ABSTRACT JOHNSON, KENDALL A. Characterization and Fungicide

ABSTRACT

JOHNSON, KENDALL A. Characterization and Fungicide Efficacy of North Carolina Colletotrichum Populations Causing Glomerella Leaf Spot and Fruit Rot on Apple. (Under the direction of Dr. Sara Villani).

Glomerella leaf spot (GLS) and fruit rot (GFR) is historically caused by members of the

Colletotrichum gloeosporioides species complex. Although the disease was first reported in the

1970s in the United States, the disease has reemerged on apples in North Carolina (NC) causing economically devastating losses. These pathogens cause necrotic leaf lesions and rots of fruit that will ultimately lead to premature defoliation, a decrease in yield, and unsaleable fruit. According to previous research, ‘Red Delicious’ and ‘Fuji’ have shown partial or complete resistance to

GLS and GFR. Cultivars from the ‘Golden Delicious’ lineage are the most susceptible which includes cvs. ‘Golden Delicious’, ‘Gala’, and ‘Pink Lady’ which are also amongst consumer favorites and most commonly grown in NC. Currently there is a paucity of information regarding the management of the disease, but fungicides, cultural practices, and the planting of resistant cultivars are primarily used to reduce disease within an orchard. Fungicide use is limited due to the risk of resistance, but has found to be the most effective form of control for this disease. The objectives of this research were to 1) characterize Colletotrichum species based on morphological and molecular techniques and evaluate in vitro QoI fungicide sensitivity shifts in

Colletotrichum populations and 2) determine the most efficacious fungicide and timing for the management of GLS and GFR.

Three hundred ninety-six isolates were collected from symptomatic apple leaves and fruit from a total of 17 orchards in North Carolina and Georgia in 2016. Three hundred and seventy- three of those recovered isolates were grown on potato dextrose agar (PDA) at 25°C under constant light for 10 d. After 10 d, isolates were characterized based on colony color, growth pattern and conidium shape and size. Six morphotypes were determined through morphological characterization in addition to multilocus sequence analysis identifying two Colletotrichum species, C. fructicola and C. fioriniae as causal agents of GLS and GFR.

Mycelial growth inhibition and conidial germination inhibition assays were conducted to determine QoI fungicide sensitivity shifts in Colletototrichum populations. This study determined C. fioriniae isolates were more sensitive to pyraclostrobin and trifloxystrobin compared to C. fructicola isolates. All isolates were found to remain sensitive to pyraclostrobin in mycelial growth and conidial germination inhibition experiments. Through conidial germination assays, five orchards were found to have reduced sensitivity to trifloxystrobin.

A field trial experiment was conducted in 2017 and 2018 to determine the most efficacious fungicide. A non-rotational fungicide program using single-site and multi-site fungicides was evaluated for the control of GLS and GFR. In a separate field experiment, applications for pyraclostrobin plus fluxapyroxad (Merivon) were evaluated to determine a critical time for application. In the non-rotational field experiment, pyraclostrobin plus fluxapyroxad and captan were observed to be the most efficacious treatments. Applications of pyraclostrobin and fluxapyroxad at eighth and ninth cover spray had significantly less disease in

2017. In 2018, no significant differences were found between pyraclostrobin plus fluxapyroxad treatments when applied at different timings.

by Kendall A. Johnson

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Plant Pathology

Raleigh, North Carolina

2019

APPROVED BY:

______Dr. Sara Villani Dr. James Kerns Committee Chair

______Dr. Marc Cubeta

DEDICATION

To my wonderful family and friends who have encouraged me throughout this journey.

BIOGRAPHY

Kendall Anderson Johnson was born on March 10, 1993 in Pensacola, Florida. In 1996, her family moved to Hamilton, OH, but then five years later moved to Raleigh, NC. This is considered home to her because she spent the majority of her life growing up and developing as a well-rounded individual. Throughout high school, Kendall was very involved in academic and extracurricular activities, holding leadership roles in Academy of Finance and Key Club; serving as captain for two years on the soccer team while playing on a travel soccer team, taking piano lessons and finding time to work at a local restaurant.

Kendall attended East Carolina University as a biology major in August 2011. Although she had a love for science beginning at a young age, she didn’t discover her love of plants until her junior year. She sought out every class she could involving plants such as plant biotechnology, plant physiology, and plant biology. She participated in a terrestrial field ecology and research problems in biology summer course with the Smithsonian Tropical Research

Institute in Gamboa, Panama. Throughout this course, she had to design an experiment and assess data to draw conclusions. This established an interest in doing her own research.

After graduation, Kendall accepted a job as Supply Chain Administration at Arysta

LifeScience, an agrichemical company. She was immersed in the agricultural industry learning about crop protection products and various aspects of the business. She found her interest in plant pathology after interacting with several plant pathologists in the office and going on site visits for product efficacy evaluations. She also volunteered at Goodwill Community Foundation

Farm. There, she carried out daily greenhouse operations as well as assisting with seeding, planting, and harvesting. Through these experiences, Kendall discovered her love for agriculture and crop protection.

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In 2016, Kendall began her graduate career in the research program under Dr. Sara M.

Villani working with the characterization of causal agents of Glomerella leaf spot and fruit rot on apple, Colletotrichum spp. and fungicide efficacy for various FRAC groups and modes of action for her Master of Science degree. Her time spent in Dr. Villani’s lab allowed her to develop as a scientist as well as gain experience running lab experiments and field trials. Kendall hopes to continue her education in plant pathology through a PhD program at University of Georgia.

ACKNOWLEDGMENTS

I would like to thank my committee members, Drs. Sara M. Villani, James P. Kerns, and

Marc A. Cubeta for their constant guidance and support. I would especially like to thank Dr. Sara

Villani for taking a chance on me as her first student. I know this was a big risk, but I could never thank you enough for taking the time to be an amazing mentor and teaching me everything

I know up to this point. Thank you for encouraging/forcing me to speak at grower and annual meetings even though you knew I was terrified of speaking in front of people, participate in educational outreach events and attend professional meetings. These experiences allowed me to develop professionally, build confidence while presenting to a wide range of audiences, as well as deepen my knowledge of plant pathology that will be beneficial throughout my career. I would like to acknowledge the funding sources for this work, NC Specialty Crops Block Grant and the various agrichemical companies. This project would not have been possible without their support.

I would like to recognize everyone who has been a part of the Villani lab over the past couple of years including Rachel Kreis, Harris Kopp, Cody Justus, Charlie Mackenzie, Rebecca

Littauer, and Alejandro Llanos. To Rachel, thank you so much for your help in the lab and field.

You had answers for every lab related question I threw at you and made sure I was always using the correct techniques. Thank you for also becoming a great friend who is always up for concerts and Taylor Swift jam sessions. To Harris, thank you for being the best undergraduate student worker I could have ever asked for. You are one of the hardest working people I know and it was truly an honor to teach you about my project and establish new laboratory skills. To Cody,

Charlie, Rebecca, and Alejandro, thank you for helping set up my field trial and rate disease.

This was always a daunting task every week during the summer and without you it would have taken days!

I could not have done this without my friends and family. I am grateful for the constant support of my friends and the new friendships I have made along the way. Finally, thank you to my supportive parents Annetta Johnson and Paul Johnson and step parents, Steve Eyman and

Karen Johnson, and my brother Kyle. Without you guys I would not be where I am today.

TABLE OF CONTENTS

LIST OF TABLES ...... ix

LIST OF FIGURES ...... xi

Chapter I: Literature Review ...... 1

Apple Cultivation and Production ...... 1

Production Practices ...... 2

Apple Disease Management Challenges in North Carolina ...... 3

Glomerella Leaf Spot and Fruit Rot ...... 5

Pathogen Biology and Disease Ecology ...... 7

Geographic Distribution of Glomerella Leaf Spot and Fruit Rot ...... 7

Colletotrichum gloeosporioides and Colletotrichum acutatum species complexes in apple .. 8

Molecular identification of Colletotrichum species in apple ...... 9

Management of GLS and GFR in apple ...... 11

Fungicides ...... 11

Multi-Site Inhibitor Fungicides ...... 11

Succinate Dehydrogenase Inhibitor (SDHI) Fungicides ...... 12

Demethylation Inhibitor (DMI) Fungicides ...... 13

Quinone Outside Inhibitor (QoI) Fungicides ...... 14

Cultural Control ...... 15

Resistant Cultivars ...... 16

Research Objectives ...... 17

References ...... 18

Chapter II: Molecular Characterization and QoI fungicide sensitivity of Colletotrichum species causing Glomerella leaf spot and fruit rot of apple in North Carolina ...... 24

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Abstract ...... 24

Introduction ...... 25

Materials and Methods ...... 28

Results ...... 39

Discussion ...... 51

Acknowledgements ...... 55

References ...... 56

Chapter III: Evaluation of Fungicide Efficacy and Critical Application Timing for

Managing Glomerella Leaf Spot and Fruit Rot of Apple in North Carolina ...... 60

Abstract ...... 60

Introduction ...... 61

Materials and Methods ...... 63

Results ...... 72

Discussion ...... 88

Acknowledgements ...... 95

References ...... 96

Chapter IV: Concluding Remarks and Future Directions ...... 100

References ...... 105

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LIST OF TABLES

Chapter 2: Molecular Characterization and QoI fungicide sensitivity of Colletotrichum species causing Glomerella leaf spot and fruit rot of apple in North Carolina

Table 2.1 Colletotrichum isolates from symptomatic leaves and fruit in North Carolina and Georgia orchard ...... 30

Table 2.2 Primers used in this study ...... 33

Table 2.3 Accession numbers for identified Colletotrichum species using CAL, GAPDH, ACT, and ITS sequences ...... 34

Table 2.4 Description of morphotypes characterized in this study ...... 41

Table 2.5 Morphotypes recovered from leaf and fruit tissue from Malus domestica orchards symptomatic of ABR, GLS and GFR ...... 42

Table 2.6 Orchard, number of leaf and fruit isolates identified as C. fructicola, number of leaf and fruit isolates identified as C. fioriniae ...... 45

Table 2.7 Mean mycelial percent relative growth of Colletotrichum spp. populations and K-S one sample test values to determine sensitivity to pyraclostrobin ...... 48

Table 2.8 Mean percent conidial germination of Colletotrichum spp. populations and K-S one sample test values to determine sensitivity to pyraclostrobin and trifloxystrobin ...... 49

Chapter 3: Evaluation of Fungicide Efficacy and Critical Application Timing for Managing Glomerella Leaf Spot and Fruit Rot of Apple in North Carolina

Table 3.1 Fungicides used for field experiments testing against Glomerella leaf spot and fruit rot ...... 66

Table 3.2 Active ingredient, rate, and fungicide application timing for 2017 and 2018 non-rotational Glomerella leaf spot and fruit rot fungicide experiment ...... 67

Table 3.3 Active ingredient, treatment number, rate, and timing for pyraclostrobin and fluxapyroxad for 2017 Glomerella fungicide evaluation experiment ...... 69

Table 3.4 Active ingredient, treatment number, rate, and timing for pyraclostrobin and fluxapyroxad for 2018 Glomerella fungicide evaluation experiment ...... 70

Table 3.5 Active ingredient and days when GLS symptoms reached 40% and days until harvest when 10% defoliation has been reached in non-rotational fungicide trial in 2017 ...... 78

Table 3.6 Active ingredient and days when GLS symptoms reached 40% and days until harvest when 10% defoliation has been reached in non-rotational fungicide trial in 2018 ...... 81

Table 3.7 Relative area under the disease progress curve (rAUDPC) values for non- rotational fungicide trial evaluating disease incidence and severity of leaves and incidence of fruit spot, rot and post-harvest rot for 2017 in Mills River, NC ...... 82

Table 3.8 Relative area under the disease progress curve (rAUDPC) values for non- rotational fungicide trial evaluating disease incidence and severity of leaves and incidence of fruit spot, rot and post-harvest rot for 2017 in Mills River, NC ...... 83

Table 3.9 Treatment number, leaf incidence, inner and outer leaf defoliation, fruit spot, rot incidence and post-harvest rot incidence were evaluated of the critical timing of pyraclostrobin plus fluxapyroxad in 2017 ...... 86

Table 3.10 Treatment number, leaf incidence, inner and outer leaf defoliation, fruit spot, rot, and post-harvest rot incidence were evaluated for the critical timing of pyraclostrobin plus fluxapyroxad in 2018 ...... 87

Table 3.11 Mean air temperature, relative humidity and daily sum of precipitation in 2017 and 2018 ...... 88

LIST OF FIGURES

Chapter 1: Literature Review

Figure 1.1 a. Traditional orchard planting. b. High-density orchard planting ...... 3

Figure 1.2 a. Leaf spot symptoms on ‘Gala’ b. Leaf chlorosis of ‘Gala’ c. Leaf defoliation of ‘Gala’ tree d. Fruit spot development on ‘Gala’ e. Fruit rot development on ‘Gala’ ...... 6

Chapter 2: Molecular Characterization and QoI fungicide sensitivity of Colletotrichum species causing Glomerella leaf spot and fruit rot of apple in North Carolina

Figure 2.1 Colony morphology of Colletotrichum isolates sampled from symptomatic leaves and fruit collected from orchards in Georgia and North Carolina. Isolates were characterized by colony color, growth pattern and conidium size and shape. Colony color of A and D, morphotype 1; B and E, morphotype 2; C and F, morphotype 3; G and J, morphotype 4; H and K, morphotype 5; I and L, morphotype 6 ...... 40

Figure 2.2 Distribution of effective concentration at which mycelial growth was inhibited by 50% (EC50) values for baseline isolates of Colletotrichum spp for A, pyraclostrobin (n = 21) GLS baseline; B, pyraclostrobin (n = 10) for ABR baseline; C, trifloxystrobin (n = 10) ABR baseline. Sensitivity was determine using mycelial growth inhibition assy. Baseline isolates were collected from apple orchards in North Carolina and Georgia that have never been exposed to single-site fungicides ...... 50

Chapter 3: Evaluation of Fungicide Efficacy and Critical Application Timing for Managing Glomerella Leaf Spot and Fruit Rot of Apple in North Carolina

Figure 3.1 2017 non-rotational fungicide program for days when GLS reached 40% A, non-treated control; B, pyraclostrobin and fluxapyroxad; C, difenoconazole and cyprodinil; D, benzovindiflupyr; E, thiophanate-methyl; F, captan and phosphorous acid; G, pyraclostrobin; H, phosphorous acid; I, fluxapyroxad; and J, captan ...... 76

Figure 3.2 2017 non-rotational treatments and days until harvest to reach 10% inner shoot defoliation. A, non-treated control; B, pyraclostrobin and fluxapyroxad; C, difenoconazole and cyprodinil; D, benzovindiflupry; E, thiophanate-methyl; F, captan and phosphorous acid; G, pyraclostrobin; H, phosphorous acid; I, fluxapyroxad; and J, captan ...... 77

Figure 3.3 Non-rotational treatments and days until 40% of leaf spot incidence development in 2018. A, non-treated control; B, difenoconazole and cyprodinil; C, captan and phosphorous acid; D, pyraclostrobin and fluxapyroxad; E, fluxapyroxad; F, pyraclostrobin; G, captan; H, benzovindiflupry; and I, thiophanate-methyl ...... 79

Figure 3.4 Non-rotational treatments and days until harvest to reach 10% defoliation in 2018. A, non-treated control; B, difenoconazole and cyprodinil; C, captan and phosphorous acid; D, pyraclostrobin and fluxapyroxad; E, pyraclostrobin; F, fluxapyroxad; G, captan; H, benzovindiflupry; and I, thiophanate-methyl ...... 80

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CHAPTER I

Literature Review

Apple Cultivation and Production

Apples (Malus domestica) are members of the Rosaceae and produced in most temperate regions throughout the world. China is currently the world leader in apple production, producing

77.3 million metric tons representing 58% of the global apple crop in 2017 (USDA 2018). The

United States (US) produced approximately 6% of the world’s apples in 2017 with a utilized production value of $3.4 billion USD (USDA 2018). According to the USDA Non-Citrus Fruit and Nuts 2016 Summary (USDA 2017), approximately 130,300 hectares (ha) of apples are produced throughout the US with the majority of production located in Washington (66,773 ha),

New York (16,187 ha), Michigan (13,354 ha), Pennsylvania (8,094 ha), and California (5,059 ha). The US exports approximately 25% of fresh market apples grown with Canada, Mexico, and

Thailand being the primary markets for US apple exports. In addition to exporting apple, the US imports an estimated five percent of the fresh market apples consumed in the US from countries in the southern hemisphere due to their alternate growing season (U.S. Apple Association 2017).

North Carolina (NC) ranks ninth in US apple production with over 200 commercial apple operations and 3,642 ha of apples (US Apple Association 2017). In 2017, approximately 51.1% of the apples harvested in NC were marketed for fresh consumption totaling $17.6 M USD, while 48.9% were used for processing to produce applesauce and juices totaling $6.65 M USD

(USDA 2017). The most common cultivars grown in NC for processing and fresh market apple production are ‘Golden Delicious’, ‘Gala’, ‘Rome Beauty’, and ‘Red Delicious’ (NCAGA 2017).

Production Practices

North Carolina apple growers are increasingly adopting and deploying modern high- density trellised rather than traditional planting systems (Figures 1.1 and 1.2). These reduced planting spaces (~1 m x 3.5 m) can be achieved by utilizing dwarfing rootstocks, most commonly those in the Budagovsky, Geneva, and Malling series. Accelerated profitability, improved fruit quality through better light penetration (Lordan et al. 2018), improved land efficiency, reduced pesticide input, and compatibility with new orchard technologies are among several economic and horticultural benefits of modern orchard systems (Fazio et al. 2015; Norelli et al. 2003; Robinson et al. 2003). In addition, dwarfing rootstocks, particularly within the

Geneva series, are tolerant or resistant to diseases including fire blight (“rootstock blight”),

Phytophthora root rot, and apple replant disease (Fazio et al. 2015; Robinson et al. 2003).

Despite having resistant rootstocks, horticultural practices are aimed to rapidly increase tree vigor, reduce leaf to fruit ratio, and reduce tree width to accommodate tighter tree spacing

(Westwood 1993). These practices in modern planting systems often promote a greater period of susceptibility to foliar pathogens and decrease systemic pathogen travel distance to the scion that can exacerbate disease development (Jurick and Cox 2017). North Carolina’s commercial apple industry has undergone changes to improve horticultural and orchard management techniques and integrated pest management strategies to increase the already profitable fruit production.

a b

Figure 1.1 a Traditional orchard planting b High-density orchard planting.

Apple Disease Management Challenges in North Carolina

As the southernmost major apple growing state in the US, sustainable management of apple diseases in North Carolina is becoming an increasing challenge for producers throughout the state. Obstacles for sustainable management of apple diseases in NC include abiotic stress due to irregular and changing climate patterns, consumer preference for highly susceptible cultivars, fungicide resistance development, and limitations to maximum annual fungicide applications. While orchard management paradigms can be adjusted to satisfy challenges imposed by agricultural chemical regulations, in established orchards there are few protective or remedial measures to alleviate losses either directly or indirectly associated with weather events.

For example, hail events near harvest predispose fruit risk for fruit rot pathogens. Late season frost events or inadequate chilling hours often result in prolonged bloom periods increasing the

time period for infection of flowers by the fire blight pathogen, Erwinia amylovora (Unterberger et al. 2018). As the climate continues to change, projections for more spring frost risks are increasing in some apple production regions as well as delayed growth stages due to warmer winter temperatures (Campoy et al. 2011). Optimal conditions during the summer can result in canopy microclimates near or at 100% relative humidity for extended intervals (≥12 hours/daily)

(S.M. Villani, unpublished).

The utilization of cultivars highly susceptible to early season diseases such as apple scab, powdery mildew, cedar apple rust, and fire blight, along with summer diseases such as bitter rot, white rot, and black rot, present management challenges for apple producers in NC. Currently,

14 apple cultivars comprise the majority of the apple production in North Carolina (NCDA

2017). Several consumer favorite cultivars are highly susceptible to Glomerella leaf spot (GLS) and fruit rot (GFR). These are two of the most problematic fungal apple diseases in NC that can cause extreme economic loss. The most susceptible cultivars to GLS have a ‘Golden Delicious’ parentage which include ‘Gala, ‘Golden Delicious’, and ‘Pink Lady’, while descendants from the

‘Red Delicious’ group like the cv. Fuji, have shown complete resistance (Araujo and Stadnik

2013).

Calendar and host phenology timed fungicide applications are the primary method for managing apple diseases in NC. Southeastern apple producers use a combination of older multi- site fungicides such as captan and mancozeb and single-site fungicides for apple disease management from early March through early October. This relatively long growing season combined with environmental conditions conducive to pathogen infection necessitates 15 to 30 fungicide applications annually for disease management in apples. These application requirements are often challenged by maximum annual application limits and the paucity of efficacious chemicals to manage apple summer diseases (Villani and Nance 2016a; Villani and

Nance 2016b). In addition, due to their high level of specificity, frequent applications of single- site fungicides select for resistance in pathogen populations. Increased prevalence of resistance has been observed in the management of apple scab and white rot diseases of apple. For example, Venturia inaequalis, the causal pathogen of apple scab, has well documented resistance to myclobutanil and insensitivity to difenoconazole (Villani et al. 2015). This pathogen has also shown resistance on apple to thiophanate-methyl and kresoxim-methyl, which was the first report of resistance to a single-site fungicide in Michigan and Indiana (Chapman et al. 2011). A study in China indicated that isolates of Botryosphaeria dothidea sampled from apple had low sensitivity to tebuconazole (Fan et al. 2016).

