This was possibly due to heat damage to new young growth on two of those cultivars in the greenhouse. The screening results may ultimately indicate that Flicker ( and possibly Scintilla) have a unique genetic susceptibility to this form of anthracnose among SHB cultivars co mmercially grown in Florida. However, screening of additional commercially available SHB cultivars is necessary before making this determination. The experiments in Chapter 3 were performed to test whether a detached stem inoculation protocol would provide results comparable to whole plant inoculations, confirming the detached stem assay as an effective screening tool. The results of the experiments were not comparable to the whole plant inoculations discussed in Chapter 2. Flicker, the known susceptible cultivar, generally had the highest mean number of lesions, but was not significantly different from certain other tested cultivars. Although
76 the detached stem assay is likely not an effective substitute for whole plant inoculations, it may be useful in screening large volumes of plants to identify potentially susceptible plants for further testing The focus of Chapter 4 was on determining segregation ratios for the a nthracnose susceptibility trait to discover additional information about the gene or genes controlling this trait. Scintilla is a direct progeny of Flicker, and its susceptibility sugge sts that this trait may be heritable. Since Flicker has been used as a parent in the UF blueberry breeding program, there is concern regarding whether there is susceptible germplasm in either advanced selections or released cultivars from this program. Segregating populations in Experiments 1 through 4, produced using either Flicker or Scintilla as a parent developed anthracnose stem lesion symptoms, suggesting that the susceptibility trait was inherited to some degree from each of these susceptible parents. The infection ratios observed in each individual experiment even when modifying environmental conditions to favor infection by C. gloeosporioides were too low to confirm the original hypothesis that the susceptibility/resistance trait is monogenically controlled. However, the result s of Experiments 1 through 4 were then accumulated, so that if an individual plant was infected in any of the four experiments it was considered to be susceptible (the same plants were not necessarily infected in each experiment) In that scenario, the inf ection ratios of five of the populations were a good fit with expected ratios for a monogenic trait ( assuming a fully recessive susceptible parent and a heterozygous assumedresistant parent) at P = 0.05 using a chi square test
77 A factor complicating a determination of the genetic structure of the susceptibility trait is the control of the growth and stage of development of the plants. It is impossible to uniformly replicate this factor due to genetic variability between and within the segregating populati ons. This lack of uniformity may have affected the infection rates in some of the populations. Therefore, the low infection rates in certain populations may be attributable to both genetic and ontogenic factors. Additional work is necessary to identify the genetic system and other factors responsible for controlling the anthracnose susceptibility trait present in Flicker and Scintilla, and passed on to their progeny Future work may include selecting multiple bi parental populations and genotyping them to identify SNP markers associated with the susceptibility trait, creating a linkage map, and developing a molecular marker screening tool to identify susceptible germplasm. However, based on this present research a defensible argument can be made that thi s trait is controlled by a single gene based on the fit of the observed infection ratios with expected single gene ratios. In summary the results indicate that the susceptibility trait is heritable to some degree from both Flicker and Scintilla, even if the specific gene action remains uncertain. T his suggest s that material in the UF blueberry breeding program should be tested before releasing a new cultivar where either of these two susceptible cultivars have been used as parents or are in the linea ge of a cultivars parents
