The Pennsylvania State University

The Graduate School

CELERY LEAF CURL DISEASE: UNRAVELING THE CAUSAL AGENT,

POPULATION GENETICS, SYMPTOMOLOGY AND FUNGICIDE

PERFORMANCE FOR IMPROVED DIAGNOSTICS AND MANAGEMENT

A Dissertation in

Plant Pathology

by

Sara R. May

© 2019 Sara R. May

Submitted in Partial Fulfilment of the Requirements

for the Degree of

Doctor of Philosophy

August 2019

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The dissertation of Sara R. May was reviewed and approved* by the following: Beth K. Gugino Professor of Plant Pathology Dissertation Adviser Co-Chair of Committee María del Mar Jiménez Gasco Associate Professor of Plant Pathology Co-Chair of Committee David M. Geiser Professor of Plant Pathology Richard P. Marini Professor of Horticulture Kari A. Peter Associate Research Professor of Plant Pathology Carolee T. Bull Professor of Plant Pathology Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School.

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ABSTRACT

Celery leaf curl disease was first reported in Australia in 1981 and first observed

in the United States (U.S.) in 2010. The pathogen was initially identified as

Colletotrichum acutatum J. H. Simmonds, which is now recognized as a species complex

with over 30 identified species. Limited research has been conducted on this disease over

the past 38 years. To expand our narrow understanding, research was conducted to

identify the causal species and determine if they cause the same or different symptoms,

explore population genetics and improve disease management strategies. Multilocus

phylogenetic analyses were used to determine that C. fioriniae is the primary causal agent

of leaf curl in North America and an Australian isolate was identified as C. godetiae. This

is the first reported association of C. godetiae with leaf curl disease. To gain a better

understanding of pathogen population structure and diversity and regional population

differentiation, microsatellite markers were used to evaluate genetic diversity and

population relationships. Analyses were conducted using C. fioriniae isolates collected

from celery in the U.S. and Canada from 2010 to 2017. Populations from celery showed

high genetic diversity with evidence of clonality, while isolates from apple showed

evidence of sexual reproduction indicating different inoculum sources for these

pathosystems. The sudden appearance of leaf curl disease, especially in locations where

celery has been cultivated for many years, indicates a potentially seedborne pathogen.

Preliminary experiments have demonstrated the potential seedborne nature of C. fioriniae

through vertical transmission. Distinctions in disease symptoms on celery have been

reported in association with different species of Colletotrichum and have resulted in

different names for the same or very similar diseases. Comparative analyses showed the

same symptomology is caused by three Colletotrichum species on two cultivars of celery

indicating these different species cause the same disease. Observations of 40 to 100%

disease incidence in natural epidemics make this a potentially devastating disease for

celery growers. Field trials evaluating conventional and biorational materials showed that

only conventional materials were effective on the highly susceptible cultivar ‘Tango’.

However, one trial comparing ‘Tango’ to the moderately resistant cultivar ‘Merengo’

revealed significant disease reduction in this cultivar indicating its potential for use in

both conventional and organic production systems.

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TABLE OF CONTENTS

LIST OF FIGURES .......................................................................................................vi LIST OF TABLES .........................................................................................................viii ACKNOWLEDGEMENTS ..........................................................................................ix CHAPTER 1. Introduction and Research Objectives ................................................1

Taxonomy and biology of Colletotrichum acutatum sensu lato ...................................1 Celery production ..........................................................................................................2 History of Colletotrichum on celery .............................................................................3 Emergence of celery leaf curl and similar diseases ......................................................4 The CLCD disease cycle ...............................................................................................7 Disease management .....................................................................................................8 Summary and justification of research .........................................................................9 Research objectives .......................................................................................................10 Literature cited ..............................................................................................................11

CHAPTER 2. Taxonomy and phylogenetic relationships of Colletotrichum species associated with leaf curl on celery .........................................16 Abstract .........................................................................................................................16

Introduction ...................................................................................................................16 Materials and methods ..................................................................................................19

Collection of isolates ................................................................................................19 DNA extraction and phylogenetic characterization .................................................20 Morphological characterization ................................................................................22

Results ...........................................................................................................................23 Phylogenetic characterization of Colletotrichum isolates ........................................23 Morphological characterization ................................................................................24

Discussion .....................................................................................................................24 Literature cited ..............................................................................................................37