Glomerella Leaf Spot and Fruit Rot

Glomerella leaf spot (GLS) and fruit rot (GFR), historically caused by members of the

Colletotrichum gloeosporioides species complex, is a reemerging fungal disease on apple in

North Carolina that causes devastating economic losses. GLS initially appears as small, red to purple colored specks, which then develop into asymmetrical, light tan and often concentric spots (Figure 1.2a) (Cannon et al. 2012). As the disease progresses, infected leaves become chlorotic and under high disease pressure prematurely abscise (Figure 1.2b, 1.2c) (Taylor 1971).

In absence of effective management, severely infected leaves begin to defoliate in June due to primary infection. During severe epidemics, GLS can cause more than 75% defoliation by harvest (mid-August) under optimal conditions and 100% defoliation in absence of disease management interventions (Sutton and Sanhueza 1998). Premature defoliation may impact early fruit ripening, reduced winter hardiness, and poor budset the following season (Araujo and

Stadnik 2013). The fruit rot stage of the disease is initially characterized by small, sunken black/brown spots that expand into larger concentric and rotted lesions prior to or following

harvest (Figure 1.2e). The black/brown spots can range from 1 to 5 mm in diameter (Figure 1.2d)

(Wang et al. 2015). These spots and rots decrease the value and marketability of fruit (Anderson

1956; Pierson et al. 1971).

a b

c d e

Figure 1.2 a. Leaf spot symptoms on ‘Gala’ b. Leaf chlorosis of ‘Gala’ c. Leaf defoliation of ‘Gala’ tree d. Fruit spot development on ‘Gala’ e. Fruit rot development on ‘Gala’.

Pathogen Biology and Disease Ecology

The basis of current knowledge regarding the life cycle of Colletotrichum species causing

GLS/GFR is based on pathogen species, apple cultivar, and environmental conditions in China and Brazil. It has been hypothesized that in these production regions, C. fructicola, the dominant species causing GLS/GFR, overwinters in mummified fruit, cankers, leaf litter, and crevices in bark and buds as conidia or perithecia, which serves as the source of primary inoculum to initiate disease epidemics (Rosenberger 2017). In NC, the relative contribution of ascospores and conidia in disease epidemics is currently being investigated. However, secondary infection and formation of conidia on fruit during the growing season occurs in low frequency (Sutton and

Shane 1983). Using Burkard volumetric spore traps, Sutton and Shane found that perithecia of G. cingulata could forcibly eject or disperse ascospores as early as petal fall (Hocking et al. 1967).

Rainwater and wind were also found to be critical in dispersal of ascospores and conidia (Sutton and Shane 1983). Although there is little to no information about when ascospores are released, this information can be used as a standard for initiating fungicide applications for management of

GLS/GFR.

Geographic Distribution of Glomerella Leaf Spot and Fruit Rot

GLS was first reported in the Piedmont and Coastal Plains of Georgia, US in 1970, but was not an economically important disease until 1998 when severe epidemics occurred in two eastern Tennessee orchards (Taylor 1971; González and Sutton 1999). In 1983, GLS first appeared on apples in Brazil and in 1988 a devastating epidemic occurred in Paraná State, Brazil

(González et al. 2006). Since the initial report of GLS in Brazil, causal Colletotrichum species of

GLS have increased in diversity. In 2012, C. karstii, a member of the C. boninense species complex was first identified as a causal agent of GLS on apple in Santa Catarina State, Brazil

and represented the first report of a pathogen outside the C. gloeosporioides species complex causing GLF or GFR on apple (Velho and Stadnik 2014, Damm et al. 2012). Scientists in Brazil discovered this disease over 30 years ago, but Wang et al. (2012) recently first reported this disease in eastern China (Wang et al. 2012). Most recently C. fructicola was identified as the causal agent of GLS in Uruguay (Casanova et al. 2017).

Colletotrichum gloeosporioides and Colletotrichum acutatum species complexes in apple

Fungal species belonging to the Colletotrichum gloeosporioides and C. acutatum complexes commonly cause disease on apple in the southern and mid-Atlantic regions while C. fioriniae (Marclino and Gouli 2008) from the C. acutatum complex is the predominant species found in Northeastern growing regions of the US (Biggs and Miller 2001; Munir et al. 2016).

Colletotrichum fioriniae and C. fructicola (Prihast., Cai & Hyde 2009) of the gloeosporioides complex have been observed to cause GLS/GFR by infecting fruit and leaves. Both pathogens thrive in subtropical regions with warm, rainy climates (25-28 °C and relative humidity near

100%) that are conducive for disease development and conidia dispersal (Velho et al. 2015).

C. fructicola and C. fiorinae are filamentous fungi that belong to the phylum Ascomycota and form hyaline to pale brown vegetative hyphae measuring 1.5-7.5 μm in diameter (Damm et al. 2012). Conidia of both species are hyaline and single-celled with a smooth-wall. C. fioriniae produces fusiform to cylindrical conidia with acute ends on branched conidiophores in contrast to C. fructicola that have rounded ends (Damm et al. 2012; Weir et al. 2012). Species in the C. acutatum complex are typically characterized by their conidia with acute ends, but this morphological characteristic can vary (e.g., only one acute end present) (Simmonds 1965). This differentiation can often be caused by repeated subculturing on nutrient medium and is found outside of the C. acutatum complex (Damm et al. 2012). These species of Colletotrichum exhibit both asexual and sexual reproduction. Progeny generated via the asexual stage having a lower

genetic diversity due to clonal reproduction, while sexual reproduction leads to recombination and creation of new genotypes. The mating system of Colletotrichum is either homothallic (self- fertile) or heterothallic (outcrossing) with two mating types or idiomorphs (MAT1- and MAT1-

2) (Vaillancourt et al. 2000).

In culture on potato dextrose agar (PDA), C. fioriniae produces pale pink, olivaceous, and white mycelium with salmon to orange acervuli (Damm et al. 2012). C. fioriniae produces a dark pink/red pigment on PDA, which is a reliable diagnostic characteristic for identification

(Munir et al. 2016). C. fructicola produces light-gray/olive to whitish aerial mycelium occasionally with orange conidia. Although macro and microscopic morphological characteristics are similar they can exhibit differences in pathogenicity and vegetative compatibility groups (VCG) (González et al. 2006). González et al. (2006) identified isolates sampled from fruit with GLS symptoms were represented by multiple VCGs suggesting that the isolates could have developed independently and may represent distinct populations. They thought that GLS may have originated from multiple fruit-infecting populations, but the origin is still unclear.

Molecular identification of Colletotrichum species in apple

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. 1884 was first classified as

Vermicularia gloeosporioides Penz. 1882. Von Arx and later, Muller validated the concept of

Glomerella cingulata as the sexual morph and C. gloeosporioides as the asexual morph (Weir et al. 2012). Colletotrichum gloeosporioides is a member of the C. gloeosporioides species complex along with 21 additional species (Weir et al. 2012). In the US, predominant species of

Colletotrichum sampled from apple are dependent on geographic region, cultivar and tissue infected (González et al. 2006; Munir et al. 2016; Velho et al. 2016; Casanova et al. 2017).

Historically, identification of Colletotrichum species has been based on conidial size, shape, growth rate, and colony color (Ramos and Talhinhas 2016; Hassan et al. 2018; Weir et al. 2012).

However, due to variability in macroscopic and microscopic morphological characteristics associated with different growth conditions (e.g., media, light and temperature), molecular techniques are becoming the standard method for species identification (Weir et al. 2012). The genetic similarity among species of Colletotrichum requires that multiple nuclear gene regions of genomic DNA be subject to multi-locus sequence analysis to differentiate species. Frequently used genes include; internal transcriber spacer regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), and actin (ACT) (Weir et al. 2012). Sequence analysis of the ITS region is useful for providing species complex resolution but is unsatisfactory for differentiating genetic relationships within a species complex (Cannon et al. 2012; Crouch et al. 2009). The genus Colletotrichum consists of several host-specific and genetically defined species and evolutionary lineages, but the phylogenetic and taxonomic relatedness of the species can only be confirmed through multi-locus phylogenetic analyses and placement (Weir et al.

2012; Crouch et al. 2009).

The Colletotrichum acutatum species complex has 31 accepted and recognized species including subgroups within the complex. Fungi in this species complex have exhibited diverse phenotypic differences, and occurrence over broad geographic regions and plant hosts (Damm et al. 2012). Damm et al. 2012 identified five main clades and 29 subclades representing different species of Colletotrichum based on molecular phylogenetic and multi-locus sequence analyses with ITS, ACT, beta-tubulin (TUB2), chitin synthase (CHS-1), GAPDH, and imidazoleglycerol- phosphate dehydratase (HIS3).

Management of GLS and GFR in apple

Management of Glomerella leaf spot and fruit rot consists primarily through three approaches: fungicide applications, cultural practices, and planting disease resistant cultivars.

Several GLS/GFR management strategies have been utilized based on disease management approaches on apple and different cultivated fruit crops such as peach, watermelon, and strawberry (Ishii et al. 2016; Keneith 2017; Smith and Black 1991). Although, the practice of adopting management strategies from different pathosystems can be beneficial, González et al.

2006 determined that management practices for apple bitter rot (ABR) may not be effective for managing GLS/GFR (González et al. 2006).

Fungicides. Application of fungicides has historically been the cornerstone for managing bitter rot of apple primarily caused by members of the C. acutatum complex. Historically in NC, growers adhered to a 14 to 21 day application interval beginning 21 days after petal fall, using a non-specific fungicide program to target summer diseases of apple. While limited research has been conducted on fungicide efficacy for GLS, GFR, or ABR, Colletotrichum diseases on other plant hosts have been successfully managed using quinone-outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI), mancozeb, captan, and phosphonate group fungicides (Ishii et al. 2016; Gopinath et al. 2006; Keinath 2017; Piccirillo et al. 2018).

Multi-Site Inhibitor Fungicides. Multi-site inhibitor fungicides have been used for decades and have broad-spectrum activity. There are nine subgroups in this class (FRAC group M) with no reports of cross-resistance between group members. Two common multi-site compounds used in apple disease management are mancozeb and captan, which must be applied protectively as they inhibit conidial germination. Mancozeb (FRAC M3) has a 77-day post-harvest interval (PHI) for

apples due to the breakdown of products. Captan is an important fungicide for control of apple diseases because it disrupts multiple cellular division pathways in addition to maintaining a low risk for resistance development (Cowgill et al. 2013). There are negative characteristics associated with use of this fungicide due to the lack of persistence on the leaf surface and poor distribution on apple leaves and fruit (Smith and MacHardy 1984). This fungicide can move through tissue causing stunted growth, premature fruit drop, and unsaleable fruit.

In Florida, captan is typically used to control various diseases on strawberries, especially

Colletotrichum crown rot. It effectively controls the disease and is regularly used in summer spray programs for strawberries (MacKenzie et al. 2009). On watermelon, mancozeb demonstrated greater efficacy than other products containing QoI compounds (Keinath 2017).

Multi-site fungicides are extremely important for disease control on many crops and will continue to be used because of their high level of activity and low risk of resistance development

(FRAC 2018).

Succinate Dehydrogenase Inhibitor (SDHI) Fungicides. Succinate dehydrogenase inhibitors were first marketed in 1966 to inhibit complex II of fungal respiration (Von Schmeling and

Kulka 1966). There are currently 17 SDHI compounds registered for managing plant diseases that belong to different chemical groups with various modes of actions (FRAC 2018). The succinate dehydrogenase protein contains two hydrophilic subunits (SDHA and SDHB) and two hydrophobic subunits (SDHC and SDHD) found within the mitochondrial membrane (Walter

2012). The ubiquinone site is located between the B and D regions (Walter 2012). SDHI compounds bind to the ubiquinone-binding (Qp) site to block access to the substrate and prevents succinate oxidation from cycling (Sierotzki and Scalliet 2013). In general, the most commonly observed mutation at the Qp site SDHB is a histidine (H) substitution with tyrosine (T) (Sierotzki

and Scalliet 2013). No additional mutations in the sdh gene have been identified as causing resistance in various fungal pathogens (Sierotzki and Scalliet 2013).

Resistance development is a concern for SDHIs and other single-site inhibiting fungicides (Sierotzki and Scalliet 2013). While testing efficacy of SDHI fungicides on four species of Colletotrichum gloeosporioides, C. acutatum, C. cereale, and C. obiculare, Ishii et al.

(2016) found that they were less sensitive to fluopyram, fluxapyroaxad and boscalid, but remained sensitive to penthiopyrad and benzovindiflupyr (Ishii et al. 2016). Benzovindiflupyr was the most efficacious compound inhibiting the four species of Colletotrichum on peach, apple, and cucumber (Ishii et al. 2016).

Demethylation Inhibitor (DMI) Fungicides. Demethylation inhibitors (DMI) were first used in agriculture in the 1970s (Xu et al. 2014). Currently, there are 36 DMI compounds available, with the majority belonging to the triazole subgroup. DMIs inhibit activity of the CYP51/ERG11 enzyme that regulates cellular membrane’s absorptivity and fluidity processes. This enzyme is also important for cell viability (Diaz-Trujillo et al. 2017).

DMI fungicides, have both curative and protective activity and are used on several crops for managing Colletotrichum diseases, particularly strawberry anthracnose (Smith and Black

1991). Gopinath et al. (2006) tested propiconazole, carbendazim and difenoconazole to measure disease reduction in the field and greenhouse as well as in vitro experiments observing inhibition activity of Colletotrichum capsici sporulation, conidial germination, and mycelial growth.

Differences in efficacy among the DMI fungicides were observed, as propiconazole reduced disease incidence on average by 70% compared to difenoconazole and carbendazim, at 58% and

44% in the field experiments, respectively (Gopinath et al. 2006). In a similar study testing DMI fungicide efficacy against C. acutatum on strawberries, propiconazole was also found to be the

most effective causing inhibition of colony development and mycelial growth (De los Santos and

Romero 2002). In a study assessing resistance risk of C. truncatum, Zhang et al. (2017) discovered M376L/H373N mutations contributed to the insensitivity of the pathogen to tebuconazole and myclobutanil (Zhang et al. 2017).

Quinone Outside Inhibitor (QoI) Fungicides. Quinone outside inhibitor (QoI) fungicides are a class of broad spectrum, single-site fungicides that have been utilized for the control of

Colletotrichum since their registration in 1996 (Bartlett et al. 2002). The strobilurin class consists of six fungicides: azoxystrobin, kresoxim-methyl, metominostrobin, trifloxystrobin, picoxystrobin, and pyraclostrobin (Bartlett et al. 2002). Despite being labeled for protective and curative control of fungal diseases in apples, QoIs are generally applied prior to infection due to their ability to inhibit conidial germination. Similar to other systemic fungicides, the site-specific mode of action of QoIs predisposes them to rapid resistance development.

QoI fungicides inhibit mitochondrial respiration by binding to the outer quinone oxidizing pocket of the cytochrome bc1 enzyme complex III (Grasso et al. 2006). An inhibitor blocks the electron transfer between cytochrome b and cytochrome c1 and prevents production of

ATP (Bartlett et al. 2002). The most common mechanism of QoI resistance is a single point mutation at amino acid position 143 within the mitochondrial cytochrome B gene. This mutation results in the substitution of glycine to alanine (G143A mutation). Other point mutations within the cytochrome b gene include a substitution of a phenylalanine to leucine at position 129

(F129L) and glycine to arginine at position 137 (G137R) (FRAC 2018). These mutations cause a protein sequence change that prevents fungicide binding.

Another resistance mechanism observed in vitro is the use of the alternative oxidase enzyme (AOX) found on the inner mitochondrial membrane (Olaya and Köller 1999). Although

it has only been seen in vitro, the mechanism may occur in the field although the impact on ATP production is not known (Piccirillo 2018). This alternative pathway enables electrons to bypass complex III and be directly transferred into oxygen (Wood and Hollomon 2003). Several phytopathogentic fungi have been identified as using this mechanism, therefore AOX inhibitors, such as salicylhydroxamic acid (SHAM) are used as an amendment in nutrient medium based bioassays to prevent fungal pathogens from using the pathway (Wise et al. 2008).

In recent studies, Picarillo et al. (2018) determined through field and in vitro studies that pyraclostrobin was effective against anthracnose on sweet orange, Citrus sinensis, and multiple isolates of C. gloeosporioides remained sensitive to this active ingredient and also trifloxystrobin and azoxystrobin. There are documented reports of QoI resistance in populations of C. acutatum

Florida strawberry fields for managing anthracnose fruit rot and anthracnose crown necrosis

(Forcelini et al. 2016). Resistance was caused by the G143 and F129L point mutations in the cytb gene and Forcelini et al. 2016 suggest that C. acutatum populations from a strawberry production nursery were carried on transplants after the amount of resistant isolates recovered from infected crowns and roots soon after planting. Due to these reports, the scientists believe that resistance is linked to plant source rather than being widespread, but disease management strategies should be implemented to control the range of QoI-resistant populations (Forcelini et al. 2016).

Cultural Control. The utilization of cultural control practices is pertinent due to the limited options for managing GLS/GFR. Although a demanding and expensive practice, orchard sanitation is a key strategy to reduce disease for future growing seasons. Some of these practices include removal of old fire blight strikes, cankers, and fruit mummies that can harbor overwintering conidia. As fruit mummies remain on the tree, rainfall disperses conidia becoming a primary

source of inoculum for the following season. Mummies can also drop to the ground where conidia survive on the surface of the mummies and soil during the fall season (Romero et al. 2017).

Removal of leaf litter underneath the canopy should be mowed and have an application of urea applied in autumn or spring prior to green tip to reduce inoculum (Walgenbach et al. 2018). This strategy has also been used for managing apple scab. As the leaf litter degrades, ascospore inoculum is reduced and potentially affecting the angle of ascospore release (Gadoury and

MacHardy 1985).

Resistant Cultivars

Apple cultivars that are descendants of ‘Red Delicious’ parentage are moderately to completely resistant to GLS and GFR (Araujo and Stadnik 2014) and planting highly susceptible cultivars such as ‘Gala’, ‘Golden Delicious’, and ‘Pink Lady’ is not recommended in regions with high GLS/GFR pressure. Similar to Brazil, North Carolina has optimal conditions (i.e. warm temperatures, high rainfall, and susceptible cultivars) for GLS/GFR disease outbreaks. Due to the lack of an available efficacious fungicide program, the development of GLS/GFR resistant cultivars is needed. Insituto Agronómico do Paraná developed a new cultivar, ‘Eva’ (‘Anna’ x

‘Gala’), that is adapted to warmer climates and produces a high yield of quality fruit. With a sweet taste and balanced acidity, this cultivar resembles ‘Gala’, the number one apple consumed in the

US (Pommer and Barbosa 2010). This new cultivar is becoming popular in planting areas with little to no chill accumulation because of the low chilling requirement of 250-400 hours. In 1979, another cultivar, IPR ‘Julieta,’ was released as being resistant to apple leaf spot disease caused by

Colletotrichum spp. (Pommer and Barbosa 2010; Crusius et al. 2001). This cultivar is a cross of

‘Anna’ x ‘Mollie’s Delicious’, which has to date produced large and good tasting fruits in several

Brazilian states (Pommer and Barbosa 2010).

Research Objectives

GLS and GFR have become the most economically important fungal diseases on apples in

North Carolina. The objective for this research project was to develop fungicide management paradigms for managing Collectotrichum species causing GLS/GFR on apple in NC. Disease specific fungicide programs need to be adopted due to different species having varying fungicide sensitivity profiles. The first goal for this study was to determine the most efficacious fungicide and spray timing for managing GLS/GFR. The most effective fungicide was observed through a non-rotational fungicide program where the same product was applied for each cover application throughout the season. In 2016, Merivon (pyraclostrobin + fluxapyroxad) was shown to be the most efficacious fungicide for management of GLS/GFR. This product was chosen for determining a critical time for application in 2017 and 2018. Critical times tested were petal fall, 1st cover, 2nd cover, and/or the last two cover applications before harvest. The second goal of this study was to characterize species of Colletotrichum sampled from apple fruits and leaves and to test for QoI fungicide sensitivity in vitro using active ingredients, pyraclostrobin and trifloxystrobin. Species identification was determined using morphological and multi-locus sequence typing. The QoI fungicide sensitivity of the isolates was compared to baseline populations collected from organic orchards. The results of this research have provided characterization of Colletotrichum species, development of a GLS/GFR disease specific fungicide program, and determination of pathogen sensitivity to QoI fungicides to offer NC growers’ new management strategies.

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CHAPTER II

Molecular Characterization and QoI Fungicide Sensitivity of Colletotrichum Species causing Glomerella Leaf Spot and Fruit Rot of Apple in North Carolina

ABSTRACT

K.A. Johnson, R.A. Kreis and S.M. Villani, Department of Entomology and Plant Pathology, Mountain Horticultural Crops Research and Extension Center, North Carolina State University, Mills River, NC

Glomerella leaf spot (GLS) and fruit rot (GFR) are caused by members of the Colletotrichum gloeosporioides species complex and is considered the most devastating fungal disease of apple in North Carolina. In total, 373 isolates of Colletotrichum were obtained from symptomatic leaves and fruit from 17 conventionally managed commercial orchards in Western North

Carolina (NC) and Georgia (GA). Each isolate was characterized through morphological and multilocus DNA sequence analyses. Morphological characterization consisted of colony color, growth pattern and conidium size and shape. Multilocus sequence analyses revealed that isolates belonged to species within the Colletotrichum gloeosporioides and C. acutatum species complexes. The isolates were identified as either C. fructicola in the C. gloeosporioides species complex or C. fioriniae in the C. acutatum species complex. The most prevalent species found,

C. fructicola, represented 75% of the isolates causing GLS and GFR. Fungicide sensitivity assays revealed that C. fioriniae isolates were more sensitive to pyraclostrobin and trifloxystrobin than C. fructicola isolates. This study revealed that trifloxystrobin did not inhibit mycelial growth of C. fructicola isolates at the highest discriminatory dose (100 ug ml-1). The findings of this study determined two Colletotrichum species causing GLS and GFR and species specific QoI fungicide sensitivities of species of Colletotrichum in NC.

