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1 EFFECT OF INOCULUM CONC ENTRATION, TEMPERATURE AND WETNESS DURATION ON ANTHRACNOSE FRUIT ROT DEVELOPMENT O N DIFFERENT STRAWBERRY CULTIVARS By BRUNA BALEN FORCELINI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 Bruna Balen Forcelini
3 To my family
4 A C KNOWLEDGMENTS I would like to express my sincere gratitude to my advisor Dr. Natalia Peres for giving me the opportunity to study under her guidance Dr. Peres has taught me to become a better scientist and researcher and to explore my thoughts and ideas about plant pathology. I also thank the staff of the University of Florida GCREC Strawberry Plant Pathology lab for all t he ir help and support. T hanks to my committee members, Dr. Megan Dewdney and Dr. Jim Marois for guiding my research with valuable comments. A special thanks to my friend Matt Mattia and boyfriend Tyler Mayo for their words of encouragement and for never de nying my requests for assistance during their free time. Most importantly, I would like to thank my family, without their unconditional degree. Also I thank my father for being m y role model. His passion for plant pathology inspires me to be outstanding in all I accomplish. Thank you all!
5 TABLE OF CONTENTS page A C KNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 11 Strawberry Production in Florida and the World ................................ ..................... 11 Anthracnose Fruit Rot of Strawberry ................................ ................................ ....... 13 Etiology ................................ ................................ ................................ ............. 15 Symptoms ................................ ................................ ................................ ........ 17 Epidemiology and Life Cycle ................................ ................................ ............ 19 Disease Management ................................ ................................ ...................... 28 Cultural and biological c ontrol ................................ ................................ .... 28 Chemical c ontrol ................................ ................................ ........................ 30 Objectives ................................ ................................ ................................ ............... 33 2 EFFECT OF INOCULUM CONCENTRATION, TEMPERATURE AND WETNESS DURATION ON ANTHRACNOSE FRUIT ROT DEVELOPMENT ON DIFFERENT STRAWBERR Y CULTIVARS ................................ ............................ 36 Materials and Methods ................................ ................................ ............................ 38 Fungal Isolates and Culture ................................ ................................ .............. 38 Effect of Inoculum Concentration on Anthracnose Fruit Rot Development ....... 38 Field Trial ................................ ................................ ................................ .......... 39 Detached Fruit Trial ................................ ................................ .......................... 41 Effect of Different Temperatures on Mycelial Growth of C. acutatum isolates ........ 42 Effect of Temperature on Anthracnose Fruit Rot Development on Detached Fruit ................................ ................................ ................................ ..................... 43 Effect of Wetness Duration and Temperature on Anthracnose Fruit Rot Development ................................ ................................ ................................ ....... 44 Statistical Analysis ................................ ................................ ................................ .. 46 3 RESULTS ................................ ................................ ................................ ............... 50 Effect of Inoculum Concentration on Anthracnose Fruit Rot Development ............. 50 Field Trial ................................ ................................ ................................ .......... 50 Detached Fruit Trial ................................ ................................ .......................... 51 Correlation between Detached Fruit and Field Trial ................................ ......... 52
6 Effect of Temperature on Mycelial Growth ................................ ....................... 52 Effect of Temperature on Anthracnose Fruit Rot Development on Detached Fruit ................................ ................................ ................................ ............... 53 Effect of Wetness Duration and Tempera ture on Anthracnose Fruit Rot Development ................................ ................................ ................................ 54 Discussion ................................ ................................ ................................ .............. 56 4 CONCLUSION ................................ ................................ ................................ ........ 73 REFERENCES ................................ ................................ ................................ .............. 76 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 82
7 LIST OF TABLES Table page 3 1 Analysis of variance of the effect s of cultivar, inoculum concentration, plant organ and all the inte ractions on the incidence of Anthracnose F ruit R ot of strawbe rry in field trials. ................................ ................................ ...................... 65 3 2 Mean disease incidence of strawberry cultivars when inoculated with concentrations of conidia from 10 3 to 10 6 conidia/ml in field trials. ..................... 65 3 3 E ffect of strawberry cultivar on mean disease incidence of detached strawberry fruit i noculated with different in oculum concentrations ..................... 68 3 4 Analysis of covariance of the parameters for field inoculation of Collet otrichum acutatum on immature strawberry fruit. ................................ ...... 69 3 5 Analysis of covariance of the parameters for detached fruit inoculation of Colletotrichum acutatum on immature strawberry fruit. ................................ ...... 69 3 6 Analysis of v ariance for the effects of cultivar, wetness duration and tem perature on the incidence of Anthracnose Fruit R ot of immature strawberry fruit. ................................ ................................ ................................ ... 72 3 7 Analysis of c ovariance for the effect of cultivar on the development of Anthracnose Fruit R ot of immature strawberry fruit at 1 5, 20 and 25 C and at all wetness durations periods ................................ ................................ ............. 72 3 8 Regression equations of A nthracnose Fruit Rot incidence on strawberry cultivars after inoculation of immature fruit with 10 6 conidia/ml of Colletotrichum acutatum and incubation at different temperatures and wetness duration periods. ................................ ................................ ................... 72
8 LIST OF FIGURES Figure page 1 1 Anthracnose fruit rot symptoms on strawberry. ................................ .................. 35 1 2 Life cycle of Colletotrichum acutatum the causal agent of Anthracnose Fruit Rot of strawberry.. ................................ ................................ .............................. 35 2 1 Field trial of i noculum concentration experiment. ................................ ................ 48 2 2 Detached fruit trial of inoculum concentration experiment ................................ 48 2 3 Controlled wetness duration and temperature experiment. ................................ 49 3 1 Regression of Colletotrichum acutatum inoculum concentration on A nthracnose F ruit R ot incidence of flower s and immature fruit on different strawberry cultivars ................................ ................................ ............................. 66 3 2 Regression of inoculum concentration of Colletotrichum acutatum on Anthracnose Fruit Rot development of strawberry cultivars on different plant organs. ................................ ................................ ................................ .............. 67 3 3 Incubation period (days from inoculation to symptom development) for immature fruit and flowers of different strawberry cult ivars inoculated with 10 6 conidia/ml of C olletotrichum acutatum . ................................ .............................. 67 3 4 Regression of inoculum concentration of Colletotrichum acutatum and A nthracnose F ruit R ot development on detached immature strawberry fru it. ..... 68 3 5 Effect of growth chamber temperature on Colletotrichum acutatum mycelial growth at seven day s after incubation .. ................................ .............................. 70 3 6 Regression of growth chamber temperatures from 5 to 35 C on Anthracnose Fruit R ot development on detached immature ................................ ................................ .................. 70 3 7 Infection of immature fruit of strawberry cultivars with diff erent levels of susceptibility to Anthracnose Fruit Rot by Colletotrichum acutatum for wetness durations between 0 and 48 hours at 15, 20 and 25 C. .. ..................... 71 3 8 Regression of wetness duration on A nthracnose F ruit R ot development on immature fruit of strawberry cultivars with different levels of susceptibility after inoculation with Colletotrichum acutatum and incubation at temperatures between 15 and 2 5 C and wetness periods f rom 0 to 48 hours. .. 71
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECT OF INOCULUM CONCENTRATION, TEMPERATURE AND WETNESS DURATION ON ANTH RACNOSE FRUIT ROT DEVELOPMENT ON DIFFERENT STRAWBERRY CULTIVARS By Bruna Balen Forcelini December 2013 Chair: Natalia A. Peres Major: Plant Pathology Florida is the largest producer of winter strawberries in the world. Anthracnose Fruit Rot (AFR), caused by Colletotrichum acutatum can greatly affect production if not controlled. The use of fungicid es in addition to cultural practices such as the use of certified pathogen free plants and less susceptible cultivars are important tools for AFR control The purpose of this study was to evaluate the effects of temperature, Wetness Duration (WD) and inoc ulum concentration on the development of AFR on strawberry cultivars with different levels of susceptibility to the disease that are commonly grown in Florida Disease incidence generally increased with increasing inoculum concentration, temperature and W were more susceptible than immature fruit for most cultivars. Detached fruit and fi eld trials we re generally correlated. In in vitro studies mycelial growth occurred from 10 to 30 C and C. acutatum infection on detached immature fruit was observed from 15 to 30 Festiv and 25 C
10 and from 0 to 48 h of WD. D isease incidence was higher for disease forecasting system to predict AFR outbreaks in Florida strawberry production fields.
11 CHAPTER 1 LITERATURE REVIEW Strawberry Production in Florida and the World Strawberry ( Fragaria x ananassa Duch.) is a perennial plant grown in many subtropi cal and temperate countries such as the USA, Turkey, S pain, Egypt, Mexico Japan, Poland, Korea, and Israel ( FAOSTAT, 2012 ). T he USA where cultivation started in the mid 180 is responsible for most of the strawberry production worldwide. In the past, b erries from California were transported to the rest of the country produce d in many other states and shipped all over the world thanks to advances in the USA following California and the largest winter producer in the world Production is centered around Plant City which is considered N) (USDA, 2013; Brown, 2003). In the 2012 2013 season in Florida, 8,700 acres of strawberries were harvested with a yield of 21,000 pounds per acre ( USDA, 2013). Strawberry is an aggregate fruit that can be vegetatively or sexually reproduced. The real fruit are the brown black achenes located on the outside of the receptacle (the edible part) (Darnell, 2003; Strand, 2008). Even though the strawberry i s considered a perennial plant, it is cultivated as an annual crop in Florida and many other locations (Brown, 2003; Strand, 2008). In Florida, the strawberry season is during the winter, d ifferent from other US states that have perennial production and h arvest during the summer (Brown, 2003). Strawberry production in Florida starts with site preparation and cultivar selecti on. The sandy soils with optimal drainage and cultivars that produce high
12 quality fruit are ideal to achieve high yields (Strand, 2008 ; Chandler and Legard 2003). The use of less susceptible cultivars and/or disease free plants is extremely important and thus transplants are grown in nurseries located in northern latitudes (Nova Scotia, Ontario, and Quebec, Canada) or at higher elevations ( California and North Carolina) where climatic conditions are not favorable for pest and pathogen survival (Mertely et al., 2005; Brown, 2003). Field preparation starts in September, with bedding, plastic laying, establishment of drip tape and overhead irrigation. B eds are fumigated and the row mid dles are sprayed with herbicide to reduce nematode and fungal communities and weeds, respectively (Brown, 2003; Strand, 2008). In October, when temperatures are usually around 30 C fresh ly dug transp lants are manually planted and overhead irrigated for most of the cultivars planted are short day cultivars which are highly stimulated to flower hours of light (Darnell 2003; Strand, 2008). Even though temperature is a secondary factor in strawberry flowering it can determine if stolons or C favor the development of stolons a nd cooler t emperatures below 15 C favor the development of inflorescences (Strand, 2008). When temperatures reach the freezing point, the use of overhead sprinklers is common for frost protection because f lower s and immature fruit are extremely sensitive to frost inj ury. Therefore, when air temperature reaches 0 C, overhead irrigation is turned on to protect flowers and fruit from frost damage (Fiola, 2003).
