1 Etiology, Epidemiology and Management of Fruit Rot Of

Etiology, Epidemiology and Management of Fruit Rot of Deciduous Holly in U.S. Nursery

Production

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

Shan Lin

Graduate Program in Plant Pathology

The Ohio State University

2018

Dissertation Committee

Dr. Francesca Peduto Hand, Advisor

Dr. Anne E. Dorrance

Dr. Laurence V. Madden

Dr. Sally A. Miller 1

Copyrighted by

Shan Lin

2018

Abstract

Cut branches of deciduous holly (Ilex spp.) carrying shiny and colorful fruit are popularly used for holiday decorations in the United States. Since 2012, an emerging disease causing the fruit to rot was observed across Midwestern and Eastern U.S. nurseries. A variety of other symptoms were associated with the disease, including undersized, shriveled, and dull fruit, as well as leaf spots and early plant defoliation. The disease causal agents were identified by laboratory processing of symptomatic fruit collected from nine locations across four states over five years by means of morphological characterization, multi-locus phylogenetic analyses and pathogenicity assays. Alternaria alternata and a newly described species, Diaporthe ilicicola sp. nov., were identified as the primary pathogens associated with the disease, and A. arborescens,

Colletotrichum fioriniae, C. nymphaeae, Epicoccum nigrum and species in the D. eres species complex were identified as minor pathogens in this disease complex. To determine the sources of pathogen inoculum in holly fields, and the growth stages of host susceptibility to fungal infections, we monitored the presence of these pathogens in different plant tissues (i.e., dormant twigs, mummified fruit, leaves and fruit), and we studied inoculum dynamics and assessed disease progression throughout the growing season in three Ohio nurseries exposed to natural inoculum over two consecutive years. Additionally, an outdoor container trial was conducted by artificially inoculating plant tissues using individual or combined pathogen inoculum at different stages of plant development (i.e., flower bud, full bloom, petal fall, immature fruit and mature fruit). In

ii nursery conditions, fruit rot pathogens were consistently isolated from all types of plant tissues analyzed. Mummified fruit and bark were found to be the main sources of primary inoculum, while leaf spots were identified as a source of secondary inoculum for fruit infections. Alternaria and

Colletotrichum had significantly higher isolation frequency after bloom, and peak inoculum capture by spore traps was observed during bloom. In the container trial, Diaporthe ilicicola inoculations during bloom and petal fall stages resulted in latent infections with symptoms developing when fruit reached maturity. In addition, all pathogens successfully infected mature wounded fruit. These results indicate that bloom is a critical stage to manage fruit infections and that implementing practices for fruit injury protection may lower disease levels in the field. This research represent the first step to understand this emerging fruit rot on deciduous holly and to build a foundation for further investigations. Further studies should be conducted to fully understand the effects of environmental parameters on seasonal inoculum dynamics and of host physiological factors contributing to disease development.

iii

Dedication

In memory of my grandmother, Cuifang Liu, whose endless love and support encouraged me to

be brave, strong, and to be myself.

Acknowledgments

I would like to express my sincere gratitude to my life-time advisor, Dr. Francesca Peduto

Hand for your guidance and support in the past six years since the establishment of your lab. Your wisdom and trust not only encouraged me to become a plant pathologist, but also to become a confident grown-up. How lucky I am to have had you as my advisor! Being your student is one of the best decisions made in my life!

I would like to thank my advisory committee members, Dr. Anne Dorrance, Dr. Larry

Madden and Dr. Sally Miller, for their guidance and constructive feedback throughout my research. I thank all the faculty, students, and staff in the Department of Plant Pathology, especially

Dr. Jason Slot and Emile Gluck-Thaler for their tremendous help on the description of the new

Diaporthe species. Special thanks to Dr. Tom Mitchell, Dr. Monica Lewandowski, Jim Chatfield,

Dr. Peg McMahon from the Department of Horticulture & Crop Science, as well as Mary Maloney from Chadwick Arboretum, whose enthusiasm and help made me spend a wonderful undergraduate time at Ohio State and become passionate about my dreams.

Many thanks to Dr. F. P. Trouillas for assistance with the new species description, and Drs.

J. R. Úrbez-Torres and G. Marchi for constructive feedback on my research manuscripts prior to submission. Additionally I would like to thank all the growers in Ohio, Pennsylvania, and

Massachusetts who provided fruit samples and field plots to conduct my research trials.

Many, many, thanks to all the members of the Hand Lab: Coralie Farinas and Isabel

Emanuel; former members Caterina Villari, Dana Martin, Maria Bellizzi, Veena Devi-Ganeshan; and the undergraduate students Paige Thrush, Nathan Gifford, Jenna Moore, Eric Warne, and

Sumner Lonseth, for their help in so many ways. Without you, I would have not been able to finish my PhD.

Last but not the least, I want thank my family and friends for their unconditional support and love. Thank you for always being by my side!

Vita

2013…………………………………. B.S. Agriculture, The Ohio State University, Magna

cum laude

2017.………………………………….M.S. Plant Pathology, The Ohio State University

2014-present………………………….Graduate Research Associate, Department of Plant

Pathology, The Ohio State University

Publications

Lin, S., and Peduto Hand, F. 2018. Determining the effects of inoculum concentration and

wounding on the development of fruit rot of winterberry holly. Phytopathology 108:S1.183.

Lin S., and Peduto Hand, F. 2018. Investigations on the timing of fruit infection by fungal

pathogens causing fruit rot of deciduous holly. Plant Disease First Look, retrieved from

https://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-06-18-0973-RE.

Lin S., Taylor, N. J., and Peduto Hand, F. 2018. Identification and characterization of fungal

pathogens causing fruit rot of deciduous holly. Plant Disease 102:2430-2445.

Lin, S., Martin, D. E., Taylor, N. J., Gabriel, C. K., Devi Ganeshan, V., and Peduto Hand F.

2018. First report of Phytophthora aerial blight caused by Phytophthora nicotianae on

Vinca, Lobelia and Calibrachoa in Ohio. Plant Disease 102:456.

vii

Lin, S., Taylor, N. J., and Peduto Hand, F. 2017. Determining the timing of host susceptibility to

infection by fungal pathogens associated with fruit rot disease of winterberry holly.

Phytopathology 107:S5.34.

Lin, S., Martin, D. E., Taylor, N. J., Gabriel, C. K., and Peduto Hand F. 2017. Occurrence of

Phytophthora chrysanthemi causing root and stem rot on garden mums in the United States.

Plant Disease 101:1060.

Lin, S., Taylor, N. J., and Peduto Hand, F. 2016. Identifying sources of inoculum and timing of

tissue infection by fungal pathogens associated with winterberry fruit rot. Phytopathology

106:S4.3.

Lin, S., Peduto Hand, Taylor, N.J., and Zondag, R. H. 2015. Understanding the emergent fruit rot

disease of Winterberry holly. Phytopathology 105:S4.109.

Fields of Study

Major Field: Plant Pathology

viii

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vii

List of Tables ...... xi

List of Figures ...... xiii

Chapter 1: Introduction ...... 1

1.1 THE FLORICULTURE INDUSTRY IN THE UNITED STATES ...... 1

1.2 USE OF ILEX SPP. IN ORNAMENTAL PLANT PRODUCTION ...... 3

1.3 FRUIT ROT: AN EMERGING DISEASE OF DECIDUOUS HOLLY ...... 8

1.4 OBJECTIVES ...... 15

Chapter 2: Identification and Characterization of Fungal Pathogens Causing Fruit Rot of

Deciduous Holly ...... 17

ABSTRACT ...... 17

INTRODUCTION ...... 18

MATERIALS AND METHODS ...... 20

RESULTS ...... 26

DISCUSSION ...... 32

ACKNOWLEDGMENTS ...... 40

Chapter 3: Investigations on the Timing of Fruit Infection by Fungal Pathogens Causing

Fruit Rot of Deciduous Holly ...... 70

ABSTRACT ...... 70

INTRODUCTION ...... 71

MATERIALS AND METHODS ...... 73

RESULTS ...... 79

DISCUSSION ...... 82

ACKNOWLEDGMENTS ...... 88

Chapter 4: Determining the Sources of Primary and Secondary Inoculum and Seasonal

Inoculum Dynamics of Fungal Pathogens Causing Fruit rot of Deciduous Holly .... 94

ABSTRACT ...... 94

INTRODUCTION ...... 95

MATERIALS AND METHODS ...... 97

RESULTS ...... 104

DISCUSSION ...... 108

ACKOWLEDGEMENTS ...... 115

References ...... 124

Appendix A. Supplementary Material for Chapter 4 ...... 137 x

List of Tables

Table 2.1. Fungal isolates recovered from symptomatic holly fruit used in this study for morphological characterization, phylogenetic analyses and pathogenicity tests...... 41

Table 2.2. Amplified loci, primers, PCR conditions and references for each fungal genus characterized in this study...... 45

Table 2.3. Reference sequences for Alternaria, Colletotrichum, Diaporthe and Epicoccum retrieved from GenBank that were included in the phylogenetic analyses...... 47

Table 2.4. Isolation frequency of the different fungi recovered from the 304 symptomatic holly fruit that yielded fungal growth between 2013 and 2017 across all sampling locations...... 55

Table 2.5. Conidial measurements and mycelial growth rate of select isolates of Alternaria, Colletotrichum, Diaporthe and Epicoccum retrieved from symptomatic winterberry fruit used in this study (Diaporthe ilicicola sp. nov. data not included)...... 56

Table 3.1. Fungal isolates used to inoculate plants in the container trial in 2016 and 2017...... 89

Table 3.2. Frequency of fungal isolation (%) from asymptomatic and symptomatic samples collected from the nursery at different stages of fruit development from 2016 to 2017...... 90

Table 3.3. Correlation between disease incidence and environmental variables in the nursery and container trials in 2016 and 2017...... 91

Table 4.1. Fungal isolation frequency from overwintering plant tissues, including bark, bud, xylem, and mummified fruit collected from nurseries A, B and C from 2015 to 2017...... 116

Table 4.2. Fungal isolation frequency from the 938 symptomatic leaves that yielded fungal growth from 2015 to 2017 across all three nurseries...... 117

Table 4.3. Measures of leaf spot incidence and severity used to compare disease development in nurseries A, B and C from 2015 to 2017...... 118

Table 4.4. Correlation between leaf spot incidence and severity and environmental variables in nurseries A, B and C from 2015 to 2017...... 119

Table 4.5. Results of the pathogenicity tests conducted on detached fruit using selected fungal isolates retrieved from symptomatic leaves collected in the three nurseries...... 120

Table 4.6. Results of the spore trapping studies conducted in the three nurseries from 2015 to 2017...... 121

Table A.1. Results of the pathogenicity tests conducted on detached leaves using selected fungal isolates retrieved from symptomatic leaves collected in the three nurseries...... 138

Table A.2. Colony forming unit of fruit rot pathogens in the spore trapping studies in nurseries A, B and C from 2015-2017...... 139

xii

List of Figures

Figure 2.1. Fruit rot symptoms observed on deciduous holly cultivar Sparkleberry in an Ohio nursery (A, healthy plants; B, overview of symptomatic berries on an entire plant; C, close-up of fruit exhibiting rot symptoms on twigs)...... 58

Figure 2.2. Maximum Likelihood phylogenetic tree based on the Kimura 2-parameter plus a gamma distribution model (Kimura 1980) obtained from combined endoPG, OPA1 and OPA10 datasets of Alternaria spp. The ML bootstrap support (BS) ≥70 and Bayesian posterior probability (PP) ≥0.95 values are indicated at the nodes (BS/PP). The tree was rooted with A. eichhorniae (CBS 489.92)...... 59

Figure 2.3. Morphology of Diaporthe ilicicola sp. nov. strain FPH2015502 (A, colony on PDA after 28 days at 23 °C; top and bottom of the plate; B, conidiogenous cells and alpha conidia; C, Alpha conidia; D, sporulation from conidiomata on PDA)...... 60

Figure 2.4. Maximum Likelihood analysis tree based on the General Time Reversible plus a gamma distribution model obtained from concatenated sequences of combined HIS, ITS and TUB datasets of Diaporthe spp. The ML bootstrap support (BS) ≥70 and Bayesian posterior probability (PP) ≥0.95 values are indicated at the nodes (BS/PP). The D. ilicicola sp. nov. isolates from this study are highlighted. The tree was rooted to Diaporthella corylina (CBS 121124)...... 61

Figure 2.5. Maximum Likelihood phylogenetic tree based on the Hasegawa-Kishino-Yano plus a gamma distribution model obtained from combined HIS, ITS and TUB datasets of Diaporthe spp. The ML bootstrap support (BS) ≥70 and Bayesian posterior probability (PP) ≥0.95 values are indicated at the nodes (BS/PP). The tree was rooted with D. nomurai (CBS 157.29)...... 64

Figure 2.6. Maximum Likelihood phylogenetic tree based on the Hasegawa-Kishino-Yano plus a gamma distribution model obtained from combined GADPH and TUB datasets of Colletotrichum spp. The tree with the highest log likelihood (-1486.05) is shown. The ML bootstrap support (BS) ≥70 and Bayesian posterior probability (PP) ≥0.95 values are indicated at the nodes (BS/PP). The tree was rooted with C. johnstonii (CBS 128532)...... 65

Figure 2.7. Phylogenetic tree from Maximum Likelihood analysis based on the Hasegawa- Kishino-Yano plus a gamma distribution model obtained from ITS datasets of Epicoccum spp. The ML bootstrap support (BS) ≥70 and Bayesian posterior probability (PP) ≥0.95 values are indicated at the nodes (BS/PP). The tree was rooted with E. keratinophilum (CBS 142455)...... 66 xiii

Figure 2.8. Symptoms and signs on detached holly fruit used in the pathogenicity experiments 10 days post-inoculation (A, Alternaria alternata; B, Colletotrichum fioriniae; C, Diaporthe ilicicola sp. nov.; D, Epicoccum nigrum)...... 67

Figure 2.9. Fruit rot incidence recorded on wounded and unwounded fruit inoculated in pathogenicity experiment C with individual and combined pathogens at all inoculum concentrations. Bars indicate 95% confidence intervals...... 68

Figure 2.10. Fruit rot severity recorded on wounded and unwounded fruit inoculated in pathogenicity experiment C with individual and combined pathogens at all inoculum concentrations. Bars indicate 95% confidence intervals...... 69

Figure 3.1. Rot symptoms on mature holly fruit developing from the stigmatic-end (A), calyx- end (B), and lenticels (C and D)...... 92

Figure 3.2. Stages of plant inoculation in the container trial that resulted in symptomatic fruit in 2016 and 2017, and corresponding disease incidence. Values with the same letter within the same inoculation timing are not significantly different according to Tukey’s HSD test (α = 0.05). Bars indicate standard error of the mean...... 93

Figure 4.1. Glass microscope slide coated with petroleum jelly used as a spore trap hanging from a deciduous holly tree...... 122

Figure 4.2. Healthy leaf (left) and examples of different degrees of leaf spot severity observed on holly leaves...... 123

xiv

Chapter 1: Introduction

1.1 THE FLORICULTURE INDUSTRY IN THE UNITED STATES

Ornamental horticulture originally arose as a hobby over 3000 years ago in Egypt

(McDaniel 1982). In the United States, the idea of gardening was introduced by western European settlers during the 17th century (McDaniel 1982). Since then, there was an increasing interest and demand for using plants for food, medicines, or decorations. The development of plant breeding and greenhouse production significantly contributed to the rapid growth of the floriculture industry

(McDaniel 1982). In 2015, the total wholesale value of floriculture crops in the U.S. was estimated at $4.37 billion, with the top five producing states (California, Florida, Michigan, North Carolina and Ohio) contributing 69 percent to the total value (NASS 2016). A total of 767 million square feet (71 million square meter) of covered production area and 5913 producers were recorded nationally (NASS 2016). The five most valuable floriculture crop categories produced were annual bedding/garden plants, potted flowering plants, foliage plants (indoor/patio use), herbaceous perennial plants, and propagative floriculture materials (NASS 2016). Cut flowers ranked 6th nationally with a wholesale value of approximately $374 million (NASS 2016). Cut flowers, which include flowers and attached plant parts but without roots, are widely used for bouquets, wreaths, or baskets etc. for weddings, funerals, holidays and many other special occasions (Armitage 1993).

There are over 200 kinds of cut flowers sold in the U.S. (Bonarriva 2003). The top three cut flowers measured in wholesale value in 2015 were lilies, tulips and gerbera daisies (NASS 2016). Woody

1 stem crops, including the subject of this study (Ilex spp.) are included in the specialty cut flower category among other floriculture crops (Larson 1992).

Cut flower crops can be propagated by seeds, bulbs, cuttings, or grafting. Whether they are grown for the fresh cut flower market or as preserved product, the crop production process is similar until the time of harvest and can take place in fields or in covered settings (e.g. greenhouse or high tunnel). For production in covered structures, growers sterilize soil or use soilless media before propagation. Plants are grown with appropriate nutrition, pruning, lighting and irrigation

(Bonarriva 2003; Teixeira da Silva 2006). Under controlled environments, air temperature and light intensity are often used to control plant quality and flowering time (McDaniel 1982; Teixeira da Silva 2006). For production in the field, soil type, drainage, water sources etc. are taken into consideration when selecting sites. Unlike controlled environments, only crops that can well adapt to the outdoor environment are selected for field production (Larson 1992). The optimal harvesting stage for the different crops depends on the plant genus, and it varies from when flowers are about to open to when they are fully open (Armitage 1993; Bonarriva 2003). The distance to the market is often taken into consideration when making harvesting decisions (Teixeira da Silva 2006). For preserved cut flowers, harvest takes place when plants reach the desired stage. After harvest, preserved cut flowers are treated with chemicals (e.g., glycerin) and dried completely, which allows them to maintain the shape and color of flowers, and makes them less perishable than fresh cut flowers (Armitage 1993; Bonarriva 2003; Larson 1992).

1.2 USE OF ILEX SPP. IN ORNAMENTAL PLANT PRODUCTION

1.2.1 Overview of the genus Ilex

Ilex L. is the only genus in the family Aquifoliaceae Bartl., comprising more than 400 evergreen and deciduous species, and is commonly called holly. The genus is widely distributed from temperate to tropical climate regions in both hemispheres (Burrows and Tyrl 2013; Galle

1997). Plants in this genus range from creeping to intermediate shrubs, to large trees (over 30 m tall). Holly is usually dioecious, and occasionally polygamodioecious [the majority of the flowers are dioecious with some bisexual or of the opposite sex flowers on the same plant (Fernald 1950)].

For female plants to produce fruit, male plants with similar flowering period are placed in the same field to ensure successful pollination (Galle 1997). The majority of holly species produce red fruit, while selected cultivars of some species have black (e.g., Ilex crenata Thunb. ‘Convexa’, and I. glabra Steyerm. ‘Shamrock’), yellow (e.g., I. aquifolium L. ‘Bacciflava’, and I. opaca Ait.

‘Canary’), or orange [e.g., I. decidua Walter ‘Red Cascade’, and I. verticillata (L.) A. Gary

‘Orange Beauty’] fruits, which are carried on the plant for three to six months of the year. Holly leaves can vary in color, shape and texture. Foliage can be bright green, dark green, to grayish, or variegated. Leaf shape varies from suborbicular to ovate with spines or serrated margins. Leaf texture can show significant variations from leathery, glabrous, rugose, to chartaceous (Galle

1997).

The diverse shapes and sizes of Ilex species along with the extensively long period of fruit display in the fall and winter seasons, make them popular for landscape use as foundation plants, hedges, topiary or bonsai. The fruit also represents an important food source for wildlife (Galle

1997). Many species of Ilex have been widely used to make drinks in different regions of the world.

In South America, leaves of Ilex paraguariensis St. Hilaire are brewed to make tea, also known as

“yerba matá” or “maté” (Dengler 1997). The tea made from I. guayusa Loes. leaves by the Indians is used as a substitute for coffee or tea (Dengler 1997). Native Americans brewed from roasted leaves of I. vomitoria Aiton to stimulate appetite, as well as to give agility and courage in war

(Dengler 1997). In addition, some species of Ilex have been used for medical purposes. For example, the Chinese holly (I. cornuta Lindl.) is used as a tonic for the kidneys, while I. verticillata is often used for intermittent fevers to substitute for the Peruvian bark, as well as for treating gangrene and skin eruptions (Dengler 1997).

Cut holly branches carrying brightly colored fruit are traditionally used for holiday decorations during Christmas and Thanksgiving in the United States. Gustav Teufel in Oregon was one of the first people to plant holly for cutting in the United States around 1890 (Galle 1997). In

2014, approximately 140,000 Ilex bunches were sold nationally by 42 operations, with an estimated combined wholesale and retail sale value of $571,279 (NASS 2014). The cut holly industry provides nursery growers with a sizable income during the off-season, while it also contributes to extended employment for seasonal employees assisting with harvesting, packing and shipping holly branches. These reasons, combined with the low maintenance requirements of the plant, make this crop highly desirable to growers.

1.2.2 Overview of deciduous holly

Deciduous holly species are classified into the subgenus Prinos L. Ilex decidua

(possumhaw holly), I. serrata Thunberg (Japanese winterberry), and I. verticillata (common winterberry) are the three most common species grown in North America, along with their many

4 cultivars and hybrids (i.e., I. serrata x I. verticillata). The male cultivar Apollo and female cultivar

Sparkleberry were the first crosses of I. serrata x I. verticillata developed by William F. Kosar in

1960 (Galle 1997).

Most deciduous holly are medium shrubs to large trees up to 30 m tall carrying red fruit, while some selected species and cultivars produce black, yellow or orange fruits (Galle 1997).

Deciduous holly is well established in sites with good drainage, full sun or partial shade, as well as fertile, slightly acidic (i.e., pH 6.0 - 6.5) and loamy soils. It is more hardy than the evergreen species in USDA Plant Hardiness Zones 4 – 9. With the proper plant selection, fruit can persist on the plant until late winter (Galle 1997).

In general, holly is a slow-growing plant with 5-15 cm growth per year. Twigs are glabrous or pubescent with conspicuous lenticels on the current season’s growth. Spurs branchlets are often found on deciduous holly. Leaves are usually ovate to elliptic, some species have obovate or lanceolate shape with leaf margin ranging from serrulate to serrate to crenate, and approximately

2 – 20 cm long and 1 – 7 cm wide. Deciduous holly has chartaceous or coriaceous leaves with prominent veins. The solitary axillary inflorescence is in simple or compound cymes. Flowers are white except for Ilex serrata, which has white to reddish pink flowers. The shape of deciduous holly fruit is globose or depressed globose, averaging 6 – 8 mm in diameter. Some species, such as I. kiangsiensis (S. Y. Hu) C. J. Tseng & B. W. Liu and I. macrocarpa Oliver, have larger fruit

10 – 16 mm in diameter. During fruit development, the outer layer of the ovary becomes chartaceous exocarp, the middle layer becomes fleshy mesocarp, and the inner layer becomes coriaceous endocarp. The endocarps develop into separate compartments. One single seed is contained in each endocarp (i.e., pyrene). The deciduous species usually have 4 – 13 pyrenes, with

5 smooth, striate (i.e., with fine longitudinal lines) or sulcate (i.e., with longitudinal grooves or furrows) surface. Therefore, from a strict botanical sense, the fruit of Ilex is not a berry nor a drupe, and there is not any accepted terminology to describe this type of fruit. Although it has been called

“multiseeded drupe” or “bacco-drupe” by some authors, there is no consent on the use of either term in the literature. Therefore, throughout this dissertation we will refer to the fruit of Ilex using the general term “fruit” (Bauers 1993; Galle 1997).

Bud break of deciduous holly occurs in early spring and is followed by formation of leaves.

Flowers are produced on the current-season’s growth from mid- to late spring. The blooming time varies depending on the species and cultivar, and can last 10 to 14 days. Therefore, it is important to have both male and female plants with similar flowering periods in the field to ensure pollination. It is recommended to have one male plant for every 10 to 20 female plants (Galle

1997). After petal fall, the fruit starts to develop from an ovary, turns color in early fall, fully matures in mid-fall, and persists on the plant throughout winter or until eaten by wildlife. However, fruit of Ilex serrata x I. verticillata can turn black or shrivel if frost or cold temperatures reaches below -12 °C (Galle 1997). Natural plant defoliation occurs in late fall. Nursery growers start to harvest branches when the fruit reaches final size and color and the majority of the leaves have fallen off. Heavy cutting could have a dwarfing effect on the plant, or even take the plant out of fruit production for years. Therefore, in the commercial deciduous holly industry, growers harvest one quarter to one third of the branches yearly. Besides branch removal by harvesting, plants can also benefit from removing dead branches or from renewal pruning during dormancy in late winter or early spring (Galle 1997).