INTRODUCTION

Glomerella leaf spot (GLS) and fruit rot (GFR), historically caused by members of the

Colletotrichum gloeosporioides complex, are the most economically devastating fungal disease of apple in North Carolina (NC). If managed ineffectively during periods of warm, humid weather (Wang et al. 2015), fruit losses on highly susceptible cultivars have approached or reached 100% in recent years in NC (Villani and Nance 2016). On apple, symptoms of GLS initially appear as small, red to purple colored specks, which develop into asymmetrical, light tan and often concentric lesions (Cannon et al. 2012). As the disease progresses, infected leaves become chlorotic and may prematurely abscise (Taylor 1971). The fruit rot stage is characterized by small, sunken brown spots ranging from 1 to 5 mm in diameter that expand as fruit matures, often developing into concentric rots (Wang et al. 2015).

In the United States, observations of GLS on apple have predominantly been reported from Southeastern production regions, including the Piedmont and Coastal Plains of Georgia

(Taylor 1971), Eastern Tennessee (González and Sutton 1999), and most recently North Carolina

(González and Sutton 2004). Globally, GLS and GFR have been reported in Brazil, Uruguay, and China (Casanova et al. 2017; Velho and Stadnik 2014; Wang et al. 2012). Early characterizations of GLS identified Glomerella cingulata (anamorph = Colletotrichum gloeosporioides) as the causal agent of the disease (González and Sutton 1999). However, in recent years, causal Colletotrichum species of GLS have increased in diversity. Since 2012, C. fructicola (Velho et al. 2015) and C. karstii (Velho et al. 2015) have been identified as causal pathogens of GLS. With the exception of C. karstii, which was identified as a causal agent of

GLS and GFR in Santa Catarina State, Brazil and is part of the C. boninense species complex, other causal species of GLS are members of the C. gloeosporioides species complex and C. acutatum species complex (Velho and Stadnik 2014).

In addition to GLS and GFR, species of Colletotrichum can also cause a similar disease, apple bitter rot (ABR), on fruit. Similar to GFR, symptoms of ABR initially appear as tan colored lesions on fruit that expand and become sunken over time. The development of fruit rot and conidial formation on lesions is more common with ABR as compared to GFR (Velho et al.

2015). GLS and GFR have been observed only on cultivars descending from the ‘Golden

Delicious’ lineage (e.g. ‘Gala’, ‘Pink Lady’), all commercially grown cultivars of apple are susceptible to ABR (Biggs and Miller 2001). In the US, several species of Colletotrichum have been identified as pathogens to apple with the majority of species isolated from fruit exhibiting symptoms of apple bitter rot (ABR). A recent survey of Colletotrichum species causing ABR in

Kentucky revealed C. fiorinae as the dominant causal agent, with C. nymphaeae, C. siamense, C. fructicola, and C. theobromicola recovered infrequently (Munir et al. 2016). In other apple productions of the US, the causal species of ABR has been less diverse, with C. fiorniae most commonly associated with ABR in New York and New Hampshire (Rosenberg and Cox 2016;

Wallhead et al. 2014).

Previous research highlight differences in fungicide sensitivity between the C. acutatum and C. gloeosporioides complexes, as well as between species within those respective complexes

(Bernstein et al. 1995; Munir et al. 2016; Shabi et al. 1994). Thus, the identification of

Colletotrichum species and species complexes causing GLS and GFR in NC is important for developing fungicide management recommendations for this disease. Historically, identification of Colletotrichum species was primarily based on colony color, growth rate, and conidial size and shape (Weir et al. 2012; Ramos and Talhinhas 2016). Due to the loss of isolate traits during repeated sub-culturing (Weir et al. 2012), the variability of environmental conditions during in vitro growth (e.g. light, temperature, humidity) (Weir et al. 2012), and similarity of morphological features among closely related species of Colletotrichum, multilocus sequence

analysis has become the primary tool for species classification. As a result, 33 and 22 species have now been identified in the C. acutatum and C. gloeosporioides species complexes, respectively (Damm et al. 2012; Weir et al. 2012). For Colletotrichum spp. identified as causal agents of GLS and GFR in apple, amplification and sequencing of the internal transcriber spacer region (ITS), and the actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes can be used to distinguish the gloeosporioides and acutatum complexes and species within those complexes (Damm et al. 2012; Weir et al. 2012).

Unfortunately, cultivars that are highly susceptible to GLS and GFR are also economically important cultivars for both processing and fresh market commercial apple producers in NC. Consequently, frequent applications of fungicides from petal fall through harvest are the primary strategy for managing this disease across the region. The Quinone outside Inhibitor (QoIs) fungicides are effective against Colletotrichum spp. in other pathosystems (Samuelian et al. 2014; Gao et al. 2017). In NC, the QoI/Succinate DeHydrogenase

Inhibitor (SDHI) formulated product, Pristine, is regularly used for managing summer diseases of apple including black rot, Alternaria leaf spot, and ABR. QoI fungicides inhibit mitochondrial respiration by binding to the outer quinone oxidizing pocket of the cytochrome bc1 enzyme complex III (Grasso et al. 2006). As a result of their highly specific mode of action, resistance to

QoI fungicides has been documented in populations of Colletotrichum from several fruit hosts including C. siamense from peach and blueberry (Hu et al. 2015), C. acutatum from strawberry

(Forcelini et al. 2016), C. gloeosporioides from grape (Nita and Bly 2016), and C. sinensis from sweet orange (Piccirillo et al. 2018). Point mutations within the cytochrome b (cytb) gene leading to amino acid substitutions within the translated protein have been associated with a range in

QoI-sensitivity phenotypes in phytopathogenic fungi. Complete resistance to QoIs has been associated with a substitution of glycine to alanine at amino acid position 143 (G143A), whereas

moderate resistance has been conferred through a point mutation at amino acid position 129 in which phenylalanine is exchanged with leucine (F129L). In other pathogens, a point mutation in which glycine is replaced by arginine (G137R) has also been shown to confer diminished QoI sensitivity (Gisi et al. 2002; Sierotzki et al. 2007).

The recent reemergence of GLS and GFR as an economically important disease in NC combined with management failures throughout Western NC prompted us to investigate the

Colletotrichum species associated with GLS and GFR in the region and to determine isolate sensitivity to QoI fungicides. More specifically our objectives were i) Identify the species associated with GLS and GFR in Western NC; ii) Characterize the sensitivity of a baseline population to two QoI fungicides registered for apple; and iii) Characterize the sensitivity of

Colletotrichum spp. associated with GLS and GFR in conventionally managed NC apple orchards and compare QoI fungicide sensitivity to Colletotrichum populations causing bitter rot on apple.

MATERIALS AND METHODS

Collection and isolation of Colletotrichum spp. from symptomatic apple leaves and fruit. During July and August 2016, 310 GLS and GFR samples were collected from 11 conventionally managed commercial orchards in Western NC, and 86 ABR samples were collected from five orchards in Georgia (GA) and one orchard in North Carolina (NC) with a history of multi- and single-site fungicide applications. In addition, 15 samples were collected from a homeowner tree with ABR and 22 samples from a certified organic orchard with GLS and

GFR from GA and NC, respectively. These samples were considered to be baseline, meaning that to the best of our knowledge, they had not been exposed to single-site conventional fungicides. For isolation of Colletotrichum spp, associated with GLS and GFR, symptomatic leaf and fruit samples were arbitrarily collected from Malus domestica ‘Gala’ and ‘Golden Delicious’

and were stored at 4°C for a maximum of 10 d prior to processing. For isolation of

Colletotrichum spp. associated with ABR, fruit samples were arbitrarily collected from Malus domestica ‘Honeycrisp’, ‘Red Delicious’, ‘Rome Beauty’ and ‘Jonagold’ and were stored as described above prior to processing (Table 2.1). From each conventionally managed orchard location, a minimum of 20 symptomatic fruit or leaves were collected, with no greater than three symptomatic leaves or fruit collected from a single tree.

Isolation from leaf tissue was accomplished through agitation in a 10% bleach (NaOCl) solution for three minutes followed by two consecutive rinses with sterile distilled H2O (Tancos et al. 2016). After sterilization, leaves were dried in a laminar flow hood prior to pathogen isolation. From each symptomatic leaf, three small pieces of tissue were excised from the outer margin of a single lesion and placed on Lima Bean Agar (LBA, HiMedia Laboratories, Mumbai,

India) amended with streptomycin sulfate (50 µg/ml, Sigma Aldrich) and chloramphenicol (50

µg/ml, Sigma Aldrich) (LBA ++), so that the adaxial portion of the leaf was in contact with the agar medium. Surface sterilization of symptomatic fruit tissue was accomplished by rubbing the lesion with a sterile paper towel dipped in 70% ethanol (EtOH, Fisher Chemical, Fair Lawn, NJ) followed by air drying in a laminar flow hood prior to pathogen isolation. From each fruit sample, a single lesion was selected and three small sections of the pulp were excised using a sterile scalpel from the outer margin of each lesion and cultured on LBA++. For both leaf and fruit isolations, a single colony of Colletotrichum spp. originating from one of the three leaf pieces was selected and consecutively transferred to obtain a pure culture for each isolate. All cultures were incubated at 25°C with constant light for 7 to 15 d.

Single conidial isolation was accomplished similar to (Ghajar et al. 2007) with the following modifications. Briefly, isolates were incubated on LBA for 10 days at 25°C under constant light. After 10 d of incubation, conidia were harvested using plastic sterile loops, placed

in 1 ml sterile distilled water, and a 100 μl aliquot was evenly distributed immediately onto potato dextrose agar (PDA, Difco Laboratories, Detroit, MI). After 24 hours at 25°C under constant light, a single conidium from each isolate was selected, and cultured onto LBA.

Following 10 d at 25°C under constant light of incubation, conidia were again harvested, suspended in a 50% sterile glycerol solution and stored and stored at -80°C prior to isolate characterization and fungicide sensitivity evaluation.

Table 2.1. Colletotrichum isolates from symptomatic leaves and fruit in North Carolina and Georgia orchards. Orchard Cultivar (state) # Leaf # Fruit DiseaseA designation Isolates Isolates A Unknown (GA) 0 12 ABR B Honeycrisp (NC) 0 16 ABR D Honeycrisp (GA) 0 27 ABR F Rome Beauty (GA) 0 16 ABR G Jonagold (GA) 0 15 ABR H Golden Delicious (NC) 19 14 GLS I Golden Delicious (NC) 16 1 GLS K Gala (NC) 29 0 GLS L Gala (NC) 9 0 GLS M Gala (NC) 20 24 GLS N Gala (NC) 29 6 GLS O Golden Delicious (NC) 0 27 GLS P Gala (NC) 21 7 GLS Q Gala (NC) 37 0 GLS S Gala (NC) 8 0 GLS T Gala (NC) 6 0 GLS Z Gala (NC) 8 29 GLS APrimary disease observed in isolates. ABR = Apple Bitter Rot; GLS = Glomerella Leaf Spot and Fruit Rot.

Morphological characterization of Colletotrichum spp. Three hundred and seventy three isolates recovered from leaves and fruit displaying symptoms of GLS and GFR were evaluated for macroscopic and microscopic morphological characteristics. Traits assessed for each isolate included colony color, growth pattern, and conidial size and shape. For evaluation of colony and growth pattern morphological characteristics, each isolate was grown on PDA for 10 d at 25°C under constant light. Growth patterns were evaluated based on observations from

previous studies (Weir et al. 2012; Du et al. 2015; Munir et al. 2016). To evaluate microscopic morphological characteristics, conidia were harvested from a 10-day-old culture of each isolate by gently scraping the surface with a sterile loop. Conidia were suspended in 1.5 ml of sterile distilled water and a 10 µl of the resulting conidial suspension was examined with an Olympus

BX41compound microscope (Olympus, Tokyo, Japan) at 40x. For each morphotype that identified using macroscopic characteristics (i.e. colony color, growth pattern), conidial size and shape were determined for five isolates. The length and width of five conidia per isolate were measured using the arbitrary line tool on the Olympus cellSens entry imaging software (version

1.16; Olympus Center Valley, PA). Conidial shape was evaluated by determining if the end of each conidium was rounded or acute (Weir et al. 2012; Damm et al. 2012).

Genomic DNA extraction and PCR. In each orchard population, a minimum of three isolates and a maximum of 13 isolates representing each morphotype were selected for multilocus sequence analysis. Following incubation for 14 d on PDA, approximately 100 to 200 mg of mycelium from each single conidial isolate was harvested using a sterile scalpel and placed in a 2 ml round bottom tube. To disrupt the tissue, frozen mycelia and one 5-mm stainless steel grinding bead (Qiagen, Valencia, CA) were shaken using a TissueLyser II (Qiagen) at 30 cycles per second for 30 s. DNA extractions were accomplished using the Omega E.Z.N.A. Plant

DNA Kit according to the manufacturer’s instructions (Omega Bio-Tek, Norcross, GA). Prior to polymerase chain reaction (PCR) quantity and quality of the extracted DNA was determined using a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, DE).

PCR amplifications were performed for variable regions of the internal transcriber spacer regions (ITS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin (ACT) genes. Sequencing of the ITS regions and 5.8s gene has previously been observed to distinguish between Colletotrichum species complexes. CAL and GAPDH regions have

previously been determined to differentiate species within the C. gloeosporioides and C. acutatum complexes (Damm et al. 2012; Weir et al. 2012). A region of the actin (ACT) gene was chosen for PCR amplifications to differentiate between C. siamense, C. fructicola and C. tropicale.

All primers used in this study are listed in Table 2.2. PCR reactions were conducted in a

Bio-Rad T100 Thermal Cycler (Bio-Rad, Hercules, CA) in a total volume of 25 µL. The PCR mixtures contained 12.5 µL of 2x EmeraldAmp GT PCR Master Mix (Takara Bio Inc., Mountain

View, CA), 9.5 µL dH2O, 1.0 µL of DNA (20 to 40 ng), and 1.0 µL of each primer (0.4 µM final concentration of each primer). The PCR conditions for amplification of the ITS region were programmed as follows: An initial denaturation of 3 min at 95°C, followed by 30 cycles of 95°C for 30 s, 50°C for 30s, 72°C for 1 min, and a final extension of 10 min at 72°C (White et al.

1990). Conditions differed for amplification of CAL, GAPDH, and ACT. PCR conditions for

CAL and GAPDH were an initial denaturation for 4 min at 95°C, then 35 cycles of 59°C (CAL) or 60°C (GAPDH) for 30 s, 72°C for 45 s, and a final extension of 7 min at 72°C. PCR conditions for ACT was an initial denaturation for 4 min at 95°C, then 25 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 45 s, and a final extension of 7 min at 72°C. PCR products were separated on a 2% agarose gel (Agarose RPA-2500, Phenix Research Products, Candler, NC) with 5 µL of GelRed Nucleic Acid Stain (Biotium, Inc., Fremont, CA) at 70 volts for 60 min. A

Gel Doc EZ Imager (Bio-Rad) was used to observe the gels through Image Lab software (version

5.2.1; Bio-Rad).

Table 2.2 Primers used in this study. Target Primer Direction Sequence (5'-3') Reference ACT512F Forward ATG TGC AAG GCC GGT Carbone & Actin TTC GC Kohn 1999 ACT783R Reverse TAC GAG TCC TTC TGG Carbone & CCC AT Kohn 1999 CL1C Forward GAA TTC AAG GAG GCC Weir et al. Calmodulin TTC TC 2012 CL2C Reverse CTT CTG CAT CAT GAG Weir et al. CTG GAC 2012 Glyceraldehyde-3- GDF Forward GCC GTC AAC GAC CCC Templeton et phosphate TTC ATT GA al. 1992 dehydrogenase GDR Reverse GGG TGG AGT CGT ACT Templeton et TGA GCA TGT al. 1992 ITS-1F Forward CTT GGT CAT TTA GAG Gardes & Internal transcribed GAA GTA A Bruns 1993 spacer ITS-4 Reverse TCC TCC GCT TAT TGA White et al. TAT GC 1990 C.gramcytb-bf1 Forward GAAGAGGTATGTACTAC cytochrome b GGTTCATATAG Forcelini et al. C. firoiniae C.gramcytb-br1 Reverse TAGCAGCTGGAGTTTGCA 2016 TAG cytochrome b CfG143A-F Forward ATCAAGACAAGACCGTC G143A region GGTTATA This paper C. fructicola CfG143A-R Reverse AACCATCTCCATCTATTA GTCCTA cytochrome b Cf129L-F Forward TATTATGAGAGATGTAA F129L region ATAATG This paper C. fructicola Cf129L-R Reverse AACATTGGATTATTATAT TGGTT

Sequencing of PCR products. PCR products were purified using the Wizard SV Gel and

PCR clean up system (Promega Corp., Madison, WI). Sequencing reactions were performed using Sanger Cycle Sequencing Reaction set-up through capillary run by the Genomic Sciences

Laboratory, North Carolina State University, on an ABI 3730 genetic analyzer. Consensus sequences were aligned and edited with CLC Main Workbench (version 7.9.1; Qiagen) software to attain a sequence for each isolate. Basic Local Alignment Search Tool (BLAST) was used to assess sequence similarity for the four selected regions. Single nucleotide polymorphisms (SNPs) for each locus were identified using reference strains recovered from Malus domestica (Table

2.3).

Table 2.3. Accession numbers for identified Colletotrichum species using CAL, GAPDH, ACT and ITS sequences. Gene Accession Number Top hit CAL KY986908 C. fioriniae JX009667 C. fructicola JX009672 C. fructicola JX009665 C. fructicola GAPDH JQ48629 C. fioriniae JQ48624 C. fioriniae JX009914 C. fructicola JX009958 C. fructicola JX009949 C. fructicola ACT JQ949620 C. fioriniae JQ949615 C. fioriniae JX009439 C. fructicola JX009458 C. fructicola JX009451 C. fructicola ITS JQ948299 C. fioriniae JQ948305 C. fioriniae JX010177 C. fructicola JX010178 C. fructicola JX010164 C. fructicola

Baseline sensitivity of Colletotrichum isolates to trifloyxstrobin and pyraclostrobin.

Isolates of Colletotrichum spp. recovered from GLS and GFR lesions from the organic orchard and from ABR fruit lesions from the homeowner tree were evaluated for baseline sensitivity to

the QoI fungicides, pyraclostrobin and trifloxystrobin. While QoI fungicides are generally applied to crops to inhibit spore germination, they also can be applied curatively due to their activity against mycelial growth (Bartlett et al. 2002). Therefore, baseline sensitivity was evaluated in vitro for both conidial germination and mycelial growth inhibition.

For conidial germination experiments, 19 isolates of Colletotrichum spp. from the organic orchard and 10 isolates from the homeowner orchard were evaluated for baseline sensitivity to technical grade trifloxystrobin and pyraclostrobin (Sigma-Aldrich, St. Louis, MO).

To prepare conidial suspensions, conidia were harvested from 10-day-old cultures of each isolate and placed in 1.5 ml of sterile distilled H2O. Concentrations of conidial suspensions were determined with a hemocytometer and adjusted to 105 conidia ml-1. The suspension (100 µl aliquot) was evenly distributed onto LBA medium amended with pyraclostrobin or trifloxystrobin at final concentrations of 0, 0.00001, 0.0005, 0.0001, 0.005, 0.01, 0.1, and 1 µg ml-1. To inhibit the alternative oxidase (AOX) pathway, salicylic hydroxamic acid (SHAM,

Sigma-Aldrich) was dissolved in methanol and added to the LBA medium at a final concentration of 100 µg/ml. Plates were stored in darkness for 8 h at 25°C. After eight hours, three squares, approximately 3 mm by 3 mm, were excised from each plate and 100 conidia on each agar square were examined using an Olympus SZX16 dissecting microscope (Olympus) at

11.5x. A conidium was considered germinated if the germ tube was longer than or equal in length to the conidium. For each replication of each isolate, probit analysis was conducted to calculate the effective concentration needed for 50% inhibition (EC50) based on the control (SAS

Version 9.4; Cary, NC).

In addition to conidial germination assays, pyraclostrobin and trifloxstrobin were also evaluated for their activity against the mycelial growth for the baseline populations. For this set of experiments, 21 isolates of Colletotrichum spp. from the organic orchard and 10 isolates from

the homeowner orchard were evaluated. Stored mycelial plugs were grown on PDA amended with 50 µg ml-1 streptomycin and 50 µg ml-1 chloramphenicol (Sigma-Aldrich) at 25°C for 7 to

15 days. For each isolate, 7-mm diam plugs were excised from the outermost edge of the developing colony and placed onto PDA medium amended with pyraclostrobin at final concentrations of 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 µg ml-1 and onto PDA medium amended with trifloxystrobin at final concentrations of 0, 0.001, 0.01, 0.1, 1, 10, 50, and 100 µg ml-1. SHAM was added to the medium as described above. All agar plugs were placed equidistant from each other with the mycelial growth in contact with the fungicide-amended medium. Cultures were incubated at 25°C for 4 to 6 days in the dark and radial growth of the colony was measured using an electronic digital caliper (Westward, Lake Forest, IL). For each isolate, there were two replicate plates. Dose response curves were constructed for each isolate using relative percent inhibition of colony growth at each log-transformed (log10) concentration for the respective fungicide to determine the value of the effective concentration that inhibited isolate growth by 50% (EC50).