78 REFERENCES 1. Abul Hayja, Z., Williams P.H., and Patterson, C.E., 1978. Inheritance of r esistance to a nthracnose and t arget l eaf s pot in cucumbers. Plant Disease Report er 6 43 5. 2. Adaskaveg, J.E., and Hartin, R.J. 1997. Characterization of Colletotrichum acutatum i solates causing a nthracnose of a lmond and peach in California. Phytopathology 87 979 87. 3. Adhikari, K. N. et al. 2009 Identification of a nthracnose r esistance in Lupinus L. and its t ransfer f rom l andraces to m odern cultivars. Crop and Pasture Science 60 4729. 4. Agrios, George N. 2005. Plant Pathology 5th ed n Burlingto n, VT, USA: Elsevier Academic Press 5. Amadeu, Rodrigo R. et al. 2016. AGHmatrix: R p ackage to construct r elationship m atrices for a utotetraploid and d iploid s pecies: A b lueberry e xample. The Plant Genome 9 1 10. 6. Andersen, Peter C. et al. 2016 Sustainability a ssessment of f ruit and n ut crops in n orth Florida and n orth central Florida. IF AS Extension, Publication HS765. 7. Baayen, R.P., and Schrama R.M., 1990 Comparison of f ive stem i noculation m ethods with r espect to p hytoalexin a ccumulation and Fusarium w ilt d evelopment in carnation. Netherlands Journal of Plant Pathology 96 : 315 20. 8. Baker, Jean Beard, Hancock, J.F., and Ramsdell, D.C. 1995. Screening h ighbush b lueberry cultivars for r esistance to Phomopsis canker. HortScience 30: 586 8. 9. Belkhadir, Youssef, Subramaniam, Rajagopal, and Dangi, Jeffrey L. 2004. Plant d isease r esistance p rotein signaling: NBS LRR p roteins and t heir partners. Current Opinion in Plant Biology 7 : 391 9. 10. B ergstrom, Gary C., and Nicholson Ralph L. 1999. The b iology of corn anthracnose. Plant Disease 83: 596 608. 11. Bertetti, D., Gullino M.L., and Garibaldi A. 2009. Effect of l eaf w etness d uration, t emperature and i noculum c oncentration on i nfection of e vergreen Azalea by Colletotrichum acutatum the Causal Agent of anthracnose. Journal of Plant Pathology 91: 763 6. 12. Boonchanawiwat, A. et al 2016 Mapping and q uantitative t rait l ock u nderlying r esistance to c assava a nthracnose d isease Journal of Agricultural Science 154 : 120917.
79 13. Buchwaldt, L. et al 2001. Genetics of r esistance to a nthracnose ( Colletotrichum t runcatum ) in lentil. Proc 4th European Conf on Grain Legume Research, Cracow, Poland: 242, Poster 14. Buck, Brad 2015. Florida r eleases n ew e vergreen b lueberry varieties. Southeast Farm Press February : n. pag. 15. Camp, W.H., 1945. The North American b lueberries w ith n otes on o ther g roups of Vacciniaceae. Brittonia 5 16. Cannon, P.F., Damm, U., and Weir, B.S., 2012. Colletotrichum Current status and f uture directions. Studies in Mycology 73 : 181 213. 17. Chandler, W.H. et al 1937. Chilling r equirements for o pening of b uds on d eciduous o rchard t rees and some o ther plants in California. University of California, Bulletin 611, July 18. Chang, Chung Jan et al., 2009. Bacterial l eaf scorch, a n ew b lueberry d isease caused by Xylella fastidiosa. HortScience 44 : 413 17. 19. Chavez, Dario J., and Lyrene, Paul M., 2009 a Production and i dentification of colchicine d erived t etraploid Vaccinium d arrowii and i ts u se in breeding. Journal of the American Society of Horticultural Science 134 : 356 63. 20. Chavez, Dario J., and Lyrene, Paul M., 2009 b Interspecific crosses and b ackcrosses b etween d iploid Vaccinium darrowii and t etraploid southern h ighbush blueberry. Journal of the American Society of Horticultural Science 134 : 273 80. 21. Cheong, Yong Hwa et al 200 2. Transcriptional p rofiling r eveals n ovel i nteractions b etween w ounding, p athogen, a biotic stress, and h ormonal r esponses in a rabido psis. Plant Physiology 129 : 661 77. 22. Costes, E et al., 2012. Plant a rchitecture, i ts d iversity and m anipulation in a gronomic c onditions, in r elation w ith p est and p athogen attacks. European Journal of Plant Pathology 135 : 455 70. 23. Cowley, Raymond B. et al. 2012. Development of a r eliable and r apid d etached l eaf a ssay to d etect r esistance to the f ungal d isease Phomopsis l eaf b light, c aused by Diaporthe toxica, in Lupinus albus Canadian Journal of Plant Pathology 34: 401 9. 24. de Bellaire, L. de Lapeyre, and Mourichon, X. 1997. The p attern of f ungal contamination of the b anana b unch during i ts d evelopment and p otential i nfluence on i ncidence of crown r ot and a nthracnose diseases. Plant Pathology 46: 481 9. 25. Dean, Ralph et al 2012. The top. Molecular Plant Pathology 13 : 414 30.