1.2.3 Common diseases of Ilex

A wide range of pathogens have been reported to cause leaf and twig diseases on multiple

Ilex species. Cylindrocladium avesiculatum D. L. Gill, Alfieri & Sobers causes leaf spots and twig dieback of multiple Ilex evergreen species, including I. cornuta, I. crenata, I. opaca, and I. vomitoria (Gill et al. 1970). Macroderma curtisii (Berk. & Ravenel) Höhn. causes tar spots on

American holly (I. opaca) leaves, which are characterized by reddish brown spots with a thin yellow border (Luttrell 1940). Phytophthora cinnamomi Rands was reported to cause leaf spots and root rot on I. glabra in Virginia (Moorman et al. 2004). Two other Phytophthora species, P. ilicis Buddenh. & Roy A., and P. citrophthora (R. E. Sm. & E. H. Sm.) Leonian, caused leaf and twig blight as well as defoliation on I. aquifolium in Mediterranean islands (Scanu et al. 2014) and the Pacific Northwest (Pirone 1978), and blight and brown rot on Ilex spp. in nursery production

(Leonberger et al. 2010). An outbreak of Fusarium acuminatum Ellis & Everh. resulted in defoliation, canker and dieback on I. aquifolium in Norway in 2015 (Strømeng et al. 2016). More recently, F. proliferatum (Matsush.) Nirenberg caused trunk canker on Ilex cornuta in China (Feng and Li 2018). In addition, other fungal pathogens such as Alternaria spp. Nees, Cercospora ilicis

Ell., Gloeosporium ilicis Dearn., and Phyllosticta terminalis Ell. & Martin etc., were reported causing leaf spots and canker on Ilex spp. (Alfieri et al. 1984; Pirone 1978).

Several root rots have been reported on Ilex. These diseases can be problematic in nursery production due to the extensive use of soil and the presence of a large number of various crops

(Baker 1957). Pathogens are readily spread through infested soil, containers or equipment, infected propagative materials, and watering (Baker 1957). Plants affected by root rot diseases often become chlorotic, and show stunted growth, twig dieback, and early defoliation (Galle 1997).

Common causal pathogens include Rhizoctonia solani Kuhn on I. cornuta, I. crenata and I. opaca

(Alfieri et al. 1984), Thielaviopsis basicola (Berk. & Broome) Ferraris on I. crenata (Lambe and

Wills 1980; Wick and Moore 1983), and species of Phytophthora de Bary and Pythium Pringsh. on I. cornuta and I. crenata (Alfieri et al. 1984; Grand 1985; Jung et al. 2016).

In addition to biotic diseases, several abiotic problems can be observed on Ilex. Proper species and cultivar selection based on the hardiness zone is critical to avoid weather injuries.

Deciduous species are generally hardier than evergreen species. Symptoms of winter damage include leaf browning, leaf scorching, twig death, or even plant death. Injury to young and succulent leaves may be caused by exposure to direct and hot sunlight or by leaf spines puncturing adjacent leaves (e.g., I. aquifolium and I. opaca), which results in the development of yellowish to light brown leaf spots on the upper leaf surface (Galle 1997). Although these wounds may not have direct effects on plant health, secondary pathogens may invade plants through these lesions. In selected cultivars of Ilex opaca, purple leaf blotch can sometime be observed due to genetic characteristics (Galle 1997).

1.3 FRUIT ROT: AN EMERGING DISEASE OF DECIDUOUS HOLLY

1.3.1 Disease symptoms and initial diagnostic work

Since 2012, many deciduous holly growers in the Midwestern and Eastern United States have been confronted with a fruit rot of unspecified etiology. Affected plants show brown to black leaf spots throughout the canopy and defoliate earlier in the year (September instead of October) compared to healthy plants. Prior to harvest, the fruit is undersized, shriveled, has failed to turn color and lost gloss, eventually becoming rotten. As a result, branches become unsaleable causing

8 a degree of damage ranging from decreased crop yield to complete crop loss, with a direct effect on nursery profitability. In some nurseries, plants failing to produce healthy fruit in consecutive seasons have to be cut back, with significant negative consequences on productivity for many years.

In the winter of 2013, at the time of branch harvest, severe fruit rot was observed across three Ohio nurseries, which prompted growers to submit symptomatic samples to the C. Wayne

Ellett Plant and Pest Diagnostic Clinic at The Ohio State University (OSU PPDC) for diagnosis.

The involvement of bacteria and oomycetes was ruled out through the diagnostic process, while multiple fungi were isolated from the fruit lesions, including species of Alternaria Nees,

Colletotrichum Corda, Diaporthe Nitschke and undetermined genera in the Botryosphaeriaceae

Theiss. & P. Syd. These findings provided the foundation for the studies described in this dissertation.

1.3.2 Literature review

Disease etiology. Species in the fungal genera initially isolated from symptomatic holly fruit are notoriously known to cause fruit rot on many economically important crops. For example, multiple species of Alternaria can cause fruit rot on apple (Gao et al. 2013), blueberry (Zhu and

Xiao 2015), and pomegranate (Luo et al. 2017). Species of Colletotrichum are reported on apple

(Munir et al. 2016), almond (McKay et al. 2009), and strawberry (Howard et al. 1992); while species of Diaporthe can affect blueberry (Milholland and Daykin 1983), citrus (Huang et al.

2015), grape (Lorenzini and Zapparoli 2018), and pepper (Zhang et al. 2016). Finally, fruit rot by

Botryosphaeriaceous fungi is reported on cranberry (Weidemann and Boone 1983), apple (Biggs

2004; Brown and Britton 1986) and peach (Brown and Britton 1986).

Studies on the etiology of cranberry fruit rot conducted in New Jersey (Stiles and

Oudemans 1999) and Michigan (Olatinwo et al. 2003; Olatinwo et al. 2004) found that the disease was caused by a fungal complex of more than 15 species, comprising some of the same fungal genera that were isolated from the holly fruit samples processed by the OSU PPDC (i.e.,

Botryosphaeria, Colletotrichum, and Diaporthe). However, no official study has been carried out on deciduous holly to further our understanding of the involvement of the aforementioned fungal genera in the disease.

Sources of inoculum. Multiple studies have identified that mummified fruit, as well as buds, twigs and leaves from the previous season can serve as sources of inoculum for fruit infections during the next growing season. For example, mummified fruit of olive (Moral and Trapero 2012), sour cherry (Stensvand et al. 2013), and almond (Förster and Adaskaveg 1999; McKay et al. 2014) affected by anthracnose served as the major source of primary inoculum of Colletotrichum spp. In the case of leaf blotch and fruit spot of apple and brown spot of mandarin caused by Alternaria spp. (Bassimba et al. 2014; Harteveld et al. 2014; Tanaka et al. 1989), as well as Phomopsis cane and leaf spot of grape (Anco et al. 2012; Cucuzza and Sall 1982), buds, leaves and twigs were also identified as sources of inoculum. Unlike other fruit crop systems, in commercial holly production only a third of the crop (i.e. whole branches carrying fruit) is harvested each winter, as heavy pruning may have a negative effect on future fruit production. If not eaten by wildlife, the remaining fruit left in the field eventually becomes mummified (Galle 1997) potentially serving

10 as a site for pathogens to overwinter and thus potentially constituting a source of inoculum to initiate infections during the new season.

Although fruit health is the primary interest of growers, extensive leaf spots and early defoliation are also consistently observed in the field throughout the growing season. The fungi isolated from the initial samples processed by the OSU PPDC could also infect leaves in addition to fruit, thus serving as a source of inoculum for other tissue infections throughout the current season (Leandro et al. 2001; Talhinhas et al. 2011; Yoshida et al. 2007). Indeed, the major site of infection for Alternaria spp. is the leaf (Rotem 1994). Leaf spots are also reported to be caused by

Colletotrichum spp. on blueberry (Yoshida et al. 2007) and almond (McKay et al. 2014), as well as by Diaporthe on grapes and Pachira glabra (Milagres et al. 2018; Wilcox et al. 2015).

Removal of infected plant tissues from the field has been suggested in many systems as a standard disease management practice to lower inoculum levels (Harteveld et al. 2014; Holb and

Scherm 2007; Moral and Trapero et al. 2012; Villarino et al. 2010). Identification of the sources of overwintering inoculum in deciduous holly fields as well as monitoring the development of leaf spots over time to understand if multiple infection events are carried out during the season, are important steps to undertake to ultimately facilitate the generation of effective disease management strategies that can be implemented by growers.

Growth stage(s) of host susceptibility to infection. The initial diseased samples sent to the

OSU PPDC were collected from holly plants at the end of the 2013 growing season. However, the stage of fruit development at which infection had occurred was unknown. The growth stages during which the host is susceptible to infection by some of the fungi isolated from the symptomatic holly fruit have been extensively studied in many crop systems. It was reported that

11 infections by Alternaria, Colletotrichum or Diaporthe could occur at any stage of fruit development, from bloom until fruit maturation, on a wide range of crops, such as almond, apple, blueberry, mango, mandarin, melon, peach, pear, and persimmon (Adaskaveg and Hartin 1997;

Binyamini and Schiffmann-Nadel 1972; Dayken and Milholland 1984; Halfon-Meiri and Rylski

1983; Harteveld et al. 2014; Johnson et al. 1992; Li et al. 2007; McKay et al. 2014; Nemsa et al.

2012; Ohsawa and Kobayashi 1989; Prusky et al. 1981 and 1983; Verma et al. 2007; Zaitlin et al.

2000). Moreover, studies on cranberry fruit rot caused by a fungal complex including some of the same fungal genera revealed that the disease was dynamic as the frequencies of the predominant fruit-rotting pathogens varied over time (Olatinwo et al. 2003; Olatinwo et al. 2004; Stiles and

Oudemans 1999). This suggests that sample collection throughout the season rather than a one- time collection at the end of the season, as well as monitoring of disease progression throughout the season, could provide valuable information in order to properly determine the timing of holly susceptibility to infections and the role of each pathogen in the disease.

Although plants might be susceptible to infections at different growth stages, species of

Alternaria, Botryosphaeria, Colletotrichum and Diaporthe were previously reported to cause latent infections on many fruit crops (Davidzon et al. 2010; Johnson et al. 1993; Munir et al. 2016;

Rotem 1994). Latent infection is defined as follows: “the actual infection has taken place, though macroscopically not yet visible, but further growth of the infection hypha is delayed” (Verhoeff

1974). Latent infections caused by Alternaria were reported on mango (Prusky et al. 1983) and persimmon (Prusky et al. 1981). Colletotrichum is well known as a postharvest pathogen causing bitter rot of apple (Munir et al. 2016), and anthracnose of blueberry (Hartung et al. 1981), strawberry (King et al. 1997) and olive (Moral and Trapero 2012), where plants infected in the

12 field prior to harvest, decay when fruit becomes mature. Species of Diaporthe including D. viticola

Nitschke (Phomopsis viticola) and P. mangiferae S. Ahmad were reported to cause latent infections on grape and mango, respectively (Johnson et al. 1992; Pscheidt and Pearson 1989).

These pathogens infected plants during bloom and remained latent until fruit ripened, at which point the fruit developed symptoms.

Holly fruit rot symptoms in the initial samples processed by the OSU PPDC were observed late in the season when the fruit was fully mature. While it is currently unknown whether the fungi causing the disease behave quiescently in deciduous holly fruit, the possibility of the above pathogens causing latent infections reinforces the importance of monitoring disease development throughout the growing season.

Inoculum dynamics and environmental conditions. There is a high risk of infection if the host is exposed to pathogen inoculum during its susceptible periods. Therefore, it is equally critical to determine inoculum dynamics throughout the growing season. Spore traps are considered a fast and simple means to monitor seasonal fungal spore abundance. Many types of spore traps have been developed to quantify airborne spores. Passive trapping is the simplest method to collect spores, which consists of a glass microscope slide or a thin glass rod or tube covered by a thin layer of sticky adhesive (e.g., petroleum jelly, silicon grease; Lacey and West 2006). However, these traps cannot be used directly to quantify spores in the air (Lacey and West 2006) and must be followed by further processing. Active spore trapping devices draw air and collect spores using a sticky substance-coated surface (e.g., cascade impactor, Burkard trap, or whirling arm trap; Hirst

1952; May 1945; Perkins 1957), culture medium (e.g., Anderson sampler; Anderson 1958), liquid

(e.g., liquid impingers; May 1966), or air (e.g., virtual impactor; Kauppinen et al. 1999). Power

13 supply in the field is needed to continuously charge these active spore-trapping devices.

Alternatively, some portable spore trap devices that are battery powered can be used, although they have a shorter period of usage and thus must be replaced frequently (Gregory 1954; Lacey and West 2006). A simple and inexpensive spore trap consisting of a glass microscope slide covered by a thin layer of petroleum jelly (Vaseline) on both sides has been widely used in epidemiological studies carried out in vineyards and fruit orchards in Mediterranean-type regions

(Bassimba et al. 2014; Eskalen and Gubler 2001; Trouillas et al. 2012; Úrbez-Torres et al. 2010).

Not only is this type of spore trap simple to use, but it also allows retrieval of the fungal isolates for further species identification, providing an advantage over volumetric spore-trap types.

Many studies have demonstrated that different environmental factors, including precipitation and temperature, have an effect on seasonal pathogen inoculum dynamics and host infection (Bassimba et al. 2014; Eskalen et al. 2013; Harteveld et al. 2014; Rotem 1994). For example, spore-trapping studies of Botryosphaeria spp. on grapevines found that there was a high correlation between spore release and occurrence of rainfall or irrigation events (Úrbez-Torres et al., 2010). Varied spore numbers over time may be due to rain events in sour cherry and olive anthracnose (Stensvand et al. 2017; Talhinhas et al. 2010). Also, rainfall and temperature had effects on infection epidemics of almond and olive anthracnose (McKay et al. 2014; Moral and

Trapero 2012), and Alternaria leaf blotch and fruit spot incidence on apple (Harteveld et al. 2014).

The relationship between environmental factors and the abundance of pathogen inoculum, as well as fruit infection in deciduous holly fields should be investigated to determine the effects of meteorological variables on seasonal inoculum dynamics and disease development.

1.4 OBJECTIVES

Disease management recommendations should rely on knowledge of the disease-causing agents as well as of disease epidemiology. To date, the different components of the disease triangle

(pathogen-host-environment) have not been investigated. However, identifying the different factors that contribute to disease development on deciduous holly are key steps to develop appropriate research-based management recommendations for growers. Thus, the major objectives and supporting hypotheses of my research project were:

Objective 1: To identify and characterize the fungal pathogen(s) associated with fruit rot

of deciduous holly in the United States;

Hypothesis: Fruit rot of deciduous holly is caused by a fungal complex, including

species of Alternaria, Botryosphaeriaceae, Colletotrichum, and Diaporthe;

Objective 2: To determine the potential source(s) of primary and secondary inoculum in

nursery fields;

Hypothesis 1: Fruit rot of deciduous holly is a polycyclic disease;

Hypothesis 2: The sources of primary inoculum for pathogens causing the disease

are the overwintering plant tissues, including twigs and mummified fruit; and the

sources of secondary inoculum are leaf spots;

Objective 3: To determine the growth stage(s) of the fruit that is/are susceptible to fungal

infections;

Hypothesis: Deciduous holly is susceptible to fruit rot pathogens’ infection

throughout the fruit development period;

Objective 4: To determine seasonal inoculum dynamics throughout the plant growing season;

Hypothesis: Field inoculum levels of fruit rot pathogens fluctuates throughout the

season and peak inoculum levels coincide with the fruit growth stages that are

susceptible to fungal infections;

Objective 5: To identify the effects of environmental variables on disease development and seasonal inoculum dynamics in the field;

Hypothesis 1: Disease progression is negatively correlated to temperature;

Hypothesis 2: Water-dispersed inoculum release is positively correlated with the

occurrence of rain events;

Objective 6: To identify appropriate management strategies;

Hypothesis 1: Removal of sources of primary inoculum will help lowering disease

levels in the field;

Hypothesis 2: Fungicides applied at susceptible fruit and leaf developmental stages

will help lowering disease levels in the field.

Chapter 2: Identification and Characterization of Fungal Pathogens Causing Fruit Rot of Deciduous Holly1

ABSTRACT

Cut branches of deciduous holly (Ilex spp. L.) harboring colorful berries are traditionally used as ornaments in holiday decorations. Since 2012, a fruit rot of unspecified cause has resulted in significant yield reduction and economic losses across Midwestern and Eastern U.S. nurseries. In this study, symptomatic fruit samples collected from nine different locations over five years were analyzed, and several fungal species were isolated. A combination of morphological characterization, multi-locus phylogenetic analyses and pathogenicity assays revealed that

Alternaria alternata and Diaporthe ilicicola sp. nov. were the primary pathogens associated with symptomatic fruit. Other fungi including A. arborescens, Colletotrichum fioriniae, C. nymphaeae,

Epicoccum nigrum and species in the D. eres species complex appeared to be minor pathogens in this disease complex. In detached fruit pathogenicity assays testing the role of wounding and inoculum concentration on disease development, disease incidence and severity increased when fruit was wounded and inoculated with a higher inoculum concentration. These findings indicate that management strategies that can protect fruit from injury or reduce inoculum may lower disease levels in the field. This research established the basis for further studies on this emerging disease

1Copyright The American Phytopathological Society. Reproduced, by permission, from Lin, S., Taylor, N. J., and Peduto Hand, F. 2018. Identification and characterization of fungal pathogens causing fruit rot of deciduous holly. Plant Disease 102:2430-2445. 17 and the design of research-based management strategies. To our knowledge, it also represents the first report of species of Alternaria, Colletotrichum, Diaporthe and Epicoccum causing fruit rot of deciduous holly.

INTRODUCTION

Members of the genus Ilex are deciduous or evergreen ornamental shrubs or trees, ranging in height from 1 to over 30 m (Galle 1997). The deciduous species are popularly used in the cut flower industry for winter holiday decorations because of the beautiful display of colorful fruit they present, varying from red to orange or yellow depending on the hybrid and cultivar, that can persist on the plants throughout the winter (Figure 2.1A). The overall sale value of Ilex spp. in the cut flower industry is estimated at $571,279 nationally (NASS, 2014). Sales of the cut woody stems allows revenue in late fall and early winter when there is little activity and little other income for this segment of the nursery industry, also contributing to extended employment for seasonal employees.

The three most common deciduous holly species grown in North America are Ilex decidua

(possumhaw holly), I. serrata (Japanese winterberry), and I. verticillata (common winterberry), along with their many cultivars and hybrids. Plants are functionally polygamodioecious

[mostly dioecious, but with either a few flowers of the opposite sex or a few bisexual flowers on the same plant (Fernald 1950)], requiring both male and female plants with similar flowering periods in the same field for pollination. The fruit of Ilex is composed of chartaceous exocarp, juicy mesocarp and separated coriaceous endocarp. From a botanical perspective, it is neither a berry nor a drupe, and there is a lack of specific terminology for its designation. Although the term

“bacco-drupe” was proposed by Hu in 1950 (Galle 1997), there has been a lack of agreement on the use of this term. Therefore, the general term “fruit” will be used throughout this manuscript to refer to the fruit of Ilex.

Since 2012, a fruit rot of unspecified cause has been challenging holly growers across the

Midwestern and Eastern United States. Affected plants defoliate earlier in the season (September rather than October) and carry undersized fruit that fails to turn color, loses its normal gloss, shrivels, and eventually becomes necrotic (Figure 2.1B and C). Some popular cultivars, including

Bonfire, Sparkleberry and Winter Red, are particularly affected. As a consequence, nursery growers are reporting decreased crop yield and, in some cases, complete crop loss. While an official value that quantifies these losses is not available in the literature, in the winter of 2014 several Ohio growers who participated in this project lost their entire crop to this fruit rot (Peduto

Hand, personal observation).

In the winter of 2013, at the time of branch harvest, a few symptomatic fruit samples from three Ohio nurseries were sent to the C. Wayne Ellett Plant and Pest Diagnostic Clinic at The Ohio

State University for diagnosis. The involvement of bacteria and oomycetes was ruled out through the diagnostic process but several different fungi were isolated from the symptomatic tissues, belonging to the genera Alternaria Nees, Colletotrichum Corda, Diaporthe Nitschke. and undetermined genera in the Botryosphaeriaceae Theiss. & P. Syd. Several species within these fungi are well known and economically important fruit rot pathogens that have been extensively studied on many fruit crops, including apple (Biggs 2004; Munir et al. 2016; Gao et al. 2013), blueberry (Milholland and Daykin 1983; Zhu and Xiao 2015), cranberry (Olatinwo et al. 2003)

19 and strawberry (Curry et al. 2002; Howard et al. 1992). To date, there is no official record of the occurrence of the above fungal genera as pathogens causing fruit rot on deciduous holly.

Understanding this disease problem, including elucidating the components of the disease triangle (pathogen-host-environment), is a critical step towards providing nursery growers with research-based recommendations for effective management. Thus, the initial objectives of our investigation were to (i) confirm identification of the fungal pathogen(s) associated with the disease from samples collected over multiple years and across multiple locations by conducting morphological and molecular studies, and (ii) determine factors that might be contributing to fruit infection. Future research will build upon these findings and will focus on elucidating the sources of pathogen inoculum in the field, the timing of host susceptibility to infection, as well as the role of several environmental factors in disease development. This information will be critical to identify potential management measures, including appropriate timing for horticultural practices and fungicide treatments.

MATERIALS AND METHODS

Sample collection and fungal isolation: Between 2013 and 2017, 623 symptomatic fruit of deciduous holly comprising cultivars Bonfire, Oosterwijk, Sparkleberry and Winter Red were received or collected from commercial nurseries, landscapes and arboreta across the Midwestern and Eastern U.S., including Ohio (n=593), Massachusetts (n=10), North Carolina (n=15) and

Pennsylvania (n=5). Samples received from out of state were processed immediately upon receipt, while those collected from field sites in Ohio were transported to the laboratory within a cooler and then kept at 4°C for a maximum of 24 hours before being processed. Samples were surface

20 disinfected with 2% Clorox® household bleach (The Clorox Company) for 2 min, then rinsed in sterile water three times. Following disinfection, samples were air dried on sterile paper towels in a laminar flow hood. Two pieces of symptomatic tissue comprising the exocarp and mesocarp were excised from the edge of each lesion with a sterile scalpel and transferred to Potato Dextrose

Agar (PDA; Difco Laboratories, Sparks, MD) amended with 1% tetracycline hydrochloride and

1.5% streptomycin sulfate (Fisher Scientific, Fair Lawn, NJ). All plates were incubated at 25°C under constant light for up to 10 days. Plates were checked daily and any observed fungal growth was sub-cultured onto PDA. Pure cultures of each isolate were obtained by transferring hyphal tips from the margin of the fungal colonies onto new PDA plates. All isolates were then grown on sterile filter paper and stored at -20°C until further use.

Morphological characterization: Isolates were first identified to genus based on comparison of morphological characteristics of the spores and spore-producing structures with descriptions available in the literature (Barnett and Hunter 1998). Two to 14 isolates of each of the most represented fungal genera (i.e. >4% isolation frequency) were then selected to include all locations (n=9) and years (n=5) of sampling and subject to further characterization (Table 2.1).

Pure cultures of all isolates except Alternaria spp. were transferred to fresh PDA and incubated at

23°C with 12h photoperiod for up to four weeks to induce sporulation. Isolates tentatively identified as Alternaria spp. were grown on V8 juice agar as described by Simmons (2007). The length and width of 30 randomly selected conidia from each isolate included in the study (Table

2.1) were measured using a Leica DM750 compound microscope (Leica Microsystems CMS

GmbH, Wetzlar, Germany) and the Leica Application Suite EZ software (version 3.0.0). Mean and standard error of the conidial measurements were calculated for each isolate. Conidial color,

21 shape, and presence or absence of septation, were also recorded. Additionally, fungal colony diameter on each of three plates per fungal isolate was measured three, five and seven days post- inoculation (DPI) to calculate mycelial growth rates (millimeters day-1). All data were analyzed independently using PROC GLM in SAS (version 9.4; SAS Institute Inc., Cary NC). Tukey’s honest significant difference (α=0.05) was used for mean comparisons among isolates.