CHAPTER 3. Population genetics analysis of Colletotrichum fioriniae isolates causing celery leaf curl disease in North America and comparison with C. fioriniae isolates from apple ........................................................41 Abstract .........................................................................................................................41

Introduction ...................................................................................................................41 Materials and methods ..................................................................................................46

Collection of isolates and DNA extraction ...............................................................46 Identification of isolates ...........................................................................................46 Microsatellite sequencing and genotyping ...............................................................47 Data analysis .............................................................................................................48

Results ...........................................................................................................................49 Identification of isolates ...........................................................................................49 Microsatellite sequence generation and analysis ......................................................49

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Discussion .....................................................................................................................51 Literature cited ..............................................................................................................67

CHAPTER 4. Comparison of symptoms caused by three Colletotrichum species associated with leaf curl on celery .........................................72

Abstract .........................................................................................................................72 Introduction ...................................................................................................................72 Materials and methods ..................................................................................................75

Group A experiments: comparison of C. fioriniae and C. godetiae .......................76 Group B experiments: comparison of C. fioriniae and C. nymphaeae ...................76 Group C experiments: comparison of three species with high leaf wetness period .........................................................................................................76 Symptom evaluation ...............................................................................................77 Isolate verification ..................................................................................................77 Data analysis ...........................................................................................................78

Results ...........................................................................................................................78 Group A experiments: comparison of C. fioriniae and C. godetiae .......................79 Group B experiments: comparison of C. fioriniae and C. nymphaeae ...................80 Group C experiments: comparison of three species with high leaf wetness period .........................................................................................................80 Isolate verification ..................................................................................................81

Discussion .....................................................................................................................82 Literature cited ..............................................................................................................88

CHAPTER 5. Integrating host resistance with biorational fungicides for management of celery leaf curl disease ..................................................................90

Abstract .........................................................................................................................90 Introduction ...................................................................................................................90 Materials and methods ..................................................................................................93

Plot layout ...............................................................................................................93 Fungicide treatments ...............................................................................................94 Inoculum preparation and application ....................................................................95 Disease rating ..........................................................................................................96 Data analysis ...........................................................................................................96

Results ...........................................................................................................................97 2015 Fungicide trial ................................................................................................97 2016 Fungicide trial ................................................................................................97 2017 and 2018 Fungicide trials ...............................................................................98

Discussion .....................................................................................................................99 Literature cited ..............................................................................................................106

CHAPTER 6. Research summary and future directions ...........................................108 Appendix. Detection of seedborne Colletotrichum fioriniae through vertical transmission from inoculated celery plants to seeds .....................................111

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LIST OF FIGURES

Fig. 1-1. Symptoms of celery leaf curl disease A) downward curling of leaves (epinasty), B) dark petiole lesions and lesion with adventitious roots forming, C) yellow to tan necrotic leaf spots, D) crown rot cross section, E) crown rot and distortion, F) whole plant symptoms, and G) stunting of infected plants compared to uninfected plant (right). .................................................................. 6 Fig. 1-2. General Colletotrichum life cycle showing the asexual and sexual life stages (De Silva et al. 2017) ................................................................................................................................... 7 Fig. 2-1. Image of herbarium dried culture specimen of isolate BRIP 17318 ............................. 20 Fig. 2-2. One of the 750 most parsimonious trees obtained from analysis of the combined ITS, GAPDH, CHS-1, ACT, HIS3 and TUB2 sequences alignment showing phylogenetic relationships of Colletotrichum isolates from celery in the CASC. Clades containing celery isolates (taxon labels in bold) are indicated by blocks of different colors. The phylogenetic placement of isolate BRIP 17318 based on ITS, GAPDH, CHS-1, and HIS3 indicated with an arrow. Numbers above branches indicate node support for MP (bold) and ML bootstrap analysis. C. orchidophilum CBS 632.80 was used as the outgroup ................................................................................................. 35 Fig. 2-3. Micrographs of conidiomata, appressoria and conidia from celery isolates A. Colletotrichum fioriniae (strain 16-1365), B. Colletotrichum fioriniae (strain 15-1327), C. Colletotrichum nymphaeae (strain MAFF 242590), D. Colletotrichum godetiae (strain VPRI 41701) .......................................................................................................................................... 36 Fig. 3-1. Maximum likelihood tree obtained from analysis of the GAPDH gene sequence alignment showing phylogenetic relationships of Colletotrichum isolates from celery in the C. acutatum species complex. The clade containing the celery isolates is indicated by a yellow color block. Maximum likelihood bootstrap support values above 70% are shown at the nodes. C. orchidophilum CBS 632.80 was used as the outgroup ................................................................ 62 Fig. 3-2. Number of multilocus geneotypes (MLG) found in each population studied showing very little overlap of genotypes between populations except for MLG.40 which appears in each population, except the Celery_Seedling population .................................................................... 63 Fig. 3-3. Genotype accumulation curve showing the number of multilocus genotypes (MLGs) observed with up to four loci. The curve does not plateau indicating that adding more loci to the analysis may reveal more MLGs in the populations studied ....................................................... 64 Fig. 3-4. Graphs of linkage disequilibrium analysis showing the distribution of expected ̅rd values assuming no linkage and the observed ̅rd value for the populations (blue dotted line) A. Celery_PA, B. Celery_MI, C. Celery_Seedling, D. Apple_PA .................................................. 65 Fig. 3-5. Discriminant analysis of principal components (DAPC) density plots and histograms for comparison of A. Apple_PA and Celery_Seedling populations and B. Celery_MI and Celery_PA (2010 to 2014 isolates). DAPC analysis shows clustering of isolates into their defined populations exhibiting differentiation between the populations ...................................................................... 66