80 26. Denman, S., and Sadie A. 2001. Evaluation of a stem i noculation t echnique for a ssessing r esistance to Phytophthora cinnamomi in Leucospermum cultivars. Australasian Plant Pathology 30 : 11 6. 27. Denner, F.D.N., Kotze J.M., and Putterill, J.F., 1986. The e ffect of t emperature on spore g ermination, g rowth and a ppressorium f ormation of Colletotrichum gloeosporioides and Dothiorella aromatica. South African Avocado Growers' Association Yearbook 9 : 19 22. 28. Dhingra, O.D., and Sinclair J.B., 1995. Basic Plant Pathology Methods 2nd edn., CRC Press, Inc 29. Dieguez Uribeondo, J., Forster H., and Adaskaveg J.E., 2011. Effect of w etness d uration and t emperature on the d evelopment of a nthracnose on selected a lmond t issues and c omparison of c ultivar susceptibility. Phytopathology 101 : 1013 20. 30. Dodd, J. C et al., 1991. The e ffect of climatic f actors on Colletotrichum gloeosporioides causal a gent of m ango a nthracnose, in the Philippines. Plant Pathology 40: 568 75. 31. Draper, A.D., and Scott D.H. 1971. Inheritance of a lbino seedlings in t etraploid h ighbush blueberry. Journal of the American Society for Horticultural Science 96: 791 2. 32. Draper, Arlen, and Hancock, Jim 2003. Florida 4B: n ative b lueberry with e xceptional b reeding value. Journal of the American Pomological Society 57 : 138 41. 33. Elgin, Jr., J.H., and Ostazewski S.A., 1985. Inheritance of r esistance to r ace 1 and r ace 2 a nthracnose in A rc and Sarenac AR alfalfa. Crop Science 25: 86 1 85. 34. Evans, Edward A. and Ballen, Fredy H. 2014. An o verview of US b lueberry p roduction, t rade, and consumption, with special reference to Florida. IFAS Extension, Publication FE952 35. Falconi, Cesar E., Visser Richard G. F., and v an Heusden, Sjaak 2015. Influence of p lant g rowth stage on r esistance to a nthracnose in Andean l upin ( Lupinus mutabilis ). Crop and Pasture Science 66 : 72934. 36. Fisher, R.A. 1947. The t heory of l inkage in p olysomic inheritance. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences 233: 55 87.
81 37. Fitzell, R.D., and Peak C.M. 1984. The e pidemiology of a nthracnose d isease of m ango: i noculum sources, spore p roduction and dispersal. Annals of Applied Biology 104 : 53 9. 38. Flor, H.H. 1971. Current status of the g ene for g ene concept. Annual Review of Phytopathology 9 : 275 96. 39. Fokunang, C.N. et al. 2002. Rapid screening m ethod of cassava cultivars for r esistance to Colletotrichum gloeosporioides f.sp. manihotis Journal of Phytopathology 150 : 6 12. 40. Freeman, Stanley, Katan, Talma, and Shabi, Ezra, 1998. Characterization of Colletotrichum species r esponsible for a nthracnose d iseases of v arious fruits. Plant Disease 82: 596 605. 41. Gallais, Andre 2003. Some g eneral a spects of a utopolyploid genetics. Quantitative Genetics and Breeding Methods in Autopolyploid Plants Paris F rance : Institut National de la Recherche Ag ronomique, 29 37. 42. Grandont, L., Jenczewski E., and Lloyd, A. 2013. Meiosis and i ts d eviations in p olyploid p lants. Cytogenic and Genome Research 140 : 171 84. 43. Gupta, Supriya, Singh, K.P., and Singh, A.K. 2015. Resistance to a nthracnose d isease in commercial cultivars and a dvanced h ybrids of mango. Plant Pathology Journal 14: 255 8. 44. Gururani, Mayank Anand et al. 2012. Plant d isease r esistance g enes: current status and f uture directions. Physiological and Molecular Plant Pathology 78 : 51 65. 45. Harmon, Philip F. 2014. Fungicide i nsensitivity in a nthracnose in blueberry. The Blueberry News October : 20,30. 46. Harmon, Philip F. 2014. Anthracnose and a lgal stem b lotch on southern h ighbush blueberry in Florida. IFAS Extension, FBGA Fall 2014 Blueberr y Short Course. 47. Harmon, Philip F., Harmon Carrie and Norman, Dave, 2016. Bacterial w ilt of southern h ighbush b lueberry caused by Ralstonia solanacearum IFAS Extensio n, Publication PP332. 48. Hartung, J. S., Burton C.L., and Ramsdell D.C. 1981. Epidemiological studies of b lueberry a nthracnose d isease caused by Colletotrichum gloeosporioides Phytopathology 71: 449 53. 49. Hopkins, D.L., and Harris J.W. 2000. A g reenhouse m ethod for screening g rapevine seedlings for r esistance to anthracnose. HortScience 35 : 89 91.