DNA extraction, amplification and sequencing: Total genomic DNA was extracted from pure cultures of isolates grown on PDA at 25°C for 7 days using the DNeasy® Plant Mini Kit

(Qiagen Inc. Hilden, Germany) according to manufacturer's instructions. All PCR amplifications were carried out in a final volume of 20 μL using the Bio-Rad T100™ Thermal Cycler (Bio-Rad

Laboratories, Inc., Hercules, CA). All amplified loci, primers, and PCR conditions are summarized in Table 2.2. Amplified PCR products were resolved by electrophoresis at 100 V/cm in 1.5% agarose gels in 1 x Tris-borate-EDTA buffer stained with 0.005% GelRed® (Biotium Inc.,

Fremont, CA) and visualized under a UV transilluminator. The size of the fragments was estimated using a 1kb DNA ladder (New England BioLabs Inc., Ipswich, MA). PCR products were purified using ExoSAP-IT® (Affymetrix, Inc., Santa Clara, CA) according to manufacturer's instructions and both strands were sequenced at the Genomics Shared Resource at the Ohio State University

Comprehensive Cancer Center using an ABI Prism 3730 DNA analyzer (Applied Biosystems,

Foster City, CA). Sequences were manually edited using the software Chromas (v 2.6.4,

Technelysium Pty Ltd, Australia) and assembled using MUSCLE in MEGA7 (Kumar et al. 2016).

Consensus sequences of each locus were exported and compared to those available in GenBank using the Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology

Information, Bethesda, MD).

Phylogenetic analyses: The same isolates selected for morphological characterization were subject to phylogenetic analyses (Table 2.1). Sequences of reference strains recently published in the literature were retrieved from GenBank and included in the analysis for comparison [Table 2.3 (Andrew et al. 2009; Damn et al. 2012; Dissanayake et al. 2017; Du et al.

2016; Gomes et al. 2013; Hung et al. 2013; Huang et al. 2015; Machingambi et al. 2015; Mostert et al. 2001; Peever et al. 2004; Rotondo et al. 2012; Tan et al. 2013; Thomopson et al. 2015;

Udayanga et al. 2012; Udayanga et al. 2014a; Udyanga et al. 2014b; Udayanga et al. 2015;

Valenzuela-Lopez et al. 2018; Woudenberg et al. 2015; Yang et al. 2017)]. Concatenated sequences of different loci for each fungal genus (endoPG, OPA1 and OPA10 for Alternaria; TUB and GADPH for Colletotrichum; ITS, HIS and TUB for Diaporthe) were aligned using MUSCLE in MEGA7 (Kumar et al. 2016). (Kumar et al. 2016). The nucleotide substitution model for each dataset was determined using FindModel

(https://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html) based on the Akaike

Information Criterion (AIC). Each concatenated dataset was subjected to Maximum Likelihood

(ML) analysis in MEGA7 and to Bayesian inference (BI) analysis using MrBayes as implemented in siMBa (Mrshra and Thines 2014; Ronquist and Huelsenbeck 2003). The heuristic method in

ML analysis used Subtree-Pruning-Regrafting with SPR level 5, and branch strength was evaluated using 1000 bootstrap replications. All gaps and missing data were deleted. For BI, a Metropolis

Coupled Markov Chain Monte Carlo (MCMCMC) analysis was carried out with two independent runs with four chains each for one million generations. The analysis was stopped when the average standard deviation of split frequencies fell below 0.01. Trees were sampled every 500 generations

23 and the first 25% of samples were discarded as “burnin”. Trees were visualized in FigTree v. 1.4.3

(Rambaut 2016).

Representative sequences for each locus and pathogen species included in this study were deposited into GenBank (Table 2.1). All isolate cultures are maintained in the fungal collection of the Department of Plant Pathology at The Ohio State University. The novel taxon described in this study was also deposited in the Westerdijk Fungal Biodiversity Institute Collection (CBS-KNAW,

Utrecht, The Netherlands) and registered in MycoBank (www.mycobank.org).

Pathogenicity tests: Three sets of pathogenicity experiments, each conducted twice, were carried out to further characterize the isolates included in Table 2.1. In the first experiment (A), healthy fruit were collected from potted plants of cultivar Sparkleberry and surface-disinfested as previously described. Ten individual fruit were used for each isolate. Each fruit was wounded using a sterile needle and point inoculated using a micropipette with eight μL of a sterile water-

Tween 20 solution (0.05% v/v) containing 105 conidia/mL of each isolate. Ten wounded fruit inoculated with the same amount of sterile water-Tween 20 solution served as controls. All fruit were placed on a plastic tray in a completely randomized design and the entire tray was enclosed in a moist chamber made of a clear plastic bag containing a wet sterile paper towel. Moist chambers were incubated at 25°C for up to four weeks and fruit rot incidence was recorded for each isolate.

Visual examination and identification of spores on symptomatic tissues as well as fungal re- isolation and morphological identification of the isolates were performed to fulfill Koch’s postulates.

One isolate per fungal species was selected from those used in experiment A. In two additional experiments (B and C), fruit were wounded, inoculated, and incubated as previously

24 described. In both experiments wounded and unwounded fruit inoculated with sterile water served as controls. Experiment B was conducted to test the effects of spore concentration and wounding on fruit infection. Experiments were carried out using a series of spore suspensions of each fungal species ranging from 103 to 106 conidia/mL. Inoculum was prepared by suspending spores from

7- to 21-day old PDA cultures in sterile distilled water and by adjusting the concentration using a hemacytometer. Ten individual fruit per treatment were arranged in a complete randomized block design with six blocks. Fruit incidence and severity were recorded weekly for up to six weeks post inoculation. Finally, experiment C was conducted to test the effects of combined pathogen inoculum, spore concentration and wounding on fruit infection. Spore suspensions at a concentration of 102, 103 and 105 conidia/mL were initially obtained for each fungal species. For each spore concentration, two pathogen species were combined (1:1 v/v) to obtain the fungal treatment. All combinations of fungal species and spore concentrations were tested using six single fruit per treatment arranged in a complete randomized block design with six blocks. Disease evaluation was the same as previously described.

In all experiments, treatment effects were analyzed using PROC MIXED in SAS (version

9.4; SAS Institute Inc., Cary NC). Combined datasets from the two runs of each experiment were analyzed when results were in congruence. All data were arcsine transformed before statistical analyses were performed. Treatment means and 95% confidence intervals were calculated in a transformed scale and back-transformed to present results. Least squares means (LS-means)

(α=0.05) were used for mean comparisons among treatments.

RESULTS

Sample collection and fungal isolation: Three hundred and four symptomatic fruit samples out of the 623 processed resulted in the isolation of a total of 121 fungal isolates. Species of Diaporthe and Alternaria were the most frequently isolated fungi (39.47% and 32.89%, respectively), followed by the genera Colletotrichum, Epicoccum Link, Cladosporium Link,

Botryosphaeriaceae, Fusarium Link and Phoma Sacc. (<10%; Table 2.4). In a few cases, two fungi were isolated from the same lesion (Table 2.4). No fungal growth was observed from the remaining 319 samples.

Isolate characterization: For clarity of reporting, combined morphological and molecular characterization of the isolates is reported below.

Alternaria spp. Fourteen isolates tentatively identified as Alternaria sp. were assigned to four morphological groups based on colony and conidial morphology as well as sporulation patterns (Barnett and Hunter 1998). Ten isolates in the first group developed distinctive concentric rings of growth and sporulation. Isolates produced a primary chain of 3-11 conidia and abundant lateral secondary chains of 2-5 conidia. Conidia were brown, ellipsoid with 3-6 transverse septa and sometimes one longitudinal septum. Characteristics were consistent with the description of A. alternata (Fr.) Keissl. (Simmons 2007). Two isolates (FPH2015470 and FPH2015591) in the second group formed long chains of 4-9 conidia and occasionally secondary chains of 2-4 conidia.

Conidia were wide ellipsoid or ovoid, constricted, with 4-7 transverse septa and rarely 1 longitudinal septum. Isolates in this group were tentatively identified as A. tenuissima (Nees)

Wiltshire (Simmons 2007). One isolate (FPH2015338) in the third group was characterized by short primary chains of 4-7 conidia and occasionally secondary chains of 1-3 conidia. Conidia

26 were wide ellipsoid or ovoid, constricted, and with 3-6 transverse septa and 1-2 longitudinal septa.

These characteristics were consistent with the description of A. toxicogenica E. G. Simmons

(Simmons 2007). One isolate (FPH2015395) in the fourth group produced long conidiophores and primary chains of 3-7 conidia and abundant secondary and tertiary chains of 1-3 conidia. Conidia were ellipsoid with 3-5 transverse septa and 0-2 longitudinal septa. This isolate was tentatively identified as A. arborescens E. G. Simmons (Simmons 2007). The concatenated datasets of three loci (endoPG, OPA1 and OPA10) were analyzed using ML and BI and similar tree topologies were obtained. Thirty-one sequences were included in the phylogenetic analysis consisting of 17 reference strains (Table 2.3; Andrew et al. 2009; Peever et al. 2004; Rotondo et al. 2012;

Woudenberg et al. 2015) and 14 isolates from this study. Isolate FPH2015395 grouped with A. arborescens reference strain CBS 102605 with a high bootstrap support value (BS=96) and a high

Bayesian posterior probability (PP=0.97). All other isolates clustered with several A. alternata reference sequences (BS=75, PP=0.96; Figure 2.2).

Diaporthe spp. In total, 13 isolates were identified as Diaporthe spp. Of these, three isolates (FPH201322, FPH2015394 and FPH2017229) were characterized by white, fluffy, aerial mycelium, grey to brown on the reverse side. Pycnidia in culture were dark brown to black, globose or subglobose, erumpent, solitary or aggregated, arranged in concentric rings. Creamy to yellow conidial masses were globose or exuded in cirrhi. Alpha conidia were hyaline, aseptate, smooth, guttulate and ellipsoid. Beta conidia were hyaline, aseptate, slightly curved, hamate or filiform and less common than alpha conidia. Even though these three isolates had similar morphological characteristics, the width of both alpha and beta conidia of isolate FPH2017229 was significantly smaller (Table 2.5; significance reference not shown). All three isolates were tentatively assigned

27 to the D. eres Nitschke species complex (Baumgartner et al. 2013; Dissanayake et al. 2015;

Udayanga et al. 2014b). The 10 remaining isolates presented morphological characteristics that differed from the three isolates previously described as well as from other Diaporthe species described in the literature, including those previously reported from Ilex spp. (Dissanayake et al.

2017; Du et al. 2016; Gomes et al. 2013; Huang et al. 2013; Huang et al. 2015; Machingambi et al. 2015; Mostert et al. 2001; Tan et al. 2013; Thomopson et al. 2015; Udayanga et al. 2015;

Wehmeyer 1933; Yang et al. 2017). Detailed morphological features of these isolates are reported in the new species description below (Figure 2.3).

Two sets of multi-locus phylogenetic analyses were conducted for Diaporthe spp. isolates.

In the first analysis, the concatenated HIS, ITS and TUB datasets consisted of 102 isolates from

82 taxa (Dissanayake et al. 2017; Du et al. 2016; Gomes et al. 2013; Huang et al. 2013; Huang et al. 2015; Machingambi et al. 2015; Mostert et al. 2001; Tan et al. 2013; Thomopson et al. 2015;

Udayanga et al. 2012; Udayanga et al. 2014a; Udyanga et al. 2014b; Udayanga et al. 2015; Yang et al. 2017). In both ML and BI analyses, the 13 isolates from this study grouped into three different clades (Figure 2.4). Three isolates (FPH201322, FPH2015394 and FPH2017229), in accordance with their morphology, clustered within the D. eres species complex, and grouped with reference strains CBS 138594, CBS 287.74, CBS 370.67 and CBS 587.79. The remaining ten isolates clustered with strong support (BS=85 and PP=1) as a unique and unnamed clade sharing a strongly supported phylogenetic relationship (BS=99 and PP=1) near the reference strain of D. cf. heveae

1 Petch (CBS 852.97; Figure 2.4). Morphological and molecular data revealed that these 10 isolates represented a unique and undescribed species of Diaporthe with no apparent species name.

Thus, we hereby propose a new species name to properly identify this unique taxon:

Diaporthe ilicicola S. Lin, Taylor & Peduto Hand, sp. nov.

MycoBank No. MB824842

Etymology – named according to the host it was first isolated from, namely Ilex verticillata x Ilex serrata.

Conidiomata pycnidial, dark brown to black, erumpent, solitary or aggregated, ostiolate, creamy to light salmon conidial mass globose or exuding in cirrhi, arranged in circle at the edge of colony. Conidiophores hyaline, subcylindrical, reduced to conidiogenous cells. Conidiogenous cells hyaline, subcylindrical, tapering towards the apex, 7.64-14.14 × 1.9-3.82 μm (av. ± SD =

11.2 ± 0.4 × 2.7 ± 0.14 μm). Alpha conidia hyaline, aseptate, smooth, ovate to ellipsoid, guttulate, on PDA 7.13-9.43 × 2.91-4.16 μm (av. ± SD = 8.41 ± 0.1 × 3.63 ± 0.05 μm). Beta conidia not observed. Sexual morph not observed.

Culture characteristics – Colonies on PDA at 23 °C with 12h photoperiod slow growing,

2.89 ± 0.08 mm/day, flat and compact, irregular edge, originally white to olive, turning to greyish brown while aging, reverse dark brown.

Host - Ilex verticillata x Ilex serrata

Distribution - United States

Specimens examined. United States, Ohio, Madison, on rotten fruit of Ilex verticillata x

Ilex serrata, cultivar Bonfire, December 2015, Shan Lin, holotype FPH2015502 (CBS 144318).

In the second analysis, the concatenated HIS, ITS and TUB datasets consisted of 39 isolates from 16 taxa that are within or closely related to the D. eres species complex (Gomes et al. 2013;

Gao et al. 2016; Udayanga et al. 2014b) and three Diaporthe isolates from this study (FPH201322,

FPH2015392 and FPH2017229). According to the new analysis, isolates FPH201322 and

FPH2015392 grouped with D. eres reference strains (BS=99, PP=1.0 and BS=97, PP=0.95, respectively), while isolate FPH2017229 grouped with a reference strain of D. cf. nobilis (CBS

587.79; BS=90, PP=0.99; Figure 2.5).

Colletotrichum spp. Eight isolates tentatively identified as Colletotrichum spp. were divided into two morphological groups. All but one isolate belonged to the first group and were characterized by cottony mycelium that was light grey to grey on the upper side of the plate, and brownish pink with abundant scattered melanized spots on the reverse side. Orange conidial masses formed in the center of the colony after 21 days. Conidia were hyaline, aseptate, fusiform to cylindrical, and slightly pointed at one or both ends. These features were consistent with the description of C. fioriniae (Marcelino & Gouli) Pennycook (Shivas and Tan 2009). The isolate in the second morphological group (FPH2015590) presented white to grey colored mycelium on the upper side of the plate and salmon on the reverse side. Conidia were hyaline, aseptate, fusiform to cylindrical with one or both ends slightly acute. This isolate was identified as C. nymphaeae (Pass.)

Aa (Velho et al. 2015). DNA from all Colletotrichum isolates was successfully amplified using C. acutatum species complex-specific primers targeting the ITS region, and sequence analysis confirmed that they belonged to the C. acutatum species complex (McKay et al. 2009). In the multi-gene phylogenetic analyses of combined GAPDH and TUB datasets using ML and BI, isolates grouped into two clades. Isolate FPH2015590 clustered with C. nymphaeae strain CBS

515.78, while the rest of the isolates clustered with C. fioriniae strains CBS 128517, CBS 200.35 and CBS 293.67 (Damn et al. 2012; Figure 2.6).

Epicoccum spp. Two isolates tentatively identified as Epicoccum sp. were characterized by yellow to orange colonies producing an orange to brown diffusible pigment in PDA. Dark

30 pigmented conidia were produced in sporodochia, and were multicellular and globose to pyriform.

These features were consistent with the description of Epicoccum nigrum Link (Mahadevakumar et al. 2014; Wu et al. 2017). Both ML and BI analyses using the ITS gene were in accordance with the results of morphological characterization as both isolates from this study clustered with E. nigrum reference strains (Chen et al. 2017; Valenzuela-Lopez et al. 2018; Figure 2.7).

Pathogenicity tests: In experiment A, all isolates of Alternaria alternata, Colletotrichum fioriniae, Diaporthe ilicicola sp. nov. and Epicoccum nigrum were able to infect detached wounded mature holly fruit. Brown to black lesions developed from the inoculation point on all fruit inoculated with Alternaria and Colletotrichum isolates two DPI (=100% incidence; data not shown). After three to five days, lesions enlarged to cover the whole fruit and sporulation occurred on the surface of the fruit (Figure 2.8A and 8B). Diaporthe ilicicola sp. nov. caused a light salmon discoloration of the fruit one week post-inoculation. Pycnidia exuding light yellow conidial masses

(Figure 2.8C) formed on the entire fruit 14 DPI (=100% incidence; data not shown). Isolates of D. eres induced similar symptoms but after a longer period of time (three to four weeks). Fruit rot incidence for D. eres isolates FPH201322, FPH2015394 and FPH2017229 was 45%, 70% and

65%, respectively (data not shown). Epicoccum isolates were able to induce similar symptoms to those caused by Alternaria and Colletotrichum; however, lesions remained limited to the point of inoculation (Figure 2.8D) and disease incidence was relatively lower overall (60%; data not shown).

In experiment B, all four pathogens, regardless of the concentration of inoculum tested, were able to infect both wounded and unwounded detached fruit (data not shown). Disease incidence and severity on wounded fruit, however, were significantly higher than on unwounded

31 fruit. On wounded fruit, disease incidence was above 88% for all pathogens 16 DPI (data not shown). Except for Epicoccum, disease severity increased along with inoculum concentration.

Epicoccum inoculations resulted in low fruit rot severity, ranging from 5% to 11% at all concentrations (data not shown). On unwounded fruit, symptoms started to develop 24 DPI. No correlation pattern was observed between disease incidence and inoculum concentration. Disease incidence and severity for all treatments 42 DPI were 33%-66% and 5%-41%, respectively.

Among all the pathogen-inoculum combinations tested, Diaporthe ilicicola sp. nov. at 106 conidia/mL caused the highest fruit rot incidence and severity.

In experiment C, all pathogen inoculum combinations at all concentrations were able to infect both wounded and unwounded fruit (Figure 2.9 and 2.10). However, disease incidence and severity of combined pathogens on wounded and unwounded fruit were not significantly different from those infected by individual pathogens (Figure 2.9 and 2.10). With the exception of E. nigrum, disease incidence and severity on wounded fruit for all the pathogen-concentration treatments were significantly higher than on unwounded fruit. Regardless of the presence/absence of wounding and the type of pathogen inoculum, fruit infected with 105 conidia/ml resulted in significantly higher fruit rot incidence and severity compared to that inoculated with 103 and 102 conidia/ml. No rot symptoms developed on fruit in the control treatments in any of the three sets of experiments.

DISCUSSION

Fruit rot is an emerging problem on deciduous holly that in recent years has been responsible for limiting crop production in ornamental nurseries across the Midwestern and

Eastern U.S. Rot symptoms are characterized by black lesions on the surface of the fruit that eventually cause the whole fruit to soften and deteriorate. This study represents the first attempt to elucidate the etiology of this disease through characterization of the fungi associated with symptomatic tissues.

Seven different fungal species including Alternaria alternata, A. arborescens,

Colletotrichum fioriniae, C. nymphaeae, Diaporthe ilicicola sp. nov., D. eres and Epicoccum nigrum that were commonly associated with symptomatic deciduous holly samples were studied using morphological, phylogenetic and pathogenicity characterizations. Among these, A. alternata and Diaporthe ilicicola sp. nov. were predominant. Overall, Colletotrichum and Epicoccum species were only occasionally isolated from fruit lesions, indicating that their role in the disease complex may be limited. Findings were supported by extensive sampling over multiple years and locations, including commercial nurseries, landscapes and arboreta, mainly in Ohio but also in a few other eastern U.S. states.

The different fungal species recovered from the symptomatic samples did not seem to be associated with a specific sampling location. Instead, multiple species were found within the same planting at all locations except one arboretum in North Carolina, where only Diaporthe ilicicola sp. nov. was isolated. In particular, A. alternata and D. ilicicola sp. nov. were found within the same planting from more than half of the sampling locations across Ohio, Pennsylvania and

Massachusetts (data not shown). These findings contribute to supporting our conclusion that A. alternata and Diaporthe ilicicola sp. nov. are the primary pathogens associated with fruit rot of deciduous holly.

Symptoms caused by the individual pathogens were not distinct. Additionally, fungi were not always recovered from rotten fruit, which indicates that some of the symptoms may have been due to other causes, possibly including bacterial infection, environmental stresses, physiological disorders, or mechanical injury. It has been observed that discoloration or blackening of deciduous holly fruit may occur due to cold weather or frost (Galle 1997). In our study, we consistently observed that early defoliation was associated with fruit rot in the field. Loss of leaves could expose fruit directly to the sunlight and possibly result in sunscald (Barber 1971). In this study, however, sunscald on fruit was not observed. Olatinwo et al. (2003) also found no fungi associated with some rotten fruit in the cranberry fruit rot disease complex, and speculated that it could be due to physiological breakdown or mechanical injury. While the focus of this study was to characterize the fungal pathogens associated with fruit rot of deciduous holly, the roles of other factors causing fruit rot should be further investigated.

Morphological characteristics were used to initially assign the isolated fungi to different genera. Within each genus, detailed morphological comparisons allowed us to separate isolates into several groups. Alternaria isolates in this study were identified as small-spored and grouped into four morphological groups: A. alternata, A. arborescens, A. tenuissima and A. toxicogenica mainly based on conidial morphology and sporulation patterns (Simmons 2007). However, it was impossible to rely solely on morphological characteristics for species identification because of the extensive number of species, overlapping morphological characteristics and variable growth conditions. Several studies emphasize the importance of using combined morphological and molecular characterization for accurate identification of Alternaria species (Andrew et al. 2009;

Armitage et al. 2015; Harteveld et al. 2013; Lawrence et al. 2013). In this study, we selected the

34 endopolygalacturonase gene (endoPG) and two anonymous genomic regions OPA1-3 and OPA10-

2 that were successfully used in previous studies to distinguish small-spored Alternaria species

(Andrew et al. 2009; Peever et al. 2004; Woudenberg et al. 2015) and we used concatenated sequences of these three loci to carry out phylogenetic analyses. Isolates morphologically identified as A. arborescens and A. toxicogenica, clustered with reference strains of these same species included in the analysis. However, isolates morphologically identified as A. alternata and

A. tenuissima distributed throughout the phylogenetic tree and were not grouped into any specific clade. These results were in agreement with other studies that used multi-locus phylogenetic analyses in which A. alternata and A. tenuissima could not be separated (Andrew et al. 2009;

Cabral et al. 2017; Luo et al 2017; Woundenberg et al 2015). Therefore, Andrew et al. (2009) proposed the separation of A. alternata and A. arborescens, but combined other morphospecies including A. alternata, A. tenuissima and A. toxicogenica into one single species, namely A. alternata. A recent taxonomic study by Woundenberg et al. (2015) using multi-locus phylogeny supported the aforementioned study and suggested merging 35 morphospecies of Alternaria into

A. alternata. Accordingly, we concluded that species of Alternaria associated with fruit rot of deciduous holly included A. alternata and A. arborescens.

The use of a combination of morphological and multi-locus phylogenetic analyses is strongly suggested in the literature to delimit species boundaries of Diaporthe (Udayanga et al.

2012 and 2014; Gomes et al. 2013; Santos et al. 2017). In our study, morphological characterization and phylogenetic studies of concatenated sequences of three loci (ITS+HIS+TUB) were used to identify Diaporthe spp. associated with fruit rot of deciduous holly. Several species of Diaporthe have been previously reported from dead twigs or leaves of Ilex spp., including D.

35 eres, D. hongkongensis R.R. Gomes, C. Glienke & Crous, D. ilicis (Ellis & Everh.) Wehm., D. oncostoma (Duby) Fuckel, D. oxyspora (Peck) Sacc., D. pardalota (Mont.) Nitschke ex Fuckel and D. rudis (Fr.) Nitschke (Cash et al. 1952; Gao et al. 2016 and 2017; Gomes et al. 2013; Guba and Stevenson 1963; Wehmeyer 1933). However, only a few sequences of reference strains of these species from Ilex are available in the literature [i.e. D. eres (CBS 370.67 and CBS 694.94);

D. oncostoma (CBS 809.85); Gomes et al. 2013]. For D. rudis and D. hongkongensis, sequences of isolates from Ilex were not available; therefore the ex-type and ex-epitype of these two species

(CBS 113201 and CBS 115448) were included in the analyses (Gomes et al. 2013). D. ilicis, D. oxyspora and D. pardalota were originally reported from Ilex in 1933. Sequences of these three species, regardless of the host on which they were found, were also not available and were not included in the phylogenetic studies. As a result of multi-locus ML and BI analyses, ten of 13 isolates included in the present study did not cluster with any of the above species, but grouped together in an unnamed monophyletic clade closely related to a reference strain of D. cf. heveae 1

(CBS 852.97) isolated from Heveae brasiliensis in Brazil. Sequences from a total of six D. cf. heveae isolates, comprising unpublished sources, were initially included in the phylogenetic analysis and subsequently removed to include published sources only. It is to be noted that even in the initial analysis all the isolates from our study grouped in a different clade. The two D. cf. heveae reference isolates that were left (CBS 852.97 and CBS 681.84), however, were reported to be sterile (Gomes et al. 2013) so morphological comparisons between isolates was not feasible.