Evaluation of sensitivity of Colletotrichum spp. isolates collected from commercial orchards to pyraclostrobin and trifloxystrobin. The sensitivity of isolates of Colletotrichum spp. sampled from commercial conventional orchards to the QoI fungicides trifloxystrobin and pyraclostrobin was evaluated for both the conidial germination and mycelial growth stage. For mycelial sensitivity assays, each isolate was evaluated for sensitivity to pyraclostrobin at the baseline EC50 value and at a discriminatory concentration representing 10x the baseline EC50 value (pyraclostrobin only). From each orchard population, five isolates were randomly selected for mycelial growth sensitivity to trifloxystrobin at a discriminatory concentration representing

100x the EC50 value (C. fructicola) and 10x the EC50 value (C. fioriniae). For each isolate, 7-mm diam plugs were excised from the outermost edge of an actively growing colony and place onto

PDA medium amended with SHAM (100 µg/ml) plus either pyraclostrobin or trifloxystrobin at the respective discriminatory concentrations. All agar plugs were placed equidistant from each other with the plug’s mycelial growth in contact with the fungicide-amended medium. Cultures were incubated at 25°C for 4 to 6 days in the dark and radial growth of the colony was measured using an electronic digital caliper. For each isolate, there were two replicate plates. Using SAS, a

Kolmogorov-Smirnov (K-S) one-sample test was performed to compare isolate sensitivity distributions to baseline orchard populations.

Using a K-S one-sample test, the sensitivity classification was determined for each orchard population by comparing the distribution of isolate pyraclostrobin responses for each orchard population (the “test orchard”) evaluated at the EC50 value to the sensitivity distribution of the baseline population. Initially, the probability that the distribution of pyraclostrobin sensitivity responses for a given test orchard was different from the distribution of the baseline orchard (PT=S) was determined. The outcome of the test was then used to classify the orchard population in one of two scenarios. For instance, if for a given test orchard, PT=S > 0.05 the test orchard would be considered “sensitive” since the distribution of the test orchard did not differ significantly from the baseline population. If for a given tests orchard, PT=S < 0.05 and the mean relative growth for the population was greater than the mean relative growth of the baseline population at the respective discriminatory concentration, the sensitivity distribution of the orchard would be classified as “reduced sensitive”. In one final scenario, the distribution of a test orchard population may differ significantly from the baseline population (PT=S < 0.05), and the mean relative growth of the test orchard population was lower than that of the baseline population. In this case, the orchard population would also be considered sensitive. Since no orchard populations with practical resistance to QoI fungicides were identified in this study and

thus there were no standard resistant populations available for comparison to “test orchard” sensitivity, no conclusions on practical resistance to pyraclostrobin were made.

Sensitivity to pyraclostrobin and trifloxystrobin for the conidial germination growth stage was also evaluated for the same populations of Colletotrichum spp. at the baseline EC50 value and a discriminatory dose representing 10x the baseline EC50 value. For each isolate, a conidial suspension was prepared as described above. The suspension (100 µl aliquot) was evenly distributed onto LBA medium amended with SHAM (100 µg/ml) plus either pyraclostrobin or trifloxystrobin, and stored in darkness for 8 h at 25°C. Percent germination values were averaged for each isolate. Using mean percent germination values evaluated at the EC50 discriminatory concentration, orchard populations were classified as sensitive or reduced sensitive to trifloxystrobin and pyraclostrobin using the K-S one sample test as described for mycelial growth assays above.

Analysis of the cytochrome b (cytb) gene sequences from reduced sensitive isolates of

C. fioriniae and C. fructicola. A portion of the cytb gene was amplified for isolates of C. fioriniae and C. fructicola demonstrating reduced sensitivity to trifloxystrobin and/or pyraclostrobin to determine putative involvement of the F129L, G137R, or G143A in sensitivity shifts towards resistance. Using the BLAST function in CLC Main Workbench, assembled contigs of C. fructicola strain 1104-7 (accession no. MVNS00000000; Liang et al. 2018) were searched for homologs to the cytb gene using the published cytb sequences of V. inaequalis

(accession no. AF047029). Based on a single region of homology, three primers were designed:

CfG143A-F, CfG143A-R, and CfF129L-R (Table 2.2). Since the F129L mutation region was located near the beginning of one contig, the cytb mitochondrial DNA sequence from C. fructicola (accession no. KM885301) was utilized for designing primer CfF129L-F (Table 2.2).

A single set of primers, C.gramcytb-bf1 and C.gramcytb-br2 were used for amplification of a region of the C. fioriniae cyt b gene (Table 2.2; Forcelini et al. 2016).

DNA was extracted using the Omega Bio-Tek E.Z.N.A. as described above from 10-day- old cultures of isolates in which mycelial relative growth exceeded 60% on pyraclostrobin amended-medium at the EC50myc value or 60% conidial germination at either the 1x discriminatory concentration of pyraclostrobin or trifloxystrobin. For each primer set, amplification of regions of the C. fioriniae and the C. fructicola cyt b gene was conducted in 25

µl reaction volumes and contained 1x EmeraldAmp GT PCR Master Mix (Takara Bio/Clontech

Laboratories Inc) 0.4 µM each primer, and 5 to 10 ng of genomic DNA. Cycling conditions for amplification of the C. fioriniae cytb gene was conducted as previously described (Forcelini et al.

2016). For amplification of the C. fructicola cytb region that contained the putative F129L mutation region, cycling conditions were as follows: 4 min at 95°C; 35 cycles of 30 s at 95°C, 30 s at 45°C, and 45 s at 72°C followed by a final extension step of 7 min at 72°C. Amplification of the putative G143A regions of the C. fructicola cyt b gene was performed under similar conditions, but with an annealing temperature of 53°C. All reactions were conducted in a T100

Thermal Cycler (Bio-Rad Laboratories, Inc.). All PCR products were separated and photographs of the gel were taken as described above. Products were purified and sequenced in both directions as described above using the same primers for amplification.

RESULTS

Morphological characterization. A total of 373 isolates of Colletotrichum spp. recovered from

17 orchards symptomatic of GLS and GFR or ABR in North Carolina and ABR in Georgia were divided into morphological types based on colony color, growth pattern, and spore size. Across the 17 orchards, six distinct phenotypes (Fig. 2.1) were identified following incubation on PDA for 10 days. Descriptions for each morphotype are presented in Table 2.4.

A B C

D E F

G H I

J K L

Figure 2.1. Colony morphology of Colletotrichum isolates sampled from symptomatic leaves and fruit collected from orchards in Georgia and North Carolina. Isolates were characterized by colony color, growth pattern and conidium size and shape. Colony color of A and D, morphotype 1; B and E, morphotype 2; C and F, morphotype 3; G and J, morphotype 4; H and K, morphotype 5; I and L, morphotype 6.

Table 2.4 Description of morphotypes characterized in this study. Total # Mean of Isolate conidium size Morphotype isolates growth Mycelial PDA medium Conidium (length x designation in group pattern growth color pigmentation shape width µm) 1 42 Form: Light Light brown Acute 13.4 ± 0.92 circular green, grey and green, ends x 5.6 ± 0.35 Elevation: light pink flat middle Margin: entire

2 50 Form: Olive and Light to Acute 12.2 ± 0.59 circular grey, light dark pink ends x 4.3 ± 0.17 Elevation: pink flat margin Margin: entire 3 71 Form: Olive and None Rounded 15.7 ± 0.49 circular grey ends x 5.8 ± 0.18 Elevation: flat Margin: entire 4 147 Form: Light gray, None Rounded 15.6 ± 0.65 circular white ends x 5.3 ± 0.14 Elevation: margin flat Margin: entire

5 55 Form: Light grey None Rounded 15.4 ± 0.39 circular to white, ends x 5.4 ± 0.19 Elevation: white flat margin Margin: entire

6 8 Form: White, None Rounded 15.7 ± 0.27 circular orange ends x 5.5 ± 0.16 Elevation: conidia flat production Margin: entire

All six morphotypes were identified in isolates recovered from orchards symptomatic of

GLS and GLR, whereas only two morphotypes were identified in isolates recovered from orchards symptomatic of ABR. Morphotype 4, characterized by light gray mycelia and white margin with a mean spore size of 15.6 µm in length and 5.3 µm in width, represented the largest group, containing 147 (39.4%) of all isolates surveyed. Morphotype 1 isolates are characterized by light to dark pink pigmentation and morphotype 2 isolates stained the medium a light green and brown. These morphotypes had similar conidia size and shape, compared to the other characterized morphotypes (3-6). Similarities in morphological characteristics were typically observed between isolates from leaf and fruit lesions within an orchard. Orchard T was the only orchard that did not follow this trend with a separate morphotype characterizing leaf and fruit isolates (Table 2.5).

Table 2.5 Morphotypes recovered from leaf and fruit tissue from Malus domestica orchards symptomatic of ABR, GLS and GFR. Morphotype(s) of Orchard Total Number of Morphotype(s) of Colletotrichum spp. Designation Morphotypes Colletotrichum spp. (leaf) (fruit) A 2 -a 1,2 B 2 - 1,2 D 2 - 1,2 F 2 - 1,2 G 2 - 1,2 H 3 3,5 3,5,6 I 2 1,3 1 K 3 3,4,5 - L 2 3,5 - M 5 1-5 1,3,4,5 N 1 4 - O 1 - 4 P 2 3,5 3,5 Q 1 4 - S 1 4 - T 2 4 6 Z 4 3,6 3-6 a No isolations recovered from the respective tissue type.

Molecular identification of Colletotrichum spp recovered from Southeastern US apple orchards. To identify the species of Colletotrichum isolates associated with GLS, GFR, and ABR in NC and GA, a minimum of three isolates and a maximum of 13 isolates representing each morphotype in each orchard were selected for multilocus sequence analysis. Genomic DNA regions of CAL (~750 bp), GAPDH (~300 bp), ACT (~300 bp), and the ITS region (~600 bp) were successfully amplified for 166 isolates. Through comparison of consensus sequences of the

ITS region with published sequences in GenBank, it was revealed that NC and GA

Colletotrichum spp. isolates were members of one of two Colletotrichum species complexes. All recovered ABR isolates belonged to the C. acutatum species complex, whereas the majority of those recovered from GLS or GFR lesions were members of the C. gloeosporioides species complex. Isolates belonging to the C. gloeosporioides complex were recovered from 97.6% and

99.0% of GLS and GFR samples, respectively, whereas those belonging to the C. acutatum complex were recovered from 2.4% and 1.0% of GLS and GFR samples, respectively. To further differentiate species, consensus sequences of ACT, CAL, and GAPDH were also compared to published sequences in GenBank. Seventy-five percent (280/373) of all isolates sequenced corresponded to published sequences of C. fructicola and 25% (93/373) of isolates sequenced matched C. fioriniae. From GLS, GFR and ABR lesions, C. fruticola was recovered from 97.8%,

99%, and 0% isolates, respectively whereas C. fioriniae was recovered from 2.2%, 1%, and

100%, respectively. For each amplified locus, alignment of consensus sequences with published reference sequences of C. fructicola or C. fioriniae was conducted. For C. fructicola, single polymorphisms (SNPs) were identified at the following nucleotide positions: CAL- 280 (C to T), and 289 (A to G) in a single isolate (orchard L) ; ITS - 540, in a single isolate (orchard P); and

ACT 130 (C to T) in 3 isolates (orchards M and Q). These same three isolates from orchards M and Q had a 7-nucleotide insertion (compared to reference strains) between nucleotides 80 and

81 relative to the reference strains. Alignment of all C. fructicola sequences from the GAPDH region revealed 100% homology with the GAPDH C. fructicola reference strain from the United

States (accession no. JX009914). For C. fioriniae, alignment of the sequenced region of CAL with the C. fioriniae reference strain from Malus domestica (accession no. KY986908.1) revealed SNPs for a single isolate the baseline orchard (A-05-CFi) at nucleotide position 341 (C to T). However with the exception of isolate A-05-Cfi, SNPs were identified positions 196 (C to

A), 211 (G to A) and 463 (C to T) for all additional isolates. In some isolates of C. fioriniae

SNPs within the sequenced CAL region were also identified at position 341 (C to T; 25% of isolates) and position 393 (G to A; 43% of isolates). Lastly, compared to the reference sequence, a single nucleotide deletion was identified for 63% of C. fioriniae isolates at position 24.

Alignment of C. fioriniae sequences resulting from amplification of GAPDH revealed SNPs at positions 54 (A to G) and 111 (T to C) for a single isolate (A.05.CFi). At position 176, the nucleotide sequenced differed in 5% of isolates compared to the other C. fioriniae, however the sequence of these isolates at that position was identical to that of one of the reference strains for the GAPDH locus (accession no. JQ948624.1). Alignment of C. fioriniae ACT and ITS sequences revealed SNPs at positions 29 (T to A; isolate I.20L.CFi) and 146 (A to G; isolate

G.22.CFi), respectively.

Table 2.6 Orchard, number of leaf and fruit isolates identified as C. fructicola, number of leaf and fruit isolates identified as C. fioriniae. C. fructicola C. fioriniae Orchard # of leaf isolates # of fruit isolates # of leaf isolates # of fruit isolates A -a 0 - 10 B - 0 - 16 D - 0 - 26 F - 0 - 16 G - 0 - 15 H 13 18 0 0 I 9 0 3 2 K 30 - 0 - L 9 - 0 - M 16 24 4 1 N 29 - - - O - 26 - 0 P 21 4 0 0 Q 34 - 0 - S 8 - 0 - T 6 - 0 - W 4 29 0 0 a No isolations recovered from the respective tissue type.

Sensitivity of baseline populations of Colletotrichum fructicola and C. fioriniae to trifloxystrobin and pyraclostrobin. In this study, a conidial germination inhibition assay was conducted to determine baseline sensitivity of C. fructicola and C. fioriniae isolates to the QoI fungicides trifloxystrobin and pyraclostrobin. For baseline isolates of C. fioriniae, pyraclostrobin

-1 -1 EC50-con values ranged from 0.0002 to 0.0036 µg ml (mean EC50-con = 0.0013 µg ml ± 0.0002)

- and EC50 values for trifloxystrobin ranged from 0.0001 to 0.0090 (mean EC50-con = 0.0032 µg ml

1 ± 0.0078) (Fig. 3.2c). By comparison, for baselines isolates of C. fructicola, pyraclostrobin

-1 -1 - EC50-con values ranged from 0.0004 to 0.0019 µg ml (mean EC50-con = 0.0011 µg ml ± 8.0 x10

5 -1 ) and trifloxystrobin EC50-con values ranged from 0.0010 to 0.0023 µg ml (mean EC50-con =

0.0015 µg ml-1 ± 9.12 x10-5).

The concentrations of pyraclostrobin and trifloxystrobin needed to inhibit mycelial growth by 50% were also determined using mycelial inhibition assays. Mean EC50-myc values

were determined seven days after plating. For baseline isolates of C. fioriniae, pyraclostrobin

-1 EC50-myc values of ranged from 0.0079 to 0.2452 (mean EC50-myc = 0.0851 µg ml ) with the

-1 majority of the values below 0.19 µg ml (Figure 3.2b). Trifloxystrobin EC50-myc values for the

-1 same baseline population ranged from 0.0079 to 1.39 (mean EC50-myc = 0.8160 µg ml ). For baseline isolates of C. fructicola, pyraclostrobin, EC50-myc values ranged from 0.1269 to 2.0859

-1 -1 (mean EC50-myc = 0.8529 µg ml ) with the majority of values occurring below 1.0 µg ml .

Trifloxystrobin EC50-myc values for the same baseline population ranged from 0.6037 to >100 µg

-1 ml . A baseline trifloxystrobin EC50-myc for C. fructicola was not identified as the majority of

-1 baseline isolates had an EC50-myc value >100 µg ml .

Mycelial growth sensitivity of Collectotrichum spp. commercial orchard populations to trifloxystrobin and pyraclostrobin. A total of 250 isolates of Colletotrichum spp. were evaluated for sensitivity to pyraclostrobin and 116 isolates of Colletotrichum spp. were evaluated for sensitivity to trifloxystrobin during the mycelial growth stage (Table 2.7). Eight orchards were represented by 20 or more isolates, while the remaining eight orchard populations were represented by a minimum of six isolates. Mean percent mycelial relative growth (%RGmyc) for the four orchard populations in which the dominant species was C. fioriniae ranged from 30.27 to 63.49, and ranged from 35.99 to 58.02 for 10 orchard populations in which C. fructicola was the dominant species. All orchard populations of C. fructicola were determined to be sensitive to pyraclostrobin when evaluated for efficacy against mycelial growth. Mean %RGmyc values decreased by a factor of 2 to 3 for pyraclostrobin from the baseline concentration to 10x the baseline concentration. For pyraclostrobin, not all isolates included in the multilocus sequencing analysis were tested for mycelial growth inhibition due to failure to regrow from plugs in long- term storage (-80°C). Due to high EC50 value outputs from the baseline mycelial growth inhibition assay, a K-S test was not conducted for mycelial growth on trifloxystrobin-amended

medium. However, mean % RGmyc of an isolate subset from populations of C. fructicola ranged from 49.3 to 59.5% with a mean of 56.34 ± 1.06 % when exposed to a concentration of 100x the baseline EC50-myc value for trifloxystrobin. Conversely, a subset of isolates from orchard populations of C. fioriniae was more sensitive to trifloxystrobin. Mean % RGmyc from an isolate subset (n = 5) from orchard populations of C. firoiniae ranged from 12.37 to 31.3% with a mean of 19.55 ± 4.21 % when exposed to a concentration of 10x the baseline EC50-myc value for trifloxystrobin.

Conidial germination sensitivity of Colletotrichum spp. orchard populations to trifloxystrobin and pyraclostrobin. A total of 248 isolates of Colletotrichum spp. were tested for conidial germination sensitivity to trifloxystrobin and pyraclostrobin (Table 2.8). One ABR commercial orchard population of C. fioriniae (orchard G) and five GLS/GFR commercial orchard populations of C. fructicola had reduced sensitivity to trifloxystrobin. The mean % germination for C. fructicola populations ranged from 50.79 to 79.45% with orchard O (50.79 ±

1.1089) and orchard P (79.45 ± 3.83) representing the minimum and maximum endpoints of the reduced sensitive designation. Surprisingly, no orchards had populations of Colletotrichum with reduced sensitivity to pyraclostrobin. All other orchard populations of Colletotrichum spp. were sensitive to pyraclostrobin and trifloxystrobin. Not all isolates that were screened for sensitivity to the QoI fungicides during the mycelial growth stage were evaluated for conidial germination sensitivity to the fungicides due to isolates not producing viable conidia after plating from long- term storage.

Analysis of cyt b sequences from isolates of C. fructicola and C. fioriniae.

Amplification of the C. fioriniae cyt b gene with primer pair C.gramcytb-bf1 and C.gramcytb-br1 yielded an 236bp product for the putative F129L, G137R, and G143A mutation regions. Based on the nucleotide sequence of the reference genome of C. fructicola, the anticipated amplified

product size for primer pairs CfF129L-F/R and CfG143A-F/R were 501bp and 530 bp, respectively. While amplification of all isolates of C. fructicola with primer pair CfF129L-F/R resulted in the expected product size, fragments of the cytb gene amplified with primer pair

CfG143A-F/R ranged from 530 to 1100 bp, with some products failing to amplify. For isolates of C. fructicola in which regions of the cytb gene containing the putative mutation regions were successfully amplified, the G143A mutation was discovered in 1 isolate, whereas no isolates of

C. fioriniae had the G137R or G143A mutation.

Table 2.7 Mean mycelial percent relative growth of Colletotrichum spp. populations and K-S one sample test values to determine sensitivity to pyraclostrobin Orchard Designation Primary Species # of isolates Mean % RG a Pr>Ksa b B C. fioriniae 16 30.87 ± 8.72a 0.0025 D C. fioriniae 21 50.44 ± 14.59 0.2931 F C. fioriniae 13 58.63 ± 22.58 0.3732 G C. fioriniae 14 34.42 ± 15.26 0.1082 H C. fructicola 37 36.97 ± 0.74b < .0001 I C. fructicola 28 58.02 ± 8.58 0.7848 K C. fructicola 30 45.97 ± 1.10 0.4109 L C. fructicola 9 37.048 ± 1.05 < .0001 N C. fructicola 26 49.21 ± 1.77 0.2850 O C. fructicola 26 39.92 ± 0.82 < .0001 P C. fructicola 24 43.34 ± 1.07 0.0110 Q C. fructicola 34 35.99 ± 0.66 < .0001 S C. fructicola 8 40.85 ± 1.76 0.0017 T C. fructicola 6 49.11 ± 1.28 0.8407 a The orchard population mean percent growth of Colletotrichum species on medium amended -1 with 1x EC50 values of analytical grade pyraclostrobin for C. fioriniae (0.0851 µg ml ) and C. fructicola (0.8529 µg ml-1) relative to that on non-fungicide medium (%RG). Values are means and standard errors of n isolates for each population. b The distribution of pyraclostrobin sensitivity response for Colletotrichum species population was determined using the K-S one-sample test (1x EC50) in SAS (version 9.4, Cary, NC).