13 From mid No vember to early March strawberry fruit is manually harvested on ave rage 2 to 3 times a week when b erries have a red color throughout the fruit H owever different cultivars have different maturity patterns (Brown 2003 ; Stra nd, 2008). After harvest, berries are placed in plastic containers (clamshells) and sent to a central cooling facility with low temperatures (1 C) to slow fruit deterioration and fungal growth. Finally strawberries are transported in refrigerated trucks to their market destination (Brown 2003). The majority of the strawberries planted in Florid a and other US states is grown usin g conventional production practices T he organic strawberry production industry is growing in states with dry climates like California. From 1997 to 2009, the number of acres of organic strawberries grew demand and innovations in fertilization and integrated pest management that are currently permitted by organic production policies (Koike et al., 2012). However, i n states with warm humid conditions like Florida, organic production has not been adopted, si nce weather i s extremely favorable for pests and diseases. Anthracnose Fruit Rot of Strawberry Anthracnose Fruit Rot (AFR) most often caused by Colletotrichum acutatum Simmond s, is a major strawberry disease that can greatly impact yield in production f ields in Florida and worldwide if not controlled ( Mertely et al., 2005; Legard, 2002 ). The disease cause s yield loss in many strawberry producing regions like North and South America and Europe ( Mertely et al., 2005; Freeman et al., 1997; Kososki et al., 2 001; Domingues et al., 2001). In the USA, AFR has been observed in states like Florida, California, New York, Ohio, P ennsylvania as well as other s ( Mertely et al., 2005; Strand, 2008; Turechek et al., 2006; Wilson et al., 1990 ). I n production areas where
14 climatic conditions are extremely favorable for AFR development infected fruit become unmarketable (Legard et al. 2003 ). For example, during the 2001 2002 season Legard and MacKenzie, 2003 ) reported an AFR disease incidence higher than 70%. A lthough A FR is most commonly caused by C. acutatum other Colletotrichum species such as C. gloe o sporioides and C. fragariae are capable of causing AFR symptoms on strawberry plants, but with lower economic loss es (Maas, 1998). Besides fruit C. acutatum can infect l eaves, petioles, flow ers, crown s and root s (Mertely et al., 2005). During favorable conditions, lesions may expand and completely cover the surface of the fruit especially on hig hly susceptible cultivars (Maas, 1998; Seijo et al., 2008). AFR was first observed in Florida in 1984, however the disease is not indigenous to Florida ( Howard et al. 1992 ) T he prima ry source of AFR inoculum in annual strawberry commercial fields is the transplants originating from production nurseries since the inoculum does not survive the high soil temperatures in Florida (Howard et al., 1992) The use of overhead irrigation is common in nurseries and favor s spore dispersal and AFR development ( Strand, 2008 ). In annual production systems like those in Florida, new transplants are established every season. Therefore, if infected, these plants are responsible for the introduction of the pathogen in to strawberry fields ( Legard, 2002 ). However, in strawberry producing states in the northern USA, where strawberry is cultivated in the summer as a perennial crop, AFR development is caused by C. acutatum inoculum that survives in the soil or attached to plant parts (Strand, 2008; Hokanson and Finn, 2000).
15 Etiology The anamorph ic stage of Colletotrichum acutatum is the major causal agent of AFR of strawberry, since the telemorph, Glomerella acutata which belongs to the phylum Ascomycota has only been observed under laboratory conditions (Damm et al., 2012; Guerber and Correll, 2001; Agrios, 2005). C. acutatum is consid ered a cosmopolitan plant pathogen and can cause yield loss of many tropic al crops such as mango, banana, avocado, papaya coffee, and citrus as well as temperate crops such as apple and grape. However, symptoms and crop loss can vary according to the host (Agrios, 2005). Simmonds ( 1965 ) differentiated C. acutatum from other Colletotrichum species based on ecological and morphological characteristics. Its s mall conidia with pointed ends differe d from those of C. gloe o sporioides However, in recent studies, Damm et al ( 2012 ) using molecular tools and ecological an d morphological features, described C. acutatum as a s pecies complex that is evolving rapidly. Many Colletotrichum sp ecies that were c onsidered morphologically similar to C. acutatum are now known to be closely related and belong to the C. acutatum species complex. Shape and size of conidia and mycelial growth rate of C. acutatum is different and usually distinguishable from other Colle totrichum species (Maas, 1998; Grahovac et al., 2012; Damm et al., 2012). However, s ince d ifferent Colletotrichum species can cause similar symptoms on strawberry, visual differentiation of symptoms is no t always reliabl e. T herefore the use of molecular tools, such as Polymerase Chain Reaction (PCR) should be incorporated when trying to distinguish Colletotrichum species ( Grahovac et al. 2012; Urea Padilla et al. 2002)
16 C olletotrichum acutatum conidia are hyaline, smooth walled and cylindrical to fusiform with both ends acute are produced in salmon pink or orange masses ( Maas, 1998; Damm et al., 2012 ). The pathogen produces acervuli which is the asexual fruiting body responsible for conidia produc tion ( Maas, 1998 ), and p roduction of setae on acervul i has not been observed ( Damm et al. 2012 ). Colonies are usually hyaline white and then turn pink or orange when sporulating. When older, colonies turn pale gray ( Maas, 1998; Grahovac et al., 2012; Damm et al., 2012 ). The rate of C. acutatum colony growth on potato dextrose agar at 27 C averages 8 to 9 mm / day and can be used to morphologically differentiat e Colletotrichum species affecting strawberries since it is slower than C. fragariae and C. gloeosporioides (Maas 1998 ). Temperatures around 20 C and relative humidity near 100% are ideal for C. acutatum spore production, germination and infection of strawberry (Maas, 1998). Tanaka and Passos (2002 ) observed that C. acutatum wa s more aggressive on strawberry fruit and flowers, whereas C. fragariae wa s more aggressive to crown and petioles. However, C. fragariae wa s capable of in fecting flowers and fruit, but at a lower level. Similarly, C. acutatum can infect and cause lesions on strawberry petioles. Urea Padilla et al. (2002) confirmed that C. acutatum was more capable of infecting strawberry fruit than crowns. Although C. acutatum can c ause plant decline and wilt, it differs from the rapid decline caused by C. gloeos porioides C olletotrichum acutatum affects many other hosts and anthracnose s ymptoms range from localized lesions (strawberry) to the l oss of an entire crop (bitter rot of apple) ( Agrios, 2005 ). In all of the diseases caused by C. acutatum the commo n cond itions that favor disase development are warm and humid weather ( Agrios, 2005 ). In Israel,
17 Freeman et al. (2001) observed cross infection between C. acutatum isolates from weeds such as Vicia spp. or Conyza spp. and strawberry. Isolates recovered from thes e weeds were inoculated on strawberry plants and were highly pathogenic. Contrarily disease symptoms were not observed when crops such as eggplant, tomato and pepper were inoculated with strawbe rry isolates because a pparently, C. acutatum has a n epiphytic nonpathogenic, symbiotic lifestyle on Solenaceous crops (Freeman et al. 2001). This is in agreement with cross inoculation studies of MacKenzie et al (2009) with C. acutatum isolates from strawberry, blueberry, fern and key lime I solates only caused di sease symptoms on the host and tissue from which they were collected and did not cause an epidemic on other crops (MacKenzie et al. 2009 ). However, according to Damm et al. (2012) there was no cross infect ion in the MacKenzie et al. (2009) study because t he isolates collected from each host were from different species in the Colletotrichum acutatum species complex and were not C. acutatum Damm et al. (2012) suggests that the Colletotrichum species that infects strawberry is C. nymphaeae the blueberry pat hogen C. fioriniae and the key lime pathogen C. limetticola The fern isolates w ere not included in the study (Damm et al., 2012). Symptoms Colletotrichum acutatum is considered to be specific to t he tissue it infects on ea ch host. However, for strawberry isolates are less specialized and can attack petioles, leaves, flowers, crown and roots with or without showing symptoms ( Mertely et al., 2005; Peres et al., 2005 ). On strawberry, the most characteristic symptoms are flowe r blight and fruit rot ( Maas, 1998; Peres et al., 2005 ) (Figure 1 1 ) however f ruit rot is the most detrimental symptom to crop yield since infected fruit are non marketable ( Peres et al., 2005 ) Strawberry t ransplants can come from production nurseries wi th
18 quiescent infections and o nce established under warm and wet conditions lesions on petioles may be observed. Flowers are highly susceptible to C. acutatum and the pathogen can infect an y part of the flower tissue such as buds, pedicels and peduncles and result in fruit abortion or flower death ( Maas, 1998 ). Infected p etals turn brown and sepals develop burnt t ips, later drying but remaining attached to the plant These symptoms are similar to blighted inflorescence s infected by Botrytis cinerea ( Maas 1998; Mertely et al., 2005; Legard et al. 2003 ). On immature fruit, AFR symptoms start as hard black or dark brown lesions that expand to slightly sunken lesions (Maas, 1998) initially averaging 1.5 mm a cross ( Mertely et al., 2005; Legard et al. 2003 ). Infected black achenes, also called b lack seeds, can be observed on infected immature fruit and can lead to misshapen fruit (Legard et al. 2003). L esions then expand quickly on ripening fruit and go from light brown, water soaked to black brown firm spot s measuring from 3 to 12 mm ( Maas 1998; Mertely et al., 2005; Legard et al. 2003 ). I n the center of the lesions, visible masses of orange pink conidia are produced in acervuli under humid conditions and serve as an inoculum source for further infections ( Maas, 1998; Legard et al. 2003 ). O ther organisms can also easily infect the fruit tissue colonized by C. acutatum and cause other symptoms. However, if only infected by C. acutatum lesions can m erge and cover the entire fruit ( Maas 1998; Mertely et al. 2005; Peres et al., 2005 ). During a severe epidemic in strawberry nurseries or commercial fruit fields, wilt and plant death from strawberry crown and root rots can be caused by C. acutatum Infected roots usually develop brown lesions and have a few fe eder roots. Such infected plants can slowly decline and die (Legard et al. 2003; Mertely and Peres, 2005)
19 Symptoms of ba sal crown rot can be observed under field and greenhouse conditions when plants do not establish well and consequently are stunt ed and die ( Mertely and Peres, 2005 ). Peres et al. (2005) suggested that root and crown infec tions can be a consequence of conidia or appressoria splashed from the foliage, flowers and fruit down t o the lower parts of the plant During warm humid conditions and when C. acutatum infection levels are high, petiole lesions covered with conidia can also be observed (Legard et al. 2003). Epidemiology and Life Cycle In annual production fields such as those in Florida, C. acutatum inoculum originates from symptomless infected transplants from nurseries where the use of overhead irrigation can lead to conidial dispersal (Strand, 2008). When transplants with infected petioles and runners are established in the field under warm humid weather, secondary conidia are produced (Maas, 1998; Mertely et al., 2005; Leandro et al., 2003 b), and disseminated to flowers and fruit by splashing water, contaminated soil on farming equipment, and harvesting operations, and consequently starting the sy mptomatic infection process (Mertely et al., 2005; Leandro et al., 2003b; Strand, 2008) (Figure 1 2 ). C. acutatum was thought to be a hemibiotrophic pathogen, with a biotrophic phase of feeding on living cells and a necrotrophic phase of killing cells to c omplete its life cycle. However, on strawberry, the biotrophic phase is very short (less than 12 hours) and for that reason, the pathogen can be considered a necrot roph ( Curry et al., 2002 ). AFR development is affected greatly by temperature and leaf wetne ss duration (Wilson et al. 1990). Wilson et al. (1990) used a regression model to describe the effects of leaf wetness and temperature on the development of AFR on immature and