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Fig. 4-1. Wire cages used to separate plants and prevent cross contamination of isolates. Each plant was placed in a plastic container and placed inside a wire cage. A 1/4 in. diameter PVC pipe was placed next to each pot in the plastic container and was used to add water to the bottom of the containers to prevent splashing of conidia ................................................................................... 85 Fig. 5-1. Boxplot showing the effect of fungicide treatments on end of the season celery leaf curl disease incidence on celery ‘CR-1’ inoculated with C. fioriniae in the 2015 field trial. Treatments are ordered by means. Light gray boxes are conventional treatments, dark gray boxes are biofungicide treatments and the white box is the inoculated untreated control. Non-inoculated untreated control plots (not shown) had 0% incidence ................................................................ 102 Fig. 5-2. Boxplot showing the effect of fungicide treatments on severity of celery leaf curl disease on 20 Aug 2016 on celery ‘Tango’ inoculated with C. fioriniae in the 2016 field trial. Treatments are ordered by means. Light gray boxes are conventional treatments, dark gray boxes are biofungicide treatments and the white box is the inoculated untreated control. No significant differences were observed between treatments (P = 0.143) ........................................................ 103 Fig. 5-3. Boxplot showing the effect of fungicide treatments on season-long CLCD severity as calculated using the AUDPC values on celery ‘Tango’ inoculated with C. fioriniae in the 2016 field trial. Treatments are ordered by means. Light gray boxes are conventional treatments, dark gray boxes are copper and biorational treatments and the white box is the inoculated untreated control. No significant differences were observed between treatments ....................................... 103 Fig. 5-4. Boxplot showing the effect of fungicide treatments on severity of celery leaf curl disease on celery ‘Merengo’ and ‘Tango’ inoculated with C. fioriniae in the 2018 field trial. Treatments are ordered by medians. Light gray boxes are ‘Merengo’ treatments, dark gray boxes are ‘Tango’ treatments and the white box is the inoculated untreated control. Means followed by the same letter within cultivars are not significantly different at P = 0.05 ................................................. 105 Fig. 5-5. Boxplot showing the effect of fungicide treatments on severity (AUDPC) of celery leaf curl disease on celery ‘Merengo’ and ‘Tango’ inoculated with C. fioriniae in the 2018 field trial. Treatments are ordered by medians. Light gray boxes are ‘Merengo’ treatments, dark gray boxes are ‘Tango’ treatments and the white box is the inoculated untreated control. Means followed by the same letter within cultivars are not significantly different at P = 0.05 .................................. 105 Fig. A-1. Images of C. fioriniae sporulating on celery A) seed coat, B) emerging root, C) emerging cotyledon. Images A and B are from lot A seeds and C is from lot B seeds ............... 114