Table 2.8. Mean percent conidial germination of Colletotrichum spp. populations and K-S one sample test values to determine sensitivity to pyraclostrobin and trifloxystrobin Orchard # of Mean % Designation Primary Species isolates Germa Pr>Ksac Mean % Germb Pr>Ksad B C. fioriniae 15 42.67 ± 9.22a 0.0325 52.13 ± 10.05 0.1667 D C. fioriniae 26 31.63 ± 13.53 0.0022 38.77 ± 13.91 0.0004 F C. fioriniae 13 45.83 ± 12.48 0.3993 58.0 ± 9.4 0.2719 G C. fioriniae 14 79.26 ± 10.78 0.0004 86.33 ± 7.91 < .0001 H C. fructicola 31 30.81 ± 18.32 0.0008 33.86 ± 24.17 0.0117 I C. fructicola 16 54.81 ± 15.88 0.5210 72.94 ± 9.62 <.0001 K C. fructicola 28 31.92 ± 13.08 0.0011 50.52 ± 9.56 0.8371 L C. fructicola 9 51 ± 12.24 0.6004 66.41 ± 6.16 0.0010 N C. fructicola 26 32.14 ± 6.18 < .0001 54.40 ± 9.94 0.1048 O C. fructicola 27 43.84 ± 5.98 0.0969 50.79 ± 9.97 <.0001 P C. fructicola 25 46.6 ± 9.81 0.5180 79.45 ± 3.83 <.0001 Q C. fructicola 34 45.88 ± 5.98 0.1335 30.97 ± 24.07 0.0003 S C. fructicola 8 51.0 ± 1.14 0.0884 68.29 ± 3.68 0.0018 T C. fructicola 6 27.67 ± 4.69 0.0068 42.56 ± 7.01 0.1031 a The orchard population mean percent germination of Colletotrichum species on medium - amended with 1x EC50 values of analytical grade pyraclostrobin for C. fioriniae (0.00111 µg ml 1) and C. fructicola (0.8529 µg ml-1) relative to that on non-fungicide medium (%RG). Values are means and standard errors of n isolates for each population. b The orchard population mean percent growth of Colletotrichum species on medium amended -1 with 1x EC50 values of analytical grade pyraclostrobin for C. fioriniae (0.0032 µg ml ) and C. fructicola (0.0015 µg ml-1) relative to that on non-fungicide medium (%RG). Values are means and standard errors of n isolates for each population. c The distribution of pyraclostrobin sensitivity response for Colletotrichum species population was determined using the K-S one-sample test (1x EC50) in SAS (version 9.4, Cary, NC). d The distribution of trifloxystrobin sensitivity response for Colletotrichum species population was determined using the K-S one-sample test (1x EC50) in SAS (version 9.4, Cary, NC).

A 7 n = 21 6 5 4 3 2

Number of Isolates 1 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 -1 EC50 values pyraclostrobin (µg ml )

B 3.5 n = 10 3 2.5 2 1.5 1

Number of Isolates 0.5 0 0.01 0.04 0.07 0.1 0.13 0.16 0.19 0.22 0.25 -1 EC50 values pyraclostrobin (µg ml )

C 8 n = 10 7 6 5 4 3 2 1 Number of Isolates 0 0.75 1.5 2.25 3 3.75 4.5 5.25 6 -1 EC50 values trifloxystrobin (µg ml )

DISCUSSION

Glomerella leaf spot and fruit rot are considered the most economically devastating summer fruit rot disease in the southeast. Growers report that this disease can cause up to nearly

100% crop loss at harvest and is becoming more challenging to manage year after year even with a fungicide spray program. In this study, we conducted a series of experiments to characterize

Colletotrichum spp. through morphological and DNA sequence analyses as well as to determine sensitivity shifts in test populations compared to baseline orchards. Due to the differences in fungicide sensitivity among isolates in the C. acutatum species complex and C. gloeosporioides species complex, the findings of this study suggest that management strategies should be implemented based on pathogen species.

Traditionally, Colletotrichum species have been identified through morphological characteristics including colony color, texture, conidia size and shape (Simmonds 1965; Sutton

1992; Smith and Black 1991). This method of characterization is not usually sufficient for consistent differentiation between species due to variation in morphology under environmental influences in addition to changed or lost features from repeated sub-culturing (Hyde et al. 2009;

Weir et al. 2012). Cannon et al. (2000) recommended that multi-locus sequencing is the most dependable technique to characterize Colletotrichum species. Six morphotypes were identified in this study and through microscopic and macroscopic characteristics we were able to separate isolates based on their respective species complex. However, when combined with multilocus sequence data, it was not reliable for species differentiation within the C. gloeosporoioides and

C. acutatum species complexes.

In previous studies, Colletotrichum spp. from C. gloeosporioides and C. acutatum species complexes have been identified as causal agents for GLS, yet species being most aggressive and prevalent from the former complex (Araujo and Stadnik 2013). In Brazil, C. gloeosporioides and

C. fructicola have been identified as causal agents for GLS on apple and Glomerella cingulata in

China and C. fructicola in Uruguay (Araujo and Stadnik 2013; González et al. 2006; Velho and

Stadnik 2014; Wang et al. 2012, Casanova et al. 2017). DNA sequence analysis of four multilocus regions revealed that Colletotrichum isolates causing GLS and GFR of apple in NC belong to two phylogenetic species: C. fructicola and C. fioriniae. Sequencing of the ITS region reliably differentiated Colletotrichum isolates into their respective clades, but was not adequate to identify to species. This region has been successful for species differentiation in previous studies (Hyde et al. 2014; Weir et al. 2012). We amplified three additional genes, actin, calmodulin, and glyceraldehyde 3-phosphate dehydrogenase to confirm species identification.

These genes were selected based on the ability to resolve species within the gloeosporioides and acutatum species complex with individual loci (Weir et al. 2012). Isolates collected from ABR orchards in Georgia were used to compare C. fioriniae isolates from North Carolina causing GLS and GFR. These isolates helped determine morphological differences between the C. gloeosporioides and C. acutatum species complexes. After sequences were aligned, several single nucleotide polymorphisms (SNPs) were detected within a single species.

In a recent study, Munir et al. (2016) characterized Colletotrichum species causing ABR in Kentucky orchards. Through morphological and multigene sequence analysis, five species causing ABR: C. fioriniae, C. nymphaeae, C. siamense, C. theobromicola, and C. fructicola were identified as causal agents of the disease. Given geographical proximity and the similarity in climates of apple production regions of North Carolina, Georgia, and Kentucky, it was hypothesized we would find similar species diversity causing ABR, and GLS and GFR.

Surprisingly, C. fioriniae was the most prevalent species associated with ABR in Kentucky, but it was the only species associated with ABR in both NC and GA. Furthermore, while Munir et

al. (2016) identified C. fructicola as a causal agent of ABR in KY, the occurrence of the pathogen was not associated with the leaf spot stage of the disease.

Management strategies for GLS, GFR and ABR are based on disease management from other cultivated fruit crops in addition to adopting management strategies from other pathosystems. In Brazil, the most common approach for disease management is the use of synthetic fungicides because curative fungicides are not currently available (Araujo and Stadnik

2013). North Carolina apple growers currently manage GLS and other apple fruit rot diseases with fungicides of varying modes of action and application timings (Walgenbach et al. 2018).

This study tested in vitro sensitivity of Colletotrichum spp. to QoI fungicides, which are commonly used to manage this disease because of their site-specific mode of action and effectiveness in managing anthracnose on other fruit crops. Rapid onset of qualitative resistance to QoI fungicides has previously been observed in other populations of phytopathogenic fungi in the Ascomycota. For example, in commercial orchard populations of Venturia inaequalis, practical resistance to trifloxystrobin was identified following 15 or more historical applications of QoI fungicides (Frederick et al. 2014). To monitor shifts in sensitivity to the QoI fungicides pyraclostrobin and trifloxystrobin, baseline sensitivity was determined for Southeastern populations of C. fioriniae and C. fructicola. Since QoI fungicides have demonstrated activity against both mycelial growth and conidial germination, fungicide sensitivity was evaluated for both growth stages. In this study, populations of C. fructicola were less sensitive to pyraclostrobin and trifloxystrobin during the both the mycelial and conidial growth stages.

Similar trends in in vitro fungicide sensitivity between Colletotrichum species and species complexes were previously documented (Munir et al. 2016). Based on results from previous studies and our findings suggest that different fungicide selections may be warranted based on causal Colletotrichum species in apple orchards.

While there are no published QoI baseline values for C. fructicola or C. fioriniae for comparison, we were able to compare our results to QoI fungicide sensitivity studies in other

Colletotrichum species. Forcelini et al. (2016) determined the mycelial growth EC50 value range for pyraclostrobin of C. acutatum isolates (strawberry) was from 0.017 to 0.092 µg ml-1 and

-1 conidial germination EC50 values ranged from 0.009 to 0.07 µg ml (Forcelini et al. 2016). The mycelial growth value found in this study falls within the range determined for C. acutatum, but the conidial germination value was lower than the range given. In a study evaluating fungicide sensitivity on C. acutatum isolates causing chili pepper anthracnose, the mean EC50 for mycelial growth and conidial germination was 0.29 µg ml-1 and 0.016 µg ml-1, respectively (Gao et al.

2017). These values were both higher than the EC50 values found in this study. Piccirillo et al.

(2018) found mean conidial germination EC50 values for C. gloeosporioides isolates tested on pyraclostrobin (0.11 µg ml-1) and trifloxystrobin (0.047 µg ml-1) in the presence of SHAM

(Piccirillo et al. 2018). In this study, both values were found to be lower than those stated previously. In comparison, the C. fructicola isolates in this study are more sensitive to pyraclostrobin and trifloxystrobin in conidial germination assays.

QoI reduced sensitivity and resistance has been reported in Colletotrichum species on different pathosystems. Reduced mycelial and conidial sensitivity were observed from certain orchards so we sequenced the mutation regions of those specific isolates to determine if point mutations occurred in the cytochrome b gene at G143A or F129L. Amplifications and mutations were similar to those found in C. fructicola isolates from a study conducted by Hu et al. (2015).

Amplifications in some isolates were not always achieved due to the designed primers hitting a non-conserved region in the intron. Genome sequencing might be needed to elucidate cytb gene sequence in these isolates. So far, only one isolate from our sample collection was observed to have the G143A mutation. Single-site fungicide use is prevalent in Nagano Prefecture, Japan to

manage a diverse set of summer fungal diseases in ABR caused by C. fructicola (Yokosawa et al. 2017). A study conducted by Yokosawa et al. (2017) discovered C. fructicola demonstrating signs of resistance development to azoxystrobin. Populations of C. acutatum on strawberry were observed to be resistant to azoxystrobin and pyraclostrobin with mutations found at G143A

(Forcelini et al. 2016). The G143A point mutation was also observed in C. graminicola on turfgrass conferring practical resistance to QoI fungicides as well as in Venturia inaequalis to kresoxim-methyl from the same point mutation (Avila-Adame et al. 2003; Zheng et al. 2000).

Pathogen identification and an understanding of species specific fungicide sensitivity are essential for the developing sustainable and effective GLS and GFR management strategy.

Previously, identification methods have consisted of morphology characterization and multilocus analyses utilizing individual genes (CAL, ITS, GAPDH) to determine species from collected symptomatic leaf and fruit tissue. Future studies should be conducted to investigate pathogenicity and aggressiveness of the two species on various cultivars grown in North

Carolina such as ‘Rome Beauty’, ‘Pink Lady’, ‘Granny Smith’, and ‘Red Delicious.’

ACKNOWLEDGMENTS

This work was supported by funds appropriated by the North Carolina Specialty Crops Block

Grant. We would like to thank Dr. Phillip Brannen for providing isolates from Georgia. We would also like to thank Dr. Wayne Jurick for providing assistance on building phylogenetic trees and useful advice on the analysis and interpretation of data.

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CHAPTER III

Evaluation of Fungicide Efficacy and Critical Application Timing for Managing Glomerella Leaf Spot and Fruit Rot of Apple in North Carolina

ABSTRACT

Kendall A. Johnson, Rachel M. Kreis, and Sara M. Villani, Department of Entomology and Plant Pathology, North Carolina State University, Mills River, 28759.

Glomerella leaf spot (GLS) and fruit rot (GFR) caused primarily by members of the

Colletotrichum gloeosporioides species complex, are the most economically devastating fungal disease on apples in North Carolina. With few management options and a paucity of GLS-related research in the U.S., NC apple growers have been relying on standard summer-disease fungicide programs to manage these diseases. To more effectively manage GLS and GFR during the growing season and at post-harvest, field experiments investigating multi- and single-site fungicide efficacy and application timing were conducted in a ‘Gala’ research orchard in 2017 and 2018. Fungicides representing different modes of action were applied in non-rotational programs from petal fall (PF) through ninth cover (2017) or thirteenth cover (2018). The incidence of GLS, pre-harvest GFR, and post-harvest GFR was significantly lower for programs in which the QoI fungicide, pyraclostrobin (Cabrio EG), the QoI/SDHI premix fungicide, pyraclostrobin and fluxapyroxad (Merivon), and captan (Captan 80 WDG) were applied. The programs of pyraclostrobin with fluxapyroxad or captan also resulted in a significantly lower

GLS severity. In a separate field experiment, the timing of pyraclostrobin and fluxapyroxad applications for GLS and GFR management was investigated. No significant differences were found among the most efficacious treatments, but applications of pyraclostrobin and fluxapyroxad at eighth and ninth cover spray had less disease in 2017. In 2018, no significant differences were observed between treatments except in post-harvest GFR incidence.

Pyraclostrobin and fluxapyroxad applied at PF provided the lowest value for GFR incidence.

INTRODUCTION

Glomerella leaf spot (GLS) and fruit rot (GFR), are emerging fungal diseases on apple

(Malus x domestica) in the southeastern United States (US). Historically caused by fungi in the

Colletotrichum gloeosporioides species complex, infection by these pathogens occurs on both fruit and leaves. GLS initially appears as small red to purple specks, which develop into asymmetrical, and often concentric lesions (Cannon et al. 2012). Throughout the season, progression of disease results in leaf chlorosis and premature abscission (Taylor 1971). During severe epidemics, more than 75% defoliation has been observed by harvest when conventional fungicide programs are used and 100% defoliation in absence of effective disease management interventions (Moreira and May De Mio 2015; Sutton and Sanhueza 1998). Small, brown spots ranging from 1 to 5 mm in diameter initially characterize the fruit rot disease stage. As fruit approach maturity, expansion of lesions occurs along the fruit surface accompanied by a ‘V’ shaped rot towards the core of the apple (Zhang et al. 2016).

The first report of Glomerella leaf spot and fruit rot in the US occurred in 1970 on Malus x domestica ‘Golden Delicious’ in the Piedmont and Coastal Plains of Georgia (Taylor 1971). In

1998, Sutton and Sanhueza identified GLS and GFR in two ‘Gala’ orchards in eastern Tennessee

(Sutton and Sanhueza 1998). In these original reports, Glomerella cingulata (anamorph =

Colletotrichum gloeosporioides) was reported to be the causal pathogen of this disease. Most recently, GLS and GFR caused devastating economic losses in Western North Carolina (NC).

Three of the most common varieties grown in NC, ‘Gala’, ‘Golden Delicious’, and ‘Pink Lady’ are highly susceptible to GLS and GFR (NCAGA 2017; Araujo and Stadnik 2013).

Due to the absence of commercially available cultivars with qualitative resistance to GLS and GFR, fungicides are the industry standard for managing these diseases (Walgenbach et al.

2018). Currently, limited research exists in regards to fungicide programs to manage GLS and

GFR on apple in the US. Historically, apple growers in the eastern US have relied on 14 to 21 day fungicide application intervals to manage several late season diseases of apple including white rot (Botryosphaeria dothidea), black rot (Botryosphaeria obtusa), bitter rot

(Colletotrichum spp.), flyspeck sooty blotch (fungal species complexes). In apple production climates similar to the southeastern US, a greater frequency in fungicide applications have been necessary to manage apple rot disease. Indeed, increasing inoculum pressure in apple orchards in

Brazil has prompted up to 15 protective fungicide applications targeting GLS and GFR per season (Katsuyama and Boneti 2012). On other hosts, diseases caused by Colletotrichum spp. have been successfully managed using fungicides with different biochemical modes of action

(Gopinath et al. 2006, Keinath 2017; Ishii et al. 216). For example, Colletotrichum crown rot on strawberries is primarily managed with QoI fungicides in addition to multi-site fungicides such as captan (Keinath 2017; MacKenzie et al. 2009), whereas Gopinath et al. (2006) evaluated

Demethylation Inhibitor (DMI) fungicides difenoconazole and propiconazole in vitro on

Colletotrichum capsici on chilies and observed inhibition of sporulation, conidial germination and mycelial growth. Other studies on peach and cucumber evaluated Succinate DeHydrogenase

Inhibitor (SDHI) fungicide efficacy against C. cereale and C. obiculare. Both species of

Colletotrichum were sensitive to the SDHI fungicides, benzovinidflupyr and penthiopyrad, but were less sensitive to fluxapyroxad and boscalid (Ishii et al. 2016).

Recent reports of up to 100% fruit loss at harvest or in storage by commercial growers in

NC has prompted the need for a re-evaluation of current and novel fungicide programs for managing of GLS and GFR in NC. The objectives of this study were to i) determine the species of Colletotrichum spp. causing GLS and GFR in a Malus x domestica ‘Gala’ research orchard in

Mills River, NC; ii) determine fungicide efficacy and application timings for managing

Glomerella leaf spot and fruit rot on apple; and iii) evaluate post-harvest management of GFR with fungicides applied prior to harvest.

MATERIALS AND METHODS

Collection and isolation of Collectotrichum spp. from symptomatic apple leaves and fruit. During July 2017, 18 samples of GLS and GFR samples were arbitrarily collected from a

17-year-old Malus x domestica ‘Gala’research orchard at the North Carolina State University

(NCSU) Mountain Horticultural Crops Research and Extension Center (MHCREC) in Mills

River, NC and stored at 4°C for a maximum of 10 d prior to processing. Isolation from apple leaves tissue was accomplished through agitation in a 10% bleach (NaOCl) solution for 3 minutes followed by two consecutive rinses with sterile distilled H2O (Tancos et al. 2016). Three small pieces of leaf tissue were excised from the outer margin of a single lesion from each symptomatic leaf and place on Lima Bean Agar (LBA, HiMedia Laboratories, Mumbai, India) amended with streptomycin sulfate (50 µg/ml, Sigma Aldrich) and chloramphenicol (50 µg/ml,

Sigma Aldrich) (LBA ++). A paper towel dipped in 70% ethanol (EtOH, Fisher Chemical, Fair

Lawn, NJ) was used to surface sterilize symptomatic fruit tissue. Three small sections of the pulp were excised using a sterile scalpel and placed on LBA++. Conidia from a single colony were harvested, suspended in sterile distilled water, and redistributed onto LBA++ to obtain a single- conidial isolate from each leaf or fruit sample.

Extraction of genomic DNA and PCR. Following incubation for 10 d on PDA, approximately 100 mg of mycelium from each single conidial isolate was harvested using a sterile scalpel and placed in a 2 mL round bottom tube. To disrupt the tissue, frozen mycelia and one 5-mm stainless steel grinding bead (Qiagen, Valencia, CA) was shaken using a TissueLyser

II (Qiagen) at 30 cycles per second for 30 sec. DNA extractions were accomplished using the

E.Z.N.A. Plant DNA Kit according to the manufacturer’s instructions (Omega Bio-Tek,

Norcross, GA). Prior to polymerase chain reaction (PCR) quantity and quality of extracted DNA were determined using a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies,

Wilmington, DE). PCR reactions were conducted in a Bio-Rad T100 Thermal Cycler (Bio-Rad,

Hercules, CA) in a total volume of 25 µL. The PCR mixtures contained 12.5 µL of 2x

EmeraldAmp GT PCR Master Mix (Takara Bio Inc., Mountain View, CA), 9.5 µL dH2O, 1.0 µL of DNA (20 to 40 ng), and 1.0 µL of each primer (0.4 µM final concentration of each primer).

The internal transcriber spacer (ITS) region was chosen initially to differentiate between C. gloeosporioides and C. acutatum species complexes. Calmodulin (CAL), glyceraldehyde 3- phosphate dehydrogenase (GAPDH), and actin (ACT) primers were used to differentiate species within each complex (Damm et al. 2012; Weir et al. 2012). PCR amplifications of the internal transcribed spacer (ITS) region were as follows: An initial denaturation of 3 min at 95°C, followed by 30 cycles of 95°C for 30 s, 50°C for 30s, 72°C for 1 min, and then a final extension of 10 min at 72°C (White et al. 1990). The PCR parameters for CAL and GAPDH were an initial denaturation for 4 min at 95°C, followed by 35 cycles of 95°C for 30 s, 59°C (CAL) or 60°C

(GAPDH/ACT) for 30 s, 72°C for 45 s, and then a final extension of 7 min at 72°C. The PCR parameters for ACT was an initial denaturation for 4 min at 95°C, followed by or 25 cycles,

60°C for 30 s, 72°C for 45 s, and then a final extension of 7 min at 72°C. PCR products were separated on a 2% agarose gel (Agarose RPA-2500, Phenix Research Products, Candler, NC) with 5 µL of GelRed Nucleic Acid Stain (Biotium, Inc., Fremont, CA) at 70 volts for 60 min. A

Gel Doc EZ Imager (Bio-Rad) was used to observe gels through Image Lab software (version

5.2.1; Bio-Rad).

Sequencing of PCR products. PCR products were purified using the Wizard SV Gel and

PCR clean up system (Promega Corp., Madison, WI). Sequencing reactions were performed

using Sanger Cycle Sequencing Reaction set-up through capillary run by the Genomic Sciences

Laboratory, North Carolina State University, on an ABI 3730 genetic analyzer. Consensus sequences of each isolate were aligned and edited with CLC Main Workbench (version 7.9.1;

Qiagen) software and Basic Local Alignment Search Tool (BLAST) was used to assess sequence similarity for the four selected regions.

Fungicide-based management of Glomerella leaf spot and fruit rot in an experimental apple orchard. In 2017 and 2018, two independent field experiments were conducted at MHCREC to determine fungicide efficacy and critical application timings for managing of GLS and GFR. The research orchard, planted in 1997, is a single-cultivar planting of Malus x domestica ‘Tenroy Gala’ grafted to M.7 rootstocks. For both experiments, a complete randomized block experimental design was used with four single-tree replicates per treatment.

Prior to the commencement of both trials at the petal fall growth stage, mancozeb (Koverall, 3.3 kg/ha) plus phosphorous acid (ProPhyt, 4.7 liter/ha) was applied at the pink bud and bloom host phenology stages to all treatments with the exception of the non-treated control. Fungicides applied during both years of the field experiments are presented in Table 3.1. Fungicides were applied using a Solo 451 Mist Blower (Newport News, VA) (1379 kPA) calibrated to deliver 935 liters/ha. Standard regional fertilizer, herbicide and insecticide, programs for apple (Walgenbach et al. 2018) were applied to both experiments in both years. Previous disease severity in this orchard was high; therefore supplemental inoculation was not conducted.