20 mature fruit of the st rawberry cultivar Midway. At same temperature and wetness periods, mature fruit were found to be more susceptible than immature. In addition, disease incidence on fruit increased with an increase of temperature (from 6 to 25 C), when the wetness period remained the same. When exposed to wetness durations of 0.5 to 51 hours and temperatures of 4 or 35 C, immature fruit did not show symptoms, however at 25 C and after 25 hours of leaf wetness disease incidence reached 100%. For mature fruit, disease incidence reached 97% after only 13 h ours at 25 C, and i nfected fruit were observed at 35 C when wetness durations were shorter than 2 hours. Minimum length of wetness period required to start disease development was different for mature and immature fruit. For immature fruit, infected strawb erries were observed after exposure to 5 hours of wetness, whereas for mature, only 1 hour was required. In addition, when fruit were exposed to wetness duration of 50 hours and low temperature (6 C), 13% of immature fruit and 25% of mature fruit showed di sease symptoms (Wilson et al., 1990). The temperature and wetness duration relationship directly affected AFR development on strawberry fruit and with higher temperatures, shorter wetness periods were required for immature and mature fruit infection. Lean dro et al. (2003a) observed that germination, secondary conidiation and appressorial development of C. acutatum on symptomless strawberry leaves of the cultivar Tristar are critically affected by wetness duration and temperature. Temperatures from 17.6 to 27.7 C with continuous wetness periods were optimal for germination and appressorial development. Temperatures from 21.3 to 32.7 C and wetness periods of more than 4 hours were required for secondary conidiation. Leandro et al. (2003b) also observed that C. acutatum secondary conidiation was stimulated
21 more by strawberry flower extracts than moisture alone. This was shown when secondary conidia were produced on coverslips where C. acutatum was exposed to dryness and then treated with flower extracts. Zulfi qar et al. ( 1996 ) also observed that p roduction of conidia of C. acutatum on citrus leaves was stimulated by flower extracts Conidia l production was seen in treatments when water was applied however l ess numerous than when exposed to flower extract s T hus presence of flowers extracts can increase inoculum for further fruit and flower infections. Di guez Uribeondo et al. (2011) also tested the effect of temperature and wetness duration on almond cultivars with different anthracnose susceptibility leve ls. The results were in agreement with those of Wilson et al. (1990): periods of moisture are necessary for disease development and the duration is highly dependent on temperature B etween 15 and 20 C, anthracnose symptoms can develop with wetness periods as short as 3 hours ( Di guez Uribeondo et al. 2011). In addition to C. acutatum conidiation and AFR development, temperature influences sporulation and the length of the incubation period of Colletotrichum species on strawberry plants (King et al., 1997 ). King et al. (1997) observed maximum sporulation at 25 C for 4 days after c onidial production. T he temperature incubation time relationship is similar to that of the temperature and wetness period, since with the increase of temperature, shorter incubati on periods are necessary for sporulation. Sporulation does not require free moisture and it is suggested that Colletotrichum conidia obtain moisture from the fruit to produce spores (King et al., 1997). Nonetheless, when compared to C. gloe o sporioides and C. fragariae C. acutatum had the shortest latent period at low temperatures (5 and 10 C). Thus, C. acutatum can be
22 favored in comparison to other Colletotrichum species in places like Florida, where the strawberry season is during the winter when temperat ures are cool (King et al., 1997). Initial disease symptom development on strawberry plants depends on concentration of inoculum present as quiescent infections Knowledge of the required inoculum concentration for initial disease development is useful es pecially in the case of AFR of strawberry, since in commercial fields the disease starts with infected transplants coming from the nurseries (Howard et al., 1992). The weather conditions during plant establishment (temperatures from 20 to 25 C and 8 hours of overhead irrigation for 7 days ) are extremel y favorable for AFR development. T herefore if transplants are already infected it is difficult to control the disease and the inoculum dispersal to healthy plants. T he minimum inoculum concentration required for symptom development on cultivars with different levels of susceptibility is important since growers plant cultivars that may be highly and less susceptible to anthracnose in close proximity and this information can be useful when deciding the control methods to use and the timing of fungicide applications ( Di guez Uribeondo et al. 2011 ) M ore susceptible cultivars may require a lower inoculum concentration for disease development than less susceptible cultivars A nthracnose d evelopment on almond cultivars wa s critically affected by inoculum concentration and a concentration of at least 10 4 conidia/ml was required for symptom development for cultivars NePlus Ultra (susceptible to anthracnose) and Nonpareil (less susceptible to anthracnose) ( Di guez Uribeondo et al. 2011 ) Trapero Casas and Kaiser (1992) studied the effect of different inoculum concentrations (4 x 10 4 2 x 10 5 1 x 10 6 and 1 x 10 7 conidia/ml) on the infection and
23 development of Ascochyta blight of chickpea cultivars with different susceptibility levels (highly susceptible, moderately susceptible, and resistant) to standardize inoculum concentration for artificial inoculations with Ascochyta rabiei Susceptibility levels of chickpea cultivars was fundamental when assessing the minim um inoculum concentration ne cessary for symptom development because disease severity of the susceptible cultivars was higher at all inoculum conce ntrations when compared to the resistan t cultivar. When the concentration increased from 4 x 10 4 to 1 x 10 7 co nidia/ml, disease severity of the cultivar Pedrosillano (moderately susceptible) increased from 32.9 to 83.5%. However, at the highest inoculum concentration of 1 x 10 7 conidia/ml, disease severity was only 12.7% for the resistant cultivar, whereas for the highly susceptible cultivars at all inoculum concentrations, disease severity did not increase significantly with increasing inoculum concentration and was greater than 50% with all treatments. In summary, inoculum concentration can highly influence disea se development on cultivars with different levels of susceptibility and it is important to assess cultivar susceptibility with other epidemiological factors (Trapero Casas and Kaiser, 1992). The effect of inoculum concentration on AFR development on strawb erry cultivars with different levels of susceptibility is currently unknown. Disease forecast systems have been developed to assist growers by providing information about the risk for disease development and offer an alternative method to control outbreaks with a significant reduction in fun gicide applications. Forecast systems for anthracnose diseases are usually built on models based on the effects of temperature and leaf wetness duration on disease progress (MacKenzie and Peres 2012 a ; Di guez Uribeondo et al. 2011). Several models have been developed to
24 predict disease s caused by C. acutatum and other pathogens such as Botrytis cinerea Phytophthora cactorum and Mycosphaerella fragariae on strawberry (Wilson et al. 1990 ; Carisse e t al. 2000 ; Grove et al. 1985 ; Sosa Alvarez et al. 1995 ; Bulger et al., 1987). Wilson et al. (1990 ) modeled AFR disease incidence of strawberry as a function of temperature and leaf wetness duration. MacKenzie and Peres (2012 a ), used the equation publis hed by Wilson et al. (1990) to test its effectiveness f or timing fungicide applications compared to the calendar application schedule (weekly) on cultivars with highly and moderately susceptible, respectively ). T he use of a disease threshold INF (proportion of infected fruit) was ideal for application of captan (Captan 80WDG; Micro Flo Company LLC, Memphis, TN) and INF 0.5 for pyraclostrobin (Cabrio 20EG; BASF Corporation, Rese arch Triangle Park, NC) These treatments provided efficient AFR control on both cultivars, reducing fungicide applications without reducing yield s compared to weekly applications. Bulger et al. (1987) developed a model to predict flower and fruit infectio n by B. cinerea also based on temperature and leaf wetness duration. Similar model evaluations were conducted by MacKenzie and Peres (2012 b ) for BFR T he info rmation acquired from the model evaluations to predict C. acutatum and B. cinerea infection on str awberry fruit and flowers was used to build the (SAS) ( http://www.agroclimate.org/tools/strawberry ), a disease forecasting system that provides recommendation s on timi ng fungicide applications for AFR and BFR control for Florida growers (Pavan et al. 2011 ). Sosa Alvarez et al. ( 1995) used a logarithmic polynomial model to describe the effects of length of wetness periods and temperature on sporulation of B. cinerea on
25 dead strawberry leaves. To estimate the potential risk of infection of Mycosphaerella fragariae on strawberry leaves, Carisse et al. (2000 ), developed a model based on the response of M. fragariae to temperature and leaf wetness durations. Grove et al. (19 85) used the same strawberry cultivar (Midway) as Wilson et al. (1990) for forecasting P. cactorum infections on immature fruit under natural conditions. In agreement with the other studies described above, P. cactorum infection is also highly influenced by temperature and wetness (Grove et al. 1985). Howev er, there are no models developed to describe these effects on AFR development on strawberry cultivars with different levels of susceptibility that are commonly grow n in Florida such as Strawberry Festival, Camarosa and Treasure. This information would help to adapt the forecasting system currently available for the Florida strawberry production. The use of polyethylene plastic mulch has been a common practice for ma ny decades in strawberry production fields in Florida and California (Madden et al., 1996). Weed suppression, maintenance of warm soils and prolonged harvest seasons, are some of the many advantages of the use of plastic mulch as a ground cover ( Ag Answer s, 2012 ). For those reasons, the long used system of matted row with straw mulch in strawberry producing states in the Northern USA is being replaced by the plasticulture system (Madden et al., 1993; Hokanson and Finn, 2000). When compared to soil and stra w cover, plastic mulch has a higher impact on AFR development since its smooth material favors conidial splash dispersal Studies have found highest number of re splashing spores hitting the surface of the mulch and greatest number of C. acutatum colonies present on the plastic mulch (Yang et al., 1990a; Yang et al., 1990b; Madden et al., 1993). Contrarily, w hen using soil and straw cover, more spores are
26 trapped in the straw and consequently dissemination of inoculum can be suppressed. Additionally, stra w cover has shown to be the most effective ground cover for control of conidial splash dispersal because it has the highest rate of loss of spores through surface infiltration (Yang et al., 1990a; Yang et al., 1990b; Madden et al., 1993). Coelho et al. (20 08) also observed a significant reduction in flower blight incidence caused by C. acutatum when using grass or straw mulch compared to plastic mulch (Coelho et al., 2008). However, the number of growers replacing straw cover with plastic mulch has increase d in the past years, because straw and grass mulch have become not feasible for growers and the fact that plastic mulch can maintain the soil warmer and extend the harvest period is a great advantage for growers (Hokanson and Finn, 2000; Ag Answers, 2012). Rain intensity and duration interact with ground cover to affect C. acutatum conidial dispersal (Yang et al., 1990a; Yang et al., 1990b; Madden et al., 1993; Madden et al., 1996). Colletotrichum acutatum conidia can spread from 30 to 60 cm from an inoculu m source and cause 100% infection on strawberry fruit when exposed to rain intensities of 15 and 30 mm/h with the use of plastic mulch ground cover (Yang et al., 1990b ). When the effects of soil and straw covers were tested, with 60 min of rain with an int ensity of 30 mm/h disease incidence was decreased significantly with an increase in distance from 30 to 60 cm ( Yang et al., 1990b ). Furthermore, Yang et al. (1990b ) Madden et al. (1996) and Yang et al. (1990a ) observed that at high rain intensities, ther e was an increase in spore wash off on the fruit, in the volume of droplets splashed and the number of conidia transported (Yang et al. 1990a ; Madden et al ., 1996). Rain intensity and duration are environmental factors that growers cannot manipulate,
27 h owever when using plastic mulch as a ground cover, growers can rely on cultural practices such as higher plant density to reduce disease spread. Inoculum dispersal can be reduced with increased plant density (Madden and Boudreau, 1997). Yang et al. (1990a ) observed that plant rows with high leaf area index (LAI) (4.9 over the canopy crown), had fewer fungal colonies than in rows with 2.72 LAI. With a higher plant density, water splashed conidia do not penetrate the plant canopy as much as when plants are f urther apart. In addition, Madden and Boudreau (1997) found a decrease in re splashing spores across the plants with an increase in plant density. Even though temperatures between 20 and 25 C with high relative humidity are optimum for C acutatum spore p roduction, germination, and strawberry infection (Maas, 1998) the pathogen is able to survive under dry conditions and low temperatures (Leandro et al. 2003a). In a greenhouse study, Leandro et al. (2003a) observed that C. acutatum survived up to 8 weeks on strawberry leaves as appressoria, and that 48 hours after inoculation, fungal hyphae and primary conidia died, while melanized appressoria, secondary conidia and melanized hyphal segments survived on symptomless leaf surfaces (Leandro et al. 200 1) S econdary conidia tion on symptomless leaves can increase inoculum levels until susceptible tissue is available. Howard et al. (1992) suggests that the high temperatures and moist soil conditions in Florida are not favorable for C. acutatum survival fr om year to year. A study by Freeman et al. (2002) evaluated C. acutatum survival in natural, autoclaved, and methyl bromide fumigated soils. They observed a rapid decline in C. acutatum conidial viability within 2 to 6 days after placement on natural soil; whereas in sterilized