Additionally, morphological characterizations of our ten isolates differed from the original descriptions of D. ilicis, D. oxyspora and D. pardalota available in the literature (Wehmeyer 1933).

At the time this manuscript is being prepared, no new record of morphological or molecular

36 characterization of these three species has been found. Additionally, these species had not been found to infect fruit of Ilex, either. Based on detailed morphological characterization and molecular analysis of our isolates we hereby propose a new species description, namely Diaporthe ilicicola sp. nov. The genus Diaporthe consists of species that range from host specific to those with a wide host range. In this study, only Ilex fruit was inoculated to test pathogenicity of our Diaporthe ilicicola sp. nov. isolates. Cross-inoculation of D. ilicicola sp. nov. on other fruit crops may provide valuable information for further species characterization.

The remaining three Diaporthe isolates included in our study were identified as belonging to the D. eres species complex based on both morphological and molecular analyses. To better understand the relationship of these three isolates within the D. eres species complex, a second phylogenetic analysis was conducted using additional reference strains that were within or closely related to the D. eres species complex. According to the new analysis, two of the three isolates

(FPH201322 and FPH2015394) clustered with D. eres reference strains, including one recovered from Ilex (CBS 370.67), while one isolate (FPH2017229) grouped with D. cf. nobilis (CBS

587.79). It is to be noted that alpha and beta conidia of FPH2017229 were significantly narrower compared to FPH201322 and FPH2015394, possibly indicating species differences. Our findings are in agreement with previous phylogenetic studies where separate clades for D. eres and D. nobilis were obtained (Baumgartner et al. 2013; Gomes et al. 2013; Lawrence et al. 2015), but are in disagreement with a study that specifically addressed the D. eres species complex (Udayanga et al. 2014b), in which D. nobilis was not recognized as a separate species. It is to be noted that in the latter case, ITS was not included in the multi-locus phylogenetic analysis as this gene was considered having too much variability within the genus and was discordant to other trees

37 generated in the study. While the analysis of additional loci may be needed to further differentiate our three isolates within the D. eres species complex, due to the relatively low frequency of isolation of these fungi in our study, we did not pursue additional identification. Both D. eres and

D. nobilis are reported as minor pathogens causing blight, cankers, leaf spots and fruit rot on a broad range of host families of woody plants worldwide (Baumgartner et al. 2013; Choi et al.

2017; Farr and Rossman 2018; Kaliterna et al 2012; Lawrence et al. 2015; Li et al. 2017; Lorenzini and Zapparoli 2018; Thomidis and Michailides 2009; Zhang et al. 2016). Indeed, in our pathogenicity experiments, D. eres isolates caused relatively lower fruit rot incidence compared to D. ilicicola sp. nov. and it took a longer period of time for the inoculated fruit to develop symptoms. All D. ilicicola sp. nov. isolates tested resulted in 100% disease incidence with symptoms visible in less than a week, compared to 45-70% incidence seen for D. eres that took

21-28 days to become evident. Based on these observations, fungi in the D. eres species complex should be considered minor pathogens causing fruit rot of deciduous holly.

Selected isolates belonging to the two predominantly isolated pathogen species (A. alternata and D. ilicicola sp. nov.), along with possibly minor pathogens such as C. fioriniae and

E. nigrum, were included in three sets of pathogenicity experiments that not only tested their ability to cause disease but also aimed to understand the role of wounding and inoculum concentration on disease development. All isolates were able to infect detached mature holly fruit and induce rot symptoms, and all were re-isolated from the symptomatic tissues, which fulfilled Koch's postulates. Disease incidence and severity levels however, were overall much higher in fruit that had been wounded compared to unwounded fruit, indicating that fruit injury, either biotic or abiotic in nature, could result in higher disease levels in the field. Higher inoculum concentration always

38 resulted in more severe disease, suggesting that practices that can decrease inoculum concentration

(e.g., removal of infected plant material, pruning or chemical applications) could lower disease levels in the field. Since occasionally multiple pathogens were isolated from the same lesion on the original field samples, individual inoculum and combination of pathogens were also tested.

Similar disease incidence and severity resulted from single vs. combined pathogen inoculations. It is to be acknowledged that all inoculation experiments in this study were conducted in the laboratory where fruit was maintained in a controlled environment at optimal temperature and humidity levels, which may have favored pathogen activity. However, a separate set of inoculations whose results are partially reported elsewhere (Lin et al. 2017), were made in the winter of 2016 on wounded and unwounded fruit of deciduous holly cultivar Sparkleberry maintained outdoor in a container trial. In that experiment, fruit was inoculated with some of the same isolates used in the detached fruit assays reported in this study, and then naturally exposed to fluctuating environmental conditions. While disease incidence was overall lower in the outdoor trial, all four pathogens (A. alternata, C. fioriniae, D. ilicicola sp. nov. and E. nigrum) successfully infected wounded mature fruit, caused the same symptoms observed on the detached fruit assays, and were re-isolated from the lesions, which fulfilled Koch's postulates. However, no infection was observed on unwounded fruit in planta. Further studies investigating the possible pathways of fruit infection in natural settings are underway and will complement these preliminary observations.

This study identified and characterized various fungal pathogens associated with fruit rot of deciduous holly, which represents a first step in the understanding of this disease complex.

Further research is being conducted to investigate the sources of pathogen inoculum in the field,

39 to understand when pathogens infect the plant during the growing season, and to identify the environmental factors that favor disease development. Precise information about the disease cycle and disease epidemiology is needed to ultimately provide nursery growers with effective management recommendations.

ACKNOWLEDGMENTS

This study was funded by the Ohio Department of Agriculture Specialty Crop Promotion

Program (Awards AGR-SCG-14-08 and AGR-SCG-16-09), the USDA-NIFA Hatch project

#1004939, the T. J. Kavanagh Foundation, and The Ohio State University Department of Plant

Pathology. The research described in this paper represents a portion of the dissertation submitted by S. Lin to the Office of Graduate Studies of The Ohio State University to partially fulfill requirements for the Ph.D. degree in Plant Pathology. The authors thank all the winterberry growers in Ohio, Pennsylvania, and Massachusetts who provided many of the fruit samples used in this study. Additionally, we would like to thank Dr. F. P. Trouillas for assistance with the new species description, and Drs. J.R Urbez-Torres and G. Marchi for critical review of this manuscript prior to submission.

Table 2.1. Fungal isolates recovered from symptomatic holly fruit used in this study for morphological characterization, phylogenetic analyses and pathogenicity tests.

Species Origina Year Isolateb Host, cultivar GenBank Accession No.c EPG OPA1 OPA10 Alternaria alternata MA 2015 FPH2015390 Ilex verticillata, Oosterwijk MH003571 MH003585 MH003599 A. alternata MA 2015 FPH2015396 I. verticillata, Oosterwijk MH003573 MH003587 MH003601 A. alternata OH 2015 FPH2015338 I. verticillata x I. serrata, MH003570 MH003584 MH003598 Sparkleberry A. alternata OH 2015 FPH2015468 I. verticillata x I. serrata, MH003574 MH003588 MH003602 Bonfire A. alternata OH 2015 FPH2015470 I. verticillata x I. serrata, MH003575 MH003589 MH003603

41 Sparkleberry

A. alternata OH 2015 FPH2015507* I. verticillata x I. serrata, MH003576 MH003590 MH003604 Sparkleberry A. alternata OH 2015 FPH2015567 I. verticillata x I. serrata, MH003577 MH003591 MH003605 Sparkleberry A. alternata OH 2015 FPH2015589 I. verticillata x I. serrata, MH003578 MH003592 MH003606 Bonfire A. alternata OH 2015 FPH2015591 I. verticillata x I. serrata, MH003579 MH003593 MH003607 Bonfire A. alternata OH 2015 FPH2015592 I. verticillata x I. serrata, MH003580 MH003594 MH003608 Bonfire A. alternata OH 2016 FPH2015597 I. verticillata x I. serrata, MH003581 MH003595 MH003609 Sparkleberry A. alternata OH 2016 FPH2016709 I. verticillata x I. serrata, MH003582 MH003596 MH003610 Bonfire Continued

Table 2.1 continued Species Origin Year Isolate Host, cultivar GenBank Accession No. EPG OPA1 OPA10 A. alternata OH 2016 FPH2016711 I. verticillata x I. serrata, MH003583 MH003597 MH003611 Bonfire A. arborescens MA 2015 FPH2015395 I. verticillata, Oosterwijk MH003572 MH003586 MH003600 GAPDH TUB Colletotrichum MA 2015 FPH2015392 I. verticillata, Oosterwijk MH003615 MH003623 fioriniae C. fioriniae OH 2013 FPH201317 I. verticillata x I. serrata, MH003613 MH003621 Sparkleberry C. fioriniae OH 2013 FPH201318 I. verticillata x I. serrata, MH003614 MH003622 Sparkleberry C. fioriniae OH 2015 FPH2015466* I. verticillata x I. serrata, MH003616 MH003624

42 Bonfire

C. fioriniae OH 2015 FPH2015564 I. verticillata x I. serrata, MH003617 MH003625 Bonfire C. fioriniae OH 2016 FPH2016720 I. verticillata x I. serrata, MH003619 MH003627 Sparkleberry C. fioriniae OH 2013 FPH201314 I. verticillata x I. serrata, MH003612 MH003620 Sparkleberry C. nymphaeae OH 2015 FPH2015590 I. verticillata x I. serrata, MH003618 MH003626 Bonfire HIS ITS TUB Diaporthe eres MA 2015 FPH2015394 I. verticillata, Oosterwijk MH003629 MH003632 MH003635 D. eres OH 2013 FPH201322 I. verticillata x I. serrata, MH003628 MH003631 MH003634 Bonfire D. eres PA 2017 FPH2017229 I. verticillata x I. serrata, MH003630 MH003633 MH003636 Bonfire Continued

Table 2.1 continued Species Origin Year Isolate Host, cultivar GenBank Accession No. HIS ITS TUB D. ilicicola sp. nov. OH 2015 FPH2015472 I. verticillata x I. serrata, MH171082 MH171062 MH171072 Sparkleberry D. ilicicola sp. nov. OH 2015 FPH2015473 I. verticillata x I. serrata, MH171083 MH171063 MH171073 Sparkleberry D. ilicicola sp. nov. OH 2015 FPH2015502* I. verticillata x I. serrata, MH171084 MH171064 MH171074 Bonfire D. ilicicola sp. nov. OH 2015 FPH2015509 I. verticillata x I. serrata, MH171085 MH171065 MH171075 Sparkleberry D. ilicicola sp. nov. OH 2015 FPH2015558 I. verticillata x I. serrata, MH171086 MH171066 MH171076 Sparkleberry D. ilicicola sp. nov. OH 2015 FPH2015598 I. verticillata x I. serrata, MH171087 MH171067 MH171077

43 Sparkleberry

D. ilicicola sp. nov. NC 2016 FPH2016671 Ilex sp. MH171088 MH171068 MH171078 D. ilicicola sp. nov. OH 2016 FPH2016702 I. verticillata x I. serrata, MH171089 MH171069 MH171079 Bonfire D. ilicicola sp. nov. OH 2016 FPH2016706 I. verticillata x I. serrata, MH171090 MH171070 MH171080 Bonfire D. ilicicola sp. nov. OH 2016 FPH2016725 I. verticillata x I. serrata, MH171091 MH171071 MH171081 Bonfire ITS Epicoccum nigrum OH 2015 FPH2015417* I. verticillata x I. serrata, MH003637 Bonfire E. nigrum OH 2015 FPH2015505 I. verticillata x I. serrata, MH003638 Bonfire

a MA = Massachusetts; NC = North Carolina; OH = Ohio; PA = Pennsylvania.

b Isolates with an asterisk (*) were selected for use in pathogenicity experiments B and C. c endoPG = endopolygalacturonase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HIS = histon H3; OPA1 and OPA10 = anonymous noncoding region; TUB = β-tubulin; ITS = internal transcribed spacer.

Table 2.2. Amplified loci, primers, PCR conditions and references for each fungal genus characterized in this study.

Fungal genus Locusa Primers (fw/rev) PCR conditions References Primers PCRb Alternaria endoPG PG3/PG2b 95°C 1 min; (95°C 30 s, 57°C Andrew et al. 2009 Andrew et al. 2009 30 s, 72°C 30 s) x 35 cycles; 72°C 7 min OPA1 OPA1-3L/OPA1-3Rb 94°C 1 min; (94°C 20 s, 56°C Peever et al. 2004 Peever et al. 2004 30 s, 72°C 40 s) x 35 cycles; 72°C 7 min OPA10 OPA10-2L/OPA10-2R 95°C 1 min; (95°C 30 s, 57°C Andrew et al. 2009 Andrew et al. 2009 30 s, 72°C 30 s) x 35 cycles;

45 72°C 7 min

Colletotrichum ITS CaInt2/ITS4 95°C 5 min; (95°C 30 s, 60°C Mckay et al. 2009; Mckay et al. 2009 30 s, 72°C 40 s) x 30 cycles; White et al. 1990 72°C 5 min TUB T1/Bt-2b 95°C 4 min; (95°C 30 s, 55°C Glass & Donaldson Damn et al. 2012 30 s, 72°C 45 s) x 35 cycles; 1995; O’Donnell & 72°C 7 min Cigelnik 1997 GADPH GDF/GDR 95°C 1 min; (95°C 30 s, 57°C Templeton et al. Damn et al. 2012 30 s, 72°C 30 s) x 35 cycles; 1992 72°C 7 min Diaporthe ITS ITS5/ITS4 94°C 2 min; (94°C 30 s, 58°C White et al. 1990 Gomes et al. 2013 60 s, 72°C 60 s) x 40 cycles; 72°C 3 min Continued

Table 2.2 continued Fungal genus Locus Primers (fw/rev) PCR conditions References Primers PCR HIS CYLH3F/H3-1b 95°C 1 min; (95°C 30 s, 62°C Crous et al. 2004; Gomes et al. 2013 60 s, 72°C 60 s) x 40 cycles; Glass & Donaldson 72°C 3 min 1995 TUB T1/CYLTUB1R 95°C 1 min; (95°C 30 s, 60°C Crous et al. 2004; Gomes et al. 2013 60 s, 72°C 60 s) x 40 cycles; O’Donnell & 72°C 3 min Cigelnik 1997 Epicoccum ITS ITS1/ITS4 94°C 5 min; (94°C 45 s, 57°C White et al. 1990 White et al. 1990 45 s, 72°C 90 s) x 35 cycles; 72°C 10 min

a endoPG = endopolygalacturonase, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, HIS = histon H3, OPA1 and OPA10 = anonymous noncoding region, TUB = β-tubulin, ITS = internal transcribed spacer. bIn this study, DreamTaq DNA Polymerase (5U/μL) (Thermo Scientific, Waltham, MA) was used in all the PCR reactions.

Table 2.3. Reference sequences for Alternaria, Colletotrichum, Diaporthe and Epicoccum retrieved from GenBank that were included in the phylogenetic analyses.

Species Straina Origin Host GenBank Accession No.b endoPG OPA1-3 OPA10-2 Alternaria alternata CBS 102596 USA Citrus jambhiri KP124030 AY295047 KP124637 A. alternata CBS 102598 USA Citrus jambhiri AY295026 AY295048 KP124638 A. alternata CBS 102600 USA Citrus reticulata KP124033 AY295042 KP124640 A. alternata CBS 102602 Turkey Minneola tangelo AY295023 AY295052 KP124641 A. alternata CBS 102604 Israel Minneola tangelo KP124035 AY631443 KP124643 A. alternata CBS 104.26 Unknown Unknown KP123995 - KP124603 A. alternata CBS 106.24 USA Malus sylvestris AY295020 JQ800572 JQ800620

47 A. alternata CBS 115152 China Psychotria serpens KP124049 - KP124658 A. alternata CBS 117143 Italy Capsicum annuum KP124055 - KP124665 A. alternata CBS 911.97 India Artemisia brevifolia KP124027 - KP124634 A. alternata CBS 916.96 India Arachis hypogaea JQ811978 AY295043 KP124632 A. alternata CBS 918.96 UK Dianthus chinensis KP124026 JQ859820 KP124633 A. alternata CBS 966.95 India Solanum lycopersicum KP124024 - KP124630 A. alternata EGS 44-159 Turkey Citrus reticulata x C. AY295022 AY295051 EF504051 paradisi A. arborescens CBS 102605 USA Solanum lycopersicum AY295028 AY295035 KP124712 A. betae-kenyensis CBS 118810 Kenya Beta vulgaris KP124123 - KP124733 A. eichhorniae CBS 489.92 India Eichhornia crassipes KP124130 - KP124740 Continued

Table 2.3 continued Species Strain Origin Host GenBank Accession No. GAPDH TUB Colletotrichum CBS 112996 Australia Canica papaya JQ948677 JQ005860 acutatum C. cosmi CBS 853.73 Netherlands Cosmos sp. JQ948604 JQ949925 C. fioriniae CBS 128517 USA Fiorinia externa JQ948622 JQ949943 C. fioriniae CBS 200.35 USA Rubus sp. JQ948623 JQ949944 C. fioriniae CBS 293.67 Australia Persea americana JQ948640 JQ949961 C. guajavae IMI 350839 India Psidium guajava JQ948600 JQ949921 C. nymphaeae CBS 515.78 Netherlands Nymphaea alba JQ948527 JQ949848 C. scovillei CBS 126529 Indonesia Capsicum sp. JQ948597 JQ949918 C. walleri CBS 125472 Colombia Coffea sp. JQ948605 JQ949926 C. johnstonii CBS 128532 New Zealand Solanum lycopersicum JQ948775 JQ950095 48 HIS ITS TUB Diaporthe acaciigena CBS 129521 Australia Acacia retinodes KC343489 KC343005 KC343973 D. acerina CBS 137.27 - Acer saccharum KC343490 KC343006 KC343974 D. alleghaniensis CBS 495.72 Canada Betula alleghaniensis KC343491 KC343007 KC343975 D. alnea CBS 146.46 Netherlands Alnus sp. KC343492 KC343008 KC343976 D. ampelina CBS 114016 France Vitis vinifera - AF230751 JX275452 D. amygdali CBS 126679 Portugal Prunus dulcis KC343506 KC343022 KC343990 D. apiculata CGMCC China Camellia sinensis - KP267896 KP293476 3.17533 D. australafricana CBS 111886 Australia Vitis vinifera KC343522 KC343038 KC344006 D. beckhausii CBS 138.27 - Viburnum sp. KC343525 KC343041 KC344009 D. betulae CFCC 50469 China Betula platyphylla KT732999 KT732950 KT733020 D. betulicola CFCC 51128 China Betula albosinensis KX024661 KX024653 KX024657 Continued

Table 2.3 continued Species Strain Origin Host GenBank Accession No. HIS ITS TUB D. bicincta CBS 121004 USA Juglans sp. KC343618 KC343134 KC344102 D. biguttusis CGMCC China Lithocarpus glabra - KF576282 KF576306 3.17081 D. brasiliensis CBS 133183 Brazil Aspidosperma KC343526 KC343042 KC344010 tomentosum D. carpini CBS 114437 Sweden Carpinus betulus KC343528 KC343044 KC344012 D. caulivora CBS 127268 Croatia Glycine max KC343529 KC343045 KC344013 D. celastrina CBS 139.27 USA Celastrus scandens KC343531 KC343047 KC344015 D. cf. heveae 1 CBS 852.97 Brazil Hevea brasiliensis KC343600 KC343116 KC344084 D. cf. heveae 2 CBS 681.84 India Hevea brasiliensis KC343601 KC343117 KC344085 D. cf. nobilis CBS 587.79 Japan Pinus pentaphylla KC343637 KC343153 KC344121

49 D. charlesworthii BRIP 54884m Australia Rapistrum rugosum - KJ197288 KJ197268 D. citri CBS 135422 USA Citrus sp. KJ420881 NR145304 KC843187 D. citrichinensis ZJUD34 China Citrus unshiu KJ490516 JQ954648 KJ490396 D. convolvuli CBS 124654 Turkey Convolvulus arvensis KC343538 KC343054 KC344022 D. crataegi CBS 114435 Sweden Crataegus oxyacantha KC343539 KC343055 KC344023 D. cynaroidis CBS 122676 South Africa Protea cynaroides KC343542 KC343058 KC344026 D. decedens CBS 109772 Austria Corylus avellana KC343543 KC343059 KC344027 D. detrusa CBS 109770 Austria Berberis vulgaris KC343545 KC343061 KC344029 D. ellipicola CGMCC China Lithocarpus glabra - KF576270 KF576294 3.17084 D. eres CBS 101742 Netherlands Fraxinus sp. KC343557 KC343073 KC344041 D. eres CBS 109767 Austria Acer campestre KC343559 KC343075 KC344043 D. eres CBS 122.82 Netherlands Skimmia japonica KC343561 KC343077 KC344045 D. eres CBS 129168 Latvia Rhododendron sp. KC343562 KC343078 KC344046 Continued

Table 2.3 continued Species Strain Origin Host GenBank Accession No. HIS ITS TUB D. eres CBS 138594 Germany Ulmus laevis KJ420850 KJ210529 KJ420799 D. eres CBS 186.37 UK Picea abies KC343563 KC343079 KC344047 D. eres CBS 250.38 UK Fraxinus excelsior KC343564 KC343080 KC344048 D. eres CBS 267.32 - - KC343565 KC343081 KC344049 D. eres CBS 267.55 Netherlands Laburmum x watereri KC343566 KC343082 KC344050 D. eres CBS 283.85 Netherlands Allium giganteum KC343567 KC343083 KC344051 D. eres CBS 287.74 Netherlands Sorbus aucuparia KC343568 KC343084 KC344052 D. eres CBS 297.77 Netherlands Osmanthus aquifolium KC343569 KC343085 KC344053 D. eres CBS 365.97 Netherlands Opuntia sp. KC343570 KC343086 KC344054 D. eres CBS 370.67 Netherlands Ilex aquifolium KC343571 KC343087 KC344055 D. eres CBS 375.61 - Malus sylvestris KC343572 KC343088 KC344056 50 D. eres CBS 422.50 Netherlands Phaseolus vulgaris KC343573 KC343089 KC344057

D. eres CBS 439.82 UK Cotoneaster sp. KC343574 KC343090 KC344058 D. eres CBS 445.62 Netherlands Alliaria officinalis KC343575 KC343091 KC344059 D. eres CBS 485.96 Netherlands Rumex hydrolapathum KC343576 KC343092 KC344060 D. eres CBS 528.83 Netherlands Wisteria sinensis KC343577 KC343093 KC344061 D. eres CBS 688.97 Netherlands Abutilon sp. KC343578 KC343094 KC344062 D. eres CBS 694.94 Netherlands Ilex aquifolium KC343579 KC343095 KC344063 D. eres CBS 791.68 Netherlands Magnolia x soulangeana KC343580 KC343096 KC344064 D. eres CBS 841.84 Netherlands Hordeum sp. KC343581 KC343097 KC344065 D. fibrosa CBS 109751 Austria Rhamnus cathartica KC343583 KC343099 KC344067 D. foeniculina CBS 187.27 Italy Camellia sinensis KC343591 KC343107 KC344075 D. fraxini- BRIP 54781 Australia Fraxinus angustifolia - JX862528 KF170920 angustifoliae D. gardeniae CBS 288.56 Italy Gardenia florida KC343597 KC343113 KC344081 Continued