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LIST OF TABLES

Table 2-1. Isolates used in phylogenetic analysis of Colletotrichum isolates from celery ......... 27 Table 2-2. Measurements of conidia, appressoria and hyphae of Colletotrichum isolates studied and measurement of growth after 10 days on SNA media .......................................................... 34 Table 3-1. Microsatellite characteristics and allelic diversity for loci used in this study ........... 55 Table 3-2. Isolate designation, Genbank accession numbers, year, host and location of isolation for Colletotrichum fiorinae isolates used in this study ................................................................ 56 Table 3-3. Genotypic diversity of populations used in this study and linkage disequilibrium on complete and clone corrected (CC) data ...................................................................................... 61 Table 3-4. Analysis of molecular variance (AMOVA) of Celery_Seedling and Celery_Apple and Celery_PA and Celery_MI (2010-2014) populations .................................................................. 61 Table 4-1. Division of experiments into three groups (A-C) to compare species on larger plants (A and B) and smaller plants (C). Treatments were replicated within each experiment and each experiment was repeated once ..................................................................................................... 85 Table 4-2. Assessment of symptoms on ‘Tango’ and ‘Tall Utah’ caused by three species of Colletotrichum (C. fioriniae, C. godetiae, and C. nymphaeae) evaluated in three groups of experiments (A, B, and C) ........................................................................................................... 86 Table 4-3. Comparison of symptoms between ‘Tango’ and ‘Tall Utah’ for each isolate studied in the group C experiments .............................................................................................................. 87 Table 5-1. Effect of fungicide treatments on end of the season celery leaf curl disease incidence on ‘CR-1’ celery plants (2015) as well as disease severity on ‘Tango’ plants (2016) ................ 101 Table 5-2. Effect of fungicide treatments on celery leaf curl disease severity on celery ‘Tango’ and ‘Merengo’ in the 2018 field trial. Research plots were inoculated with Pennsylvania isolates of C. fioriniae ............................................................................................................................... 104

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ACKNOWLEDGEMENTS

I would like to thank my co-advisers Dr. Beth Gugino and Dr. María del Mar Jiménez

Gasco for their guidance, support, and encouragement. My unique situation as a part-time

graduate student with a full-time job was sometimes a challenge and my co-advisers provided

their continued support and understanding throughout this journey. I also wish to thank my

supervisors Dr. Frederick Gildow, Dr. David Geiser, and Dr. Carolee Bull for their consistent

encouragement of my educational goals. I am very grateful to my committee members Dr. Kari

Peter, Dr. David Geiser, and Dr. Richard Marini and I thank them for their time and guidance of

my research.

I wish to thank Timothy Grove for his assistance in my field experiments. I would also

like to thank the many members of the Gugino lab, Jiménez Gasco lab, and the Penn State Plant

Disease Clinic for their contributions and support of my research. I am continually grateful for the

support of the Department of Plant Pathology and Environmental Microbiology and the

wonderful community of people in this department that have been cheering me on since the

beginning of my educational journey. This research has been supported by several funding

sources from the Penn State College of Agricultural Sciences including the Leonard J. Francl

Memorial Endowment, the Larry J. Jordan Memorial Endowment, and the College of

Agricultural Sciences Graduate Student Competitive Grant.

I want to thank my amazing family for their love and unwavering support, especially my

husband, John May, for his constant support and encouragement and for being there for me every

step of the way. I also thank my parents, William and Cynthia Mahoney for always believing in

me and instilling in me a drive to help others and to constantly better myself. I thank my in-laws,

Blaine and Anna May for their love and support and I thank my brother, William Mahoney and

my sister, Ann Androsky for being there when I needed someone to listen or to make me laugh.

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CHAPTER 1:

Introduction and Research Objectives

Taxonomy and biology of Colletotrichum acutatum sensu lato:

As with many fungal genera, Colletotrichum has a long and confusing taxonomic history.

Despite being one of the most significant and intensely-studied plant pathogens, its taxonomy and

classification has been challenging until the recent development of modern molecular

technologies that allowed for rapid and relatively inexpensive evaluation of multiple gene regions

for the purpose of taxonomic classification.

C. acutatum J. H. Simmonds is the anamorph (or non-sexual stage) name for this fungus,

which is classified in the Ascomycota phylum and class Sordariomycetes. It is often referred to as

a Coelomycete, an older classification term which is now used informally to describe fungi that

produce spores or conidia in a fruiting body, which in the case of Colletotrichum is called an

acervulus. The species C. acutatum was mainly distinguished from other Colletotrichum species

by the acute ends of the conidia. The teleomorph (or sexual) state, Glomerella acutata, has only

been found twice in nature, and has been produced in culture (Guerber and Correll 1997, 2001;

LoBuglio and Pfister 2008; Talgø et al. 2007). The teleomorph was also observed in artificial

crosses conducted with the species C. acutatum var. fioriniae, which is now recognized as the

species C. fioriniae (Marcelino & Gouli) Pennycook (Marcelino et al. 2008; Pennycook 2017).