82 50. Hyde, K.D. et al., 2009. Colletotrichum n ames in current use. Fungal Diversity 39: 147 82. 51. Jamil, Farhat F., and Nicholson, Ralph L. 1987. Susceptibility of c orn to i solates of Colletotrichum graminicola p athogenic to o ther grasses. Plant Disease 71 : 809 10. 52. Jeffries, P. et al., 1990. The b iology and control of Colletotrichum species on t ropical f ruit crops. Plant Pathology 39: 343 66. 53. Kelly, J.D., and Young R.A. 1996. Proposed symbol for a nthracnose r esistance genes. Annual Report Bean Improvement Cooperative 39 : 20 4. 54. Kim, Wan Gyu et al., 2009. Occurrence of a nthracnose on h ighbush b lueberry caused by Colletotrichum gloeosporioides in Korea. Mycobiology 37: 310 2. 55. King, W. T. et al. 1997. Effects of t emperature on sporulation and l atent p eriod of Colletotrichum Spp. i nfecting strawberry fruit. Plant Disease 81: 77 84. 56. Krebs, S.L., and Hancock J.F., 1989. Tetrasomic i nheritance of i soenzyme m arkers in the h ighbush b lueberry, Vaccinium corymbosum L. Heredity 63: 11 8. 57. Leandro, L.F.S. et al ., 2003 Influence of t emperatures and w etness d uration on conidia and a ppressoria of Colletotrichum acutatum on symptomless strawberry leaves. Phytopathology 93: 513 20. 58. Lee, DuHyung, 1993. Effect of t emperature on the conidium g ermination and a ppressorium f ormation of Colletotrichum acutatum C. dematium and C. gloeosporioides The Korean Journal of Mycology 21 : 2249. Abstract. 59. Lewers, K.S. et al., 2007. Evaluation of el ite n ative strawberry g ermplasm for r esistance to a nthracnose crown r ot d isease caused by Colletotrichum species. Journal of the American Society for Horticultural Science 132 : 842 9. 60. Linde, D.C., Bridges W.C., and Rhodes B.B., 1990. Inheritance of r esistance in cucumber to r ace 2 of Colletotrichum lagenarium Theoretical Applied Genetics 79: 13 6. 61. Lindhout, Pim, 2002. The p erspective of p olygenic r esistance in b reeding for d urable d isease resistance. Euphytica 124 : 217 26. 62. Lloyd, Andrew, and Bomblies Kirsten, 2016. Meiosis in a utopolyploid and allopolyploid arabidopsis Current Opinion in Plant Biology 30: 116 22.