Table 2.3 continued Species Strain Origin Host GenBank Accession No. HIS ITS TUB D. goulteri BRIP 55657a Australia Helianthus annuus - KJ197290 KJ197270 D. helianthi CBS 592.81 Serbia Helianthus annuus KC343599 KC343115 KC344083 D. helicis AR 5211 France Hedera helix KJ420875 KJ210538 KJ420828 D. hickoriae CBS 145.26 USA Carya glabra KC343602 KC343118 KC344086 D. hongkongensis CBS 115448 China Dichroa febrifuga KC343603 KC343119 KC344087 D. impulsa CBS 114434 Sweden Sorbus aucuparia KC343605 KC343121 KC344089 D. juglandicola CFCC 51134 China Juglans mandshurica KX024622 KU985101 KX024634 D. longicicola CGMCC China Lithocarpus glabra - KF576267 KF576291 3.17089 D. longicolla ATCC 60325 USA Glycine max KJ659188 NR144924 KJ610883 D. longispora CBS 194.36 Canada Ribes sp. KC343619 KC343135 KC344103 51 D. lusitanicae CBS 123212 Portugal Foeniculum vulgare KC343620 KC343136 KC344104 D. macintoshii BRIP 55064a Australia Rapistrum rugosum - KJ197289 KJ197269 D. mahothocarpus CGMCC China Lithocarpus glabra - KC153096 KF576312 3.15181 D. manihotia CBS 505.76 Rwanda Manihot utilissima KC343622 KC343138 KC344106 D. masirevicii BRIP 54256 Australia Glycine max - KJ197277 KJ197257 D. mayteni CBS 133185 Brazil Maytenus ilicifolia KC343623 KC343139 KC344107 D. melonis CBS 507.78 USA Cucumis melo KC343626 KC343142 KC344110 D. miriciae BRIP 54736j Australia Helianthus annuus - KJ197282 KJ197262 D. musigena CBS 129519 Australia Musa sp. KC343627 KC343143 KC344111 D. neilliae CBS 144.27 USA Spiraea sp. KC343628 KC343144 KC344112 D. neoarctii CBS 109490 USA Ambrosia trifida KC343629 KC343145 KC344113 D. nomurai CBS 157.29 Japan Moru sp. KC343638 KC343154 KC344122 Continued

Table 2.3 continued Species Strain Origin Host GenBank Accession No. HIS ITS TUB D. nothofagi BRIP 54801 Australia Nothofagus - JX862530 KF170922 cunninghamii D. novem CBS 127270 Croatia Glycine max KC343640 KC343156 KC344124 D. ocoteae CBS 141330 France Ocotea obtusata - KX228293 KX228388 D. oncostroma CBS 809.85 Germany Ilex aquifolium KC343647 KC343163 KC344131 D. oxe CBS 133186 Brazil Maytenus ilicifolia KC343648 KC343164 KC344132 D. padi CBS 114200 Sweden Prunus padus KC343653 KC343169 KC344137 D. paranensis CBS 133184 Brazil Maytenus ilicifolia KC343655 KC343171 KC344139 D. penetriteum CGMCC China Camellia sinensis KP714493 KP267879 KP293459 3.17532 D. perjuncta CBS 109745 Austria Ulmus glabra KC343656 KC343172 KC344140

52 D. phaseolorum AR 4203 USA Phaseolus vulgaris KJ659220 KJ590738 KJ610893 D. pseudomangiferae CBS 101339 Dominican Mangifera indica KC343665 KC343181 KC344149 D. pulla CBS 338.89 Yugoslavia Hedera helix KC343636 KC343152 KC344120 D. pustulata CBS 109742 Austria Acer pseudoplatanus KC343669 KC343185 KC344153 D. pustulata CBS 109760 Austria Acer pseudoplatanus KC343670 KC343186 KC344154 D. pustulata CBS 109784 Austria Prunus padus KC343671 KC343187 KC344155 D. raonikayaporum CBS 133182 Brazil Spondias mombin KC343672 KC343188 KC344156 D. rudis CBS 113201 Portugal Vitis vinifera KC343718 KC343234 KC344202 D. sackstonii BRIP 54669b Australia Helianthus annuus - KJ197287 KJ197267 D. salicicola BRIP 54825 Australia Salix purpurea - JX862531 KF170923 D. schini CBS 133181 Brazil Schinus terebinthifolius KC343675 KC343191 KC344159 D. scobina CBS 251.38 UK Fraxinus excelsior KC343679 KC343195 KC344163 D. subclavata ZJUD95 China Citrus sp. KJ490572 KJ490630 KJ490451 D. terebinthifolii CBS 133180 Brazil Schinus terebinthifolius KC343700 KC343216 KC344184 Continued

Table 2.3 continued Species Strain Origin Host GenBank Accession No. HIS ITS TUB D. toxica CBS 534.93 Australia Lupinus angustifolius KC343704 KC343220 KC344188 D. vaccinii CBS 160.32 USA Oxycoccus macrocarpos KC343712 KC343228 KC344196 D. vawdreyi BRIP 57887a Australia Psidium guajava - KR936126 KR936128 D. virgiliae CMW 40755 South Africa Virgilia oroboides - KP247573 KP247583 D. wollworthii CBS 148.27 - Ulmus americana KC343729 KC343245 KC344213 Diaporthella corylina CBS 121124 China Corylus sp. KC343488 KC343004 KC343972 ITS Epicoccum CBS 120105 Brazil Amaranthus Sp. GU237760 brasiliense E. camelliae CGMCC China Camellia sinensis KY742091 3.18343

53 E. keratinophilum CBS 142455 USA Human superficial tissue LT592930 E. latusicollum CGMCC China Sorghum bicolor KY742101 3.18346 E. nigrum CBS 125.82 Netherlands Human toe nail FJ426995 E. nigrum CBS 173.73 USA Dactylis glomerata FJ426996 E. nigrum LC 8158 USA Poa annua KY742111 E. nigrum LC 8159 USA Poa annua KY742112 E. ovisporum CBS 180.80 South Africa Zea mays FJ427068 E. pimprinum CBS 246.60 India Soil KY742113 E. pimprinum PD 77/1028 India Soil KY742114

a Ex-holotype, ex-epitype, ex-type, authentic or representative cultures are in bold (AR = Collection of Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville; ATCC = American Type Culture Collection; BRIP = Plant Pathology Herbarium, Dutton Park, Queensland, Australia; CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The

Netherlands; CFCC = China Forestry Culture Collection Center, Beijing, China; CGMCC = China General Microbial Culture Collection Center, China; CMW = Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; EGS = Collection of E.G. Simmons; IMI: Culture collection of CABI Europe UK Centre, Egham, UK; LC: Collection of L. Cai; PD: Plant Protection Service, Wageningen, the Netherlands; ZJUD = Zhejiang University, China). b endoPG = endopolygalacturonase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HIS = histon H3; OPA1 and OPA10 = anonymous noncoding region; TUB = β-tubulin; ITS = internal transcribed spacer.

Table 2.4. Isolation frequency of the different fungi recovered from the 304 symptomatic holly fruit that yielded fungal growth between 2013 and 2017 across all sampling locations.

Fungal species Isolation frequency (%) Diaporthe spp. 39.47 Alternaria spp. 32.89 Colletotrichum spp. 7.24 Epicoccum spp. 4.61 Cladosporium spp. 3.68 Alternaria sp. + Diaporthe sp. 2.34 Botryosphaeriaceae 2.01 Alternaria sp. + Colletotrichum sp. 1.67 Fusarium spp. 1.00 Phoma spp. 1.00 Epicoccum sp. + Diaporthe sp. 1.00 Alternaria sp. + Fusarium sp. 0.33

Morphospecies Isolate Conidium length (μm) Conidium width (μm) Mycelial growth rate (mm/day) Alternaria FPH2015390 32.58 ± 0.63 (27.86-42.8)v 11.09 ± 0.26 (8.79-13.9) 5.39 ± 0.06 (5.31-5.54)w alternata FPH2015396 31.85 ± 0.67 (31.49-40) 9.39 ± 0.24 (6.93-12.28) 5.4 ± 0.04 (5.3-5.49) FPH2015468 30.85 ± 0.92 (23.84-39.71) 11.11 ± 0.3 (8.29-13.75) 5.52 ± 0.03 (5.48-5.59) FPH2015507 32.9 ± 0.65 (25.79-40.7) 10.05 ± 0.2 (80.5-12.17) 5.32 ± 0.004 (5.32-5.33) FPH2015567 31.66 ± 0.74 (25.2-40.46) 11.87 ± 0.24 (8.01-13.56) 5.17 ± 0.05 (5.06-5.25) FPH2015589 31.31 ± 0.73 (25.36-41.1) 11.08 ± 0.32 (8.25-13.67) 5.28 ± 0.02 (5.25-5.31) FPH2015592 31.26 ± 0.55 (27.26-38.06) 10.07 ± 0.27 (7.95-13.54) 5.63 ± 0.02 (5.6-5.67)

56 FPH2015597 32.92 ± 0.83 (25.46-42.75) 10.37 ± 0.2 (8.76-13.72) 5.16 ± 0.05 (5.06-5.27)

FPH2016709 32.98 ± 0.77 (26.88-42.28) 11.8 ± 0.29 (9.15-15.19) 5.23 ± 0.02 (5.19-5.28) FPH2016711 31.95 ± 0.84 (25.85-43.78) 10.85 ± 0.23 (8.19-13.5) 5.58 ± 0.02 (5.53-5.62) Mean 32.09 ± 0.23 Cx 10.77 ± 0.09 B 5.37 ± 0.03 A A. arborescens FPH2015395 29.05 ± 0 .67 (22.47-35.37) 11.84 ± 0.21 (9.21-15.11) 5.26 ± 0.05 (5.2-5.37) Mean 29.05 ± 0 .67 D 11.84 ± 0.21 A 5.26 ± 0.05 A A. tenuissima FPH2015470 43.19 ± 1.1 (35.73-59.01) 10.76 ± 0.28 (8.46-13.52) 5.58 ± 0.02 (5.54-5.62) FPH2015591 39.04 ± 0.88 (32.57-51.34) 10.3 ± 0.18 (8.66-12.3) 5.31 ± 0.02 (5.29-5.35) Mean 41.11 ± 0.52 A 10.53 ± 0.2 B B 5.45 ± 0.06 A A. toxicogenica FPH2015338 37.29 ± 0.66 (30.38-44.79) 10.7 ± 0.23 (8.81-13.88) 5.31 ± 0.01 (5.29-5.32) Mean 37.29 ± 0.66 B 10.7 ± 0.23 B 5.31 ± 0.01 A Continued

Table 2.5 continued Morphospecies Isolate Conidium length (μm) Conidium width (μm) Mycelial growth rate (mm/day) Colletotrichum FPH201314 10.57 ± 0.21 (8.9-13.24) 3.71 ± 0.11 (2.94-5.72) 3.02 ± 0.03 (2.97-3.08) fioriniae FPH201317 9.02 ± 0.19 (7.32-10.61) 4.01 ± 0.11 (3.15-5.47) 2.83 ± 0.02 (2.77-2.85) FPH201319 9.86 ± 0.13 (8.25-11.93) 4.39 ± 0.07 (3.51-5.12) 2.51 ± 0.08 (2.39-2.69) FPH2015392 6.13 ± 0.17 (4.37-8.41) 3.74 ± 0.08 (3.06-5.17) 2.69 ± 0.11 (2.52-2.96) FPH2015466 11.17 ± 0.14 (9.77-12.74) 4.81 ± 0.09 (4.03-5.77) 2.99 ± 0.01 (2.97-3.01) FPH2015564 11.17 ± 0.13 (9.88-12.5) 4.81 ± 0.09 (4.03-5.75) 3.02 ± 0.02 (2.97-3.06) FPH2016720 10.45 ± 0.24 (8.01-12.38) 4 ± 0.11 (2.94-5.52) 3.25 ± 0.1 (3.02-3.43) Mean 9.77 ± 0.13 A 4.21 ± 0.05 B 2.9 ± 0.06 A A C. nymphaeae FPH2015590 10.13 ± 0.17 (8.31-13.6) 4.54 ± 0.1 (3.65-6.31) 2.87 ± 0.11 (2.59-3.02) Mean 10.13 ± 0.17 A 4.54 ± 0.1 A 2.87 ± 0.11 A Diaporthe eres FPH201322 7.58 ± 0.09 (6.74-8.61)y 2.99 ± 0.04 (2.59-3.53) 4.7 ± 0.19 (4.25-4.96) 57 24.73 ± 0.52 (18.91-29.65)z 1.68 ± 0.05 (1.27-2.13) FPH2015394 6.38 ± 0.12 (5.04-8.01) 2.89 ± 0.06 (2.21-3.82) 5.59 ± 0.12 (5.31-5.8) 24.39 ± 0.38 (21.66-29.79) 1.8 ± 0.03 (1.48-2.09) FPH2017229 6.87 ± 0.08 (6-7.58) 2.59 ± 0.05 (2.15-3.32) 4.89 ± 0.09 (4.76-5.05) 24.81 ± 0.44 (18.76-29.66) 1.63 ± 0.03 (1.27-2.05) Epicoccum FPH2015417 20.27 ± 0.27 (17.37-23.26) 18 ± 0.3 (14.12-22.18) 3.43 ± 0.02 (3.38-3.45) nigrum FPH2015505 19.85 ± 0.31 (17.05-23.78) 17.23 ± 0.32 (13.17- 3.57 ± 0.05 (3.48-3.69) 21.71)

vValues reported are Mean ± standard error (minimum-maximum size) of 30 randomly selected conidia. wValues reported are Mean ± standard error (minimum-maximum mycelia growth rate). xValues with the same letter in the same column within each fungal genus are not significantly different (α=0.05). yalpha conidia zbeta conidia

Continued

Figure 2.4 continued

Chapter 3: Investigations on the Timing of Fruit Infection by Fungal Pathogens Causing Fruit Rot of Deciduous Holly2

ABSTRACT

Fruit rot of deciduous holly is an emerging fungal disease that is affecting plant production across Midwestern and Eastern U.S. nurseries. To determine the growth stage(s) of host susceptibility to infection by the major pathogens associated with the disease, Alternaria alternata and Diaporthe ilicicola, and minor pathogens such as Colletotrichum fioriniae and Epicoccum nigrum, we conducted two sets of experiments over two consecutive seasons. In the first case we monitored the presence of the pathogens as well as disease progression in a commercial nursery under natural conditions by collecting plant tissues from the flower bud stage until fruit maturity.

The target pathogens were consistently isolated from asymptomatic samples at all stages of fruit development and from symptomatic samples at fruit maturity across the two years of collection.

A significant increase in fungal isolation frequency, primarily species of Alternaria and

Colletotrichum, was observed right after flowering, but fruit rot symptoms only developed on mature fruit. In the second case we artificially inoculated containerized plants maintained outdoor at our research farm with individual or combined pathogens at different fruit developmental stages, and we assessed disease incidence on mature fruit to determine the time of host susceptibility to

2Copyright The American Phytopathological Society. Reproduced, by permission, from Lin, S., and Peduto Hand, F. 2018. Investigations on the timing of fruit infection by fungal pathogens causing fruit rot of deciduous holly. Plant Disease First Look, retrieved from https://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-06-18-0973-RE. 70

infection and, indirectly, of whether pathogens in the fungal complex carry out latent infections.

Diaporthe ilicicola could cause latent infection on deciduous holly fruit when inoculated at the full bloom and petal fall stages, and all inoculations made on wounded mature fruit resulted in fruit rot. These findings suggest that flowering represents a critical period to manage D. ilicicola infections, and that mature fruit should be protected from any injury to avoid disease. In both experiments a negative correlation between disease incidence and temperature was found, however the decrease in temperature also coincided with fruit ripening. The effects of temperature and changes in physiological properties of the fruit during maturation on disease development should be further investigated to fully interpret these findings.

INTRODUCTION

Deciduous hollies (Ilex spp. L.) are dioecious ornamental shrubs popularly used in the cut flower industry (Galle 1997). Their branches carrying brightly colored fruit are harvested in late fall to be used in winter holiday decorations, representing an important source of revenue for nursery growers in a period of the year in which business is otherwise at low ebb. We previously reported that deciduous holly production in Midwestern and Eastern U.S. nurseries has been affected in recent years by a fruit rot causing plant material to become unsalable (Lin et al. 2018).

The disease is characterized by a variety of symptoms, including early plant defoliation and production of fruit that is undersized, loses its gloss, fails to turn color, shrivels and eventually becomes rotten (Lin et al. 2018).

Disease management recommendations should rely on knowledge of the disease-causing agents as well as of disease epidemiology. Previous studies conducted over multiple years and

across different locations have revealed that a fungal complex is associated with this disease (Lin et al. 2018). Alternaria alternata (Fr.) Keissl. and a recently described Diaporthe Nitschke species, namely D. ilicicola S. Lin, Taylor & Peduto Hand, were identified as the primary pathogens.

Several other fungal species, including A. arborescens E. G. Simmons, Colletotrichum fioriniae

(Marcelino & Gouli) Pennycook, C. nymphaeae (Pass.) Aa, fungi in the D. eres Nitschke species complex and Epicoccum nigrum Link, were identified as minor pathogens (Lin et al. 2018). While all these pathogens were isolated to a greater or lesser extent from mature holly fruit at or around the time of branch harvest (late fall), it is not known exactly when in the growing season fruit infections may occur.

Bud break of deciduous holly occurs in early spring and is followed by formation of leaves.

Flowers are produced in mid- to late spring and the blooming period lasts between 10 and 14 days depending on the species and cultivar (Galle 1997). After petal fall occurs, the fruit starts to develop, turning color in early fall and being fully mature by mid-fall (Galle 1997). Some of the pathogens involved in the fungal complex have been extensively studied on other crops to understand which growth stage(s) of the host is/are susceptible to infection. It has been reported that infections caused by A. alternata on apple, mango, mandarin, persimmon, Pingguoli pear

(Pyrus bretschneideri), and sweet pepper could occur at different stages of fruit development, from bloom until fruit maturation (Halfon-Meiri and Rylski 1983; Harteveld et al. 2014; Li et al. 2007 and Nemsa et al. 2012; Prusky et al. 1981 and 1983). Likewise, several species of Colletotrichum and Diaporthe have been reported to be able to infect almond, blueberry, mango, melon and peach from the flowering stage until fruit maturation (Adaskaveg and Hartin 1997; Binyamini and

Schiffmann-Nadel 1972; Dayken and Milholland 1984; Johnson et al. 1992; Mckay et al. 2014;

Ohsawa and Kobayashi 1989; Verma et al. 2007; Zaitlin et al. 2000).

No study has been yet conducted to identify the factors contributing to the development of fruit rot on deciduous holly. However, proper identification of all the components of the disease triangle, including factors related to the susceptibility of the host to infection are needed to fully understand this disease and provide holly growers with research-based recommendations for effective disease management. To this extent, the specific objective of this study was to determine the growth stage(s) of deciduous holly fruit that is/are susceptible to infection by different pathogen(s) in the fungal complex.

MATERIALS AND METHODS

Assessment of fruit stage susceptibility under nursery conditions: A field survey was conducted in 2016 and 2017 in a nursery in Wooster, OH, which was exposed to natural pathogen inoculum and in which fruit rot had been observed yearly since 2014. Plant material included the deciduous holly hybrid Ilex verticillata x Ilex serrata cultivar Bonfire, which was previously reported being susceptible to fruit rot (Lin et al. 2018). The surveyed area of the nursery comprised

20 trees, which were located in one individual row within the production field that had been established in 1994. No management practices were carried out in the entire nursery, including the experimental plot, at any point of the growing season throughout the duration of the experiments.

Plants were sampled weekly or biweekly from May to December of each year at five different stages of fruit development to assess infection and monitor for disease symptoms development.

The aforementioned five stages, the corresponding sampling periods of each year, and the

combined total number of samplings are defined as follows: stage 1 (flower bud) – more than 90% of flower buds fully developed, with expanded petals that are white but still closed (from May 27 to June 9, 2016 and from May 26 to June 9, 2017; total samplings = 6); stage 2 (full bloom) – more than 90% of flowers open (June 16, 2016 and June 16, 2017; total samplings = 2); stage 3 (petal fall) – petals of more than 90% of flowers fallen and small green fruit set (June 23, 2016 and June

23, 2017; total samplings = 2); stage 4 (immature fruit) – fruit from 1-2 mm to 6-8 mm in diameter and not completely red (from June 30 to Sep 21, 2016 and from June 30 to Sep 29, 2017; total samplings = 27); stage 5 (mature fruit) – fruit fully developed and completely red (from Sep 29 to

Dec 1, 2016 and from Oct 6 to Dec 1, 2017; total samplings = 17). On each sampling date, 10 individual plant tissues (i.e., flower buds, flowers or fruit) were randomly collected from the entire canopy of each tree in the experimental plot to maximize randomization and then pooled together inside a plastic bag. Samples were then transported to the laboratory inside a cooler and stored at

4°C for a maximum of 24 hours before being processed. One hundred plant tissues were randomly extracted from the bag and processed as described herein: samples collected from stage 1 through

3 were surface disinfected for 90 sec with 0.5%, 0.5% and 1% household bleach (6% sodium hypochlorite; The Clorox Company), respectively. Samples were then rinsed in sterile water three times and placed on a sterile paper towel inside a laminar flow hood to allow air-drying. Each plant tissue was then plated on Potato Dextrose Agar (PDA; Difco Laboratories, Sparks, MD) amended with 0.1% lactic acid, 0.01% tetracycline hydrochloride and 0.015% streptomycin sulfate (Fisher

Scientific, Fair Lawn, NJ) and plates were incubated on a laboratory bench at 25°C for up to 10 days. Fungal colonies developing from the tissues were counted and any fungal growth was sub- cultured onto a new PDA plate. Pure cultures of each isolate were obtained by transferring hyphal

tips from the margin of the fungal colonies onto new PDA plates. All isolates were then preserved as mycelial plugs in sterile distilled water at 4°C (Humber 1997). All isolates were tentatively identified to species using morphological features (Barnett and Hunter 1998; Baumgartner et al.

2013; Dissanayake et al. 2015; Lin et al. 2018; Mahadevakumar et al. 2014; Shivas and Tan 2009;

Simmons 2007; Udayanga et al. 2014; Velho et al. 2015; Wu et al. 2017). To confirm species identification, at least half of the isolates collected at each stage of fruit development that showed similar morphology were randomly selected and subject to molecular characterization as described in Lin et al. (2018).

Fruit collected from stage 4 through 5 was visually inspected for symptoms of fruit rot and disease incidence and severity were recorded from each fruit. Fruit rot incidence was assessed as presence or absence of symptoms on each fruit, while disease severity was assessed as the percentage of fruit surface area affected. Total disease incidence and severity were then calculated for each sampling time. Subsequently, fruit was sorted into two groups: symptomatic and asymptomatic. Fungal isolation from the lesions on symptomatic fruit and culture identification were conducted as described in Lin et al. (2018). Asymptomatic fruit were surface disinfected as previously described but by using 2% household bleach (6% sodium hypochlorite; The Clorox

Company) for 2 min. Once dry, fruit were cut in half and plated cut-side down on V8 juice agar

(Olatinwo et al. 2003) rather than PDA to facilitate growth of slower-growing fungi. Plate incubation conditions, identification and preservation of fungal colonies developing from the asymptomatic tissue were the same as previously described.

Container trial locations and experimental design: An outdoor container trial was established at The Ohio State University Agricultural and Natural Resources Laboratory

(Waterman Farms) in Columbus, OH to conduct artificial inoculations with the fungal pathogens associated with fruit rot of deciduous holly and assess the timing of fruit infection. Experiments were conducted over two consecutive growing seasons (2016-2017) from spring to late fall (from

April 19 through December 10, 2016 and from April 26 through December 8, 2017). Plant material included two-year old (2016) or three-year old (2017) plants of Ilex verticillata x Ilex serrata female cultivar Sparkleberry, and male pollinator cultivars Apollo, Jim Dandy and Southern

Gentleman, which were placed among females to facilitate pollination. All plants were maintained in 3-gallon (11.35 L) nursery containers containing a mix of 10-15% Com-Til and 85-90% pine bark (Kurtz Bros., Columbus, OH). Plants were fertilized at the time of trial establishment with

55g of Osmocote® 14-14-14 (Everris NA Inc. Dublin, OH) and watered as needed throughout the duration of the trial by a drip irrigation system. Plants were arranged in rows in a randomized complete block design with six blocks. Rows were 15 m long and 0.3 m apart. In 2016, when plants were younger, two Sparkleberry plants per treatment were included in each block and were treated as a single experimental unit (average fruit number = 91). In 2017, plants grew bigger and only one plant per treatment was included in each block (average fruit number = 101). Between seasons (December 2016 through April 2017) plants were overwintered in a covered polyhouse.

Container trial inoculations and data collection: Isolates of Alternaria alternata,

Colletotrichum fioriniae, Diaporthe ilicicola and Epicoccum nigrum originally isolated from symptomatic holly fruit and whose virulence was previously confirmed through the fulfillment of

Koch’s postulates (Lin et al. 2018) were used in the container trials and are listed in Table 3.1.