The anamorph name Colletotrichum is the older and more commonly used name and is the

recommended name for use under the one fungus, one name taxonomic system (Réblová et al.

2016).

The species C. acutatum was first described by Simmonds in Australia in 1965 and by the

early 1990s taxonomists began to recognize that variations existed among isolates in C. acutatum

and suggested that it was more likely comprised of a complex of species, C. acutatum sensu lato

(s.l.). It was also noted that resolving this genus would require data in addition to traditional

morphological taxonomic characters (Aa et al. 1990). Between 1996 to 2007 several publications

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divided the species complex into subclades with varying designations (Guerber et al. 2003;

Johnston and Jones 1997; Lardner et al. 1999; Sreenivasaprasad et al. 1996; Sreenivasaprasad and

Talhinhas 2005; Whitelaw-Weckert et al. 2007). In 2012, Damm et al. published a multilocus

phylogenetic analysis evaluating six loci. This analysis resulted in the division of the complex

into five clades with 29 species including the description of 19 new species. Since then five

additional species have been added to this species complex (Bragança et al. 2016; Crous et al.

2015; De Silva et al. 2017; Huang et al. 2013; Uematsu et al. 2012).

Celery Production:

In 2018, growers in the U.S. harvested 30,200 acres of fresh market and processing celery

(Apium graveolens var. dulce) valued at $444 million (NASS 2019). Most of the U.S. celery crop

is planted in California with 94% of the crop or 28,300 acres planted there in 2018. While celery

is a minor crop in Pennsylvania, it is grown on many small diversified farms that sell produce

direct-to-consumers in several ways including directly on-farm, through community supported

agriculture operations (CSAs) or roadside produce stands, and at farmers markets or produce

auctions across the state. Pennsylvania ranks 4th in the nation in direct farm sales and 3rd in direct-

to-consumer sales and, as part of the northeast region, ranks #1 in the U.S. in farmers market

sales (NASS 2015).

There are two basic types of celery cultivars, self-blanching (yellow) and Pascal (green)

celery (Rubatzky et al. 1999). Most celery cultivars grown in the U.S. are the Pascal (green) type.

Celery is typically grown from seed in greenhouses and transplanted into the field. For seed

production celery is a biennial plant and requires a vernalization period where plants are exposed

to temperatures between 5 to 10°C before bolting and flower formation occurs (Rubatzky et al.

1999). Commercial seed production for celery occurs where winter temperatures are low enough

for vernalization, but not cold enough to kill the plants which cannot tolerate extended freezing

temperatures. Celery and other Apiaceae crops are dry-seeded crops that are grown in temperate

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regions with dry summers for optimal, high-quality seed production. Cultivation of celery seeds

used for crop production (rather than as a culinary seasoning) occurs in various locations

including the coastal valleys of California near San Luis Obispo, the south of France, and Italy

(Navazio 2012; Rubatzky et al. 1999).

History of Colletotrichum on Celery:

The earliest reports of Colletotrichum on celery took place in 1956 with an unidentified

species causing a foliar disease in Florida (Cox 1956, 1957). Leaf symptoms were described as

“small reddish-brown spots that gradually enlarge to 1/25 to 3/25 inch in diameter”. The author

referred to this disease as anthracnose on celery and growers in Florida referred to the disease as

“nailhead” due to the small leafspot symptoms. Isolations were made from symptomatic plant

material and Koch’s postulates were completed. Morphological characteristics of the fungus

included falcate conidia and the development of setae on the acervuli in culture and on infected

host tissue. The morphological features were thought to be similar to Colletotrichum truncatum,

however, the author suggested more research was needed confirming the pathogen’s taxonomic

identity (Cox 1957). An isolate of the fungus was deposited in the American Type Culture

Collection (ATCC) (Accession No. 12880, unidentified Colletotrichum), however, this item has

been de-accessioned and is no longer available (ATCC, personal communication).