28 soils C. acutatum and C. gloe o sporioides conidia survived at up to 1 year. This may be due to low microbial competition in sterilized soils compared to natural soils. Furthermore, in soils fumigated with methyl bromide conidia survi ved up to 2 to 4 months at 11% soil moisture. At field capacity (22% soil moisture content), the fungal population declined rapidly within 6 to 7 days after placement in fumigated soils (Freeman et al. 2002). In American strawberry fields, the use of meth yl b romide has been bann ed and other fu migants such as the mixture of d ichloropropene and chloropicrin are used to reduce nematodes and the fungal community in the soil. Climatic factors and the type of production system used in commercial strawberry fiel ds can influence the survival and dispersal of C. acutatum on strawberry plants and in the soil. Thus, u nderstanding the life cycle of C. acutatum and the conditions adequate for disease development are useful when managing AFR. Disease Management Anthrac nose Fruit Rot (AFR) can be managed in nurserie s and commercial fields with a combination of cultural, chemical and biological control. Cultural and b iological c ontrol AFR control should start prior to strawberry planting ( Maas, 1998 ). The best AFR control strategy is to avoid introduction of the pathogen into commercial fields by planting anthracnose free transplants. Unfortunately, that is difficult to achieve since infected plants may not show symptoms (quiescent infections) at plantin g ( Mertely et al., 2005). In the few anthracnose free areas where strawberries are grown quarantine regulations ca n be an effective preventive measure ( Maas, 1998 ). In Florida, where weather conditions are favorable for AFR, growers rely in part on plant ing less susceptible cultivars to suppress disease outbreaks and reduce
29 inoculum dissemination ( Chandler et al., 2006 ). Chandler et al. (2006) observed in three strawberry seasons the least susceptible ediate in susceptibility), whereas ost susceptible. Unfortunately, one of the most planted cultivars in Florida is less susce ptible to AFR but high ly susceptible to Botrytis Fruit Rot (BFR), a nother major strawberry di sease However, the opposite w less susceptible to BFR, but highly susceptible to AFR and which is susceptible to both AFR and BFR ( Mertely and Peres, 2006 ). Choosing strawberry cultivars that are less susceptible to major strawberry diseases is an important cultural control strategy, since it can reduce the need for fungicide applications (Chandler et al., 2006). However, the difficul ties of planting only cultivars with low susceptibility levels are that many of these cultivars have low fruit quality and yield. Also, because cultivars have different fruiting periods, growers plant a variety of cultivars to have fruit available all seas on long. Drip irrigation is also an important cultural control method since conidia are w ater splashed dispersed (Maas, 1998). Coelho et al. (2008) observed that flower blight appeared earlier on strawberry plants when overhead irrigation was used than on drip irrigat ed plants Another advantage of drip irrigation is that fertilization can also be done simultaneously. However, irrigation and fertilization should be well controlled since nutrients may favor disease development. Maas ( 1998 ) reported that high levels of nitrogen favor AFR development, whereas calcium applications in the form of calcium sulfate (CaSO 4 ) and calcium chloride (CaCl 2 ) may delay infection of greenhouse grown strawber ries (Smith and Gupton, 1993 ).
30 H eat treatments by submerging bare r oot transplants in 49 C water for 5 minutes have been tested to eliminate quiescent infections However, the long term effects of heat treatments on plant vigor and development may be detrimental to the plant and deserve s more study ( Freeman et al., 1997 ) In addition to the cultural control methods cited above, growers should scout their fields after prolonged leaf wetness periods looking for AFR symptoms such as flower blight or immature fruit with blackened achenes ( Mertely et al., 2005 ) to monitor disease outbreak s and remove symptomatic fruit and flowers from the field to prevent inoculum build up and infection ( Maas, 1998 ). The concern about fungicide resistance, environmental safety and sustainable production has triggered researchers to study biological AFR control methods. Freeman et al. (200 4 ) found that the fungus Trichoderma at high concentrations (0.8%) was effective as a biocontrol agent for control of AFR and BFR in strawberries in Israel Thorpe et al. (2003) tested the suppressive ab ility of several strawberry fungi and bacteria to C. acutatum Five fungal isolates of the genera Cephalosporium Myrioconium and Paecilomyces and two bacterial isolates from the Enterobacteriaceae inhibited in vitro fungal growth and reduced the number of appressoria and conidia on strawberry leaves. Unfortunately, there are not many more positive results in AFR bio control on strawberries. Thus, m ore studies about the efficacy of biocontrol agents on AFR on strawberries are needed and if successful, will p rovide growers alternatives to chemical control. Chemical c ontrol The use of fungicides to control A FR and other strawberry diseases is a popular practice among growers and is usually based on a calendar schedule of weekly
31 applications (Mertely et al., 2005; Turecheck et al., 2006). This practice can cause some negative environmental im pact, increa se the possibility of fungicide resistance and the cost of production. Early in the season, from November to December, inoculum levels are low, temperatures in Florida are cool and usually not favorable for C. acutatum and consequently infected plants do not show symptoms. At this time low label rate s of broad spectrum protectant fungicides l ike c aptan are sprayed to p revent disease outbreaks ( Mertely et al., 2005). F rom Januar y to March, inoculum levels increase and climatic conditions approach the optimum temperature and humidit y for AFR development, and t he weekly use of higher label rates of broad spectrum fungicide s i s essential to control conidial production and disseminat ion from diseased to healthy plants ( Mertely et al., 2005 ). In t he USA, many fungicides are labeled and effective in controlling AFR of strawberry such as c aptan Amercia, Cary, NC), c yprodinil + f luodioxonil (Switch; Syngenta Crop Protection, Greensboro, NC), pyraclostrobin ( Cabrio, BASF Corporation, R esearch Triangle Park, NC), and a zoxystrobin (Abound, Syngenta Crop Protection) (Mertely et al., 2005). However some of these fungicides are only registered for strawberry but not labeled for AFR such as the mixture of fluodioxonil and cyprodinil, or thiophanate methyl (Topsin M 70WP, UPI, King of Prussia, PA) The active ingredients p yraclostrobin and azoxystrobi n are registered for strawbe rry and labeled for AFR They have curative and pr otective properties thereby provide a high level of control when sprayed pre and post infection ( Turechek et al., 2006 ). Turechek et al. (2006) observed that when pyraclostrobin was
32 spr ayed within 3 hours and up to 8 hours after wetting events of 24 hours or less it controlled AFR on strawberry plants under experimental conditions. However, pyraclostrobin was more effective when sprayed as a protectant ( Turechek e t al., 2006 ). When using moderately susceptible strawberry culti vars, like Strawberry Festival growers can take more risks and pyraclostrobin applications can be made up to 24 hours after a wetting event. The advantage of spraying fungicides when weather conditions are conducive for AFR development instead of following a calendar based spraying schedule is that the number of applications can be reduced and thus prevent ing the risk of fungicide resistance development ( Turechek et al., 2006) When the disea se i s detected in the field, single site fungicides are recommended along with the usual multi site products T ank mixing a single site with a multi site can help to prevent fungicide resistance ( Mertely et al., 2005 ). In plants where C. acutatum infection is already established, the use of specific single site fungicides such as azoxystrobin, boscalid + pyraclostrobin, and cyprodinil + fludioxonil is a good control method to reduc e disease incidence (Daugovish et al., 2009 FRAC, 2013 ). Furthermore, an adv antage of the use of some single site fungicides i s that they also control BFR a nother major strawberry disease in Florida ( Mertely et al., 2005 ). Other active ingredients have been shown to control AFR of strawberry worldwide. As in the USA, Brazilian strawberry production suffers from AFR. Kososki et al. (2001) evaluat ed different active ingredients and observed that under controlled conditions, prochloraz at 100 g/ml and tebu conazole at 50 g/ml reduced C. acutatum mycelial growth Moreover, prochloraz and tebuconazole were also the most effective in controlling conidial germination when compared to iprodione, thiophanate methyl
33 propiconazole, mancozeb, folpet and copper sulfate In field trials, prochloraz showed fewer bl ighted f lowers than benomy l, a standard treatment in controlling AFR in Brazil, before it was eliminated from the market in the early 2000 s Domingues et al ( 2001 ) and Freeman et al. (1997) also confirmed the efficacy of prochloraz in controlling C. acutatum in f ield and in vitro studies Freeman et al (1997) observed that p rochloraz Mn, prochloraz Zn and the combination of prochloraz Zn + folpet had the hi ghest in vitro inhibition of C. acutatum compared to captan, propiconazole prochloraz and difenoconazole. Unfortunately prochloraz is not registered for strawberries in the USA or Brazil ( Kososki et al., 2001 ). The use of fungicides by itself is not enough to control AFR in strawberries. Timing fungicide applications is also of extreme importance With the o bjective of reducing the number of fungicide applications during a strawberry season, MacKenzie and Peres (2012 a ) tested the effectiveness of timing fungicide applications based on temperature and leaf wetness duration adequate for AFR development T his in formation was used t o build the a disease forecasting system based on temperature and leaf wetness duration to forecast disease outbreaks and advise strawberry growers on timing fungicide applications (Pavan et al., 2011 ). Objectives Strawberry cultivars planted in Florida differ in susceptibility to C. acutatum however not enough information is known about the effects of inoculum concentration, temperature and wetness duration on AFR development on these cultivars. Ther efore, the objectives of this project were to evaluate the effects of inoculum concentration, temperature and wetness duration on the development of AFR on flowers and immature fruit of strawberry cultivars with different susceptibility levels under field and laboratory
34 conditions. The information collected will be used to adapt models used in a disease forecasting system to predict disease outbreaks in Florida commercial fields.
35 Figure 1 1 Anthracnose fruit rot symptoms on strawberry A ) Symptomatic flower. B ) Symptomatic immature fruit. C ) Symptomatic m ature fruit. Credits: University of Florida, GCREC Strawberry Plant Path olog y. Phot o s courtesy of University of Florida UF/ IFAS GCREC. Figure 1 2. Life cycle of Colletotrichum acutatum the causal agent of Anthracnose Fruit Rot of strawberry. Figure by Peres et al. in: Lifestyles of Colletotrichum acutatum 2005.
36 CHAPTER 2 EFFECT OF INOCULUM CONCENTRATION, TEMPERATURE AND WETNESS DURATION ON ANTHRACNOSE FRUIT ROT DEVELOPMENT ON DIFFE RENT STRAWBERRY CULTIVARS The United States of America is the major producer of strawberries ( Fragaria x ananassa Duch) in the world followed by Turkey, Spain, Egypt and Mexico ( F AOSTAT 2012 ). In the USA, California is the largest strawberry producer dur ing the summer (USDA, 2013) and Florida is the main producer and distributor of fresh winter strawberries D uring the 2012 2013 season, yield was 182.7 billion pounds and 8,700 acres of strawberries were harvested (USDA, 2013). AFR of strawberry, most commonly caused by the fungus C olletotrichum acutatum Simmonds is a major disease in Florida and wor ldwide (Maas, 1998; Legard, 2002 ). Transplants i nfected with C. acutatum are respons ible for introducing the pathogen in annual str awberry production commercial fields (Strand, 2008; Mertely et al., 2005). Comm on symptoms of AFR are flower blight and fruit rot, however during severe epidemics crown and root rots as well as petiole lesions may be observed (Legard et al. 2003; Mertely and Peres, 2005; Peres et al., 2005). Additionally, AFR development is highly dependent Fes the most common cultivars g rown in Florida, are considered moderately susceptible susceptible to AFR (Chandl er et al., 2006 ; Seijo et al., 2008 ). S ince infected transplants are the main source of inoculum, the amount of quiescent infections o n transplants may influence initial AFR symptom app earance in commercial fields depending on cultivar susceptibili ty, since more susceptible cultivars may require lower inoculum concentrations for symptom development.