Inoculum was prepared by flooding 2-4 weeks-old PDA cultures using a sterile distilled water-

Tween 20 solution (0.05% v/v) and by adjusting the concentration to 105 conidia/mL using a

hemacytometer. Inoculum of each pathogen species was obtained by combining spore suspensions from all isolates (2-7) of each species (Table 3.1).

In 2016, only individual pathogen species inoculum was used in the experiments, while in

2017, additional treatments were included to assess the effects of combined pathogens on disease development. The additional treatments in 2017 were obtained by combining inoculum of two pathogens (i.e., A. alternata + C. fioriniae; A. alternata + D. ilicicola; and A. alternata + E. nigrum;

1:1 v/v), each initially prepared as previously described. In 2016, plants at the fruit development stage 1 (flower bud), 2 (full bloom), 3 (petal fall), 4 (immature fruit), and 5 (mature fruit) were inoculated on June 3, June 14, June 26, Aug 17, and Oct 11, respectively. In 2017, inoculation dates from stage 1 to 5 were May 30, June 5, June 13, Aug 16 and Oct 10, respectively. At stage

5, a subset of fruit was wounded using a sterile needle prior to inoculation to determine the effects of wounding on infection. Prior to each inoculation, plants from the appropriate treatments were moved inside a head house and plant tissues that were not at the specific inoculation stage were removed (e.g. newly developed flowers on plants to be inoculated at stage 4). On the inoculation day, every flower bud (stage 1), flower (stage 2 and 3) or fruit (stage 4 and 5) on each plant were point inoculated using a micropipette with a volume of the different spore suspensions that could be held on the tissue without dropping (i.e. 6μL on stage 1-3; 12μL on stage 4-5). In the case of wounded fruit at stage 5, a drop of spore suspension was placed directly on the wound. In all cases, inoculum was allowed to dry on the inoculated tissues for approximately 15 minutes to prevent dislodging the spore suspension droplets. Subsequently, plants were enclosed in a plastic bag and kept in a climatized head house at 25±2°C for 24 hours to favor infection prior to being moved back in the outdoor trial. A subset of fruit at stage 5 in the control group were wounded using a

sterile needle and inoculated with sterile distilled water to serve as controls. Due to space limitations, only one set of control plants was used for all inoculation stages. These plants remained undisturbed and non-inoculated in the trial throughout the duration of the experiments. Incidence of fruit rot in each treatment was evaluated weekly (from June 26 to December 7, 2016 and from

June 13 to December 5, 2017) by recording the number of fruit exhibiting rot symptoms in each plant. Re-isolation of the inoculated pathogen from the symptomatic fruit and isolate identification was carried out as previously described (Lin et al. 2018).

Meteorological data: Meteorological data, including minimum, maximum and average daily temperature, as well as precipitation were downloaded from the USDA-ARS weather research network (http://www.oardc.ohio-state.edu/weather1/) available for Columbus and

Wooster, OH for the entire disease assessment periods (2016-2017).

Statistical analysis: Frequency of fungal isolation from asymptomatic plant tissues collected from the nursery was calculated for each sampling stage. To test the effects of stages of fruit development (stage 1-5) on fungal isolation frequency, a repeated measures ANOVA analysis was performed for each individual pathogen. All data were arcsine transformed and analyzed using

PROC GLIMMIX in SAS (version 9.4; SAS Institute Inc., Cary NC) and then back-transformed to present results. The number of samplings, which differed for each fruit developmental stage, was specified in the WEIGHT option. The stage of fruit development was treated as a fixed factor, while the year was treated as a random factor. Least squares means (LS-means; α = 0.05) were used for comparisons among different stages. As far as the container trial is concerned, fruit rot incidence data from the final disease assessment in both 2016 and 2017 was subject to one-way

ANOVA analysis. Each inoculation stage was analyzed independently using PROC GLM in SAS

(version 9.4; SAS Institute Inc., Cary NC). Tukey’s HSD test was used for mean separation among treatments (α = 0.05).

Mean minimum, maximum and average daily temperature, and accumulated precipitation between consecutive disease assessment dates were calculated for both nursery and container trials from the period of one week before the onset of symptoms until the last disease assessment.

Pearson’s correlation test (α = 0.05) was used to analyze correlation between different meteorological factors and disease incidence using PROC CORR in SAS (version 9.4; SAS

Institute Inc., Cary NC).

RESULTS

Assessment of fruit stage susceptibility under nursery conditions: From 2016 to 2017, a total of 600 flower buds, 200 flowers and 4819 fruit (4507 asymptomatic and 312 symptomatic) at different developmental stages were collected and processed. Multiple fungi, including species of Alternaria, Diaporthe, Colletotrichum, Epicoccum, Botrytis P. Micheli ex Haller, Cladosporium

Link, Fusarium Link, Pestalotia De Not., Phoma Sacc. and fungi in the Botryosphaeriaceae

Theiss. & P. Syd., were isolated from the plant tissues. Among these, species of Alternaria,

Diaporthe and Epicoccum were consistently isolated from asymptomatic samples at all stages of fruit development (1 through 5; Table 3.2). With the exclusion of stage 3, species of Colletotrichum sp. were also consistently isolated from these samples (Table 3.2). Based on morphological and molecular identification of selected isolates of Alternaria (n = 27), Diaporthe (n = 27),

Colletotrichum (n = 15), and Epicoccum (n = 26), recovered from asymptomatic tissues, the most prevalent species included A. alternata, A. arborescens, C. fioriniae, C. nymphaeae, D. ilicicola,

fungi in the D. eres species complex and E. nigrum (data not shown). In general, the isolation frequency of all four fungal genera increased along with the progression of fruit development and maturation (Table 3.2). When fruit became mature (stage 5), species of Alternaria and Epicoccum had higher isolation frequency from asymptomatic fruit (85.83% and 68.21%, respectively), followed by Diaporthe (14.13%) and Colletotrichum (4.38%; Table 3.2). For Alternaria and

Colletotrichum, a significant increase in isolation frequency was observed from stage 3 to 4, while for Epicoccum, it increased significantly from stage 1 to 4. No significant difference in isolation frequency was observed for Diaporthe (Table 3.2).

In both 2016 and 2017, fruit rot symptoms were first observed in November (average temperature 5.5°C and 6.5°C, respectively) when the fruit was fully mature (stage 5). Symptoms mostly developed from the side of the fruit corresponding to the location of the stigma on the flower. Less frequently, symptoms were observed from the corresponding calyx and from the lenticels naturally distributed on the fruit surface (Figure 3.1). Diaporthe spp. had the highest isolation frequency (25.64%) from symptomatic fruit, followed by species of Alternaria,

Epicoccum and Colletotrichum (Table 3.2). The disease incidence from the final disease assessment was 40% and 60% in 2016 and 2017, respectively, while severity was 21.78% and

48.21%, respectively. Frequently, it was observed that a combination of factors (e.g., insect and bird activity, hail etc.) caused wounds to form on mature fruit (stage 5). Occasionally, rot symptoms developed from the wounds and species of Alternaria and Epicoccum were isolated

(data not shown).

In 2016, a negative correlation between mean maximum temperature and disease incidence was observed. No significant correlation was observed between other meteorological factors and disease incidence (Table 3.3).

Container trials. Fruit rot symptoms were first observed in the container trials at the beginning of November 2016 (average temperature 12.8°C), and at the end of October 2017

(average temperature 15.6°C). In both years, symptoms developed on fruit inoculated with D. ilicicola at stage 2 and 3, and on fruit inoculated with all pathogens at stage 5, but only on the wounded treatments (Figure 3.2). Symptoms developed from the side of the fruit corresponding to the location of the stigma on the flower when inoculated at stage 2 and 3, or from the wound when inoculated at stage 5. In 2017, fruit inoculated with the combined D. ilicicola + A. alternata inoculum at stage 2 and 3, also developed symptoms (Figure 3.2). However, disease incidence was significantly lower than on fruit inoculated with D. ilicicola only at the same inoculation stages.

Disease incidence on wounded fruit inoculated at stage 5 ranged from 25.47% to 68.54%, with

Epicoccum nigrum or A. alternata + E. nigrum showing significantly lower values (Figure 3.2).

An average of 1.15% and 2.61% of wounded fruit at stage 5 from non-inoculated control plants developed rot symptoms in 2016 and 2017, respectively (Figure 3.2) and species of Alternaria and

Epicoccum were retrieved from the lesions. No disease was observed on unwounded fruit from non-inoculated control plants in either year.

In both 2016 and 2017, disease incidence in all symptomatic treatments was negatively correlated to mean maximum and average temperature (Table 3.3). In 2017, disease incidence in symptomatic treatments inoculated at stage 2 or 3 was also negatively correlated to mean minimum

temperature (Table 3.3). No significant correlation was observed between accumulated precipitation and disease incidence (Table 3.3).

DISCUSSION

Fruit rot of deciduous holly is an emerging disease that is affecting plant production across

Midwestern and Eastern U.S. nurseries. Several fungal pathogens, including Alternaria alternata,

A. arborescens, D. ilicicola, Colletotrichum fioriniae, C. nymphaeae, Epicoccum nigrum and fungi in the D. eres species complex, were found to be the disease causal agents (Lin et al. 2018). To advance our understanding of this disease, in this study we focused on identifying the timing of host susceptibility to infection by the fungal pathogens by monitoring the presence of the disease causal agents in the fruit at different stages of development (from flower bud to mature fruit) as well as disease progression on plants exposed to natural inoculum (i.e. “nursery assessment”) or artificially inoculated (i.e. “container trials”).

In the nursery, the disease causal agents were consistently isolated from asymptomatic samples with isolation frequency increasing along with the progression of fruit development and maturation. At stage 5 (mature fruit), Alternaria spp. had the highest isolation frequency from asymptomatic fruit, but relatively lower frequency of isolation from symptomatic fruit compared to Diaporthe spp. Isolation frequency of different pathogens may vary in a given location based on the presence of relative inoculum, which may be influenced by the history of disease management practices, the presence of alternate hosts, as well as environmental factors (Harteveld et al. 2014; Holb and Scherm 2007; McKay et al. 2014; Moral and Trapero 2012). While only one nursery was included in this study, a more extensive sampling of symptomatic fruit conducted over

multiple years and locations revealed that these two genera are equally associated with diseased fruit (Lin et al. 2018). A survey to determine the relative distribution of different species in a specific nursery could be valuable to optimize the design of disease management strategies.

Disease incidence and severity in the nursery were lower in 2016 compared to 2017. This may have been due to less pathogen inoculum being present in the field during the 2016 season, or to less favorable environmental conditions. Many studies have identified that infected fruit left in the field can serve as a source of inoculum for diseases caused by the same pathogen genera involved in fruit rot of deciduous holly (Harteveld et al. 2014; McKay et al. 2014; Stensvand et al.

2017). Before the establishment of our trial (2016), although disease had occurred in the nursery in the previous year, branches were harvested and many infected fruit, potentially harboring pathogen inoculum, were removed. However, after trial establishment, no branches, including those carrying infected fruit, were harvested. This may explain why higher disease levels were observed in 2017. In addition, several studies have demonstrated that different environmental factors have effects on the relative availability of pathogen inoculum and on host infection

(Bassimba et al. 2014; Granke and Hausbeck 2010; King et al. 1997). Environmental conditions in 2016 may have been less conducive to inoculum production or infection development in the field. Further investigations on the identification of potential sources of inoculum in nurseries and the effects of important environmental factors on disease progression are underway and should provide valuable information on understanding the disease cycle(s) and further developing disease management strategies.

In the nursery, a significant increase in fungal isolation frequency (Alternaria and

Colletotrichum) was observed right after flowering. Disease symptoms became visible only at the

end of the season when the fruit was fully mature and mostly developed from the side of the fruit corresponding to the location of the stigma on the flower. This suggests that infections are initiated during bloom but fruit colonization occurs only later in the season during fruit ripening, a process that is consistent with the description of a latent infection (Verhoeff 1974).

In the container trials, Diaporthe ilicicola was the only pathogen that caused disease on mature fruit when inoculated during bloom or petal fall stage. As observed in the symptomatic fruit samples collected from the nursery, the majority of the rot symptoms developed from the stigmatic-end, and in a few cases from the calyx-end of the fruit. It was also observed that the pathogen could mostly be recovered from the fruit exocarp instead of the mesocarp (data not shown). Thus, the data suggests that D. ilicicola infects holly fruit during bloom and remains quiescent in the exocarp until yet undetermined triggering conditions activate the pathogen and cause symptoms to become visible. We speculate that host physiological changes during fruit maturation could play a role in in quiescent pathogen activation.

In our container trials, no disease developed on mature fruit that had been inoculated at any of the flower stages (stage 1 through 3) with A. alternata. However, fewer fruit were present on plants that had been inoculated with A. alternata during bloom compared to other treatments (data not shown). We suspect that inoculation of flowers with A. alternata may have caused flowers to drop, thereby resulting in fewer of the surviving fruit with infections at the end of the season.

Similar results were obtained by Luo et al. (2017) as no Alternaria heart rot disease developed on pomegranate fruit because most flowers dropped after being inoculated.

Another possible explanation could be that the host environment conditions of deciduous holly fruit did not support Alternaria from exiting quiescence. Even though the majority of the

mature fruit samples were found to be colonized by Alternaria spp., the pathogen was retrieved from the lesions on symptomatic fruit in only approximately 4% of the cases. Other studies have shown or suggested that changes in host physiology during fruit ripening may be related to the activation of A. alternata. On mango fruit Droby et al. (1987) found an association of the antifungal compounds (5-substituted resorcinols) in unripe fruit with the quiescence of A. alternata. In a study on apple core rot caused by A. alternata, Shtienberg (2012) speculated different calcium concentrations in the locule walls of two apple cultivars may be associated with different levels of host susceptibility to infection. Previous studies demonstrated that calcium had inhibitory effects on the activity of polygalacturonase (Biggs et al. 1997; Chardonnet et al. 2000), an enzyme that is secreted by Alternaria during infection (Rotem 1994). All these findings suggest correlations between host physiology and fungal infections.

In both the nursery and container trials, rot symptoms were observed at cool temperatures

(below 6.5°C and 15.6°C, respectively) and disease incidence was negatively correlated, in one or both years of the study, to mean maximum temperature (nursery) or mean maximum, minimum or average temperature (container trials). Although these findings might suggest that exposure to low temperatures could be involved in pathogen activation, the decrease in temperature also coincided with fruit ripening. Based on the data collected in this study it is not possible to fully understand the role of temperature in disease development. Changes in the physiological properties of deciduous holly fruit during ripening are also currently unknown. Studies to further investigate the factors (host and environment) that terminate quiescence of A. alternata and D. ilicicola in deciduous holly fruit are underway and will contribute to complementing the observations reported in this study and further understanding this disease.

We previously reported that occasionally two pathogens in the fungal complex, one most frequently being Alternaria, could be isolated from the same fruit lesion (Lin et al. 2018). In the current study, when plants were inoculated with combined Alternaria treatments infections occurred on wounded mature fruit but disease incidence was never significantly greater than that caused by the more virulent of the pathogens when it was used alone. Similar results were also observed in a detached fruit inoculation study we previously conducted (Lin et al. 2018). For combined pathogens inoculated at the bloom or petal fall stage, only plants inoculated with A. alternata + D. ilicicola developed fruit rot symptoms; however, disease incidence was significantly lower than when D. ilicicola was inoculated alone. Alternaria alternata and

Phomopsis mangiferae S. Ahmad were studied on other fruit crops to demonstrate their ability of modulating ambient pH in the host to enhance pathogenicity (Davidzon et al. 2010; Eshel et al.

2002). A. alternata can secrete ammonia to elevate ambient pH in melon, pepper, persimmon, and tomato fruit (Eshel et al. 2002). Similarly, gluconic acid is secreted by P. mangiferae to lower pH and enhance its pathogenicity in mango fruit (Davidzon et al. 2010). A. alternata and D. ilicicola in deciduous holly may have contradictory mechanisms to manipulate the host environmental and favor infection, which could be a possible explanation for why lower disease incidence was observed when they were inoculated together. Further studies investigating the factors that activate these pathogens in deciduous holly fruit, including plant nutrition, may provide valuable information for understanding the mechanisms of fungal quiescence and developing appropriate disease management strategies.

In the nursery we observed that mature fruit (stage 5) was frequently injured by either biotic or abiotic factors (e.g., hail, insects, birds etc.). Therefore, in the container trial, we included

a wounded treatment on mature fruit to assess the effects of wounding on disease development.

All four pathogens caused fruit rot on wounded fruit but no disease was observed on unwounded fruit. Although a low level of fruit rot was also observed on non-inoculated control plants that had been wounded, disease incidence was negligible compared to all other treatments in both years.

Species of Alternaria and Epicoccum were isolated from those lesions, which may have been caused by inoculum drift from the inoculated treatments or the presence of natural inoculum in the outdoor environment. This reinforced the concept that some pathogens in the complex could take advantage of the presence of wounds to infect fruit. In the detached fruit inoculation study previously mentioned (Lin et al. 2018), wounded fruit developed significantly higher disease incidence and severity compared to unwounded fruit. Although in that study pathogens were able to also infect unwounded fruit, it should be acknowledged that the assay was conducted under optimal laboratory conditions as opposed to fluctuating environmental conditions of this study.

Both studies however are in agreement that mature fruit in the field should be protected from any type of wounding to reduce disease levels.

This study provides the first report of latent infection caused by Diaporthe ilicicola on deciduous holly fruit. Our findings suggest that flowering represents a critical moment to implement disease management practices that target D. ilicicola, and that mature fruit should be protected from injury to avoid disease. While many aspects of this disease remain to be investigated, including identification of the sources of pathogen inoculum in the field and of host factors and environmental conditions that are conducive to disease development, this research advanced our understanding of the epidemiology of this emerging disease and ultimately facilitates the development of research-based disease management strategies.

ACKNOWLEDGMENTS

This study was funded by the Ohio Department of Agriculture Specialty Crop Promotion

Program (Awards AGR-SCG-14-08 and AGR-SCG-16-09), the USDA-NIFA Hatch project

#1004939, the T. J. Kavanagh Foundation, and The Ohio State University Department of Plant

J.R Urbez-Torres and G. Marchi for critical review of this manuscript prior to submission.

Table 3.1. Fungal isolates used to inoculate plants in the container trial in 2016 and 2017.

Fungal species Isolate No. Origin Year of isolation Reference FPH2015416 Ohio 2015 Lin et al., 2018 FPH2015419 Ohio 2015 Lin et al., 2018 FPH2015468 Ohio 2015 Lin et al., 2018 Alternaria alternata FPH2015507 Ohio 2015 Lin et al., 2018 FPH2015508 Ohio 2015 Lin et al., 2018 FPH2015593 Ohio 2015 Lin et al., 2018 FPH2015597 Ohio 2015 Lin et al., 2018 Colletotrichum FPH2015466 Ohio 2015 Lin et al., 2018 fioriniae FPH2015564 Ohio 2015 Lin et al., 2018 FPH2015473 Ohio 2015 Lin et al., 2018 FPH2015502 Ohio 2015 Lin et al., 2018 Diaporthe ilicicola FPH2015509 Ohio 2015 Lin et al., 2018 FPH2015598 Ohio 2015 Lin et al., 2018 FPH2015417 Ohio 2015 Lin et al., 2018 Epicoccum nigrum FPH2015505 Ohio 2015 Lin et al., 2018

Table 3.2. Frequency of fungal isolation (%) from asymptomatic and symptomatic samples collected from the nursery at different stages of fruit development from 2016 to 2017.

Stage of fruit Isolation frequency (%)w Pathogen development Asymptomatic samples Symptomatic samples Alternaria 1 4.16 ax NAy 2 23.53 a NA 3 29.58 a -z 4 76.75 b - 5 85.83 b 3.85 Diaporthe 1 12.98 a NA 2 18.50 a NA 3 11.98 a - 4 11.14 a - 5 14.13 a 25.64 Colletotrichum 1 0.08 a NA 2 0.25 ab NA 3 0.00 a - 4 3.36 b - 5 4.38 b 0.32 Epicoccum 1 3.82 a NA 2 18.45 ab NA 3 26.50 ab - 4 42.26 b - 5 68.21 b 2.88

wColumns represent the value of pooled data across years of observation (n = 2). xValues with the same letter within each pathogen are not significantly different according to LS- means multiple comparisons (α = 0.05). yNA = not applicable. zThe sign (-) indicates that no symptoms were observed on the fruit at the specific stage.

Table 3.3. Correlation between disease incidence and environmental variables in the nursery and container trials in 2016 and 2017.

Pearson Correlation Coefficient (r) b, c Experiment Correlation Factor a P Max. °T Min. °T Avg. °T 2016 -0.98 Nursery 2017 2016 D. ilicicola (stage 2) -0.86 -0.83 2016 D. ilicicola (stage 3) -0.85 -0.89 Container 2017 D. ilicicola (stage 2) -0.82 -0.83 -0.83 trial 2017 D. ilicicola + A. alternata (stage 2) -0.79 -0.80 -0.79 2017 D. ilicicola (stage 3) -0.79 -0.83 -0.81 2017 D. ilicicola + A. alternata (stage 3) -0.80 -0.84 -0.83

aDisease incidence observed each year in the nursery trials or according to the different inoculation treatments that resulted in symptomatic fruit in the container trials. bP = accumulated precipitation (mm); Max. °T = mean maximum temperature (°C); Min. °T = mean minimum temperature (°C); Avg. °T = mean average temperature (°C). cBlank column = not significant (p > 0.05).

Figure 3.1. Rot symptoms on mature holly fruit developing from the stigmatic-end (A), calyx-end (B), and lenticels (C and D).

Chapter 4: Determining the Sources of Primary and Secondary Inoculum and Seasonal Inoculum Dynamics of Fungal Pathogens Causing Fruit rot of Deciduous Holly3

ABSTRACT

Fruit rot of deciduous holly, caused by species of Alternaria, Colletotrichum, Diaporthe and

Epicoccum, is affecting plant production in Midwestern and Eastern U.S. nurseries. To determine the source(s) of inoculum, dormant twigs and mummified fruit were collected, and leaf spot development was monitored throughout the season from three Ohio nurseries over two consecutive years. Mummified fruit was the main source of primary inoculum for species of Alternaria and

Epicoccum, while mummified fruit and bark were equally important for species of Colletotrichum and Diaporthe. Brown irregular leaf spots developed in the summer, and disease incidence and severity increased along with leaf and fruit development. Coalesced leaf spots eventually resulted in early plant defoliation. When tested for their pathogenicity on fruit, leaf spot isolates were able to infect wounded mature fruit and induce rot symptoms, which indicated that leaf spots could serve as a source of secondary inoculum for fruit infections. In addition, spore traps were used to monitor seasonal inoculum abundance in the nurseries. Fruit rot pathogens were captured by the spore traps throughout the season with peak dissemination occurring during flowering. In this study we also attempted to understand the role of environmental factors on leaf spot development.

3 This chapter was accepted with revision by Plant Disease. Lin, S., and Peduto Hand, F. 20XX. Determining the Sources of Primary and Secondary Inoculum and Seasonal Inoculum Dynamics of Fungal Pathogens Causing Fruit Rot of Deciduous Holly. 94

While leaf spot incidence and severity were negatively correlated to mean maximum, minimum and average temperature, a decrease in temperature also coincided with leaf senescence. The role of temperature on leaf spot development should be further studied to fully interpret these results.

INTRODUCTION

Fruit rot of deciduous holly (Ilex spp. L.) was recently reported as an emerging threat to plant production across Midwestern and Eastern U.S. nurseries (Lin et al. 2018). It was determined that multiple fungal pathogens can cause the disease, primarily Alternaria alternata (Fr.) Keissl., and the recently described fungus Diaporthe ilicicola S. Lin, Taylor & Peduto Hand, as well as other minor pathogens, such as A. arborescens E. G. Simmons, Colletotrichum fioriniae

(Marcelino & Gouli) Pennycook, C. nymphaeae (Pass.) Aa, fungi in the D. eres Nitschke species complex, and Epicoccum nigrum Link (Lin et al. 2018). Seasonal symptoms of the disease include leaf spots that eventually lead to early plant defoliation (September rather than October), and dull, shriveled, undersized fruit, that fails to turn color and eventually becomes rotten (Lin et al. 2018).

On older plants, cankers and dieback can also occasionally be observed.