83 63. Luo, Z.W., Zhang R.M., and Kearsey M.J 2004. Theoretical b asis for g enetic l inkage a nalysis in a utotetraploid species. Proceedings of the National Academy of Sciences of the United States of America 101 : 7040 5. 64. Lyrene, P.M. 1993. Some p roblems and o pportunities in b lueberry breeding. Acta Horticulturae 346 : 63 71. 65. Lyrene, Paul M. 1997. Value of v arious t axa in b reeding t etraploid blueberries in Florida. Euphytica 94 : 15 22. 66. Lyrene, Paul M. 2002. Development of h ighbush b lueberry cultivars a dapt ed to Florida. Journal of the American Pomological Society 56: 79 85. 67. Lyrene, Paul M. 2005. Breeding l ow chill b lueberries and p eaches for subtropical areas. HortScience 40 : 1947 9. 68. Ma, H. X. et al. 2006. Main e ffects, e pistasis, and e nvironmental i nteractions of q uantitative t rait l oci for Fusarium h ead b light r esistance in a r ecombinant i nbred population. Phytopathology 96 : 534 41. 69. McNew, G.L 1960. The n ature, o rigin, and e volution of parasitism. Plant Pathology: An Advanced Treatise. Ed. Horsfall, J.G. and Dimond, A.E. New York, NY, USA: Academic Press, 19 69. 70. Mei, Jiaqin et al 2012. Screening r esistance a gainst Sclerotinia sclerotiorum in Brassica crops w ith u se of d etached stem a ssay u nder controlled environment. European Journal of Plant Pathology 134: 599 604. 71. Mignouna, H.D. et al., 2002. A genetic linkage map of water yam ( Dioscorea alata L.) Bbased on AFLP markers and QTL analysis for anthracnose resistance. Theoretical and Applied Genetics 105: 726 35. 72. Miles, T.D. et al. 2013. The e ffect of e nvironmental f actors on i nfection of b lueberry f ruit by Colletotrichum a cutatum Plant Pathology 62 : 1238 47. 73. Monroe, J.S., Santini, J.B., and Latin, R., 1997. A m odel d efining the r elationship between t emperature and l eaf w etness d uration, and i nfection of w atermelon by Colletotrichum orbiculare. Plant Disease 81 : 739 42. 74. Moraes, Gomes et al. 2015. Prepenetration and p enetration of Colletotrichum gloeosporioides into g uava f ruit (Psidium guajava L.): e ffects of t emperature, w etness p eriod and f ruit age. Journal of Phytopathology 163 : 149 59. 75. Moral, Juan 2012. Effect of t emperature, w etness d uration, and p lanting d ensity on o live a nthracnose c aused by Colletotrichum spp. Phytopathology 102 : 975 81.
84 76. M nch Steffen et al., 2008. The h emibiotrophic l ifestyle of Colletotrichum species. Journal of Plant Physiology 165 : 41 51. 77. OConnell, R. et al. 2000. Dissecting the c ell b iology of Colletotrichum i nfection processes. In: Prusky, Dov ed. Colletotrichum: Host Specificity, Pathology, and Host Pathogen Interaction. St. Paul, MN USA : American Phytopathological Society, 57 77. 78. Olmstead, James W. et al. 2015. Floral b ud c hill r equirement of l ow chill southern h ighbush b lueberry germplasm. Journal of the American Pomological Society 69: 4 10. 79. Peres, N.A. et al. 2005. Lifestyles of Colletotrichum acutatum Plant Disease 89: 784 96. 80. Perfect, Sarah E. et al. 1999. Colletotrichum : a m odel g enus for studies on p athology and f ungal p lant interactions. Fungal Genetics and Biology 27: 186 98. 81. Pettitt, T. R. et al. 2011 A simple d etached l eaf a ssay p rovides r apid and i nexpensive d etermination of p athogenicity of Pythium i solates to 'All Year Round' (AYR) chrysanthemum r oots. Plant Pathology, 60: 946 9 6. 82. Poethig, R. S. 2003. Phase c hange and the r egulation of d evelopmental t iming in plants. Science 301 : 334 6. 83. Poland, Jesse A. et al. 2009. Shades of g ray: t he w orld of q uantitative d isease resistance. Trends in Plant Science 14: 21 9. 84. Polashock, James J. et al. eds. 2017. Compendium of B lueberry, C ranberry, and L ingonberry D iseases and P ests. St. Paul, MN, U.S.A.: APS Press, American Phytopathological Society 85. Qu, Luping, and Hancock James F., 1995. Nature of 2n g amete f ormation and m ode of i nheritance in i nterspecific h ybrids of d iploid Vaccinium darrowii and t etraploid V. corymbosum Theoretical Applied Genetics 91: 1309 15. 86. Qu Luping, Hancock James F. and Whallon J.H., 1998. Evolution in an a utotetraploid g roup d isplaying p redominantly b ivalent p airing at m eiosis: g enomic similarity of d iploid Vaccinium darrowii and a utotetraploid V. Corymbosum (Ericaceae). American Journal of Botany 85 : 698 703. 87. Quito Avila, Diego F. et al. 2013. Genetic characterization of b lueberry n ecrotic r ing b lotch v irus, a n ovel RNA v irus with u nique g enetic features. Journal of General Virology 94: 1426 34.