37 C olletotrichum acutatum the anamorph stage of Glomerella acutata produces conidia that are responsible for plant infection (Damm et al., 2012; Guerber and Correll, 2001; Agrios, 2005). The fungus is a hemibiotroph ic pathogen and on strawberries the necrotroph ic phase is predominant (Curry et al., 2002). Conidial dispersal occurs mainly through splashing water, harvesting operations and contaminated soil on machinery and farming equipment (Mertely et al., 2005, Leandro et al., 2003 b ; Strand, 2008). S econdary coni dial production, germination, appressorial formation and fruit infection of C. acutatum are highly affected by temperature and wetness duration (WD) (Maas, 1998; Leandro et al., 2003 a ). Secondary conidia l germination and appressorial development on sympto mless leaves may occur with continuous wetness and temperatures from 17 to 27 C (Leandro et al., 2003 a ). S econdary conidiation is stimulated more by the presence of strawberry flower extracts than by water, and thus the presence of flowers may increase ino culum on strawberry plants for subsequent organ infections. In addition, AFR incidence on immature and mature strawberry fruit increase s with increasing temperature s from 6 to 25 C and WD from 1 to 51 hour s. (Maas, 1998; Wilson et al., 1990 ). The effects of temperature and WD on AFR development have been used to bui ld SAS ) The SAS provides growers with information about weather conditions that are favorable for AFR and BFR development and recommendations on timing fungic ide applications. The main advantage of using such system is the reduction o f fungicide application s during the season without reducing plant yield (MacKenzie and Peres, 2012 a ). This is extr emely impor tant in Florida where humid conditions favor disease d evelopment and the use of preventive
38 fungicide applications is a common practice among strawberry growers. However, environmental safety and the increase in fungicide resistance is a concern among growers and researchers. To improve and adapt the SAS, more information was needed about the effects of temperature, WD a nd inoculum concentrations on commonly grown strawberry cultivars in Florida and plant organs with different levels of susceptibility. M aterials and Methods Fungal Isolates and Culture Four isolates of C. acutatum 2 163 2 1 79 3 32 and 98 324 that were sequenced ( G3PD GS and ITS regions) in a previous study (except for 98 324) (MacKenzie et al. 2009) were used for plant inoculation and cultural characterization. The GenBank accessio n numbers for sequences from the isolates are for the ITS G3PD and GS regions respectively, EU647302, EU647315, and EU647328 for the 2 163, EU647303, EU647316, and EU647329 for 2 179, and EU647304, EU647317, and EU647330 for 3 32 (MacKenzie et al., 2009) Strains were isolated from strawberry petiole (2 163), fruit (2 179 and 98 324) and crown (3 32) and m aintained on filter paper in a sterile envel ope in airtight container with D rierite (W.A. Hammond Drierite Company, LTD) at 20 C. To revive the isolates, a small piece of the filter paper ( 4 mm 2 ) c ont ai ning the isolate was placed on 90 mm plates with potato dextrose agar (BD grown for 8 days at 22 C. Effect of Inoculum Concentration on Anthracnose Fruit Rot Development To determi ne the effect of different inoculum concentrations on AFR development on different strawberry cultivars, a field and a detached fr uit experiment were carried out The field experiment was conducted during the 2011 2012 and 2012
39 2013 strawberry seasons, whi le the detached fruit experiment was done during the 2012 2013 season. Field Trial Bare root straw berry transplants of the cultivars C amarosa, Strawberry Festival and Treasure were established in the field in mid October 2011 at the University of Florida Gulf Coast Research and Education Center. In mid October 2012, transplants of were established in the same location. Before transplant, plastic mulch covered beds were fumigated with 1,3 d ichloropropene and chloropicri n (Telone C 35 Dow AgroSciences Indianapolis, IN) in both seasons The bed s were 91.4 m long, 71 cm wide and 15 cm high at the edges and were 18 cm hi gh at the center. Beds were 1.2 m apart, measuring from their center s Transplants were placed 30 cm apart in two rows. For plant establishment, transplants were irrigated with overhead sprinklers for 10 to 12 days and for the rest of the season plants were irrigated and fertilized daily through drip irrigation F or f our da ys during the 2011 2012 season (January 4, 5 and 15 and February 13 ) temperatures were below 0 C and the use of overhead irrigation was necessary for freeze protection. Overhead irrigation was not necessary for freeze protection during the 2012 2013 seaso n. Four inoculum concentrations (10 3 10 4 10 5 and 10 6 conidia/ml) of C. acutatum and a control (deionized water) were chosen as the treatments for the 2011 2012 season. In the 2012 2013 season, the inoculum concentration 10 2 conidia/ml was added. Flowers, immature, pink and mature fruit were inoculated to compare organ susceptibility. For standardization at the time of inocu lation, flowers had to be open have intact petals and fresh yellow pollen; immatu re fruit had chlorophyll and were not starting to turn white; pink fruit had lost chlorophyll and started to turn pink; and mature
40 fruit were beginning to turn red. During the 2011 2012 season, flowers, immature, pink and mature fruit of Treasure Camarosa and Strawberry Festival were inoculated, whereas in the 2012 inoculated. Isolates 2 179, 3 32 and 98 324 were revived as previously described and grown for eight days, until fungal mycelium covered 2/3 of the plate. Coloni es were scraped using sterile water and a glass rod to obtain conidial suspensions The conidial suspension of each isolate was poured in to an Erlenmeyer flask through a double layer of cheesecloth. Conidial s uspensions were counted with a h emocytometer (B right Line, Hausser Scientific) and then adjusted to 10 6 conidia/ml for each isolate Then all isolates of the same conidial concentration were mixed The final c onidial concentrations were achieved th rough serial dilution and refrigerated at 5 C until in oculation time. All inoculum suspensions were prepared on the same day. In the 2011 2012 season, ten pl ants per treatment were chosen arbitrarily and flagged. A total of 50 plants per cultivar were used (10 plants x 5 treatments). No AFR symptoms were obse rved prior to inoculation. On the days of inoculation, January 21 and February 14, all the flowers, immature, pink and mature fruit present on the selected plants were tagged with different tape colors to identify the plant organs that would be inoculated (flowers = yellow; immature fruit = green; pink fruit = orange and mature fruit = red) (Figure 2 1A). Mean temperat ure at the time of inoculation and mean daily temperature were 21.5 and 16.5 C (January 21) and 23.2 and 16.4 C (February 14, 2012). Dur ing the 2012 2013 season, the experimental unit were flowers and fruit instead of plants like the previous season. F our replications of 10 flowers and immature
41 fruit per inoculum concentration were inoculated on March 22. The mean temperature at time and day of inoculation were 21.1 and 15. 6 C respectively On inoculation days, tagged plant organs were mist sprayed with approximately 250 l of the conidial suspensions at the designated concentration with an atomizer (Spra tool, Crown). Immediately after inocu lation, p lants were covered with 46 x 61 cm plastic bags (Uline) containing a small amount of deionized water to maintain humidity (Figure 2 1B). Sixteen hours after inoculation, plastic bag s were removed and plants were allowed to dry. Tagged plant organs were eva luated for disease incidence over 21 days starting five days after inoculation dur ing the 2011 2012 season and over 10 days starting five days after inoculation in the 2012 2013 season. T he experiment was co n ducted twice for each cultivar i n the on. In the experiment conducted on plants under a high tunnel (Figure 2 1C) whereas immature fruit were from plants in the open field. The experiment was a completely randomized design. Detached Fruit Trial Five inoculum concentrations (10 2 10 3 10 4 10 5 and 10 6 conidia/ml) of the C. acutatum isolates 2 163, 2 179, and 3 32, plus a control (deionized water) were tested in the detached fruit complete block design consisting of four replications of 4 immature fruit/treatment (16 fruit/treatment). During the 2012 th at were on average 2 to 3 cm 2 and had receptacle with chlorophyll were harvested from the field In the laboratory fruit went through a second triage (Figure 2 2A) for better standardization and were surface sterilized using 0.7% sodium hypochlorite for
42 six minutes and then rinsed four times with sterile water. Four plastic boxes measuring 31.5 x 25 x 10 cm and eight egg car tons (one dozen wells) per cultivar were sprayed with alcohol and sterilized inside a fume hood with ultraviolet light for 20 mi nutes. Then, fruit were placed in the egg cartons inside the plastic boxes according to replication and treatment (Figure 2 2B) and were surface dried inside the hood for 20 minutes. To maintain humidity inside the boxes, 75 ml of deionized water w ere placed under the egg cartons. Inoculum was prepared as described for the field experiment. Fruit were inoculated with a 5 l droplet of the inoculum suspension at the top of the fruit for standardization and to facilitate disease evaluation. Plastic boxes were covered and maintained at room temperature (23 C 1) for 9 days. Fruit were evaluated for disease incidence and severity for 9 days starting fi ve days after inoculation. The experi ment was repeated four times fo r each cultivar. In addition to this experiment, another trial (days after flowering). Op en flowers with fresh pollen were tagged and fruit harvested 8 or 12 days after wards were used as the experimental unit. This trial was repeated twice for each fruit age Effect of Different Temperatures on Mycelial Growth of C. acutatum isolates A mycelia l growth assay was conducted to test the influence of temperature on the growth rate of C. acutatum isolates 2 163, 2 179 and 3 32. Six millimeter diameter mycelial plugs of each of the three isolates whe re cut from the margin of an 8 day old co lony (when 2/3 of the plate was covered by mycelia) and placed on PDA Plates were closed with parafilm and placed in growth chambers set at 5, 10, 15, 20, 25, 30 and 35 C and in the dark. After 7 days, C. acutatum colonies were measured in one
43 direction. The size of the plug (6cm) was subtracted from the measurement to give the final colony size. The experiment was conducted as a split plot design with five plates per isolate (subplots) per temperature (whole plots) and was repeated three times. Effect of Temperatur e on Anthracnose Fruit Rot Development on Detached Fruit A growth chamber study using detached immature fruit was conducted to determine the effect of temperature on AFR development on Camarosa and Strawberry Festival during the 2012 2013 strawberry se ason. A total of seven temperatures (5, 10, 15, 20, 25, 30 and 35 C) were evaluated. Isolates 2 163, 2 179 and 3 32 were selected for fruit inoculation and inoculum was prepared as described above except that the inoculum concentration selected in this ass ay was 10 5 conidia/ml. Nine fruit (6 inoculated and 3 control s ) per temperature per cultivar were harvested from the field according to size and color (as described above) and brought to the laboratory for additional triage. Fruit were surface sterilized u sing 0.7% sodium hypochlorite for six minutes and then rinsed four times with sterile water. Seven boxes and 14 egg cartons (1 egg carton per cultivar/temperature) were sterilized with ultraviolet light. Boxes we re considered as the replications and there was 1 box per temperature. Fruit were placed in egg cartons (9 per egg carton) inside boxes and left to air dry inside a fume hood for 20 minutes. Each box contained both cultivars (1 egg carton/cultivar). Seventy five ml of water was added to boxes to sim ulate a humid chamber as described in the previous experiment. A 5 l droplet of the conidial suspension was placed on the upper surface of the fruit and boxes were closed to prevent droplet movement and were only moved to growth chambers 5 minutes after i noculation. One growth chamber was set to each temperature AFR incidence on inoculated fruit was evaluated o ve r 9 days, starting on
44 the fifth day after inoculation. For the lower temperatures (5, 10 and 15 C) the evaluation period was extended to 19 days after inoculation, because at lower temperatures disease progress is slower. This experiment was conducted in a split plot design where the different temperatures were the whole plots and the cultivars were the subplots. This experiment was repeated four times and i n the fourth experiment, the number of fruit used per treatment increased to 7 Effect of Wetness Duration and Temperature on Anthracnose Fruit Rot Development To determine the effect of Wetness Duration ( WD) and temperature on AFR development, an environmental growth chamber study was conducted during the 2011 2012 and 2012 2013 strawberry seasons. For that, growth chamber temperatures 15, 20 and 25 C and wetness periods of 0, 3, 6, 12, 24 and 48 hours plus a control (water treatment + 48 h ours of WD) were evaluated. During the 2011 2012 season, two hundred bare originat ing from Nova Scotia, Canada were planted in 15 x 15 x 16 cm plastic pots filled with soil containing Canadian sphagnum peat (65%), perlite and vermiculite (Fafard) in October of 201 1. Plants were fertilized with Osmocote Plus granular slow release (Scotts, 15 9 12) irrigated daily and remained in a greenhouse until they were ready fo r inoculation. In the 2012 2013 season n October 2012 and January 2013, following the same steps as in the previous season. However plants were then fertilized with Miracle Gro Water Soluble All Purpose Plant Food (24 8 16) every 10 days.