Cut branches of deciduous holly are traditionally used for winter holiday decorations, and the brightly colored fruit is the main interest for customers (Galle 1997). Due to the fruit turning necrotic, in the last several years it was not economically feasible for growers to harvest the crop; therefore, diseased plant tissues were left in the field. The presence of infected host tissues in the field has been identified as a source of inoculum for infection initiation and continuation throughout the growing season in many crop systems. For example, mummified fruit was identified as the main primary inoculum source for almond and olive anthracnose caused by

Colletotrichum spp. (Förster and Adaskaveg 1999; Moral and Trapero 2012), and brown rot on stone fruit caused by Monilinia spp. Honey (Casals et al. 2015; Holb and Scherm 2007).

Additionally, twigs, buds or leaves could serve as sources of inoculum for apple leaf blotch and fruit spot disease caused by Alternaria spp. Nees (Harteveld et al. 2014; Tanaka et al. 1989), as well as Alternaria brown spot of mandarin (Bassimba et al. 2014) and Phomopsis cane and leaf spots of grapes (Anco et al. 2012; Cucuzza and Sall 1982). Consequently, removal of infected plant tissues from fields has been suggested in different systems as a standard disease management practice to lower inoculum levels (Harteveld et al. 2014; Holb and Scherm 2007; Villarino et al.

2010).

Previous studies on the epidemiology of fruit rot of deciduous holly have identified susceptible developmental stages of the fruit to infections. Fruit infections caused by D. ilicicola occurred during the full bloom or petal fall stages, with the pathogen remaining quiescent until fruit maturation (Lin and Peduto Hand 2018). Also, wounded mature fruit was susceptible to infection by all pathogens tested (Lin and Peduto Hand 2018). However, inoculum availability in the field during these susceptible periods is currently unknown. Therefore, it is critical to monitor seasonal inoculum dynamics to formulate effective management strategies. Spore trapping studies have been conducted to determine seasonal spore dynamics for avocado branch canker caused by fungal species in the Botryosphaeriaceae and Diaporthaceae families (Eskalen et al. 2013), as well as Alternaria brown spot of mandarin caused by A. alternata (Bassimba et al. 2014). The environmental factors, including rainfall and temperature, were reported to be influencing inoculum abundance, spore production and infection in different climatic regions (Bassimba et al.

2014; Eskalen et al. 2013; Harteveld et al. 2014; Rotem 1994). The effects of these environmental

factors on seasonal inoculum dynamics and on disease development in deciduous holly fields is currently unknown.

While important aspects of this fruit rot disease have been revealed, there is still lack of knowledge on many components of the disease cycle, including potential sources of pathogen inoculum and seasonal inoculum dynamics, as well as the effects of environmental factors on disease development and inoculum availability. Therefore, the objectives of this study were to (i) identify the plant tissues where pathogens causing fruit rot overwinter (i.e. primary inoculum); (ii) identify the potential source of secondary inoculum during plant development; (iii) determine the timing of spore dissemination during the growing season; and (iv) identify the effects of temperature and precipitation on leaf spot development and seasonal inoculum dynamics in the field.

MATERIALS AND METHODS

Trial locations and characteristics. Field trials were initially established in two nurseries in Madison, OH (nurseries A and B) in 2015 and 2016, which were exposed to natural pathogen inoculum and in which fruit rot had been observed yearly since 2014 (Lin et al. 2018). Due to low disease incidence (<5%) observed at the end of the 2015 season, a third nursery (nursery C), located in Wooster, OH, in which fruit rot had also been recorded (Lin et al. 2018), was added in

2016. Trials were conducted in each nursery for 2 consecutive seasons (2015 and 2016 in nurseries

A and B; 2016 and 2017 in nursery C). Plant material included two susceptible cultivars of the deciduous holly hybrid Ilex verticillata x Ilex serrata (22 and 24 years-old cv. Bonfire in nurseries

A and C, respectively; 27 years-old cv. Sparkleberry in nursery B). At all locations, the

experimental plot comprised 20 trees from one individual row within the production field. In nurseries A and B, the rows surrounding the trial plots served as buffer zones, and both trial plots and buffer zones were not disturbed by any management practice at any point of the growing season throughout the duration of the trial. The rest of the field was managed according to the grower’s standard practices. In nursery C, the entire nursery, including the experimental plot, was not disturbed by any management practice throughout the duration of the trial.

Assessment of sources of primary inoculum. One-year old twigs and mummified fruit from the previous growing season were collected on April 1 of each year from each nursery

(nurseries A and B in 2015; nurseries A, B and C in 2016; nursery C in 2017), except that no mummified fruit was available in nurseries A and B in 2016 due to bird activity. During each sampling, six individual twigs with no visible cankers, and 10 mummified fruit, were randomly collected from the entire canopy of each of the 20 trees in the experimental plots and then pooled together inside a plastic bag (one bag per type of tissue sampled per nursery). Bags containing the samples were transported to the laboratory inside a cooler and stored at 4°C for a maximum of 24 hours before further processing. Sixty overwintering twigs and 100 mummified fruit were then randomly extracted from the respective bags from each nursery and processed as described herein: overwintering twigs were surface disinfected for 5 sec in 70% ethanol, followed by 1 min in 0.5% household bleach (6% sodium hypochlorite; The Clorox Company, Oakland, CA), while mummified fruit were surface disinfected for 2 min in 2% household bleach (6% sodium hypochlorite; The Clorox Company, Oakland, CA); both types of samples were then rinsed in sterile water three times. All samples were allowed to air dry on sterile paper towels inside a laminar flow hood. Buds, bark and xylem of each twig were separated using a sterile scalpel. Bark

and xylem tissues were further cut into 2 mm × 5 mm sections. One individual bud, bark and xylem sections per twig were randomly selected and plated on Potato Dextrose Agar (PDA; Difco

Laboratories, Sparks, MD) amended with 0.1% lactic acid, 0.01% tetracycline hydrochloride and

0.015% streptomycin sulfate (Fisher Scientific, Fair Lawn, NJ). Each mummified fruit was cut horizontally in half and plated cut-side down on V8 juice agar rather than PDA to facilitate recovery of some fungi with slower growth (Olatinwo et al. 2003). All plates were incubated on a laboratory bench under constant light at 25°C for up to 10 days. Fungal colonies developing from the tissues were counted and sub-cultured onto a new PDA plate. Pure cultures of each isolate, obtained by hyphal tip transfer, were preserved as mycelial plugs in sterile distilled water at 4°C

(Humber 1997). All isolates were tentatively identified to species based on morphology (Barnett and Hunter 1998; Baumgartner et al. 2013; Dissanayake et al. 2015; Lin et al. 2018;

Mahadevakumar et al. 2014; Shivas and Tan 2009; Simmons 2007; Udayanga et al. 2014; Velho et al. 2015; Wu et al. 2017). For isolates exhibiting similar morphological characteristics, at least half of those recovered from each nursery and each type of plant tissue were subject to molecular characterization to confirm identity as described in Lin et al. (2018).

Assessment of source of secondary inoculum. Holly leaves were sampled weekly or biweekly from bud break until plants were more than 30% defoliated to monitor leaf spot development and progression. Leaves were collected from May 15 to October 29, 2015 from nursery A (total samplings, i.e. n = 12) and B (n = 12); from May 12 to October 29, 2016 from nursery A (n = 16) and B (n = 16); and from May 12 to October 6, 2016 (n = 13) and May 12 to

September 29, 2017 (n = 13) from nursery C. At each sampling time, ten individual leaves were randomly collected from the entire tree canopy of each of the 20 trees in the experimental plots

and pooled together and mixed thoroughly inside a plastic bag. One hundred leaves were then randomly extracted from the pool, and leaf spot incidence and severity were visually assessed on each leaf. Disease incidence was assessed as presence or absence of leaf spots, while disease severity was assessed as the percentage of leaf surface affected by leaf spots. Total leaf spot incidence and severity were then calculated for each nursery. Subsequently, symptomatic leaves were subject to fungal isolation as described herein: leaves were surface disinfected in 2% household bleach (6% sodium hypochlorite; The Clorox Company, Oakland, CA) for 30 sec, followed by 50% ethanol for 30 sec, and rinsed three times in sterile water. Leaves were then placed on sterile paper towels inside a laminar flow hood to air-dry. Up to 30 pieces of leaf tissues

(2 mm × 2 mm) were cut from the edge of randomly selected lesions on each leaf, and then transferred to amended PDA previously described. Plate incubation, fungal isolation, identification and preservation of the fungal isolates were the same as described above.

Pathogenicity tests. Two sets of experiments, each being conducted twice, were conducted to determine pathogenicity of the fungal isolates recovered from leaf spots, on different plant tissues. One isolate of each of the major fungal species causing fruit rot (i.e., A. alternata, D. ilicicola, C. fioriniae, and E. nigrum; Lin et al. 2018) recovered from each nursery and year (if applicable), was randomly selected and used in each experiment. In the first experiment (A), one- month-old healthy leaves were collected from three-year-old potted Ilex verticillata x Ilex serrata cv. Sparkleberry plants and immediately surface disinfected as previously described. Ten individual leaves were wounded on eight spots per leaf using a sterile needle; six μL of a sterile water-Tween 20 solution (0.05% v/v) containing 105 conidia/mL of each isolate were then individually placed on each wound, as well as on eight spots on 10 unwounded leaves. Ten

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wounded and ten unwounded leaves inoculated with the same amount of sterile water-Tween 20 solution (0.05%, v/v) served as controls. Leaves were arranged in a completely randomized design on a plastic tray enclosed in a clear polyethylene bag containing wet sterile paper towels. The tray was incubated on a laboratory bench under constant light at 25 °C for up to five weeks. Leaf spot incidence was recorded as described above for each isolate. Fungal re-isolation from leaf spots and morphological identification of the isolates were carried out to prove their pathogenicity.

In experiment B, the same isolates used in experiment A were tested for pathogenicity on holly fruit. Healthy mature fruit was collected from the same plants used in experiment A and surface disinfected as described above. Eight individual fruit were used for each isolate. Each fruit was wounded using a sterile needle and point inoculated using a micropipette with six μL of a sterile water-Tween 20 solution (0.05% v/v) containing 105 conidia/mL of each isolate. Eight wounded fruit inoculated with six μL of sterile water-Tween 20 solution (0.05%, v/v) were used as controls. Experimental design, fruit incubation conditions, disease assessment, re-isolation and identification of the fungal isolates were the same as described above.

Spore trapping studies. Spore trapping studies were carried out in nurseries A and B in

2015 and 2016, and in nursery C in 2016 and 2017. Spore traps consisted of glass microscope slides (25 mm × 75 mm × 1 mm, Fisher Scientific, Fair Lawn, NJ) coated on both sides with a thin layer of petroleum jelly (TopCare, Elk Grove Village, IL), which were secured on holly branches using a clip binder and a zip tie (Figure 4.1). Two spore traps were placed at approximately 70 cm and 140 cm above ground, respectively, on the 5th, 11th and 16th tree within the experimental plot of each nursery (i.e. every 5 or 6 trees). Spore traps were changed weekly from April 1 to

November 12, 2015; from April 1 to November 28, 2016; and from April 1 to November 22, 2017,

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and collected individually in sterile 50 mL screw-cap tubes. Upon collection, spore traps were transported to the laboratory inside a cooler and stored at 4 °C for a maximum of 24 hours before being processed. For each spore trap, a spore suspension was obtained by adding 10 mL of sterile distilled water into the tube and shaking vigorously for 1 min by hand. Two, 200 μL suspension aliquots were placed on two replicate amended PDA plates previously described, and spread using a sterile 90° curved glass cell spreader (Chemglass Life Sciences LLC, Vineland, NJ). Plates were incubated on a laboratory bench at 25 °C under constant light. Fungal colony-forming units (CFUs) were counted for up to three weeks (Eskalen and Gubler 2001; Úrbez-Torres et al. 2010). Fungal isolate purification, identification and preservation were the same as previously described.

Meteorological data. Meteorological data, including maximum, minimum and average daily temperature, as well as precipitation were downloaded from the USDA-ARS weather research network (http://www.oardc.ohio-state.edu/weather1/) available for Madison and

Wooster, OH for the entire disease assessment periods (2015 - 2017).

Data analysis. To determine the sources of primary inoculum, the number of each overwintering plant tissue (i.e., bark, bud, xylem and mummified fruit) that yielded fungal growth in each nursery each year was counted. Fungal isolation frequency was calculated for each type of plant tissue at each sampling location, as well as for pooled data across locations. To test the effects of type of plant tissue on fungal recovery, a chi-square goodness of fit test (α = 0.05) was carried out for each individual pathogen on the pooled data using PROC FREQ in SAS (version 9.4; SAS

Institute Inc., Cary, NC).

The area under the disease progress curve (AUDPC) was calculated independently for each nursery and year using leaf spot incidence and severity data collected throughout the growing

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season (Shaner and Finnery 1977). Values of relative AUDPC (rAUDPC) were used to standardize the AUDPCs among nurseries and years, which were calculated using AUDPC values divided by the total number of days between the first (i.e., bud break) and the last assessment (Fry 1978). Due to intrinsic differences in trial locations and years of sampling, no statistical comparison was performed among rAUDPCs.

In the pathogenicity tests, leaf spot and fruit rot incidence data was subject to ANOVA analysis using PROC GLM in SAS (version 9.4; SAS Institute Inc., Cary NC). Datasets from the two experiment runs were combined prior to analysis when results were in congruence. All data were arcsine transformed before statistical analyses were performed and then back-transformed to present results. Tukey’s HSD test (α=0.05) was used for mean comparisons among pathogens.

In the spore trapping studies, the CFUs per mL of spore suspension of each holly fruit rot pathogen (i.e., Alternaria, Colletotrichum, Diaporthe and Epicoccum) were calculated for each sampling location and time, as well as its corresponding area under the spore production curve

(AUSPC) (Scherm et al. 2008). In order to determine seasonal spore abundance at different phenological stages of plant development in the field, values of AUSPC across the two years of data collection in each nursery were calculated for each of six plant developmental stages as indicated herein: stage 0 (leaf emergence) – from the establishment of the trial with plants that are still in dormancy to the onset of flower bud development – April to June; stage 1 (flower bud) –

June; stage 2 (full bloom) – June; stage 3 (petal fall) – June; stage 4 (immature fruit) – June to

September; and stage 5 (mature fruit) – September to December. To standardize AUSPC in each stage, relative AUSPC (rAUSPC) was calculated using values of AUSPC divided by the number of days in the corresponding stages (Fry 1978). A repeated measures ANOVA analysis was

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performed to compare rAUSPC of different stages of plant development (stage 0-5) for each individual pathogen. All data were arcsine transformed and analyzed using PROC GLIMMIX in

SAS (version 9.4; SAS Institute Inc., Cary NC) and then back-transformed to present results. The number of days of each plant developmental stage was specified in the WEIGHT option. The stage of plant development and nursery were treated as fixed factors, while the year was treated as a random factor. Least squares means (LS-means; α=0.05) were used for comparison among factors.

Finally, to determine the relationship between leaf spot development or seasonal spore abundance, and environmental variables, mean maximum, minimum and average daily temperature and accumulated precipitation between consecutive disease assessments from the period of one week before visible symptoms until the last disease assessment, or between consecutive spore traps collections, were calculated for each nursery and year during the entire experiment periods. Pearson’s correlation test (α = 0.05) was used to analyze the correlation between meteorological factors and leaf spot incidence and severity or spore counts using PROC

CORR in SAS (version 9.4; SAS Institute Inc., Cary, NC).

RESULTS

Primary inoculum. From 2015 to 2017, a total of 360 twigs as well as 400 mummified fruit from three nurseries were processed. While no fungi were isolated from the twigs’ xylem, species of Alternaria, Cladosporium Link, Colletotrichum, Diaporthe, Epicoccum, Fusarium

Link, Phoma Sacc. and fungi in the Botryosphaeriaceae Theiss. & P. Syd., were isolated from the bark, buds or the mummified fruit. Among these, the two most frequently isolated genera were

Alternaria and Diaporthe from both bark and buds, and Alternaria and Epicoccum from mummified fruit (data not shown). Selected isolates recovered from bark (n = 9), buds (n = 7) and 104

mummified fruit (n = 13) were identified as A. alternata, A. arborescens, C. fioriniae, C. nymphaeae, D. ilicicola, fungi in the D. eres species complex and E. nigrum based on morphological features and molecular characterization as described in Lin et al. (2018; data not shown). All fungi were recovered from bark, buds and mummified fruit, except for Colletotrichum, which was not recovered from buds (Table 4.1). The highest isolation frequency of Alternaria,

Colletotrichum and Epicoccum was obtained from mummified fruit, while that of Diaporthe was obtained from bark (Table 4.1).

Secondary inoculum. Over the three years of sampling from the three nurseries, 938 out of the 1532 symptomatic leaves processed yielded fungal growth. Species of Alternaria and

Diaporthe were the most frequently isolated pathogens from leaf spots (59.38% and 25.48%, respectively), followed by Epicoccum, Colletotrichum, Phoma, fungi in the Botryosphaeriaceae and Pestalotia De Not. (<10%; Table 4.2). Of the 75 isolates that were selected for morphological and molecular identification, the most prevalent species were the same as those recovered from the overwintering plant tissues, including A. alternata, A. arborescens, C. fioriniae, C. nymphaeae,

D. ilicicola, fungi in the D. eres species complex and E. nigrum (data not shown).

Leaf spot symptoms were first observed on June 26, 2015 and August 18, 2016 in nursery

A; on June 26, 2015 and August 25, 2016 in nursery B; and on July 15, 2016 and July 7, 2017 in nursery C. Leaf spots were initially light brown and irregularly shaped. As disease progressed over the season, lesions turned dark brown to black and coalesced causing blighted areas (Figure 4.2) and subsequent leaf drop. Leaf spot incidence and severity increased along with leaf and fruit development, and ranged between 70-100% and 10.79-33.37%, respectively by the final disease assessment (Table 4.3). For nursery A and C, incidence and severity rAUDPC values in the first

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sampling year (i.e. 2015 for nursery A, and 2016 for nursery C) were lower than those in the second year (i.e. 2016 for nursery A, and 2017 for nursery C; Table 4.3). The opposite trend was observed in nursery B.

No correlation was found between leaf spot incidence or severity and cumulative precipitation in any nursery-year combination (Table 4.4). Leaf spot incidence was negatively correlated to the mean average temperature in all observations except for nurseries A and B in

2015, and nursery C in 2017. Mean maximum and minimum temperatures were always negatively correlated to leaf spot incidence in nurseries A and B, while for nursery C only in the combined year (2016 + 2017) observation (Table 4.4). Except for nurseries A and B in 2015 and nursery C in 2017, a negative correlation was found between leaf spot severity and mean maximum, minimum and average temperatures (Table 4.4).

Pathogenicity tests. All isolates of A. alternata, C. fioriniae, D. ilicicola and E. nigrum originally isolated from leaf lesions were able to infect detached wounded and unwounded holly leaves, which proved their pathogenicity (experiment A; Table A.1). Leaf spots symptoms similar to those observed in the field started developing from the wounded treatments one week post- inoculation. Unwounded treatments started developing symptoms two weeks post-inoculation.

Pycnidia formed on the lesions on leaves inoculated with D. ilicicola three weeks post-inoculation.

No symptoms were observed on the control leaves. All aforementioned isolates also successfully infected wounded mature fruit (experiment B; Table 4.5). Fruit inoculated with A. alternata, C. fioriniae, and E. nigrum developed brown lesions from the wound, which enlarged to cover the entire fruit after three to five days, except for lesions caused by E. nigrum, which remained limited to the point of inoculation. Salmon discoloration of the fruit was observed on those inoculated with

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D. ilicicola one week post-inoculation, followed by production of pycnidia on the entire fruit surface. Mean fruit rot incidence values reached 91.25% or above for all pathogens (Table 4.5).

Fruit rot incidence caused by C. fioriniae was significantly lower than that of other inoculations

(Table 4.5). No fruit rot developed on the control fruit.

Spore trapping studies. Species of Alternaria, Cladosporium, Colletotrichum, Diaporthe,

Epicoccum, Fusarium, and Phoma, which were consistently isolated from the plant tissues processed in this study, were also retrieved from the spore traps. Spores were produced throughout the growing season. For Alternaria and Colletotrichum, the stage of plant development was a significant factor influencing spore abundance (p = 0.0044, and 0.0272, respectively); specifically, rAUSPC of Alternaria at stage 2 (full bloom) was significantly higher than at stages 0 (leaf emergence), 1 (flower bud), 4 (immature fruit) and 5 (mature fruit); and rAUSPC of

Colletotrichum at stage 2 was significantly higher than at stage 0 and 5 (Table 4.6). For Diaporthe, nursery, stage of plant development, and their interaction were all significant factors (p = 0.0211,

0.0477, and 0.005, respectively); specifically, rAUSPC at stage 2 in nursery C was significantly higher than in all other stage-nursery combinations (Table 4.6). No significant effect was found for Epicoccum.

A significant negative correlation was found between mean maximum, minimum and average daily temperatures and number of Alternaria (r = -0.18, p < 0.05) and Epicoccum spores

(r = -0.18, p < 0.05) only, across three years of studies in all nurseries (data not shown). No correlation was found between spore counts and accumulated precipitation.

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DISCUSSION

In this study we identified potential sources of primary and secondary inoculum of fruit rot pathogens of deciduous holly in Ohio nurseries. We also monitored seasonal inoculum dynamics and leaf spot progression, and described their relationship to environmental factors.

The pathogens causing fruit rot on deciduous holly were consistently isolated from the different overwintering host tissues analyzed, including buds, bark and mummified fruit. Among these, mummified fruit was the main source of overwintering inoculum for Alternaria and

Epicoccum, while mummified fruit and bark were equally important for Diaporthe. Colletotrichum was also equally isolated from bark and mummified fruit, although at very low levels, but never retrieved from buds. Based on these findings, bark, buds and mummified fruit should all be considered as potential sources of primary inoculum for these fruit rot pathogens.

Diaporthe is a common canker pathogen on many woody plants, including almond (Varjas et al. 2017), grape (Baumgartner et al. 2013), and pear (Bai et al. 2015). Although Diaporthe is one of the primary pathogens causing fruit rot of deciduous holly, cankers were rarely observed on the plants included in this study, which, at the time this disease was first found in 2012 (Lin et al. 2018), were 19-24 years old. This may be due to the fact that trees might be more susceptible to cankers as they grow older (Amponsah et al. 2017; Arnold and Straby 1973). Indeed, just recently (2017) a canker was observed on a twig in the experimental plot, from which D. ilicicola was isolated (data not shown). Additionally, cankers and dieback were observed in older fields on plants that were more than 40 years old (Lin, personal observation). Continuing to monitor canker development in nurseries will be important to identify if/when infected limbs should be pruned.

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As suggested in other crop systems, infected host tissues should be removed from the field to decrease inoculum levels (Harteveld et al. 2014; Holb and Scherm 2007; Villarino et al. 2010).

In deciduous holly commercial production, only one quarter to one third of the branches carrying the fruit are harvested from the plants to be sold (Galle 1997). In some years, branch harvest could be even lower due to decreased product demand or poor fruit quality, in which cases twigs and mummified fruit remain in the field until early spring. A previous study carried out to determine the effects of inoculum concentrations on holly fruit infection using detached fruit suggested that higher inoculum concentration could result in more severe disease (Lin et al. 2018). Therefore, removal of overwintering infected twigs and mummified fruit potentially harboring fruit rot pathogens should be recommended to decrease inoculum concentration in the field. Although complete mummified fruit removal is not practical for growers to implement due to the small fruit size and large number of fruit per plant, pruning infected twigs with attached diseased fruit could be feasible.

Over three years of leaf collection from three nurseries revealed that Alternaria and

Diaporthe were the pathogens most frequently associated with leaf spots (>25%), followed by

Epicoccum and Colletotrichum (>5%). Additionally, the species A. alternata and D. ilicicola were consistently isolated from each sampling location and year. This was in agreement with the results of symptomatic fruit isolations carried out in previous studies (Lin et al. 2018), and reinforces our conclusions that A. alternata and D. ilicicola are the primary pathogens in the fungal complex, while A. arborescens, C. fioriniae, C. nymphaeae, fungi in the D. eres species complex and E. nigrum are minor pathogens causing both leaf spots and fruit rot.

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Leaf spot symptoms caused by the individual pathogen were not distinct, and in approximately one third of the cases fungi were not recovered from leaf lesions. Therefore, some leaf symptoms may have been caused by other factors, such as insect activity, mechanical injury, herbicide drift, or extensive periods of leaf wetness (Galle 1997). While in the current study we focused on the role leaf spots caused by fungal pathogens might have in the disease cycle, the effects of other factors causing leaf spots should be further investigated.