Table 3.1 Fungicides used for field experiments testing against Glomerella leaf spot and fruit rot. FRAC Active Ingredient (%)a Trade nameb Code Formc Manufacturer Pyraclostrobin (21.26) and Merivon 11, 7 SC BASF Fluxapyroxad (21.26) Difenoconazole (8.4) and Inspire Super 3, 9 EW Syngenta Cyprodinil (24.1) Benzovindiflupyr (10.27) Aprovia 7 EC Syngenta Thiophanate-methyl (85) Thiophanate 1 WDG ADAMA Methyl 85 WDG Mancozeb (75) Koverall M3 DF FMC Phosphorus acid (34.3) Prophyt 33 SC Helena Captan (78.2) Captan 80 WDG M4 WDG ADAMA Pyraclostrobin (20) Cabrio EG 11 EG BASF Fluxapyroxad (26.55) Sercadis 7 EC BASF Ziram (76) Ziram 76DF M3 DF UPI a Number in parentheses indicates percentage of active ingredient in commercial products. b Products used in this study. c Formulation: EW = Emulsion, oil in water, EC = Emulsifiable concentrate, EG = Emulsifiable granule, DF = Dry flowable, and WDG = Water dispersible granule.

Non-rotational fungicide field trial. To directly compare the fungicide efficacy with different biochemical modes of action (i.e. different FRAC groups) a non-rotational fungicide experiment was conducted in 2017 and 2018 (table 3.2). In 2017, non-rotational fungicide programs began at 50% petal fall (PF), which occurred on 25 April and continued on approximately 10 to 21 day cover applications until 2 days prior to harvest on 21 August. In

2018, fungicide applications began on 27 April (50% PF), but were applied on approximately 7 to 10 day intervals due to high precipitation during the growing season. While identical fungicides were applied in 2017 and in 2018, no fungicide was applied to the same tree

(treatment replicate) in both years.

Table 3.2 Active ingredient, rate, and fungicide application timing for 2017 and 2018 non- rotational Glomerella leaf spot and fruit rot fungicide experiment. Active Ingredient Rate (amt./ha) 2017 Timinga 2018 Timing Non-treated control Not applicable (N/A) (N/A) (N/A) Pyraclostrobin and 402 ml PF-9Cb PF-13Cc fluxapyroxad Difenoconazole and cyprodinil 877 ml PF-9C PF-13C Benzovindiflupyr 512 ml PF-9C PF-13C Thiophanate-methyl 897 g PF-9C PF-13C Mancozeb and phosphrous acid 3.3 kg + 4.7 liter PF-2C PF-2C Captan and phosphrous acid 4.3 kg + 4.7 liter 3C-9C 3C-13C Pyraclostrobin 645 g PF-9C PF-13C Phosphrous acid 7 liter PF-9C PF-13C Fluxapyroxad 241 ml PF-9C PF-13C Captan 5.6 kg PF-9C PF-13C a PF = petal fall, #C = cover spray number b Dates of applications were 25 April, 3 May, 16 May, 26 May, 9 June, 28 June, 13 July, 27 July, 4 August, and 19 August, 2017. c Dates of applications were 8 May, 17 May, 25 May, 1 June, 8 June, 15 June, 22 June, 28 June, 9 July, 20 July, 26 July, 6 August, and 17 August, 2018.

Critical application timing of pyraclostrobin and fluxapyroxad (Merivon).

Preliminary research conducted at MHCREC in 2016 determined that Merivon (BASF, Research

Triangle Park, NC) a premixed formulated product that contains two active ingredients: pyraclostrobin and fluxapyroxad, was the most efficacious commercially available product for apples for managing GLS and GFR (Villani and Nance 2016). While both active chemistries inhibit mitochondrial respiration, pyraclostrobin, a Quinone outside Inhibitor (QoI, FRAC 11) fungicide and fluxapyroxad, a Succinate DeHydrogenase Inhibitor (SDHI, FRAC 7) fungicide both disrupt electron transport at two different locations. SDHI compounds bind to the ubiquinone (Qp) site in complex II, while QoIs bind to the Qo site in the cytochrome bc1 enzyme complex. Due to its efficacious activity against GLS and GFR in 2016, pyraclostrobin and fluxapyroxad was applied at different growth stages and cover sprays to determine if there were critical timings for pyraclostrobin plus fluxapyroxad applications to reduce GLS and GFR incidence and/or severity during the pre- and post-harvest period. Eight different programs and an non-treated control were evaluated for critical pyraclostrobin plus fluxapyroxad application

timing in 2017 (Table 3.3). The three most efficacious programs from 2017 and those most likely to be adopted by growers were evaluated in 2018 (Table 3.4). As with the non-rotational experiment, fungicide programs to evaluate critical application timings for GLS and GFR management commenced at 50% PF. Applications were conducted on approximately 10 to 21 and 7 to 10 d intervals in 2017 and 2018, respectively, as described above, with the final application in both years occurring two days prior to harvest.

Table 3.3 Treatment program, active ingredient, rate, and timing for pyraclostrobin and fluxapyroxad for 2017 Glomerella fungicide evaluation experiment. Active Ingredient and Rate of formulated product (treatment number) (amt./ha) Timinga Non-treated control (1) N/A N/A Captan and mancozeb (2) 2.8 kg, 3.3 kg PF-2Cb Captan and ziram 2.8 kg, 3.3 kg 3C-7C Pyraclostrobin and fluxapyroxad 402 ml 8C-9C

Captan and mancozeb (3) 2.8 kg, 3.3 kg PF-2C Captan and ziram 2.8 kg, 3.3 kg 3C-7C,9C Pyraclostrobin and fluxapyroxad 402 ml 8C

Captan and mancozeb (4) 2.8 kg, 3.3 kg PF-2C Captan and ziram 2.8 kg, 3.3 kg 3C-8C Pyraclostrobin and fluxapyroxad 402 ml 9C

Captan and mancozeb (5) 2.8 kg, 3.3 kg PF Pyraclostrobin and fluxapyroxad 402 ml 1C,2C Captan and ziram 2.8 kg, 3.3 kg 3C-9C

Pyraclostrobin and fluxapyroxad (6) 402 ml PF,1C Captan and mancozeb 2.8 kg, 3.3 kg 2C Captan and ziram 2.8 kg, 3.3 kg 3C-9C

Captan and mancozeb (7) 2.8 kg, 3.3 kg PF,1C Pyraclostrobin and fluxapyroxad 402 ml 2C Captan and ziram 2.8 kg, 3.3 kg 3C-9C

Pyraclostrobin and fluxapyroxad (8) 402 ml PF Captan and mancozeb 2.8 kg, 3.3 kg 1C,2C Captan and ziram 2.8 kg, 3.3 kg 3C-9C

Captan and mancozeb (9) 2.8 kg, 3.3 kg PF,2C Pyraclostrobin and fluxapyroxad 402 ml 1C Captan and ziram 2.8 kg, 3.3 kg 3C-9C a PF = petal fall, #C = cover spray number b Dates of applications were 25 April, 3 May, 16 May, 26 May, 9 June, 28 June, 13 July, 27 July, 4 August, and 19 August, 2017.

Table 3.4 Treatment program, active ingredient, rate, and timing for pyraclostrobin and fluxapyroxad for 2018 Glomerella fungicide evaluation experiment. Active Ingredient and (treatment Rate of formulated product number) (amt./ha) Timinga Non-treated control (1) N/A N/A

Pyraclostrobin and fluxapyroxad (2) 402 ml PF,1Cb Captan and mancozeb 2.8 kg, 3.3 kg 2C Captan and ziram 2.8 kg, 3.3 kg 3C-13C

Captan and mancozeb (3) 2.8 kg, 3.3 kg PF Pyraclostrobin and fluxapyroxad 402 ml 1C,2C Captan and ziram 2.8 kg, 3.3 kg 3C-13C

Pyraclostrobin and fluxapyroxad (4) 402 ml PF Captan and mancozeb 2.8 kg, 3.3 kg 1C,2C Captan and ziram 2.8 kg, 3.3 kg 3C-13C a PF = petal fall, #C = cover spray number b Dates of applications were 8 May, 17 May, 25 May, 1 June, 8 June, 15 June, 22 June, 28 June, 9 July, 20 July, 26 July, 6 August, and 17 August, 2018.

Foliar Evaluation of Glomerella Leaf Spot. For each treatment replicate, eight mid- shoot leaves on 20 terminal shoots were evaluated in each experimental unit. To gain a better understanding of spatial disease progression within a tree, 10 shoots were selected for evaluation from the inner portion of the tree canopy < 50 cm from the trunk) and 10 shoots were selected for evaluation from the outer canopy ( > 50 cm from the trunk). Prior to disease assessment, shoots and leaves were individually labeled to allow for repeated measurements throughout the experiment. Disease incidence was determined by counting the number of leaves with GLS symptoms from eight total leaves and multiplying by 100%. Disease severity was determined by counting the number of leaves defoliated due to GLS infection from a total of eight original leaves and multiplying by 100%. In 2017 and 2018, disease incidence and severity were evaluated at six and nine times, respectively.

Evaluation of Glomerella fruit spot and Glomerella fruit rot prior to harvest. Five fruit were evaluated with 10 collections assessed per treatment in each replicate. A spot was characterized as a lesion greater than 1 mm in diameter and not causing symptoms to the flesh,

while a rot was characterized by a large, sunken lesion causing fruit tissue maceration. Due to high disease pressure in both years of each fungicide evaluation experiment, fruit with greater than one spot caused by Colletotrichum spp. were included in the incidence rating. The incidence of fruit spots and rots caused by Colletotrichum spp. was assessed one day after the final fungicide application (and one day prior to harvest) in 2017 and 2018.

Post-harvest evaluation of Glomerella fruit rot. To evaluate the efficacy of pre-harvest fungicide applications for post-harvest disease control, apples were harvested on 21 August and

20 August in 2017 and 2018, respectively. For each treatment, the development of GFR was evaluated by collecting 80 fruit with Glomerella fruit spot symptoms with 20 apples assessed per treatment replicate and a total of 4 replications per treatment. Fruit were stored on 20 cell pad cushion insert trays (Glacier Valley Enterprises Inc., Baraboo, WI), with four trays stored in one bushel covered cardboard boxes (Glacier Valley Enterprises Inc.) in the dark at room temperature 20°C to 25°C. Fruit rot was rated 14 days after harvest in both years.

Data analyses. Incidence and severity ratings for GLS were used to calculate the relative area under disease progress curve (rAUDPC) for each fungicide treatment using incidence or severity percentages from each rating date. Glomerella fruit spot and rot incidence at harvest and post-harvest rot incidence were calculated using Microsoft Excel. The data was subjected to an analysis of variance (ANOVA) one-way table using Arcsin to transform the data. AUDPC was calculated according to the following formula:

n AUDPC = å i = 1 ((yi + yi +1/2)(ti + 1 – ti))

Where yi = disease incidence/severity at the i-th observation, ti = time (days) at the i-th observation, and n = total number of observations. The rAUDPC was calculated as:

rAUDPC = AUDPC / (tf – to) x 100

Where tf = the number of days at the final rating and t0 = the time symptoms were first observed. Inner and outer diseased leaf data were initially analyzed separately as a combined dataset to determine significant differences with respect to sample location. Beginning at the first observation of GLS on an untreated tree (day 0), the number of days until GLS/GFR symptom development reached 40% and days until 10% inner shoot defoliation for selected treatments was calculated using regression analysis in order to gain better understanding of disease development under different fungicide programs. Both incidence and defoliation data was subjected to analyses of variance (ANOVA) and the means were compared using LSMEANS procedure in

SAS 9.4 with an adjustment for Tukey’s HSD to control for family-wise error. All data were analyzed separately for each year of the experiment.

NC Climate Retrieval and Observations Network of the Southeast Database.

Weather was monitored at the Mountain Horticultural Crops Research Station (35.42721, -

82.55888) by an ECONET – tower. NC Climate Retrieval and Observations Network of the

Southeast Database (CRONOS) is supported by NC Agricultural Research Service. Monthly mean daily temperature, relative humidity, and daily sum of precipitation were recorded.

RESULTS

Molecular identification of Colletotrichum spp. in research orchard. Genomic DNA sequences of CAL (~750 bp), GAPDH (~300 bp), ACT (~300 bp), and the ITS region (~600 bp) were generated for 18, 11, 11, and 11 isolates, respectively. Comparison of consensus ITS sequences with published sequences in GenBank, suggested that all recovered isolates were members of the Colletotrichum gloeosporioides species complex. To further differentiate species, consensus sequences of ACT, CAL, and GAPDH were compared to published sequences in GenBank (ACT = JX009458, JX009451, JX009439; CAL = JX009672, JX009665

JX009667; GAPDH = JX009958, JX009949, JX009914). These genes were selected based off

the performance of the individual genes at resolving species within the C. gloeosporioides species complex (Weir et al. 2012). All isolates matched published sequences of C. fructicola.

Evaluation of non-rotational fungicide field experiment for management of pre-and post-harvest Glomerella leaf spot. The location of the leaf was not significant for GLS spot incidence in either 2017 or 2018 (P = 0.3221 and P = 0.6771, respectively) and so inner and outer shoot incidence data was combined for analysis. In the 2017 experiment, the non-rotational fungicide programs of pyraclostrobin plus fluxapyroxad and captan plus phosphorous acid provided the greatest protections against GLS and leaf defoliation. If a treatment had 40% spot incidence £ 20 days and 10% defoliation > 40 days until harvest, it would typically be considered a fungicide control failure. The non-rotational program of pyraclostrobin plus fluxapyroxad suppressed GLS development below 40% for 64.4 days whereas GLS incidence in the thiophanate-methyl and the non-treated control programs reached 40% incidence of GLS at

21.4 and 19.8 days, respectively (Figure 3.1, Table 3.5).

Non-rotational programs consisting of pyraclostrobin plus fluxapyroxad, captan plus phosphorous acid, and captan at the 5.6 kg/ha rate, most effectively reduced defoliation compared to other treatments. Although the most effective treatments were not significantly different from each other, they were statistically different (P ≤ 0.0001) from poorly performing treatments. For the most effective treatments, mean rAUDPC values of inner canopy defoliation ranged from 26.0 ± 3.3 to 39.0 ± 3.9, whereas outer defoliation rAUDPC values up to 13.7 ± 2.4.

Pyraclostrobin plus fluxapyroxad delayed 10% defoliation until 0 days to harvest compared to difenoconazole plus cyprodinil and the untreated control which reached 10% defoliation with

63.6 and 56.1 days prior to harvest, respectively (Figure 3.2, Table 3.5).

In the 2018 experiment, applications of captan (5.6 kg/ha) and pyraclostrobin plus fluxapyroxad resulted in the lowest spot incidence on leaves, with mean rAUDPC values of 6.8 ±

2.1 and 12.6 ± 1.0, respectively. Similar to 2017, the least effective treatments included fluxapyroxad, difenoconazole plus cyprodinil, and the non-treated control. Captan applied without a mix partner (5.6 kg/ha) was the only treatment that suppressed spot incidence below

40% at harvest compared to fluxapyroxad, difenoconazole plus cyprodinil, and the untreated control developed 40% GLS spot incidence at 18.1, 21.5, and 24.9 days, respectively (Fig. 3.3,

Table 3.6).

For the majority of treatments (six of nine), no significant differences were observed for inner and outer shoot defoliation, but treatments of fluxapyroxad and difenoconazole plus cyprodinil were the least effective in preventing inner and outer defoliation (fluxapyroxad inner and outer defoliation rAUDPC = 29.9 ± 6.0 and 13.1 ± 0.7, respectively) throughout the season.

The most efficacious treatments delaying defoliation included captan (5.6 kg/ha) and pyraclostrobin plus fluxapyroxad. Both treatments did not reach 10% defoliation on inner shoots throughout the season in 2018 (Figure 3.4, Table 3.6).

Evaluation of non-rotational fungicide field trial for management of pre- and post- harvest Glomerella fruit rot.

In 2017, five treatments were observed to have greater than 99% spot development on fruit at harvest while pyraclostrobin plus fluxapyroxad provided the greatest protection against spot and fruit rot development at harvest with an observed incidence of 43.0 ± 15.4 % and 6.0 ±

3.8 % , respectively. Phosphorous acid, applied as a standalone product, and the non-treated control had a harvest rot incidence of 86.0 ± 7.8 % and 100.0 ± 0.0%, respectively (Table 3.7).

Post-harvest fruit rot incidence ranged from 45.0 ± 8.66% to 100 ± 0.0% with pyraclostrobin plus fluxapyroxad, pyraclostrobin, and captan plus phosphorous acid providing the greatest protection against fruit rot development in storage.

At harvest in 2018, four treatments resulted in greater than 95% fruit spot incidence compared to treatments containing captan, which provided the greatest level of protection against fruit spot (Table 3.8). The incidence of Glomerella fruit rot was less than 2% in non-rotational programs of pyraclostrobin plus fluxapyroxad, captan plus phosphorous acid, pyraclostrobin, and captan. Three treatments were excluded in the post-harvest rot analysis due to 100% fruit rot development on the sampled fruit at harvest. Although captan provided the greatest level of protection against fruit rot development at harvest, after 14 days in storage, this product was no longer as effective with 30.6 ± 8.9% fruit rot incidence. Treatments of pyraclostrobin, captan plus phosphorous acid, and pyraclostrobin plus fluxapyroxad were most efficacious treatments against post-harvest fruit rot development with 8.75 ± 2.39%, 17.50 ± 4.79%, and 17.50 ± 7.77% rot incidence, respectively.

A 120 y = -0.0107x2 + 2.1219x - 0.4478 120B y = -0.0099x2 + 2.1237x - 4.0163 R² = 0.9887 R² = 0.975 100 100 80 80 60 60 40 40 20 20 0 0 -50 -20 0 50 100 -20 0 50 100

C80 y = 0.0138x2 - 0.2781x + 0.7452 120D y = 0.0037x2 + 0.943x - 3.259 R² = 0.9915 100 R² = 0.9752 60 80 40 60 40 20 20 0 0 0 20 40 60 80 100 -20 0 50 100 -20 y = -0.0144x2 + 2.4705x - 3.2052 120E 100F 2 R² = 0.9862 y = 0.0135x - 0.1295x + 0.5306 100 80 R² = 0.9606 80 60 60 40 40 20 20 0 0 -20 0 50 100 0 50 100

2 100G y = 0.0153x2 - 0.1223x + 0.0848 150H y = -0.0102x + 2.2106x - 7.4119 R² = 0.947 80 R² = 0.9717 100 60 40 50 20 0 0 0 50 100 -20 0 50 100 -50

y = -0.0006x3 + 0.0718x2 - 0.6261x + 2 120I 120J y = 0.0099x + 0.4388x - 3.2965 0.1103 R² = 0.9298 100 R² = 0.9981 100 80 80 60 60 40 40 20 20 0 0 -20 0 50 100 -20 0 50 100

Figure 3.1 2017 non-rotational fungicide program for days when GLS reached 40% A, non- treated control; B, pyraclostrobin and fluxapyroxad; C, difenoconazole and cyprodinil; D, benzovindiflupyr; E, thiophanate-methyl; F, captan and phosphorous acid; G, pyraclostrobin; H, phosphorous acid; I, fluxapyroxad; and J, captan.

2 A 2 B3.5 y = 0.0003x + 0.0074x - 0.0915 y = 0.0074x + 0.6843x - 6.053 R² = 0.6724 R² = 0.9503 3 150 2.5 2 100 1.5 50 1 0.5 0 0 0 20 40 60 80 100 -50 -0.5 0 20 40 60 80 100

3 2 120C 35D y = 0.0001x - 0.0088x + 0.2193x - y = 0.013x2 + 0.1483x - 3.397 0.0625 100 30 R² = 0.9694 25 R² = 0.9986 80 20 60 15 40 10 20 5 0 0 0 20 40 60 80 100 -20 0 50 100 -5 3 2 100E y = 0.0129x2 - 0.0835x - 1.3992 15F y = 8E-05x - 0.0063x + 0.139x - R² = 0.995 0.1134 80 R² = 0.9939 10 60 40 5 20 0 0 0 50 100 0 20 40 60 80 100 -20 -5

15G y = 0.0028x2 - 0.1174x + 0.4096 100H y = 0.02x2 - 0.5563x + 0.2173 R² = 0.9807 80 R² = 0.9806 10 60 5 40 20 0 0 0 50 100 -5 -20 0 50 100

30 y = 0.006x2 - 0.2133x + 0.4846 100I y = 0.0198x2 - 0.4989x + 0.0912 J R² = 0.9908 R² = 0.9945 25 80 20 60 15 40 10 20 5 0 0 -20 0 50 100 -5 0 50 100

Table 3.5 Active ingredients and days when GLS symptoms reached 40% and days until harvest when 10% defoliation has been reached in non-rotational fungicide trial in 2017. Symptom Days until 40% Days until Active Ingredient GLS Harvest Non-treated control 21.4 63.6 Pyraclostrobin and fluxapyroxad 64.4 N/Aa Difenoconazole and cyprodinil 23.3 56.1 Benzonvindiflupyr 39.7 6.5 Thiophanate-methyl 19.8 49.9 Captan and phosphorous acid 59.1 N/A Pyraclostrobin 55.2 N/A Phosphorous acid 24.1 43 Fluxapyroxad 34.8 44.7 Captan 47.6 23 a Treatment did not reach 10% defoliation.