45 During the 2011 2012 season, s trawberry flowers, immature, pink an d mature fruit were inoculated and during the 2012 2013 season o nly immature fruit. In the 2011 2012 season, four plants/ WD/temperature/cultivar (84 plants per cultivar) were identified according to treatment and plant organs were tagged (flower = yellow; immature fruit = green; pink fruit = orange and mature fruit = red). During the 2012 2013 season, only immature fruit were used for i noculation and t en immature fruit/ WD/temperature/cultivar w ere tagged and considered as a replication. However for the third experiment conducted with there was limited number of immature fruit, there fore only 4 fruit/ WD/temperature combinations were used One environmental growth chamber was set for each temperature with a 12 h light/12 h dark photoperiod (2011 2012) or 24 h dark (2012 2013), and t wo growth rooms were adjusted to 25 C with 12h light/12 h dark photoperiod D ata loggers (WatchDog A Series, Spec trum) were placed inside growth chamber s and rooms to monitor temperature. Eight day old cultures of C. acutatum isolates 2 163, 2 179 and 3 32 w ere scraped with sterile water using a glass rod as desc ribed above. Conidial suspension s of each isolate were filtered through double layer cheesecloth and concentration adjusted to 10 6 conidia/ml with the use of a h emocytometer. Subsequently, conidial suspension of the three isolates were mixed and stored in a 236ml container for inoculation. Tagged plant organs were mist sprayed with a 150l suspension or sterile water (control) using an atomizer (Spra tool, Crown). Plants were carefully bagged and closed with a zip tie. To maintain humidity inside the plasti c bags, two moist cotton balls sprayed with deionized water were placed on th e bottom of each bag Bagged plants were then transferred to growth chamber s according to temperature and WD (F igure 2
46 3A), except for the 0 hour of WD treatment. These were inoculated and immedi ately placed in front of fans (F igure 2 3B) until plant organs were visually dr y (approximately 30 minutes). Thirty minutes prior to the end of the wetness period plants were removed from the bags, dried as described above and trans ferred to growth rooms at 25 C for 21 days in the first season and for 9 days in the second season. Plants were irrigated daily by adding water directly on the soil to avoid leaf, flower or fruit wetness. Starting five days after plant inoculation, tagged organs were evaluated daily for disease incidence. Trials during the 2011 2012 season were conducted at the same time for the three cultivars, however during the 2012 2013 season, cultivars Camarosa and Strawberry Festival were inoculated on different date s since growth chamber space was limitat ed In th e 2011 2012 season, plant organ were evaluated up to 21 days after inoculation, whereas in the la t ter season they were evaluated up to 9 days after inoculation. The experiment was conducted twice for each c ultivar in the 2011 2012 season and three times in the 2012 2 013 with a split plot design, wh ere the temperatures were the whole plots and the wetness periods the subplots. Statistical Analysis Arcsine transformation was used for incidence values because t here were many dot points with 0 and 100% disease incidence For the statistical analysis both disease incidence and arcsine transformed values were used An Analysis of Variance (ANOVA) was conducted to test the effects of experiment i noculum concentrati on, cultivar temperature, wetness duration and their interactions (SAS, version 9.3; SAS Institute, Cary, NC). Data of homogenous trials for each cultivar were combined and used to analyze treatment effect s and plant organ susceptibility using the Least Significant Difference (LSD) or Tukey (SAS). The c orrelation between field and detached fruit
47 assays and cultivars were analyzed using analysis of covariance (SAS) for the inoculum c oncentration and temperature x WD assays, respectively. There were many mi ssing values i n the 2011 2012 inoculum concentration field trials, because of the absence of flowers or fruit on the inoculated plants. Therefore, data from the 4 pl ants that had the most flowers/fruit instead of 10 plants/treatment were used In addition, f or the statistical analysis of that experiment, disease incidence a t 10 days after inoculation was used
48 Figure 2 1. Field trial of i noculum concentration experiment. A ) Tagged strawberry flowers, immature, pink and mature fruit B ) Inoculated strawberry plants covered with plastic bags to maintain humidity C ) Overview of field plot under the tunnel. All treatment s and replications were arranged in a single bed. Photos courtesy of Bruna B Forcelini. Figure 2 2 Detached fruit trial of inoculum concentration experime nt. A ) Selection of immature strawberry fruit. Fruit located on the far left wer e selected for inoculation. B ) A rrangement of inoculated detached fruit for each replication and cultivar. Photos courtesy of Bruna B. Forcelini.
49 Figure 2 3 Controlled wetness duration and temperature experiment. A) Inoculated and bagged plants inside the growth chamber set at the design ated treatment temperature. B ) Use of fans to dry strawberry plants after different wetness periods Photos courtesy of Bruna B. Forcelini.
50 CHAPTER 3 RESULTS For all of the experiments, the statistical anal ysis using disease incidence or the arcsine transformed data showed similar results. Effect of Inoculum Concentration on Anthracnose Fruit Rot Development Field Trial Disease incidence increased with increa sing inoculum concentration from 0 to 10 6 conidia/ml for the three cultivars in the 2011 2012 and 2012 2013 strawberry seasons. The effects of strawberry cultivar, inoculum concentration and plant organ were significant ( P < 0.05), wh ile all interactions (cultivar x concentration, cultivar x plant organ and concentration x plant organ) were not (Table 3 1). For a low disease incidence was observed on flowers and immature fruit on non inoculated plants (Figure 3 1 high er disease incidence and did not differ from each other (Table 3 2). Inoculum concentration was highly significant ( P < 0.0001) for the three cultivars and the highest disease incidences were observed for all cultivars when flower s and fru it were inoculated with conidia at 10 6 /ml. A second order polynomial regression indicated that disease incidence on immature fruit and f lower s for the three cultivars increased with an increase in inoculum concentration from 0 to 10 6 conidia/ml. H owever the lowest inoculum concentration required for disease development differed among cult ivars and plant organs (Figure 3 2 ). First AFR symp tom appearance for
51 3 conidia/ml, whereas for immature fruit a higher inoculum concentration (10 4 conidia/ml) was needed. F lowers were more susceptible to AFR than immature fruit ( P =0.0147). Disease incidence for flowers reached 83% on the more susceptible cultivars (Camarosa and Treasure), whereas for immature fruit the maximum disease incidence was about 40%. F or incidence on flower s and immature fruit were not signifi cantly different ( P = 0.2037). AFR incubation period ( days from inoculation to symptom appearance) differed between trial one and two. Since trial one produced inconsistent results and had a low R 2 only the results of the second trial are shown. The AFR i ncubation period was shorter for flowers than for immature fruit f or all three cultivars However, when or h plant organs (Figure 3 3 ) The incubation period days for immature fruit and 7.25 days for flowers. Data from pink and mature fruit were not used in the stat istical analysis because fruit was over ripened before the end of the evaluation period. Detached Fruit T rial In the detached fruit experiment, disease incidence also increased with er disease at 10 6 conidia/ml (Table 3 3). The highest disease incidence was observed at the highest inoculum concentrations (10 5 and 10 6 conidia/ml) for both cultivars (Figure 3 4 ). In addition, the minimum inoculum
52 4 conidia/ml was sufficient for symptom symptoms were only o bserved at 10 5 are from the first two trials (fruit selected according to size and color), since the se trials did not differ. Data from the additional trials (fruit selected according to age) are not sho wn because trials were different among each other and from the first two experiments. Correlation between Detached Fruit and Field T rial For each cultivar, a polynomial regression of second order was used to fit the curves for the detached fruit and field experiments. Disease incidence was lower for and field experiments were similar to each other and between cultivars A value equivalent or less than 1. 96 is require d to determine if the curves are similar and t he analysis of covariance indicated a value of 0.6 (Tables 3 4 and 3 5). Therefore, the trials were correlated and similar results can be achieved in laboratory and in field trials. Even though trials are corre lated, inoculated detached fruit had a shorter incubation period detached fruit, the period was less than 7 days. Effect of Temperature on Mycelial Growth Temperature affected mycelial growth of the three C. acutatum isolates (2 163, 2 179 and 3 32) similarly. At 5 and 35 C, no mycelial growth was observed for any of the three isolates (Figure 3 5 ). Growth of C. acutatum increased with increasing temperature from 10 to 25 C and then rapidly decreased at 30 C. Highest growth was
53 observed at 25 C and colony diameter ranged from 49.2 to 53.4 mm and averaged 7.32 mm/da y. Seven days after mycelial plug was transfer red to the culture media, colony diameter averaged 4.8, 22.3, 43.8 and 34 mm at 10, 15, 20 and 30 C, respectively. Effect of Temperature on Anthracnose Fruit Rot Development on Detached Fruit The effect of tem perature was highly significant ( P < 0.0001), whereas cultivar and the interaction between temperature and cultivar were not. Cultivars were analyzed separately for homogeneity within the four experiments and there was no statistical difference between the m ( P P Therefore, experiments were combined and disease incidence was averaged for each treatment. At 5, 10 and 35 C no AFR symptoms were observed on detached immature fruit tion period of 19 days (Figure 3 6 ). A second order polynomial regression showed an increase and subsequent decline on disease incidence when temperatures ranged from 15 to 30 C. However at 15 C disease in cidence was low (4.15 %) compared to 20, 25 and 30 C. Disease incidence increased rapidly when fruit were exposed to 20 C, but at 25 C cultivars were different. Disease incidence was the same at 20 and 25 (91.6 7 % was 84.5 and 47% respectively Out of the four experiments, two had high disease incidence (83.3 and 71.4%) and two had low incidence (16.6 and 16.6% at 25 C ). Therefore, when averaged, the mean incidence for the 25 trawberry At 30 C
54 At the highest temperature (35 C), immature fruit dried out rapidly and no fruit were infected. The maximum disease incidence was at 20 and 25 C (91.7%) for e reas the with immature fruit at 20 C. Even though cul tivars were not significantly different from a disease incidence equal to or greater than the mean incidence for Disease severity values were not analyzed and are not shown because infected fruit of with inoculum concentrations of 10 5 and 10 6 conidia/ml and therefore the only severity data available were at the two highest concentrations. Effect of Wetness Duration and Temperature on Anthracnose Fruit Rot Deve lopment The ANOVA showed a sign ificant effect of temperature, wetness d uration (WD) and their interaction on the development of anthracnose fruit rot (Table 3 6). In addition to the ANOVA an analysis of covariance confirmed that cultivars did not differ within temperatures and at all WDs. The data of all three experiments of each cultivar were combined. D isease incidence increased non Festi wetness for most temperatures (Table 3 7 and Figure 3 7 ) A second order polynomial regression best fit the data and resulted i n high R 2 values for both C (Figure 3 8 ). The R 2 .8997, 0.9819 and 0.9435 C, respectively (Table 3 8). No diseased fruit were observed in the control treatments (inoculum + 0 hour WD, and no inoculum + 48 hour s of WD) at any temperature With 3 hours of WD, mean
55 C than at the other the highest mean disease incidence (16.6%) was at 25 C. Mean d hours was the same at 20 and 25 C (11.1%) and for was higher at 20 C (30%) than at 25 C (23.3%). At 12 hours of WD, mean inc idence was 58.8% at 25 C for mean disease inciden ce with 24 h of WD ranged fro m 46.83% to 76.6% from 15 to 25 and from mean disease incidences were observed with 48 h of WD a t 20 3 7 ). The 24 and 48 h WD treatments did not differ, but were statistically different from the for the treatments with 6, 12 and 24 hours o f WD disease incidence increased with the increase in temperature, whereas in the 3 h treatment incidence decreased at 20 C followed by an increase at 25 C. At 48 h disease incidence increased rap idly from 15 to 20 C, but declined slightly at 25 C. Unlike the other treatments, disease incidence for treatments with 3 and 12 hours of WD from 15 to 20 C and then increased at 25 C (Figure 3 8 ). During the 2011 20 12 season, a 12 h light/ 12 h dark photoperiod was used in the growth chambers Later, it was found that the temperature inside the bags with inoculated plants were on average 5 C above the selected temperature treatment when the lights were on Therefore, data from this ex periment was not used in the statistical analysis.