Sources of primary and secondary pathogen inoculum are known to be associated with the level of disease developing in fields (Harteveld et al. 2014; Holb and Scherm 2007; Villarino et al. 2010). Thus, differences in rAUDPC observed across nurseries in this study may have been due to the amount of inoculum present in each field. For example, in 2015, rAUDPC values in nursery

A were lower than in nursery B, which may have been due to a lower level of primary inoculum present in nursery A. Indeed, even though in 2014 both nurseries experienced severe fruit rot, and the non-marketability of the fruit led growers to not harvest any branches, at the beginning of 2015 nursery A pruned infected twigs and removed some mummified fruit, a practice that could have been responsible for decreased inoculum concentrations. In 2016, rAUDPC in nurseries A and B were both lower than in nursery C. One possible explanation could be that although the trial plot and buffer zones in all nurseries were not disturbed by any management practice, the rest of nurseries A and B was subject to growers’ standard practices, which, in the previous season (2015), included multiple chemical applications that resulted in little leaf spot observed outside of the trial plot (Lin, personal observation). This in turn could have been responsible for decreased inoculum levels in the entire field. In addition, low fruit rot incidence was observed in the entire field in nurseries A and B in 2015 (< 5%; data not shown) and the fruit that was not harvested, which could

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have potentially served as a source of primary inoculum, was likely eaten up by birds by the end of 2015. Moreover, the first leaf spot symptoms in nurseries A and B in 2015 were observed in

June, while in 2016 they were observed in August. Delayed leaf spot development may have been the consequence of decreased field inoculum levels in 2016.

Similar considerations can be made regarding nursery C, where rAUDPC in 2016 was lower than in 2017. Although no management practices were carried out in the entire nursery throughout the study period, branches were harvested at the end of 2015 (before the establishment of our trial) even though fruit rot was observed in the field. Therefore, some infected tissues potentially harboring pathogens were removed. Similar to what was observed for leaf spots, we previously reported that fruit rot incidence and severity in nursery C in 2016 was also lower compared to 2017 (Lin and Peduto Hand 2018). All these observations reinforced the conclusion that removal of inoculum could contribute to decreasing infections during the growing season.

That being said, while similar grower standard practices were carried out in nurseries A and B, rAUDPC of leaf spot incidence and severity in nursery A in 2015 was lower than in 2016, while in nursery B was the opposite. This indicates that decreasing inoculum levels might not be the only factor influencing disease development.

Both leaf spot incidence and severity were found to be negatively correlated to mean maximum, minimum and average daily temperature across sampling locations and years. This might suggest that low temperature could play a role in leaf spot development. However, a decrease in temperature coincided with leaf senescence, which may also be a factor influencing host susceptibility to infections (Häffner et al. 2015). Therefore, conclusions on the role of temperature on disease development cannot be made based on the data collected in this study.

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All four major fruit rot causal agents, i.e. A. alternata, D. ilicicola, C. fioriniae and E. nigrum recovered from leaf spots were pathogenic to both wounded and unwounded young leaves, but it took longer for unwounded leaves to develop symptoms, which suggested that any biotic or abiotic injury to the leaves should be avoided to minimize leaf infection. Furthermore, all these leaf spot pathogens also successfully infected holly fruit in detached fruit assays and produced the same symptoms observed in the field (Lin et al. 2018), which suggested the role of leaf spots as a source of secondary inoculum during the growing season. Consequently, any practices (e.g., chemical application, sanitation) that can reduce leaf spot occurrence might be helpful in reducing fruit infections.

The same group of fungal pathogens consistently isolated from host tissues (Lin et al. 2018;

Lin and Peduto Hand 2018; and this study) was also recovered from the spore traps used in this study. Peak rAUSPC values of spore counts for Alternaria, Diaporthe and Colletotrichum across three nurseries mostly occurred during the full bloom stage. This supported the results of previous studies on the timing of fruit susceptibility to infection, in which the isolation frequency of

Alternaria, Colletotrichum and Epicoccum from fruit exposed to natural inoculum increased after flowering, and where fruit artificially inoculated with Diaporthe ilicicola at the full bloom and petal fall stages successfully developed symptoms (Lin and Peduto Hand 2018). Spores of

Diaporthe, one of the primary pathogens in the disease complex, were mainly caught in nursery C but not frequently from nursery A and B. Even though Diaporthe was identified as a main player in the disease based on extensive sampling across multiple years and locations (Lin et al. 2018), the relative distribution of this pathogen in a specific nursery could be different, which may explain the differences in inoculum abundance observed in this study across nurseries.

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A correlation analysis was conducted to understand the role of environmental factors on spore production. Spore counts of Alternaria and Epicoccum were negatively correlated to mean maximum, minimum and average temperature; however, the Pearson correlation coefficient (r) was very low (-0.18). This could be due to the fact that only a few spores were caught by the spore traps across all locations and years of sampling, making it difficult to correlate with weather data.

Spore traps made of glass slides coated with petroleum jelly (used in this study) have been successfully used to monitor seasonal spore abundance in vineyards, mandarin and cherry orchards in Mediterranean-type climates (Bassimba et al. 2014; Eskalen and Gubler 2001; Trouillas et al.

2012; Úrbez-Torres et al. 2010) but their performance in colder climates was unknown. Although other spore trapping devices are available for monitoring spore abundance (i.e. Hirst-Burkard volumetric spore trap) they mostly rely on morphological identification of the spores directly from the traps, or, alternatively, can be coupled with PCR-based assays using species-specific primers

(Calderon et al. 2002). However, some pathogens of interest in this study (i.e., Alternaria,

Diaporthe, and Epicoccum) cannot be identified to species level solely using morphology (Lin et al. 2018). Also, as one of the primary pathogens in the fungal complex (D. ilicicola) was only recently described, no species-specific primers are currently available. The spore traps used in this study not only allowed us to obtain fungal colonies and further identify isolates to the species level using both morphological and molecular characterization, but were also low cost, thus representing a useful exploratory tool to monitor inoculum dynamics in the field. The low spore numbers caught in this study were possibly due to the efficacy of the adhesive in capturing spores in our specific climatic conditions (Jenkyn 1974). Another possible reason for the low spore numbers caught could be that low levels of inoculum was available in the field during the period of the study due

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to the aforementioned removal of primary and secondary inocula. Indeed, nurseries A and B, which were regularly managed by growers, had relatively fewer spore counts compared to nursery C, which was rarely disturbed. Even though the overall spore abundance was low, the above observations still support our conclusion that removal of primary and secondary inoculum would help to decrease field inoculum concentrations. Further studies to determine the effects of environmental factors on inoculum production should be carried out in nurseries with high levels of inoculum.

Based on our previous research (Lin and Peduto Hand 2018; Lin et al. 2018) and findings from the current study, we hypothesize that the disease cycle of fruit rot on deciduous holly is as follows: the disease causal agents overwinter in buds, bark of woody tissues, as well as mummified fruit left on the trees at the end of the growing season. Spores start to discharge from the primary inoculum sources after bud break. The majority of the spores are disseminated during flowering by means of wind, rain, or through the activity of pollinators. Open flowers as well as the developing fruit at petal fall, and leaves, are susceptible to primary infections. Pathogens, such as

D. ilicicola, cause latent infections remaining inactive in the developing fruit until yet undetermined changes in host physiological properties occurring during fruit maturation activate it, resulting in rot symptoms development from the stigma- or calyx-end of mature fruit. Leaves infected by primary inoculum develop leaf spot symptoms during summer. Conidia from the primary lesions infect other healthy leaves and establish secondary infections. Conidia produced on the infected leaves act as secondary inoculum to continue infecting newly emerged healthy leaves throughout the growing season as well as injured mature fruit at the end of the season.

Expanded and coalesced leaf lesions result in early plant defoliation. Infected fruit is undersized,

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shriveled, loses gloss and becomes rotten in November, just prior to harvest. Conidia produced from the infected fruit disseminate and infect other healthy fruit in the presence of open wounds.

This study contributed to deepen our understanding of this disease by identifying the sources of primary and secondary inoculum in nurseries and their dissemination throughout the growing season. The information generated through this study is of direct relevance to the design of disease management strategies. Experiments testing the efficacy of chemical treatments at different stages of fruit development are ongoing, and will likely result in improved management of this emerging disease.

ACKOWLEDGEMENTS

This study was funded by the Ohio Department of Agriculture Specialty Crop Promotion Program

(Awards AGR-SCG-14-08 and AGR-SCG-16-09), the USDA-NIFA Hatch project #1004939, the

T. J. Kavanagh Foundation, and The Ohio State University Department of Plant Pathology. The research described in this paper represents a portion of the dissertation submitted by S. Lin to the

Office of Graduate Studies of The Ohio State University to partially fulfill requirements for the

Ph.D. degree in Plant Pathology. The authors thank all the holly growers in Ohio who provided many of the samples used in this study. The authors also thank Dr. G. Marchi for critical review of this manuscript prior to submission.

115

Table 4.1. Fungal isolation frequency from overwintering plant tissues, including bark, bud, xylem, and mummified fruit collected from nurseries A, B and C from 2015 to 2017.

Pathogen Overwintering Isolation frequency (%) plant tissue Nursery Aw Nursery Bw Nursery Cw Pooledx Alternaria Bark 12.50 13.33 5 10.28 bz Bud 8.33 10 0.83 6.39 b Xylem 0 0 0 0 c Mummified fruit 70y 100 y 86.5 85.75 a

Diaporthe Bark 4.17 22.50 31.67 19.44 a Bud 1.67 10 24.17 11.94 b Xylem 0 0 0 0 c Mummified fruit 9 y 7 y 20 14 b

Colletotrichum Bark 0 0 3.33 1.11 a Bud 0 0 0 0 b Xylem 0 0 0 0 b Mummified fruit 0 y 0 y 2.5 1.25 a

Epicoccum Bark 18.33 2.5 3.33 8.06 b Bud 4.17 0 0.83 1.67 c Xylem 0 0 0 0 d Mummified fruit 45 y 17 y 41.5 36.25 a

wColumns represent the value of pooled data across two years of observations in each nursery, unless otherwise noted. xColumn represents the value of pooled data across all years and nurseries. yData include only one year of observations (mummified fruit was not present in 2016 due to bird activity) zValues with the same letter within each pathogen are not significantly different according to Chi- square goodness of fit test (α = 0.05).

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Table 4.2. Fungal isolation frequency from the 938 symptomatic leaves that yielded fungal growth from 2015 to 2017 across all three nurseries.

Fungal species Isolation frequency (%) Alternaria spp. 59.38 Diaporthe spp. 25.48 Epicoccum spp. 5.76 Colletotrichum spp. 5.12 Phoma spp. 3.62 Botryosphaeriaceae 2.03 Pestalotia spp. 0.21

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Table 4.3. Measures of leaf spot incidence and severity used to compare disease development in nurseries A, B and C from 2015 to 2017.

Mean final assessment (%)a rAUDPCb Year Nursery Incidence Severity Incidence Severity 2015 A 70.00 10.79 15.90 1.90 B 100.00 27.14 32.80 5.80 2016 A 94.00 16.33 28.30 2.80 B 100.00 33.37 28.50 4.10 C 92.00 21.67 32.90 6.10 2017 C 76.00 12.33 40.10 7.60

aColumns represent the mean disease value (%) on the final assessment. brAUDPC = relative area under the disease progress curve. Columns represent the AUDPC values divided by the total number of days between the first (i.e., bud break) and the last assessment. Each nursery and year represent one data point.

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Table 4.4. Correlation between leaf spot incidence and severity and environmental variables in nurseries A, B and C from 2015 to 2017.

Pearson correlation coefficient (r) a, b Correlation factor Nursery Year P Max. °T Min. °T Avg. °T Leaf spot incidence A 2015 -0.64* -0.74* 2016 -0.87** -0.84** -0.82* B 2015 -0.75* -0.79* 2016 -0.89** -0.86* -0.82* C 2016 -0.57 2017 A 2015+2016 -0.51* -0.62* -0.57* B 2015+2016 -0.73** -0.78** -0.47 C 2016+2017 -0.41 -0.49* A+B+C 2015+2016+2017 -0.42** -0.58** -0.47** Leaf spot severity A 2015 -0.81* -0.87** 2016 -0.96** -0.96** -0.91** B 2015 -0.9** -0.93** 2016 -0.95** -0.93** -0.91** C 2016 -0.7* -0.63 -0.74* 2017 -0.62 -0.56 A 2015+2016 -0.79** -0.87** -0.57* B 2015+2016 -0.9** -0.92** -0.44 C 2016+2017 -0.54** -0.61** -0.67** A+B+C 2015+2016+2017 -0.53** -0.69** -0.45**

aBlank cell = not significant (p > 0.05); *= significant at p < 0.01; **= significant at p < 0.001. bP = accumulated precipitation (mm); Max. °T = mean maximum temperature (°C); Min. °T = mean minimum temperature (°C); Avg. °T = mean average daily temperature (°C).

119

Table 4.5. Results of the pathogenicity tests conducted on detached fruit using selected fungal isolates retrieved from symptomatic leaves collected in the three nurseries.

Fungal species Isolate No.v Isolate origin w Fruit rot incidence (%)x, y Alternaria alternata FPH2015319 A 100.00 FPH2016506 A 100.00 FPH2015335 B 100.00 FPH2016534 B 100.00 FPH2016587 C 100.00 FPH2017129 C 100.00 Mean 100.00 az Diaporthe ilicicola FPH2015307 A 81.25 FPH2015314 B 93.75 FPH2016464 B 100.00 FPH2016557 C 93.75 FPH2017127 C 100.00 Mean 93.75 a Epicoccum nigrum FPH2015327 A 100.00 FPH2016571 A 93.75 FPH2015321 B 100.00 FPH2016576 B 100.00 FPH2016582 C 100.00 Mean 98.75 a Colletotrichum fioriniae FPH2015326 A 100.00 FPH2015339 B 93.75 FPH2016410 B 81.25 FPH2016484 C 100.00 FPH2017157 C 81.25 Mean 91.25 b

vThe first four digits in the isolate number refer to the year of isolation. wNursery from which the isolate was retrieved. xColumns represent the back-transformed value of pooled data from two experiment runs. yInoculations were done on wounded fruit only. zValues with the same letter are not significantly different according to Tukey’s HSD test (α = 0.05).

120

Table 4.6. Results of the spore trapping studies conducted in the three nurseries from 2015 to 2017.

v Stage of plant rAUSPC Pathogen development Nursery Aw Nursery Bw Nursery Cw Pooledx Alternaria 0 0.24 0.03 0.33 0.20 ay 1 1.63 0.25 0.00 0.63 a 2 3.32 3.27 6.00 4.20 b 3 0.25 1.69 0.82 0.92 ab 4 0.07 0.39 0.32 0.26 a 5 0.07 0.71 0.10 0.29 a Colletotrichum 0 0.00 0.00 0.13 0.04 a 1 0.00 0.00 0.44 0.15 ab 2 1.25 5.00 2.00 2.75 b 3 0.00 0.71 4.36 1.69 ab 4 0.34 0.21 0.07 0.21 ab 5 0.00 0.11 0.12 0.08 a Diaporthe 0 0.00 0.00 0.00 0.00 NAz 1 0.00 0.00 0.00 0.00 NA 2 0.00 0.00 3.50 1.17 NA 3 0.00 0.00 0.00 0.00 NA 4 0.00 0.00 0.00 0.00 NA 5 0.00 0.35 0.00 0.12 NA

vrAUSPC = relative area under the spore production curve. wColumns represent the value of pooled data across two years of observations in each nursery. xColumn represent the value of pooled data across all years and nurseries. yValues with the same letter within each pathogen are not significantly different according to least squares means (LS-means; α = 0.05). zNA= not applicable. rAUSPC values for Diaporthe were not compared across nurseries due to the significant interaction between nursery and stage of plant development (p = 0.005). The rAUSPC value at stage 2 in nursery C is significantly higher than in all other nursery-stage combinations.

121

Figure 4.1. Glass microscope slide coated with petroleum jelly used as a spore trap hanging from a deciduous holly tree.

122

Figure 4.2. Healthy leaf (left) and examples of different degrees of leaf spot severity observed on holly leaves.

123

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Appendix A. Supplementary Material for Chapter 4

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Table A.1. Results of the pathogenicity tests conducted on detached leaves using selected fungal isolates retrieved from symptomatic leaves collected in the three nurseries.

Leaf spot incidence (%)y Fungal species Isolate No.w Isolate Origin x Wounded Unwounded Alternaria alternata FPH2015319 A 100.00 100.00 FPH2016506 A 80.00 90.00 FPH2015335 B 80.00 95.00 FPH2016534 B 70.00 100.00 FPH2016587 C 65.00 95.00 FPH2017129 C 100.00 100.00 Mean 82.50 az 96.67 az Diaporthe ilicicola FPH2015307 A 100.00 90.00 FPH2015314 B 75.00 75.00 FPH2016464 B 80.00 80.00 FPH2016557 C 70.00 95.00 FPH2017127 C 70.00 75.00 Mean 79.00 a 83.00 b Epicoccum nigrum FPH2015327 A 55.00 50.00 FPH2016571 A 50.00 55.00 FPH2015321 B 80.00 85.00 FPH2016576 B 65.00 60.00 FPH2016582 C 90.00 85.00 Mean 68.00 b 67.00 b Colletotrichum fioriniae FPH2015326 A 90.00 90.00 FPH2015339 B 100.00 100.00 FPH2016410 B 90.00 90.00 FPH2016484 C 85.00 95.00 FPH2017157 C 90.00 90.00 Mean 91.00 a 93.00 a

wThe first four digits in the isolate number refer to the year of isolation xNursery from which the isolate was retrieved yColumns represent the back-transformed values of pooled data from two experiment runs. zValues with the same letter within the same column are not significantly different according to Tukey’s HSD test (α = 0.05). 138

Table A.2. Colony forming units of fruit rot pathogens in the spore trapping studies in nurseries A, B and C from 2015 - 2017.

Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. A 2015 0 7-Apr 0 0 0 0 17-Apr 1 0 0 0 24-Apr 0 0 0 0 1-May 0 0 0 0 8-May 0 0 0 0 15-May 0 0 0 0 21-May 0 0 0 0 29-May 1 0 0 0 5-Jun 0 0 0 0

139 1 12-Jun 0 0 0 0 19-Jun 0 13 0 1 2 26-Jun 0 0 0 1 3 2-Jul 0 0 0 0 4 10-Jul 0 0 0 0 16-Jul 1 0 0 0 30-Jul 0 0 0 0 6-Aug 0 20 0 1 13-Aug 2 0 0 0 21-Aug 0 0 0 0 28-Aug 0 0 0 0 4-Sep 1 0 0 0 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 12-Sep 0 0 0 0 26-Sep 3 0 0 0 5 2-Oct 0 0 0 0 10-Oct 1 2 0 0 16-Oct 1 0 0 0 22-Oct 0 0 0 0 29-Oct 0 0 0 0 5-Nov 1 0 0 1 12-Nov 2 0 0 0 2016 0 8-Apr 0 0 0 0 15-Apr 0 0 0 0 140 22-Apr 0 0 0 0

29-Apr 1 0 0 0 6-May 0 0 0 0 12-May 0 0 0 0 20-May 0 0 0 0 27-May 0 0 0 0 3-Jun 1 0 0 0 1 9-Jun 0 0 0 0 16-Jun 0 0 0 0 2 23-Jun 2 0 0 0 3 30-Jun 0 0 0 0 4 7-Jul 0 0 0 0 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 15-Jul 0 0 0 0 21-Jul 0 0 0 0 28-Jul 0 1 0 0 11-Aug 0 0 0 0 18-Aug 0 0 0 0 25-Aug 0 0 0 0 1-Sep 0 0 0 0 7-Sep 1 0 0 0 15-Sep 0 0 0 0 21-Sep 0 0 0 0 5 29-Sep 0 0 0 0

141 6-Oct 0 0 0 0

13-Oct 1 0 0 0 20-Oct 1 0 0 1 29-Oct 1 0 0 0 3-Nov 0 0 0 1 10-Nov 1 0 0 0 17-Nov 1 0 0 1 28-Nov 2 0 0 3 B 2015 0 7-Apr 1 0 0 2 17-Apr 0 0 0 1 24-Apr 1 0 0 1 1-May 0 0 0 0 8-May 0 0 0 0 15-May 0 0 0 0 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 21-May 0 0 0 0 29-May 0 0 0 0 1 5-Jun 3 0 0 0 2 19-Jun 1 0 0 0 26-Jun 0 0 0 0 3 2-Jul 0 0 0 0 4 10-Jul 0 0 0 0 16-Jul 0 0 0 0 30-Jul 0 0 0 0 6-Aug 0 0 0 0

142 13-Aug 0 0 0 0

21-Aug 0 0 0 0 28-Aug 1 0 0 0 4-Sep 0 0 0 0 12-Sep 0 0 0 2 18-Sep 1 0 0 2 26-Sep 3 0 0 0 5 2-Oct 1 1 0 2 10-Oct 0 0 0 0 16-Oct 1 1 0 0 22-Oct 0 0 0 0 29-Oct 0 1 0 0 5-Nov 0 0 0 1 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 12-Nov 1 0 0 0 2016 0 8-Apr 1 0 0 0 15-Apr 1 0 0 0 22-Apr 1 0 0 0 29-Apr 0 0 0 1 12-May 0 0 0 0 20-May 2 0 0 2 27-May 0 0 0 0 3-Jun 0 0 0 1 1 9-Jun 0 0 0 0 16-Jun 0 0 0 0 143 23-Jun 0 0 0 0 2 30-Jun 0 0 0 0 3 7-Jul 0 0 0 0 4 15-Jul 0 0 0 0 21-Jul 0 0 0 0 28-Jul 1 0 0 0 7-Aug 0 0 0 0 11-Aug 0 0 0 0 18-Aug 0 0 0 0 25-Aug 0 0 0 0 1-Sep 0 0 0 0 7-Sep 0 0 0 0 15-Sep 1 0 0 0 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 21-Sep 0 0 0 0 29-Sep 0 0 0 0 5 6-Oct 0 0 3 0 13-Oct 0 0 3 0 20-Oct 1 1 0 1 29-Oct 0 0 0 0 10-Nov 0 0 0 4 17-Nov 1 0 0 0 28-Nov 1 1 0 0 C 2016 0 8-Apr 0 1 0 0 15-Apr 0 0 0 0

144 21-Apr 0 0 0 1

29-Apr 0 1 0 0 6-May 0 0 0 0 12-May 0 1 0 0 19-May 0 0 0 0 1 27-May 0 0 0 0 2-Jun 0 3 0 1 9-Jun 0 3 0 0 2 16-Jun 0 0 0 0 3 23-Jun 0 0 0 0 4 30-Jun 0 1 0 0 7-Jul 1 1 0 0 15-Jul 0 0 0 0 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 21-Jul 0 0 0 0 28-Jul 0 1 0 0 7-Aug 0 0 0 0 11-Aug 0 1 0 0 18-Aug 0 0 0 0 25-Aug 0 0 0 0 1-Sep 0 0 0 0 15-Sep 0 0 0 0 21-Sep 0 0 0 0 29-Sep 1 1 0 0

145 5 6-Oct 0 0 0 0 13-Oct 1 0 0 0

20-Oct 0 0 0 1 29-Oct 0 0 0 1 3-Nov 0 0 0 0 10-Nov 0 0 0 0 17-Nov 0 0 0 0 28-Nov 0 0 0 0 2017 0 7-Apr 0 1 0 0 14-Apr 0 0 0 0 21-Apr 0 1 0 0 28-Apr 0 0 0 0 5-May 2 0 0 0 12-May 2 0 0 0 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 19-May 0 0 0 0 1 26-May 1 0 0 0 2-Jun 2 0 5 0 9-Jun 0 0 0 0 2 16-Jun 0 0 0 0 3 23-Jun 0 0 0 1 4 30-Jun 2 1 0 2 7-Jul 1 1 0 1 21-Jul 0 0 0 0 28-Jul 0 0 0 1

146 3-Aug 0 0 0 1

11-Aug 3 2 0 0 18-Aug 0 0 0 2 25-Aug 0 0 0 0 1-Sep 1 0 2 0 8-Sep 1 0 0 1 15-Sep 0 0 0 2 22-Sep 0 0 0 1 29-Sep 2 0 0 2 5 6-Oct 2 1 0 3 13-Oct 0 0 0 5 21-Oct 2 0 0 2 29-Oct 1 1 0 4 4-Nov 2 0 0 1 Continued

Table A.2 Continued Stage of plant Date of Colony forming unit/mL Nursery Year development Collection Alternaria spp. Colletotrichum spp. Diaporthe spp. Epicoccum spp. 10-Nov 1 0 0 3 17-Nov 0 0 0 2 22-Nov 0 0 0 3

147