2 150A y = -0.0062x + 1.9759x - 5.4653 150B y = -0.0108x2 + 2.274x - 3.8651 R² = 0.9462 R² = 0.9679 100 100

50 50

0 0 0 20 40 60 80 0 20 40 60 80 -50 -50

3 2 80C D80 y = 0.0007x - 0.0558x + 1.2443x - y = 0.0133x2 - 0.2994x + 3.9542 1.4422 R² = 0.934 60 60 R² = 0.9783

40 40 20 20 0 0 0 20 40 60 80 0 20 40 60 80 -20

120E y = -0.0155x2 + 2.5882x - 3.022 100F y = 0.0218x2 - 0.7573x + 5.5778 R² = 0.978 100 80 R² = 0.9337 80 60 60 40 40 20 20 0 0 -20 0 20 40 60 80 -20 0 20 40 60 80

3 2 30G y = 0.0002x - 0.0226x + 0.6053x - 100H y = 0.0159x2 - 0.1523x + 5.3682 0.0929 R² = 0.9561 25 R² = 0.9938 80 20 60 15 10 40 5 20 0 0 -5 0 20 40 60 80 0 20 40 60 80

2 150I y = 0.0147x + 0.1472x + 2.8343 R² = 0.9898 100

0 0 20 40 60 80

100A y = 0.027x2 - 0.8656x + 2.4316 150B R² = 0.9901 y = 0.0255x2 - 0.5132x - 1.9943 80 R² = 0.9519 100 60 40 50 20 0 0 0 20 40 60 80 -20 0 20 40 60 80 -50

C5 y = -2E-05x3 + 0.0032x2 - 0.0845x + D4 y = 2E-05x3 - 0.0009x2 + 0.0085x + 4 0.1737 0.0195 R² = 0.9146 3 R² = 0.9359 3 2 2 1 1 0 0 0 20 40 60 80 -1 0 20 40 60 80 -1

E2.5 y = 3E-05x3 - 0.0022x2 + 0.0427x - 120F y = -0.0004x3 + 0.0719x2 - 1.6947x + 0.0571 2 100 2.7188 R² = 0.9536 R² = 0.9596 1.5 80 60 1 40 0.5 20 0 0 -0.5 0 20 40 60 80 -20 0 20 40 60 80

G0.8 y = 8E-06x3 - 0.0007x2 + 0.0139x - 25H y = 0.0002x3 - 0.0167x2 + 0.3115x - 0.3972 0.0185 20 0.6 R² = 0.9536 R² = 0.9828 15 0.4 10 0.2 5 0 0 0 20 40 60 80 -0.2 -5 0 20 40 60 80

20I y = 0.0001x3 - 0.0109x2 + 0.1877x - 0.1586 15 R² = 0.9886

0 0 20 40 60 80 -5

Table 3.6. Active ingredients and days when GLS symptoms reached 40% and days until harvest when 10% defoliation has been reached in non-rotational fungicide trial in 2018. Active ingredient Symptom Days until 40% GLS Days until harvest Non-treated control 24.97 36.8 Difenoconazole and cyprodinil 21.48 38.8 Captan and phosphorous acid 64.52 N/A Pyraclostrobin and fluxapyroxad 66.37 N/A Fluxapyroxad 18.73 39.7 Pyraclostrobin 60.74 N/A Captan N/Aa N/A Benzovindiflupyr 51.7 N/A Thiophanate-methyl 45.5 4.1 a Treatment did not reach 40% GLS symptoms or treatment did not reach 10% defoliation.

Table 3.7. Relative area under the disease progress curve (rAUDPC) values for non-rotational fungicide trial evaluating disease incidence and severity of leaves and incidence of fruit spot, rot and post-harvest rot for 2017 in Mills River, NC. Inner Leaf Outer Leaf Fruit Spot Fruit Rot PH Fruit Rot Active Ingredient Leaf Incidence Severity Severity Incidence (%) Incidence (%) Incidence (%) Non-treated control 62.69 ± 6.01 ab 38.97 ± 3.89 a 8.36 ± 1.46 100 ± 0.0 a 100 ± 0.0 a 100 ± 0.0 a abcde Pyraclostrobin and 21.25 ± 4.09 f 0.91 ± 0.30 d 1.88 ± 1.08 f 43.0 ± 15.44 d 6.0 ± 3.83 d 45.0 ± 8.66 e fluxapyroxad Difenoconazole and 59.38 ± 5.66 ab 32.42 ± 5.65 ab 11.96 ± 1.96 ab 100 ± 0.0 a 77.5 ± 4.92 b 100 ± 0.0 a cyprodinil Benzonvindiflupyr 43.80 ± 4.22 c 7.62 ± 2.37 d 5.64 ± 0.78 cdef 89.5 ± 4.99 a 41.0 ± 5.74 c 78.75 ± 9.66 bc Thiophanate-methyl 62.96 ± 2.27 a 25.74 ± 2.95 bc 9.13 ± 2.41 abcd 99.5 ± 0.5 a 83.0 ± 5.80 b 97.50 ± 1.44 a Captan and 27.54 ± 4.46 ef 2.47 ± 0.65 d 3.74 ± 1.41 def 54.43 ± 13.5 cd 21.72 ± 6.08 d 73.33 ± 8.08 cd phosphorous acid Pyraclostrobin 31.42 ± 6.62 de 2.11 ± 0.44 d 3.36 ± 1.30 ef 69.0 ± 8.88 b 17.5 ± 2.63 d 57.50 ± 5.20 de Phosphorous acid 58.99 ± 3.15 ab 24.22 ± 4.95 c 13.67 ± 2.44 a 100 ± 0.0 a 86.0 ± 7.75 ab 98.75 ± 1.25 a Fluxapyroxad 53.61 ± 3.03 b 26.16 ± 3.25 bc 9.31 ± 2.29 abc 100 ± 0.0 a 72.11 ± 10.31 b 100 ± 0.0 a Captan 38.34 ± 3.35 cd 5.94 ± 2.96 d 6.63 ± 3.06 bcdef 60.5 ± 14.59 bc 45.33 ± 4.37 c 91.67 ± 3.33 ab a rAUDPC values are the means of four replicates. Means followed by the same letter within each rating are not significantly different using the Tukey-Kramer honestly significant different (P £ 0.05). b Values within a followed by the same column letter are not significantly different according to LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD control for family-wise error.

Table 3.8. Relative area under the disease progress curve (rAUDPC) values for non-rotational fungicide experiment evaluating disease incidence and severity of leaves and disease incidence of fruit spot, rot and post-harvest fruit rot for 2018 in Mills River, NC. Leaf Inner Leaf Outer Leaf Fruit Spot Fruit Rot PH Fruit Rot Active Ingredient Incidencea,c Severity Severity Incidence (%)b Incidence (%) Incidence (%) Non-treated control 58.87 ± 2.76 b 22.26 ± 3.00 b 11.42 ± 3.27 a 100 ± 0.0 a 75.5 ± 11.24 b - Pyraclostrobin and 12.63 ± 0.98 ef 0.51 ± 0.26 c 0.91 ± 0.41 b 14 ± 3.27 c 0.5 ± 0.5 d 17.50 ± 7.77 bc fluxapyroxad Difenoconazole and 62.27 ± 1.91 ab 29.40 ± 0.60 a 11.48 ± 2.36 a 100 ± 0.0 a 89 ± 2.65 a - cyprodinil Benzovindiflupyr 30.01 ± 6.12 d 2.28 ± 1.57 c 1.95 ± 0.97 b 64.38 ± 12.90 b 12.75 ± 7.82 cd 61.25 ± 3.15 a Captan and 17.96 ± 2.33 e 1.28 ± 0.55 c 0.12 ± 0.05 b 5.33 ± 4.37 cd 0.67 ± 0.67 d 17.50 ± 4.79 bc phosphorous acid Thiophanate Methyl 37.18 ± 1.56 c 3.05 ± 0.64 c 1.52 ± 0.31 b 95 ± 2.38 a 19.5 ± 6.40 c 65.00 ± 7.07 a Pyraclostrobin 18.89 ± 1.60 e 0.06 ± 0.06 c 0.16 ± 0.16 b 13.5 ± 6.08 c 1.5 ± 0.95 d 8.75 ± 2.39 c Fluxapyroxad 65.60 ± 3.48 a 29.85 ± 5.98 a 13.09 ± 0.66 a 100 ± 0.0 a 82.67 ± 2.91 ab - Captan 6.78 ± 2.10 f 0.20 ± 0.04 c 0.0 ± 0.0 b 0.5 ± 0.5 d 0.0 ± 0.0 d 30.63 ± 8.92 b a rAUDPC values were calculated for leaf incidence and severity. rAUDPC values are the means of four replicates. Means followed by the same letter within each rating are not significantly different using the Tukey-Kramer honestly significant different (P £ 0.05). b Fruit spot, rot and post-harvest (PH) rots are percent incidence. c Values within a column followed by the same letter are not significantly different according to LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD control for family-wise error.

Evaluation of critical application timing of premix product, pyraclostrobin + fluxapyroxad (Merivon). To assess critical application timings for management of GLS and

GFR, the application timing of pyraclostrobin plus fluxapyroxad was altered during both the

2017 and 2018 experiments. In 2017, different application timings of pyraclostrobin plus fluxapyroxad included PF, 1C, 2C, 8C, and 9C. In the 2017 field experiment no significant differences in leaf disease incidence, expressed as rAUDPC values, were observed between treatments, excluding the untreated control (P ≤ 0.0001). Relative AUDPC values for leaf disease incidence ranged from 22.85 ± 3.93 to 62.69 ± 6.01.

Relative AUPDC values for defoliation of inner shoots ranged from 1.22 ± 0.71 to 38.97

± 3.89 with applications at 8C and 9C and the non-treated control providing the greatest and lowest efficacy, respectively. With the exception of the untreated program, no significant differences in rAUDPC values for inner defoliation were observed between the fungicide timing treatments. Relative AUDPC values for outer leaf defoliation ranged from 1.81 ± 0.50 to 8.39 ±

2.38, with pyraclostrobin plus fluxapyroxad applications at 8C and 9C and providing the greatest efficacy against outer leaf defoliation (Table 3.9).

Significant differences were observed in fruit spot incidence. Percent incidence of fruit spot ranged from 40.0 ± 13.0% (applied at 8C and 9C) to 100.0 ± 0.0% (non-treated control).

Although not significantly different from other treatments with alternating timings of pyraclostrobin plus fluxapyroxad, when applied at 1C and 2C, the fruit were observed to have

10.0 ± 3.6% fruit rot incidence before harvest. After 14 days post-harvest, applications of pyraclostrobin plus fluxapyroxad were observed to be most effective when applied at 8C

(treatment 2) and 8C and 9C (treatment 3) (Table 3.9).

Based on results of the 2017 field experiment, in 2018, three programs were selected to evaluate application timing of the fungicide for managing GLS and GFR (Table 3.10).

Pyraclostrobin plus fluxapyroxad timings included PF, PF and 1C, and 1C and 2C. Although numerical differences were observed, there were no statistically significant differences observed between the treatments for leaf incidence, leaf severity or fruit spot and rot incidence, excluding the untreated control. After 14 days post-harvest, a significant difference was observed when pyraclostrobin plus fluxapyroxad was applied at just PF (treatment 4) compared to applications at PF and 1C (treatment 2).

Table 3.9. Treatment number, leaf incidence, inner and outer leaf defoliation, fruit spot, rot incidence and post-harvest rot incidence were evaluated for the critical timing of pyraclostrobin plus fluxapyroxad in 2017. Treatment number Inner leaf Outer leaf Fruit spot Fruit rot PH Rot (fungicide timing) Leaf Incidence defoliation defoliation Incidence (%) incidence (%) Incidence (%) 1 (N/A) 62.69 ± 6.01 aa 38.97 ± 3.89 a 8.37 ± 1.46 a 100.0 ± 0.0 a 100.0 ± 0.0 a - 2 (8C,9C) 22.85 ± 3.93 c 1.22 ± 0.71 b 1.81 ± 0.50 c 40.0 ± 12.96 c 18.5 ± 4.79 bcd 23.75 ± 9.66 c 3 (8C) 33.80 ± 1.41 b 2.47 ± 0.66 b 5.82 ± 0.36 ab 56.5 ± 9.43 bc 25.5 ± 6.18 bc 32.50 ± 7.77 c 4 (9C) 29.52 ± 3.66 bc 2.33 ± 0.86 b 3.37 ± 0.87 bc 47.5 ± 3.94 bc 12.0 ± 3.74 cd 72.5 ± 5.95 b 5 (1C,2C) 27.75 ± 1.24 bc 1.54 ± 0.40 b 4.92 ± 1.48 abc 49.0 ± 4.04 bc 10.0 ± 3.56 d 77.5 ± 6.29 b 6 (PF, 1C) 31.75 ± 8.74 bc 1.20 ± 0.38 b 5.08 ± 1.88 abc 65.0 ± 11.68 b 28.0 ± 6.93 b 77.5 ± 5.20 b 7 (2C) 35.35 ± 6.62 b 2.81 ± 1.41b 8.39 ± 2.38 a 63.0 ± 8.19 b 24.5 ± 3.77 bc 78.75 ± 6.25 b 8 (PF) 27.30 ± 4.28 c 1.44 ± 0.58 b 6.04 ± 6.04 b 62.0 ± 15.45 b 20.0 ± 8.04 bcd 85.0 ± 4.56 a 9 (1C) 29.85 ± 2.80 bc 1.27 ± 0.85 b 5.55 ± 0.55 ab 62.0 ± 7.79 b 22.5 ± 2.50 bcd 82.5 ± 5.20 a arAUDPC values are the means of four replicates. Means followed by the same letter within each rating are not significantly different using the Tukey-Kramer honestly significant different (P £ 0.05). b Values within a column followed by the same letter are not significantly different according to LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD control for family-wise error.

Table 3.10. Treatment number, leaf incidence, inner and outer leaf defoliation, fruit spot, rot, and post-harvest rot incidence were evaluated for the critical timing of pyraclostrobin plus fluxapyroxad in 2018. Treatment number Inner leaf Outer leaf Fruit Spot Fruit rot PH Rot Leaf Incidence (fungicide timing) Defoliation Defoliation incidence (%) incidence (%) Incidence (%) 1 (N/A) 58.87 ± 2.76 a 22.26 ± 3.00 a 11.42 ± 3.27 a 100.0 ± 0.0 a 75.50 ± 11.24 a - 2 (PF, 1C) 15.96 ± 2.32 b 0.30 ± 0.22 b 0.28 ± 0.12 b 13.0 ± 5.26 b 1.0 ± 0.58 b 47.5 ± 3.22 a 3 (1C,2C) 13.29 ± 1.14 b 0.45 ± 0.10 b 0.97 ± 0.68 b 12.5 ± 7.89 b 2.0 ± 1.15 b 33.75 ± 4.27 ab 4 (PF) 11.63 ± 0.54 b 0.51 ± 0.32 b 0.25 ± 0.25 b 12.5 ± 7.13 b 2.0 ± 0.82 b 27.5 ± 4.79 b arAUDPC values are the means of four replicates. Means followed by the same letter within each rating are not significantly different using the Tukey-Kramer honestly significant different (P £ 0.05). b Values within a column followed by the same letter are not significantly different according to LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD control for family-wise error.

NC Climate Retrieval and Observations Network Of the Southeast Database (CRONOS).

In 2017, the mean air temperature during the growing season ranged from 15°C to 22.7°C. As the growing season progressed, the mean relative humidity increased and reached a mean of 81% during harvest period in Aug. Rainfall recorded during Apr and May was greater than 200 mm.

In Apr 2018, mean temperature was 11.3°C, and increased 8 degrees at petal fall and fruit set growth stages occur. Mean rainfall in May and Aug was 389.1 mm and 272.5 mm, respectively

(Table 3.11).

Table 3.11. Mean air temperature, relative humidity and daily sum of precipitation in 2017 and 2018. 2017 2018 Mean Mean Mean Air Sum of Mean Air Sum of Relative Relative Month Temperature Precip. Month Temperature Precip. Humidity Humidity ° (mm) ° (mm) ( C) (%) ( C) (%) April 15 221.2 70 April 11.3 118.4 63 May 17.3 200.7 71 May 19.4 389.1 76 June 20.5 81 74 June 22.0 90.7 74 July 22.7 118.1 79 July 22.4 177.3 78 August 21.3 193.3 81 August 21.4 272.5 80

DISCUSSION

Fungicides play a critical role in the management of Glomerella leaf spot and fruit rot of apple. A fungicide efficacy experiment conducted in 2017 and 2018 in Mills River, NC, revealed that there are few effective fungicides available for the management of GLS and GFR diseases.

A premixed QoI plus SDHI product and fungicides with multi-site modes of action reduced GLS and GFR greater than SDHIs, DMIs, MBC or phosphonate group fungicides used as individual products. Results also demonstrated that the application timing of pyraclostrobin plus fluxpyroxad in general did not influence pre-harvest disease incidence, when applied in rotation with another highly efficacious product. In this study, we found that the combined use of pyraclostrobin and fluxapyroxad had the greatest impact on reducing GLS and GFR diseases and

was comparable to the commonly applied protectant fungicides captan (Captan 80 WDG) and ziram (Ziram 76 DF).

Before this study, there was no comprehensive information on fungicide application programs for the managing of GLS and GFR in Western NC. Traditionally, NC apple growers would apply fungicides on a 14 to 21 day intervals from first cover until harvest. In our study, this interval did not adequately suppress disease, especially during a growing season with temperatures greater than 20°C and heavy rainfall (Villani and Nance 2016). Other apple producing regions that experience GLS and GFR including southern Brazil and eastern China have been unsuccessful in managing these disease with chemical control. Wang et al. (2015) determined that a traditional spray system and extensive applications of the fungicides tebuconazole, thiophanate-methyl and prochloraz did not effectively suppress disease in susceptible cultivars in China (Wang et al. 2015). In this study, fungicides were applied on a minimum 10-day interval (2017), but in 2018, the interval was reduced to a minimum 7-day interval. With a tighter spray interval, disease leaf incidence and severity were lower for majority of the treatments.

Pathogen identification essential for the development of a sustainable GLS and GFR fungicide application program. Causal Colletotrichum spp. have increased in diversity over the years with C. karstii, C. gloeosporioides, and C. fructicola identifying as causal agents of GLS and GFR (Velho et al. 2014; Velho et al. 2015; González and Sutton 1999). Through multilocus sequencing, the symptomatic apple fruit and leaf samples collected from our research orchard all identified the fungus causing the leaf and fruit spot as C. fructicola. Conserved regions including

CAL, ACT, ITS, and GAPDH confirmed the isolates from matched published sequences.

When utilizing a premix product or two different products in a tank mix, it is important to determine the most efficacious active ingredient for disease reduction. Pyraclostrobin (Cabrio

EG) and fluxapyroxad (Sercadis) are the active ingredients in Merivon. Based on the disease incidence and severity results from the two individual products, the QoI component, pyraclostrobin, was the more effective a.i. in reducing GLS and GFR diseases compared to the

SDHI component, fluxapyroxad. Although both are single-site fungicides, their mode of action differs. QoIs inhibit the mitochondrial respiration by binding to the outer quinone oxidizing pocket of the cytochrome bc1 enzyme in complex III, while SDHIs bind to the ubiquinone- binding (Qp) site to block access to the substrate which prevents succinate oxidation from cycling (Grasso et al. 2006; Sierotzki and Scalliet 2013). Pyraclostrobin has protective and curative activity because of its systemic properties making it persistent and providing even distribution within leaves and fruit (Gao et al. 2017). Through previously reported SDHI fungicide efficacy experiments, authors concluded Colletotrichum spp. are less sensitive to fluxapyroxad (Ishii et al. 2016). Although pyraclostrobin plus fluxapyroxad demonstrated efficacy against GLS and GFR diseases, a major concern is the active ingredient’s site-specific nature that predisposes them to rapid resistance development.

It has been observed that repeated use of a fungicide class with a single-site mode of action could increase the frequency of resistance within a population (Beckerman et al. 2013).

Single-site fungicides are widely used to manage GLS and GFR as well as other Colletotrichum spp on various pathosystems (Smith and Black 1991; Ishii et al. 2016). Reports of C. gloeosporioides and C. acutatum on peach, apple and cucumber, were found to be less sensitive to fluopyram and boscalid, which are SDHI fungicides in FRAC group 7 (Ishii et al. 2016). QoI resistance has also been reported in C. acutatum populations in Florida strawberry fields

(Forcelini et al. 2016). To minimize resistance development in the Colletotrichum populations in

NC, we recommend rotating protectant and single-site fungicides throughout the growing season.

The evaluated tank mix was the combination of phosphorous acid (ProPhyt) and captan

(Captan 80 WDG). Phosphorous acid is hypothesized to enhance the activity of captan to become a more efficacious treatment for disease management (DeMarsay 2012). Captan is a non-systemic fungicide that will remain on the leaf surface and become redistributed through a rain event. Phosphorous acid acts as a systemic fungicide with eradicant, anti-sporulant, and post-infection activity. The compound moves through the xylem to reach parts of plants not directly sprayed. It has been shown to enhance host defense mechanism while being toxic to some plant pathogens (Rosenberger and Cox 2009). Phosphorous acid will extend residual fungicide activity to slow symptom development when mixed with captan. In 2017, a tank mix of phosphorous acid and captan, reduced disease significantly compared to applying the products individually. In 2018, this treatment provided the best level of fruit protection before harvest with 0% rot development. Our results suggest captan provides protective activity on the outer tissue while the addition of phosphorous acid stimulates host defense mechanism throughout the plant allowing movement of captan. Not only can the combination of phosphorous acid and captan reduce disease in the field, it will decrease the rate of captan (max = 40 lbs/season) for the prolonged use during the growing season.