56 Discussion Development of AFR of strawberry is greatly affected by the concentration of inoculum on the plant, temperature and wetness duration. The knowledge of the minimum inoculum concentration, optimum temperature and wetness duration required for C acutatum infection on cultivars with different le vels of susceptibility such as Strawberry Festival, Camaros a and Treasure are necessary to adapt disease forecasting systems for to develop more efficient disease management programs. A forecasting system to predict AFR incidence based on temperature and wetness duration has been developed to advise Florida strawberry growers on timing fungicide applications when weather conditions are favorable, instead of following a preventive program of calendar (weekly) applications (MacKenzie and Peres, 2012 a ) However, information about th e effect of temperature and wetness duration for the strawberry cultivars common ly grown in Florida was not available ; hence, the importance of this research project. Anthracnose fruit rot incidence increase d with the increasing inoculum concentration for all strawberry cultivars and plant organs. Di guez Uribeondo et al. (2011), Trapero Casas and Kaiser (1992) and Chungu et al. (2001) reported similar results with anthracnose of almond, Ascochyta blight on c hickpea and Septoria tritici blotc h on wheat, respectively, where d isease severity increased non linearly with increasing inoculum con centration Di guez Uribeondo et al. (2011 ) observed that when inoculating C. acutatum on almond cultivars with different levels of susceptibility, minimum inoculum concentration of 10 4 conidia/ml for disease development. In our study, the highly susceptibl e cultivars Camarosa and Treasure required a lower
57 inoculum concentration for initial disease development and had higher percentage of infected immature fruit and flower s considered less susceptible to AFR. strawberry cultivars worldwide, due to its high berry quality and low susceptibility to common strawberry diseases like AFR and Botrytis fruit rot. Thus, o ur results are different than those of Di guez Uribe ondo et al. ( 2011 ) who reported that the less and more susceptible almond cultivars required the same inoculum concentration for anthracnose development. The difference in cultivar susceptibility and the minimum inoculum concentration required for disease development is extremely important to understand since strawberry transplants may arrive from nurseries already infected with C. acutatum. I f transplants arrive at production fields infected with as little as 10 3 conidia/m g it would be enough for the more susceptible cultivars like Camarosa and Treasure to start developing symptoms after planting. However, symptoms would not develop on by secondary conidiation, to approximately 10 4 conidia/m g Polymerase Chain R eaction ( PCR ) or quantitative Polymerase Chain Reaction (qPCR) could be used to detect C. acutatum o n st rawberry transplants. The use of qPCR would be more e fficient since it not only detects the presence or absence of the fungus but a lso quantifies the pathogen when compared to a DNA standard ( Postollec et al. 2011 ). Unfortunately, qPCR is an expensive method to use on a regular basis when testing a large number of transplant s Recently, r esearchers have been working on developing an inexpensive, highly specific, efficient assay, called the loop mediated isothermal amplification (LAMP), which is an alternative
58 to qPCR (Zhang et al 2013; Notomi et al., 2000). Preliminary results have shown that th e LAMP method is able to detect C. acutatum on symptomless plants within one hour by using two sets of primers to amplify the internal transcribed spacer (ITS) such as G1 and the tubulin 2 genes (tub2) (Zhang et al 2013). With the use of these molecular tools, r esearchers and extension ag ent s will be able to detect the amount of inoculum concentration present in samples of transplants and recommend initial spray applications according to that and to the level of susceptibility of the strawberry cultivar planted. In a ddition to cultivar s usceptibility, plant organ is an important factor for AFR development. Strawberry flowers were more susceptible than immature fruit of the high susceptibility of strawberry flowers to C. acutatum. Furthermore, a study conducted from 2002 to 2004 to evaluate the susceptibility of flower s and fruit of to C acutatum according to their age (open flower, 4, 8, 12, 16 and 20 day old fruit) showed that disease incidence for flower s reached sharply declined when fruit were 8 days old ( J. Mertely, unpublished ). In our study open flowers and 8 day old f ruit with no petals attached were also used Wilson et al. (1990 ) as well as Mertely ( unpublished ) observed that disease incidence increased with fruit maturati on. Therefore, immature fruit are less susceptible to AFR than flowers and mature fruit. However even though our results showed that AFR symptoms on flowers of all three cultivars were observed with a lower inoculum concentration than the required for infection of immature fruit, there was no significant
59 difference between the susceptibility levels of plant organs. I nformation about t he higher susceptibility of flower versus immature fruit can help gro wers to schedule fungicide growers should follow a strict spraying sc hedule when flowers are predominant compared to immature fruit, and use fungicides with protective and curative effects, such as the QoI fungicides azoxystrobin and pyraclostrobrin. Strawberry cultivar and plant organ highly affect AFR incubation period. Flowers shorter incubation period s than immature fruit. This may be correlated with the higher susceptibility of flowers compared to immature fruit but not f These findings are also in agreement w that 8 day old fruit had a longer incubation period than open J. Mertely, unpubli shed ). In our study, incubation period was shorter for for of pla nt organ. These results may be associated with C. acutatum and also agree with Mertely flowers of had a longer incubation period than those of Thus, plant organs and cultivars with shorter incubation periods may harbor more disease cycles and build up inoculum quicker f or subsequent tissue infection. The same trend obser ved in the detached fruit trials was observed in the field trials. D isease in cidence in detached fruit was highly affected by inoculum concentration and increased with increasing concentration for both cultivars. A non linear increase in disease incidence occurred from 10 4 and 10 5 conidia/ml to 10 6 Camarosa had higher disease incidence than
60 the minimum inoculum concentration re quired for AFR symptom development was higher in detached fruit than field trials for both cultivars. T his may be explained by the difference in fruit surface exposed to the inoculum. In the field trials, fruit were inoculated with a higher quantity of ino culum suspension and over the whole fruit surface The r esults from detached fruit a nd field trials were correlated and show that similar results can be generated in laboratory conditions using detached fruit. Some of the advantages of the d etached fruit assay are: the control of environmental conditions in the laboratory, since potted plants in growt h rooms and the greenhouse do not flow er and fruit well; and there can be space limitation s in greenhouse s and growth room/chamber s for potted plants T herefo re the use of detached fruit in the laboratory occupies l ess space and biotic and abiotic factors such as pests, wind, and temperature fluctuation are better controlled. D etached fruit senesced and matured faster than attached fruit S ymptoms developed on average 7 days after fruit inoculation on detached fruit trials and 8 days in the field. This may be because fruit were under ideal conditions (23 1 C and high humidity) in the l aboratory, whereas fruit inoculated in the field were exposed to fluctuatin g temperatures. Temperature highly affected C. acutatum mycelial growth and infection of strawberry fruit. Mycelial growth of the three C a cutatum isolates used in our study was only observed when they were exposed to temperatures ranging from 10 to 30 C. The optimum t emperature for growth was at 25 C which agrees with Wilson et al. (1990). Even though Wilson et al. (1990) used a different isolate of C. acutatum they reported
61 no mycelial growth at 5 and 35 C and observed an increase in growth from 10 t o 25 C and a sudden decline at 30 C. In our trials with detached fruit, disease incidence generally increased with the increase in temperature from 15 to 30 C for both cultivars. This is in partial agreement with Wilson et al. (1990), who reported that AF R incidence increased with increasing temperatures from 6 to 25 C. The difference of temperature ranges at which symptoms were seen can be rel ated to the strawberry cultivar used Wilson et al. (1990) used the cultivar Midway ( not commonly grown in the Flo rida annual production system ) and evaluated dis ease incidence under different wetness periods, whereas our laboratory study used continuous wetness for the nine day period. Although disease incidence increased rapidly from 15 to 20 C for both cultivars, optim um temperature was different between cultivars. The highest disease incidence for for fruit exposed to 20 and 25 C and was 20 Strawberry et al. (1997), wh o obs erved maximum C. acutatum sporula tion on strawberry plants at 25 C up to 4 days after production of conidia and that the increase in temperature decrea sed the incubation period. I ncubation period was not evaluated in these trials; however, we did observe that symptoms appeared first on the treatments 20, 25 and 30 C (data not shown). W etness duration (WD) has been reported in many studies to influence C. acutatum infection on stra wberry and other hosts (Wilson et al., 1990; Leandro et al., 2003 a ; Di guez Uribeondo et al., 2011 ). Our results confirmed that it directly affects AFR dev elopment on Camarosa and Strawberry Festival However, the effect of WD
62 also depended on t emperat ure, and the interaction of wetness duration temperature (Wilson et al., 1990). Results of our study and those of Di guez Uribeondo et al. (2011) with almond showed that infected fruit were only observed when plants were exposed to a wetness period after i noculation. Disease incidence on immature fruit generally increased non linearly from 15 to 25 C and from 0 to 48 hours of wetness for both cultivars. Similar results were observed by Wilson et al. (1990) with C. acutatum infection on immature and mature s trawberry fruit of the cultivar Midway. They reported that the increase of disease incide nce was positively correlated with an incre ase in temperature from 6 to 25 C and wetness duration from 0.5 to 51 hours. At 15 C, disease symptoms had developed with only 3 hours of WD f or of the wetness periods at 20 and 25 C. At 15 linearly with the increase of at 3 hours than at 6 hours of WD, likely an artifact of the experiment, and this caused a low coefficient of determination ( R 2 ). Moreover, even t h ough for most of the tre higher disease incidence, no difference in the covariance analysis was observed between the two cultivars. This was also seen in the study testing the effects of A second order polynomial regression was used to describe the effect of temperature and WD for both cultiva at 20 and 25 eq uation s described well th e effect of temperature at all wetness durations for both
63 cultivars. Wilson et al. (1990) developed an equation to predict AFR incidence on immature and mature strawberry fruit b ased on temperature and wetness duration for the cult ivar Mid way. MacKenzie and Peres (2012 a ) monitored temperature and wetness duration in Florida and evaluated the equation by Wilson et al. (1990) to predict AFR Different thresholds for the predicted proportion of infected fruit (INF) were evaluated for timing fungicide applications with captan and pyraclostrobin on two strawberry cultivars handler et al., 2006 ), two INF thresholds, a low er ( INF INF were evaluated to determine whether INF and require fewer fungicide application s H igh disease incidence was observed when strawberry fruit were sprayed when the IN F exceeded 0.5 for both cultivars and thus a low threshold was selected for both the web based SAS that collects data from weather stations at different locations in Florida and automat ically calculates the risk for AFR development based on the INF (MacKenzie and Peres 2012 a ) Our study aimed to determine whether the infection threshold used in the model built in the SAS should be the same for a highly susceptible cultiv ar such as Camarosa and a less susceptible cultivar such as Strawberry Festival. Our findings suggest that the disease threshold used in the model built in the SAS (MacKenzie and Peres, 2012 a ) should be the same for cultivars with different levels of susceptibility since weather conditions (wetness duration and temperature) required for
64 However, disease incidence curves were for most W D and temperature combinations This research indicates that inoculum concentration influences disease outcome depending on the cultivar susceptibility. In pra c tical terms, it means that even low levels of latent infection coming with the strawberry transplants can induce symptom development on highly susceptible cultivars. Even though final disease incidence was f different cultivars did not differ with different wetness durations and temperatures. Therefore, the same proportion of diseased fruit threshold can be used for the highly and moderately susceptible In a ddition to cultivar susceptib ility, plant organ susceptibility and incubation period should be taken into account when using disease forecasting syst ems to control AFR Since flowers are more susceptible than immature fruit o f most cultivars, growers should follow the fungicide spray recommendations strictly when flowers are predominant. Finally, AFR incidence values for detached fruit inoculations agreed with field trials which allows better control of the environmental conditions and is more practical to p erform than field experimen ts. Even though the SAS can use the same thresholds for cultivars with different susceptibility levels, growers could take more risks when planting less susceptible cultivars since disease incidence is unlike to reach an epidemic when using the system. How ever, for highly susceptible cultivars, a preventative spraying schedule may be more efficient when controlling AFR.