In 1959 in Queensland Australia, a disease outbreak described as anthracnose on celery

was identified (Burden 1969; Simmonds 1966). The main symptom of this disease included

lesions on the petioles that resulted in leaflet death, as well as, leaf spots. The causal pathogen

was identified as Colletotrichum orbiculare and a description of the disease is provided in the

book A Handbook of Plant Diseases in Colour (Vock 1978). Also, in Queensland in 1964, a

fungal pathogen identified as C. acutatum was noted as causing a leaf spot on celery (Simmonds

1966). However, no descriptions of the symptoms or fungus were provided in the report. A third

report in 1965 noted six Colletotrichum isolates representing two species isolated from celery in

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Queensland (Simmonds 1965). One isolate was identified as C. acutatum while the other five

were C. orbiculare. This research did not describe the diseases caused by these fungi on celery,

but the study used one of the C. orbiculare isolates to artificially inoculate papaw fruit (Carica

papaya) and no visible symptoms developed. Note that in all the above cases a range of

Colletotrichum species are described associated with celery diseases but the symptoms described

do not include the characteristic leaf epinasty that is later observed with celery leaf curl disease

(CLCD).

Emergence of celery leaf curl and similar diseases:

The first report of leaf curl on celery, where the disease received its common name,

occurred in 1981 when the pathogen was isolated from symptomatic celery and identified

morphologically as C. acutatum and a specimen was submitted to the Queensland Plant

Pathology Herbarium (BRIP 13638) in Queensland, Australia (Wright and Heaton 1991). The

first formal description of CLCD is in the second edition of the book A Handbook of Plant

Diseases in Colour (Vock 1982). This edition also describes anthracnose on celery, caused by C.

orbiculare, which was originally noted in the 1978 edition of this book mentioned above.

Between 1991 to 1993 two manuscripts and a Master’s thesis were published on CLCD

in Queensland, Australia (Davis 1992; Heaton and Dullahide 1993; Wright and Heaton 1991).

This research was in response to a severe epidemic of the disease in 1990, which resulted in

significant yield losses for celery crops in southeast Queensland. This work is noted in the

Compendium of Umbelliferous Crop Diseases where both CLCD and anthracnose of celery are

described under the heading “Colletotrichum Diseases of Celery” (Davis and Raid 2002).

In the book Diseases of Vegetable Crops in Australia published in 2010, the authors state

that CLCD is caused by the fungi C. acutatum and C. orbiculare, further obscuring species and

disease association (Persley et al. 2010). A description of the disease is provided which makes

note of bending, distortion and curling of the leaves, as well as, the development of small

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translucent spots on the leaf blades. Anthracnose is not described at all and the authors appear to

have combined the two diseases and pathogens under the name of leaf curl.

The story becomes more intricate when in 2000 there was a brief report of anthracnose on

celery in Japan caused by C. acutatum and then in 2007 another disease very similar to celery leaf

curl was found in Japan however the authors chose to name the disease celery stunt anthracnose

(Fujinaga et al. 2011; Takeuchi et al. 2000). Based on ITS and β-tubulin sequence data, the

anthracnose pathogen from 2000 was identified as C. fioriniae and the celery stunt anthracnose

pathogen was identified as C. simmondsii but later re-identified as C. nymphaeae (Pass) Aa based

on a broader multi-gene analysis (Fujinaga et al. 2011; Sato and Moriwaki 2013).

Symptoms of CLCD on celery were first found in the U.S. in June of 2010, when a celery

sample with leaf curl symptoms was submitted to the Penn State Plant Disease Clinic from

Franklin County, PA. An isolate from another sample with the same symptoms that was

submitted to the clinic in 2011 from Dauphin County, PA was used to complete Koch’s postulates

on celery plants ‘Sonora’, ‘Tango’ and ‘Tall Utah’ (Pollok et al. 2012). Sequencing of the Internal

Transcribed Spacer region of the rDNA (ITS) was done using the primers ITS-1F and ITS4

(Gardes and Bruns 1993; White et al. 1990). The isolate was found to have 99 to 100% sequence

similarity to other known isolates of C. acutatum deposited in GenBank. The ITS sequence from

the 2011 Dauphin County, PA isolate was deposited in GenBank (Accession No. JQ794875).

Since then CLCD symptoms have also been reported on celery in Michigan, Georgia, New York

and Ontario, Canada (Jordan et al. 2018; McDonald and Van der Kooi 2015; Rodriguez-

Salamanca et al. 2012; Sharma et al. 2019). Samples displaying celery leaf curl have been

submitted to the PSU Clinic each year since the first sample in 2010. Symptoms of the disease

include downward curling of the leaves (epinasty), leaf spots, petiole lesions often with

adventitious root formation, crown rot and stunting (Fig. 1-1).