Hydrolysis and pH should always be taken into consideration to ensure the effectiveness of a spray solution. The breakdown of a pesticide is dependent on the pH, temperature of water and time in the spray tank. The pH of spray water can reduce the efficacy of pesticides that can result in poor crop protection. Captan degrades quickly under alkaline conditions with a half-life of 3 hours at a pH of 7.1 and 10 minutes at a pH of 8.2 (Halcomb 2012). Sartoretto (1991) indicates a pH range of 5.0 to 6.0 is safe for a spray solution. The ProPhyt label recommends the pH of a spray solution be greater than 5.5 to prevent phytotoxicity to the crop as well as ensuring effective disease management (Luxembourg-Pamol, inc). To achieve this optimal pH range, the

addition of phosphorous acid to a spray solution will lower the pH and in turn, slow the hydrolysis rate of captan.

The premix product of pyraclostrobin plus fluxapyroxad was applied at different cover sprays throughout the season. Differences in disease incidence and severity were not observed among various treatments. Indeed, differences may not have been noticed due to the products sprayed in between critical timing applications; captan and mancozeb or captan and ziram performed well in the non-rotational field experiment providing efficacious activity against disease symptoms. Another explanation for lack of observed differences could be due to the half- life of pyraclostrobin. Pyraclostrobin has a dissipation half-life ranging from 51 to 99 d with a mean of 75 d (NYSDEC 2004). In 2017 and 2018, our harvest day was at 83 and 76 d, respectively, so with pyraclostrobin’s half-life at maximum of 99 d, it suggests the product is not breaking down and having residual activity throughout the season with just one or two applications. An application at PF or 1C could potentially remain effective up until harvest and one application before harvest could be beneficial in reducing rot development in storage.

This study evaluated GLS and GFR disease progression in treatments throughout a season to determine when a tree reached 40% leaf spot incidence and 10% disease severity

(defoliation). If plants in a treatment had 40% leaf spot incidence £ 20 days and 10% defoliation

> 40 days until harvest, it would typically be considered a fungicide control failure. Treatments that never reached 10% defoliation were characterized as the most effective treatments that included, captan and the premix product, pyraclostrobin and fluxapyroxad. Least effective treatments defoliated as early as 37 days until harvest. Delaying symptom development increases the likelihood of maintaining high yields of marketable fruit at harvest. Early defoliation has been reported to have negative implications at harvest affecting fruit yield, quality, and bud break and fruit set the following growing season (Mohamed 2008).

GLS and GFR epidemics are likely related to temperature, humidity, and rainfall. The climate in Western NC is characterized by warm temperatures, high humidity, and moderate rainfall during the apple growing season and is similar to the subtropical climate of Brazil

(Becker et al. 2000; González et al. 2006). Ideal apple growing conditions need an estimated 500 to1,000 chilling hours (the number of hours below 7.2°C) needed during the winter to allow for an adequate bloom in the spring. Rainfall of 1016 to 1270 mm per year is optimal for growth, but too much rain during the spring or summer months can increase fruit disease susceptibility.

Based on CRONOS, MHCREC received 814.2 mm of precipitation in 2017 and 1048 mm in

2018. Although the amount of precipitation in 2018 is within the range for optimal growth of apple trees, this is potentially too much rainfall during the spring and summer months for keeping apple diseases, including GLS and GFR to a minimum.

Fungicide use is important throughout the season to manage GLS and GFR, but another increasingly crucial time period and practice is post-harvest management. To maintain high yield from harvest for marketability of fruit, disease development in storage should be evaluated (El-

Ramady et al. 2015; Farinati et al. 2017). Various abiotic and biotic stresses, such as disease can affect the fruit and result in reduced quality (Ansari and Tuteja 2013). A fungicide disease management program should be maintained until harvest to protect against disease development during storage to extend periods of time without fruit deterioration as well as being careful during handling and transport; a time when harvested fruits can become infected due to sustained injuries (Snowdown 2008; El-Ramady et al. 2015; Sôyu et al. 2003).

In 2008, researchers conducted an experiment to determine the efficacy of pre-harvest fungicide applications and cold storage for post-harvest control of Botrytis fruit rot caused by

Botrytis cinerea. They concluded pre-harvest fungicide applications were necessary to provide control of post-harvest Botrytis fruit rot (Ellis et al. 2008). Cold storage proved to not be

adequate enough for disease control with 12% of fruit displaying symptoms compared to fruit with pre-harvest fungicide applications with 0% fruit rot incidence. The researchers determined disease control was enhanced when pre-harvest fungicide applications and cold storage was utilized. Another study assessed the control of post-harvest diseases on Florida citrus by pre- harvest fungicide applications. Ritenour et al. (2004) tested four compounds, pyraclostrobin, phosphorous acid, thiophanate methyl, and benomyl before harvest to observe the percentage of fruit with visible symptoms of decay, stem-end rot (SER), and anthracnose post-harvest.

Researchers concluded thiophanate methyl (4.1%)and benomyl (7.9%) were the most efficacious products to apply before harvest to reduce post-harvest diseases on citrus (value in parenthesis is the percentage of fruit with SER post-harvest) (Ritenour et al. 2004). Although these studies were conducted in different pathosystems and on various fungi, it does suggest that pre-harvest fungicides are beneficial and can be effective in reducing post-harvest disease development in storage.

Current commercial apple storage standards in North Carolina include, reduction of room temperatures as low as 0°C to -1.1°C, lower oxygen levels to 1.5 to 3.0% and monitored and controlled CO2 levels (Watkins 2003; ncagr.gov). To preserve fruit quality, 1- methylcyclopropene (1-MCP) applications are widely used in the apple industry before fruit is placed in storage. 1-MCP blocks ethylene receptors to delay ethylene dependent responses such as ripening (Watkins 2008; Lee et al. 2011; Liu et al. 2016). Rot development could have been more prevalent in this study because the apples were stored at a range of 20°C to 25°C which is considered optimal temperatures for Colletotrichum growth. 1-MCP technology was not utilized in this experiment, but if used, rot development could have been reduced and shelf life extended.

GLS and GFR are important disease of apples that can cause severe defoliation and spot and rots on leaves and fruit. Based on the results of these two fungicide experiments, there are

few fungicides effective in managing these diseases. A multi-site protectant fungicide should be used in addition to a maximum of four applications of pyraclostrobin and fluxapyroxad during a summer spray program to reduce GLS and GFR disease symptoms. Although we did not find a critical time to apply the single-site fungicide, continued research is needed to optimize product application timing. The disease cycle has not been the subject of comprehensive investigation, and the time when ascospores of Colletotrichum are released is still unknown. Determining when initial infection occurs is crucial for disease management. A single-site fungicide should be applied at this time, in order to inhibit spore germination, infection and further spread of disease.

Until this information is determined, we suggest applying a single-site fungicide at petal fall, before harvest, and saving two applications at the highest rate for when there is the potential of heavy rainfall for several days. The higher rate will extend the residual period and have a lasting effect to delay disease progression. The application of protectant fungicides such as captan or ziram for cover sprays can reduce resistance development of single-site fungicides.

ACKNOWLEGMENTS

We would like to thank the Mountain Horticulture Center for Research and Extension for land used in these field experiments. A big thank you to the undergraduate workers in the lab that helped set up the field experiments and collect data. We would also like to recognize the farm crew for their assistance in field and tree maintenance and herbicide and insecticide applications.

Funding for this work was provided by the NCDA&CS Specialty Crop Block Grant Program.

We thank BASF, Syngenta, FMC, Dupont, Certis, and Luxembourg-Pamol for chemical donation to our research program.

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4. Beckerman, J.L., G.W. Sundin, and D.A. Rosenberger. 2013. ‘Do some IPM concepts contribute to the development of fungicide resistance? Lessons learned from the apple scab pathosystem in the United States.’ Pest Management Science, 71:331-342.

5. Cannon, P.F., U. Damm, P.R. Johnston, and B.S. Weir. 2012. ‘Colletotrichum – current status and future directions. Studies in Mycology, 73:181-213.

6. Damm, U., P.F. Cannon, J.H.C Woudenberg, and P.W. Crous. 2012. ‘The Colletotrichum acutatum species complex.’ Studies in Mycology, 73.1: 37-113.

7. DeMarsay, A. 2012. ‘The New Guidelines for Developing an Effective Fungicide Spray Program for Wine Grapes in Maryland.’ University of Maryland Extension.

8. Ellis, M.A., L.V. Madden, and L.L. Wilson. 2008. ‘Efficacy of Pre-harvest Fungicide Applications and Cold Storage for Postharvst Control of Botrytis Fruit Rot (Gray Mold) on Red Raspberyy.’ Plant Health Progress.

9. El-Ramady, H.R., E. Domokos-Szabolcsy, N.A. Abdalla, H.S. Taha, and M. Fári. 2015. ‘Postharvest Management of Fruits and Vegetables Storage.’ Sustainable Agriculture Reviews, 65-152.

10. Farinati, S., A. Rasori, S. Varotto, and C. Bonghi. 2017. ‘Rosaceae Fruit Development, Ripening and Post-harvest: An Epigenetic Perspective.’ Frontiers of Plant Science, 8:1247.

11. Forcelini, B.B., T.E. Seijo, A. Amiri, and N.A. Peres. 2016. ‘Resistance in Strawberry Isolates of Colletotrichum acutatum from Florida to Quinone-Outside Inhibitor Fungicides.’ Plant Disease, 100.10:2050-2056.

13. González, E and T.B. Sutton. 1999. ‘First Report of Glomerella Leaf Spot (Glomerella cingulata) of Apple in the United States.’ Plant Disease, 83.11:1074.

14. González, E., T.B. Sutton, and J.C. Correll. 2006. ‘Clarification of the etiology of Glomerella leaf spot and bitter rot of apple caused by Colletotrichum spp. based on morphology and genetic, molecular, and pathogenicity tests.’ Phytopathology, 96.9:982-992.

15. Gopinath, K., Radhakrishnan, N.V., and Jayaraj, J. 2006. ‘Effect of propiconazole and difenoconazole on the control of anthracnose of chilli fruits caused by Colletotrichum capsici.’ Crop Protection, 25.9:1024-1031.

16. Grasso, V., S. Palermo, H. Sierotzki, A. Garibaldi, and U. Gisi. 2006. ‘Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens.’ Pest Management Science, 62:465-472.

17. Halcomb, M. 2012. ‘The pH of the Spray Water is Very Important.’ The University of Tennessee Extension.

18. Ishii, H., F. Zhen, M. Hu, and G. Schnabel. 2016. ‘Efficacy of SDHI fungicides, including benzovindiflupyr, against Colletotrichum species.’ Pest Management Science, 72.10: 1844–1853.

19. Keinath, A.P. 2017. ‘Minimizing yield and quality losses in watermelon with multi-site and strobilurin fungicides effective against foliar and fruit anthracnose.’ Crop Protection, 106:72-78.

20. Lee, J., D.R. Rudell, P.J. Davies, and C.B. Watkins. 2012. ‘Metabolic changes in 1- methylcyclopropene (1-MCP)-treated ‘Empire’ apple fruit during storage.’ Metabolomics, 8.4:742-753.

21. Liu, Ruiling, Y. Wang, G. Qin, and S. Tian. 2016. ‘Molecular basis of 1- methylcyclopropene regulating organic acid metabolism in apple fruit during storage.’ Postharvest Biology and Technology, 117:57-63.

22. MacKenzie, S.J., J.C. Mertely, and N.A. Peres. 2009. ‘Curative and protectant activity of fungicides for control of crown rot of strawberry caused by Colletotrichum gloeosporioides. Plant Disease, 93:815-820.

23. Mohamed, A.K.A. 2008. ‘The effect of chilling, defoliation and hydrogen cyanamide on dormancy release, bud break and fruiting of Anna apple cultivar.’ Scientia Horticulturae, 118.1:25-32.

24. Moreira, R.R. and L.L. May De Mio. 2015. ‘Potential biological agents isolated from apple fail to control Glomerella leaf spot in the field.’ Biological Control, 87:56-63.

25. New York State Department of Environmental Conservation. 2004. ‘Pyraclostrobin – NYS Registrations: Insignia, Headline, and Cabrio.’ http://pmep.cce.cornell.edu/

26. North Carolina Apple Growers Association. 2018. https://www.ncapplegrowers.com.

27. Ritenour, M.A., R.R. Pelosi, M.S. Burton, E.W. Stover, H. Dou, and T.G. McCollum. 2004. ‘Assessing the Efficacy of Preharvest Fungicide Applications to Control Postharvest Disease of Florida Citrus.’ HortTechnology, 14.1:58-62.

28. Rosenberger, D.A. and K.D. Cox. 2009. ‘Using Phosphite Fungicides to Control Apple Disease.’ New York Fruit Quarterly, 17.2:9-13.

29. Sartoretto, P. 1991. ‘The pH Factor.’ WA Cleary Chemical Corp. 4 pg handout.

30. Sierotzki, H. and G. Scalliet. 2013. ‘A Review of Current Knowledge of Resistance Aspects for the Next-Generation Succinate Dehydrogenase Inhibitor Fungicides.’ Phytopathology, 103:880-887.

31. Smith, B.J. and L.L. Black. 1991. ‘Greenhouse efficacy of fungicides for control of anthracnose crown rot of strawberry,’ The Strawberry into the 21st Century, 221-223.

32. Snowden, A.L. 2008. ‘Post-Harvest Diseases and Disorders of Fruits and Vegetables: Volume 1: General Introduction and Fruits.’ CRC Press, 1:174.

33. Sutton, T.B. and R.M. Sanhueza. 1998. ‘Necrotic Leaf Blotch of Golden Delicious- Glomerella Leaf Spot: A Resolution of Common Names.’ Plant Disease, 82.3: 267- 68.

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35. Tancos, K.A., S. Villani, S. Kuehne, and E. Borejsza-Wysocka, D. Breth, J. Carol, H.S. Aldwinckle, and K.D. Cox. 2016. ‘Prevalence of Streptomycin-Resistant Erwinia amylovora in New York Apple Orchards.’ Plant Disease, 100.4:802-809.

36. Taylor, Jack. 1971. ‘A necrotic leaf blotch and fruit rot of apple caused by a strain of Glomerella cingulata.’ Phytopathology, 61.2: 221.

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38. Velho, A.C., S. Alaniz, L. Casanova, P. Mondino, and M.J. Stadnik. 2015. ‘New insights into the characterization of Colletotrichum species associated with apple disease in southern Brazil and Uruguay’, Fungal Biology, 119.4:229-244.

39. Villani, S.M. and D.A. Nance. 2016. ‘Evaluation of non-rotational fungicide programs for the management of Glomerella leaf spot and fruit rot on ‘Gala’ apple in NC, 2016.’ Plant Disease Management Report, 11.

40. Walgenbach, J., Parker, M., Villani, S., Mitchem, W., and Lockwood, D. 2018. Integrated Orchard Management Guide for Commercial Apples in the Southeast.

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CHAPTER IV.

CONCLUDING REMARKS AND FUTURE DIRECTIONS

Glomerella leaf spot (GLS) and fruit rot (GFR) are reemerging fungal diseases on apple causing devastating environmental and economic impacts on apple growing regions around the world, including the southeastern United States. Management has become increasingly challenging for producers due to changing climate patterns, abiotic stresses, limitations to annual fungicide applications and plantings of highly susceptible cultivars. Currently, commercial growers primarily apply single-site and protectant fungicides to maintain yield and fruit quality for consumers. However, a maximum of four applications of single-site fungicides per season is mandated to reduce pathogen resistance development (FRAC 2018). Previous work has demonstrated repeated applications of single-site fungicides will inevitably lead to resistance

(Forcelini et al. 2016; Grasso et al. 2006). The work presented in this thesis addresses management strategies through chemical control and characterization of Colletotrichum species through morphological and multilocus DNA sequence analysis. Additionally, in vitro experiments focused on Quinone outside Inhibitor (QoI) fungicide sensitivity shifts in

Colletotrichum populations using baseline populations were observed. The most efficacious fungicides are determined to develop a management program for southeastern apple growers as well as gathering baseline sensitivity information to enable rapid screening for resistance in future studies.

In chapter 2, three hundred and seventy-three isolates collected from seventeen orchards in NC and GA were characterized through morphological and multilocus sequence analysis. This objective was important because as stated previously, Colletotrichum species have various levels of sensitivity to fungicides and different management strategies may need to be implemented based on accurate identification of the pathogen. Six morphotypes were characterized based on

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colony color, growth pattern, and conidium shape and size. Characterizing isolates through morphological characteristics has proved to be an unreliable technique because of the changed or lost features from environmental influences and repeated subculturing (Hyde et al. 2009; Weir et al. 2012). We determined 75% of the isolates collected causing GLS and GFR identified as C. fructicola belonging to C. gloeosporioides species complex. Fungicide resistance has increasingly become a major concern within our agriculture systems. The effective concentration that inhibits 50% (EC50) of mycelial growth and spore germination was calculated from in vitro fungicide assays. A range of sensitivities were observed from these values that will now serve as a baseline for future studies of C. fructicola and C. fioriniae isolates. A Kolomogorov-Smirnov

(K-S) one-sample test was performed to compare isolate sensitivity distributions to baseline orchard populations. The sensitivity classification was determined for each population by comparing the distribution of isolate pyraclostrobin or trifloxystrobin responses for each test orchard population evaluated at the EC50 value to the sensitivity distribution of the baseline population. All orchard populations of C. fructicola and C. fioriniae were determined to be sensitive to pyraclostrobin when evaluated for efficacy against mycelial growth and conidial germination. Due to high EC50 value outputs from the baseline mycelial growth inhibition assay,

K-S test was not conducted on trifloxystrobin-amended medium. One commercial orchard population of C. fioriniae and four orchard populations of C. fructicola had reduced sensitivity to trifloxystrobin in conidial germination assays. Primers were designed for amplification of the cytb gene. This is the region were point mutations, G143A and F129L, can be observed indicating resistance to QoI fungicides. Currently, only one isolate in our sample collection was found to have the G143A mutation. Orchard populations should continue to be monitored for sensitivity shifts and mutations in the cytb gene to make sure management strategies continue demonstrating efficacious activity against GLS and GFR.

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Fungicide applications has historically been the foundation for managing Colletotrichum disease on other plant hosts. They have been successfully managed using captan, mancozeb, succinate dehydrogenase inhibitor (SDHI), QoIs, and phosphonate group fungicides (Gopinath et al. 2006; Ishii et al. 2016; Keinath 2017; Piccirillo et al. 2018). QoI fungicides have been utilized for the control of Colletotrichum spp. since their registration in 1996 (Bartlett et al. 2002).

Although resistance to QoIs has been reported in Colletotrichum species in various pathosystems, the QoIs as a class has remained one of the most efficacious groups for crop protection (Forcelini et al. 2016; Yokosawa et al. 2017; Avila-Adame et al. 2003). Currently, there is little to no research in the U.S on these diseases and it was imperative to develop a management program for southeastern apple growers. During severe epidemics, GLS can cause more than 75% defoliation by harvest (mid-August) under optimal conditions and 100% defoliation in absence of disease management interventions on susceptible cultivars (Sutton and

Sanhueza 1998). Various Colletotrichum species have demonstrated different levels of sensitivity to fungicides in a field setting, which was important for us to determine what species we were trying to manage.

In chapter 3, we determined through multilocus sequence analysis, leaf and fruit samples collected from the research orchard identified C. fructicola as the causal agent of the leaf and fruit spot within our orchard. Two fungicide efficacy field experiments were conducted in consecutive years to determine the most efficacious treatments reducing GLS and GFR. We concluded spray programs containing QoI fungicides and protectant fungicides (i.e. captan, ziram) were most efficacious in managing the diseases. A QoI is a single-site fungicide that inhibits mitochondrial respiration by binding to the outer quinone oxidizing pocket of the cytochrome bc1 enzyme in complex III (source). Because of its systemic properties, the fungicides are persistent and provide even distribution within the leaves and fruit (Gao et al.

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2017). Protectant fungicides, like captan, are non-system fungicides that disrupt multiple cellular division pathways (Cowgill et al. 2013). Multi-site compounds are applied protectively to inhibit conidial germination. We recommend applying a single-site fungicide at petal fall, before harvest, and saving two applications at the highest rate for when there is the potential of heavy rainfall for several days. When single-site fungicides are not applied, protectant fungicides such as captan or ziram should be applied to delay disease progression and reduce resistance development of single-site fungicides.

In summary, GLS and GFR are major concerns for apple growers in southeastern United

States, particularly North Carolina because of its ability to cause 100% crop loss on susceptible cultivars if not properly managed. This research was critical to determine the most efficacious fungicides and will continue to be of importance for the sustainability of effective chemical management for these diseases throughout the southeastern United States. QoIs and protectant fungicides should be implemented into summer fungicide spray programs to reduce disease within the orchard as wells as maintaining good cultural control practices to reduce inoculum. A fungicide resistance monitoring system has been developed to assess Colletotrichum populations for sensitivity shifts if growers in the region have concerns about fungicide efficacy.

There is still much needed research to be conducted in regards to determining the initial point of infection in the field as well as pathogenicity tests on various cultivars. To understand the timing of ascospore release, spore traps should be utilized to provide a clear timeline of when to start preventively spraying for GLS and GFR. Pathogenicity tests on a wide range of cultivars grown in western NC should be conducted in the future. This will provide an understanding of susceptible cultivars to GLS and GFR in addition to the aggressiveness of the pathogen on each cultivar.

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In conclusion, when I began my Master of Science degree in plant pathology, I wanted to become a well-rounded scientist that was equipped with skills and knowledge to conduct applied field research, run laboratory experiments, as well as successfully communicate the results and recommendations to a wide variety of audiences, including scientists, growers, and local communities. Because of the paucity of information regarding fungicide activity and lack of effective management programs, I designed a fungicide field experiment for two consecutive seasons for the development of a management program controlling GLS and GFR. It was important that I provide apple disease and orchard management education to benefit the growers in western North Carolina. Through my laboratory experiments, I gained new skills and established a baseline sensitivity to pyraclostrobin and trifloxystrobin that will aid in screening for resistance in the future studies. Overall, this program has instilled a strong foundation of field and laboratory research skills that I will use as I begin my next chapter continuing my education in plant pathology.

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