85 88. Rahman, Mahfuzur, Ojiambo, Peter and Louws Frank, 2015. Initial i noculum and spatial d ispersal of Colletotrichum gloeosporioides the causal a gent of strawberry a nthracnose crown rot. Plant Disease 99: 80 6. 89. Sanogo, S. et al. 1997. Weather v ariables a ssociated with i nfection of t omato f ruit by Colletotrichum coccodes Plant Disease 81 : 753 6. 90. Schilder, Annemiek 2010. Cane a nthracnose f ound in some b lueberry fields. Crop Advisory Team Alerts Department of Plant Pathology. Michigan State University, June. 91. Sharpe, R.H. 1953. Horticultu ral d evelopment of Florida blueberries. Florida St ate Horticultural Society: 188 90. 92. Sharpe, R.H., and Darrow, G.M., 1959. Breeding b lueberries for the f lorida climate. Florida St ate Horticultural Society: 308 11. 93. Stewart, Philip J, Clark John R. and Fenn Patrick, 2005. Detached cane a ssay of r esistance to Botryosphaeria c ane canker ( Botryosphaeria dothidea ) in e astern U.S. b lackberry genotypes. International Journal of Fruit Science 5 : 57 64. 94. Talg V., and Stensvand, A., 2013. A simple and e ffecti ve i noculation m ethod for Phytophthora and f ungal species on w oody plants. EPPO Bulletin 43: 276 9. 95. Thatcher, Louise F., Anderson, Jonathan P., and Singh, Karam B. 2005. Plant d efence r espo nses: w hat h ave w e l earnt from arabidopsis? Functional Plant Biology 32: 1 19. 96. Thekke Veetil, Thanuja et al. 2014. A n ew o phiovirus i s a ssociated with b lueberry m osaic virus. Virus Research 189 : 92 6. 97. Tooley, Paul W., Browning Marsha and Leighty Robert M 2014. Effects of i noculum d ensity and w ounding on stem i nfection of t hree e astern US f orest species by Phytophthora ramorum Journal of Phytopathology 162 : 683 9. 98. Tullu, A. et al. 2003. Genetics of r esistance to a nthracnose and i dentification of AFLP and RAPD m arkers l inked to the r esistance g ene in PI 320937 g ermplasm in l entil ( Lens culinaris Medikus). Theoretical and Applied Genetics 106 : 428 34. 99. Twizeyimana, M. et al. 2012. A cut stem i noculation t echnique to e valuate soybean for r esistance to Macrophomina phaseolina. Plant Disease 96: 1210 5. 100. Van Loon, L.C. 1997. Induced r esistance in p lants and the r ole of p athogenesis r elated proteins. European Journal of Plant Pathology 103 : 753 65.