65 Table 3 1 Analysis of variance of the effect s of cultivar, inoculum concentration, plant organ and all the interactions on the inciden ce of A nthracnose F ruit R ot of strawberry in field trials Effect Num DF F Value Pr > F Cultivar 2 5.97 0.0259 Inoculum Concentration 5 19.08 0.0003 Plant organ 1 9.59 0.0147 Cultivar*Concentration 8 1.08 0.4591 Cultivar*Plant organ 2 1.90 0.2118 Concentration*Plant organ 5 1.68 0.2444 Table 3 2 Mean disease incidence of strawberry cultivars when inoculated with concentrations of conidia from 10 3 to 10 6 conidia/ml in field trials. a Mean separation within rows followed by the same letter are not significantly different according to F test (LSD) ( P b Percentage data were transformed by arcsine prior to analysis, but non transformed data are presented Cultivar Disease Incidence (%) Camarosa 54.35 A a Treasure 51.64 A b Strawberry Festival 39.53 B
66 Figure 3 1 Regression of Colletotrichum acutatum inoculum concentration on A nthracnose F ruit R ot incidence of flower s and immature fruit on different strawberry cultivars. A ) Cultivar Camarosa B ) Cultivar Strawberry Festival C ) Cultivar Treasure. Results represent the mean incidence of three experiments f o r or
67 Figure 3 2 Regression of inoculum concentration of Colletotrich um acutatum on A nthracnose F ruit R ot development of strawberry cultivars on different plant organs. A ) Inoculated immature fruit B ) Inoculated flowers Data are the Figure 3 3 Incubation period (days from inoculation to symptom development) for immature fruit and flowers of different strawberry cultivars inoculated with 10 6 conidia/ml of C olletotrichum acutatum Data is the mean of one field trial.
68 T able 3 3 E ffect of strawberry cultivar on m ean disease incidence of detached strawberry fruit inoculated with different inoculum concentrations Cultivar Disease Incidence (%) Camarosa 2 3.96 A a Festival 13.54 B b a Mean separation with rows followed by the same letter are not significantly different according to t test (LSD) ( P b Percentage data were transformed by arcs ine prior to analysis, but non transformed data are presented Figure 3 4 Regression of inoculum concentration of Colletotrichum acutatum and A nthracnose F ruit R ot development on detached immature strawberry fruit. Plan ts were inoculated with concentrations from 0 to 10 6 conidia/ml and maintained at continuous wetness and room temperature for nine days Data are the means of four trials.
69 Table 3 4 Analysis of covariance of the parameters for field inoculation of Colletotrichum acutatum on immature strawberry fruit Effect Estimate Standard Error DF t Value Pr > |t| Intercept 0.06449 0.1122 2 0.57 0.6234 a Treatment 0.07335 0.01120 96 5.76 <0.0001 a Percentage d ata were transformed by a rcsine prior to analysis, but non transformed data are presented. Table 3 5 Analysis of covariance of the para meters for detached fruit inoculation of Colletotrichum acutatum on immature strawberry fruit Effect Estimate Standard Error DF t Value Pr > |t| Intercept 0.08366 0.07658 1 1.09 0.4719 a Treatment 0.1107 0.01402 93 7.90 <0.0001 a Percentage d ata were transformed by a rcsine prior to analysis, but non transformed data are presented.
70 Figure 3 5 Effect of growth chamber temperature on Colletotrichum acutatum mycelial growth a t seven day s after incubation Results represent the mean incidence of three experiments for each fungal isolate. Figure 3 6 Regression of growth chamber temperatures from 5 to 35 C on Anthracnose Fruit Rot development on detached immature strawberry fruit for using a second order polynomial regression Results represent the mean incidence of four experiments.
71 Figure 3 7 Infection of immature frui t of strawberry cultivars with different levels of susceptibility to Anthracnose Fruit Rot by Colletotrichum acutatum for wetness durations between 0 and 48 hours at 15, 20 and 25 C. A ) Cultivar Strawberry Festival B ) Cultivar Camarosa Results represent the mean incidence of three experiments. Figure 3 8 Regression of wetness duration on A nthracnose F ruit R ot development on immature fruit of strawberry cultivars with different levels of susceptibility after inoculation with Colletotrichum acutatum and incubation at temperatures between 15 and 2 5 C and wetness periods from 0 to 48 hours. A ) Cultivar Strawberry Festival B) Cultivar Camarosa Results represent the mean disease incidence of three experiments.
72 Table 3 6 Analysis of Variance f or the effects of cultivar, wetness duration and temperature on the incidence of Anthracnose Fruit R ot of immature strawberry fruit. Effect DF F Value Pr > F Cultivar 1 3.37 0.0706 Wetness Duration 4 32.98 <0.0001 Temperature 2 11.1 <0.0001 Cultivar x Wetness Duration 4 0.25 0.9105 Cultivar x Temperature 2 0.01 0.9937 Wetness Duration x Temperature 8 2.56 0.0167 Table 3 7 Analysis of Covariance for the effect of cultivar on the developmen t of A nthracnose F ruit R ot of immature st rawberry fruit at 15, 20 and 25 C an d at all wetness duration periods Temperature ( C) Estimate Standard Error DF T Value Pr>t 15 0.09135 0.1097 32 0.38 0.4114 a 20 0.09712 0.1472 32 0.66 0.5142 25 0.07407 0.1517 32 0.49 0.6287 a Percentage d ata were transformed by a rcsine prior to analysis, but non transformed data are presented. Table 3 8 Regres sion equations for A nthracnose Fruit Rot incidence on strawberry cultivars after inoculation of immature fruit with 10 6 conidia/ml of Colletotrichum acutatum and incubation at different temperatur es and wetness duration periods. Cultivar Temperature ( C) Regression equation R 2 Festival 15 y= 0.031x 2 + 2.4968x 3.6487 0.8997 20 y= 0.0462x 2 + 3.9761x 4.852 0.9819 25 y= 0.071x 2 + 5.0873x 2.237 0.9435 Camarosa 15 y= 0.0178x 2 + 1.7647x + 7.4172 0.6069 20 y= 0.0453x 2 + 4.3664x 3.9687 0.9493 25 y= 0.081x 2 + 5.8447x 2.2328 0.9195
73 CHAPTER 4 CONCLUSION Anthracnose fruit rot has affected strawberry production in commercial fields in Florida and other areas where strawberry is grown for many decades. Weather conditions, such as moderate temperatures ( 20 and 25 C) and humidity are present in Florida and are extremely conducive for AFR development. Growers usually rely on fungicides and cultural practices to control AFR. However the excessive use of f ungicides can be detrimental to the environment and lead to fungicide resistance. Therefore, a disease forecasting system that predicts AFR outbreaks and provides growers with spray recommendations has been developed. However, mor e in formation about the weather effects on AFR development on strawberry cultivars and plant organs with different susceptibility levels was necessary to better production systems. In this project, we evaluated the effect of temperature, wetness duration and inoculum concentration on AFR development on flowers and fruit of highly and moderately susceptible strawberry cultivars. Increases in temperature, wetness duration and inoculum concentration generally increased disease incidence non linearly for both cultivar and plant organ. The highly susceptible cultivars, Treasure and Camarosa had required a lower inoculum concentration for initial AFR symptom development. Flowers we re more susceptible to C. acutatum different in susceptibility T he minimum inoculum concentration necessary for symptom development was lower for flowers than for im
74 for immature fruit than for flowers for all cultivars. Since infected transplants are the main source of inoculum in strawberry commercial fields, the quantification of C. acutatum on strawberry transplants wi th the aid of molecular tools will be helpful to determine initial spray applications. Furthermore, detached fruit trials generated similar results to th ose conducted in the field and allow the control of environmental conditions a nd require less space. Mycelial growth of C. acutatum and AFR development on detached fruit of were directly affect ed by increasing temperature s from 10 to 30 C Wetness duration and temperature were confirmed to be imp ortant microclimatic factors for AFR development on immature strawberry fruit. AFR exposed to wetness durations after inoculation at all temperatures. A non linear increase in disease incidence was observed for both cultivars as wetness duration periods and temperatures increased. Regression curves for AFR development f o r the different cultivar s were not different and minimum wetness duration for symptom development was 3 hours at all temperatures. Even though there was no difference among the slopes of the curves for the two cultivars, disease incidence was always Based on our results, the disease infection thresh old used in the model adapted for should A FR is a major threat to Florida strawberry production and the excessive number of fung icide spra ys and possibility of fungicide resistance concerns growers and
75 researchers. The use of a disease forecasting system adapted to Florida climatic conditions helps to limit fungicide applications to only when conditions are conducive to AFR develop y growers to establish spray schedules according to plant organ susceptibility.
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82 BIOGRAPHICAL SKETCH Bruna Balen Forcelini was born in Passo Fundo, Brazil. She lived in Gainesville, FL from 1994 to 1997 where her father got his Doctor of Philosophy degree in plant p athology at the University of Florida. In 2006, Bruna enrolled in the University of Passo Fundo where she would get her b gronomy. In the fall of 2010, she decided to return to Gainesville for her final undergraduate internship at UF, where she started working with Dr. Natalia Peres. Th en, in May, 2011 she began to pursue her graduate degree in plant p athology under the supervision of Dr. Peres. Her project consisted of the evaluation of the effects of temperature, wetness duration and inoculum concentration on anthracnose fruit rot d evelopment on flowers and fruit of strawberry cultivars with different levels of susceptibility.