86 101. Vander Kloet, S.P. 1988. The Genus Vaccinium in North America. Agriculture Canada. 102. Vijaya, Suzianti Iskandar et al. 2015. Characterization and p athogenicity of Colletotrichum truncatum causing stem a nthracnose of r edf leshed d ragon f ruit ( Hylocereus polyrhizus ) in Malaysia. Journal of Phytopathology 163 : 67 71. 103. Voorrips, Roeland E., Finkers Richard and Sanjaya, Lia, 2004. QTL m apping of a nthracnose ( Colletotrichum spp.) r esistance in a cross b etween Capsicum annuum and C. chinense Theoretical Applied Genetics 109 : 127582. 104. Whalen, Maureen C. 2005. Host d efence in a d evelopmental context. Molecular Plant Pathology 6 : 34760. 105. Wharton, P.S., and Schilder, A.C. 2008. Novel i nfection strategies of Colletotrichum acutatum on r ipe b lueberry fruit. Plant Pathology 57 : 122 34. 106. Williamson, J.G. et al. 2014. Southern h ighbush b lueberry cultivars from the University of Florida. IFA S Extension, Publication HS1245. 107. Williamson, J.G., Olmstead, J.W., and Lyrene, P.M., 2015. Floridas commercial b lueberry industry. IFAS Extensio n, Publication HS742 108. Williamson, J.G., Olmstead, J.W., and Lyren e, P.M., 2015. Reproductive g rowth and d evelopment of blueberry. IF AS Extension, Publication HS976 109. Williamson, Jeffrey G. et al., 2016. 2017 Florida b lueberry i ntegrated p est m anagement guide. IFAS Extension, Publication HS1156 110. Wilson, L.L., Madden, L.V., and Ellis M.A., 1990. Influence of t emperature and w etness d uration on i nfection of i mmature and m ature strawberry f ruit by Colletotrichum acutatum Phytopathology 80 : 111 6. 111. Wright, A.F., 2010. Identification of species in the Botrysphaeria f amily causing stem b light on southern h ighbush blueberry in Florida. Plant Disease 94 : 966 71. 112. Xu, C.N. et al. 2013. First r eport of stem and l eaf a nthracnose on b lueberry caused by Colletotrichum gloeosporioides in China. Plant Disease 97 : 845. 113. Yang, Huaan, et al., 2009. Development of sequencespecific PCR m arkers a ssociated with a p olygenic controlled t rait for m arker a ssisted selection u sing a m odified s elective g enotyping strategy: a case study on a nthracnose d isease r esistance in w hite l upin ( Lupinus albus L.). Molecular Breeding 25 : 23949.
87 114. Yoshida, Shigenobu, and Tsukiboshi, Takao 2002. Shoot b light and l eaf s pot of b lueberry a nthracnose caused by Colletotrichum acutatum Journal of General Plant Pathology 68 : 246 8. 115. Young, R.A., and Kelly J.D., 1997. RAPD m arkers l inked to t hree m ajor a nthracnose r esistance g enes in common bean. Crop Science 37 : 940 6. 116. Yun, Hae Keun et al. 2006. Evaluating the r esistance of g rapevines a gainst a nthracnose by p athogen i noculation, v ineyard i nspection, and b ioassay with culture f iltrate from Elsinoe ampelina. Journal of the American Pomological Society 60: 97 103. 117. Zhao, J. 2004. Evaluation of Sclerotinia stem r ot r esistance in o ilseed Brassica napus u sing a p etiole i noculation t echnique u nder g reenhouse conditions. Plant Disease 88 : 1033 8. 118. Commodities and p roducts blueberries. Ag Marketing Resource Center Iowa State University, July 2015. https://www.agmrc.org/commodities products/fruits/blueberries/ 119. 201 5 b lueberry statistics. U.S. Department of Agriculture, National Agricultural Statistics Service 2016 https://www.nass.usda.gov/Statistics_by_State/New_Jersey/Publications/Blueberry _Statistics/NJ%20Blueberry%20Summary %202015.pdf
88 BIOGRAPHICAL SKETCH Douglas A. Phillips earned a Bachelor of Science in horticultural sciences, with a minor in soil sciences, from North Carolina State University in May 2015. After gra duation he joined the blueberry breeding program at the University of Florida to complete a Master of Science degree in horticultural sciences. While at the University of Florida he worked with James Olmstead and Patricio Munoz, and studied anthracnose susceptibility in southern highbush blueberry.