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Fig. 1-1. Symptoms of celery leaf curl disease A) downward curling of leaves (epinasty), B) dark petiole lesions and lesion with adventitious roots forming, C) yellow to tan necrotic leaf spots, D) crown rot cross section, E) crown rot and distortion, F) whole plant symptoms, and G) stunting of infected plants compared to uninfected plant (right).

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The CLCD Disease Cycle:

There are still a lot of gaps in knowledge regarding the CLCD disease cycle and the C.

acutatum s.l. life cycle on celery and other hosts. A general Colletotrichum life cycle is shown in

Fig. 1-2 (De Silva et al. 2017). The main source of initial inoculum for CLCD disease epidemics

is unknown and could include host crop

debris in soil, alternative cultivated or

environmental hosts, and/or seedborne

inoculum. In perennial systems C.

acutatum s.l. is thought to overwinter as

mycelium or appressoria on the host and

has been found in blueberry twigs and

flower buds (Wharton and Diéguez-

Uribeondo 2004). Perennial hosts like

blueberries could serve as an inoculum

source as could other environmental

hosts such as weeds and forest plants

where C. acutatum s.l. has been found as an endophyte or pathogen on plants such as hemlock,

tulip poplar, barberry and poison ivy (Kasson et al. 2014; Marcelino et al. 2009).

The celery stunt anthracnose pathogen, C. nymphaeae, has been found on celery seeds in

Japan and there are other reports of C. acutatum s. l. on seeds of safflower, zinnia, cowpea and

lupin (Kim et al. 1999; Kulik et al. 2005; Kulshretha 1976; Prasanna 1986; Yamagishi et al.

2015). Therefore, seed is a potential source of inoculum for CLCD epidemics and could explain

the sudden emergence of this disease in North America.

Extensive research has been done to elucidate the ideal conditions for disease

development. Several studies have shown that warmer temperatures > 25°C and high humidity

Fig. 1-2. General Colletotrichum life cycle showing the asexual and sexual life stages (De Silva et al. 2017).

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are ideal for the development of disease symptoms (Davis 1992; Pavel 2016; Rodriguez-

Salamanca et al. 2015).

Disease Management:

Celery leaf curl has the potential to cause significant economic losses for celery growers

with reports of yield losses ranging from 25 to 50% in severe CLCD outbreaks in Australia

(Wright and Heaton 1991) and 99% disease incidence observed in a natural epidemic reported in

this dissertation (Chapter 5). Investigating management options including cultivar resistance,

fungicide efficacy and crop rotation is essential for helping growers and home gardeners to

minimize the damage caused by CLCD and produce healthy celery crops.

To date, all celery cultivars evaluated are considered susceptible to CLCD, however,

research has shown some variation in susceptibility indicating the use of less susceptible cultivars

could be beneficial for disease management (Davis 1992; McDonald and Vander Kooi 2015;

Reynolds et al. 2016, 2017, 2018; Wright and Heaton 1991). Fungicide research related to celery

has identified many conventional fungicides that reduce disease incidence and yield loss from

CLCD, however more research is needed to identify options that are effective for organic

production systems and to optimize conventional management options to reduce the risk of

fungicide resistance developing (Heaton and Dullahide 1993; Raid et al. 2013, 2014; Rodriguez-

Salamanca et al. 2015).

Among conventional fungicides, those classified as Quinone outside Inhibitors (QoI) and

found in the Fungicide Resistance Action Committee (FRAC) codes group 11 have been the most

effective against CLCD (Raid et al. 2013, 2014; Rodriguez-Salamanca et al. 2015).

Unfortunately, these site-specific fungicides which include azoxystrobin (Quadris 2.08SC) and

pyraclostrobin (Cabrio) are at high risk for the development of fungicide resistance in pathogen

populations. To prevent the development of resistance only limited applications of these

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fungicides can be applied and they need to be rotated or combined with other products to reduce

the risk of resistance developing.

The efficacy of biorational products available for organic production has been variable.

These products are either microbial-based or biochemically-based and function in various ways

including competing with pathogens, disrupting pathogen cell walls and inducing host resistance

responses. More research is needed to evaluate the efficacy of biorational products to provide

management options for organic growers.

Summary and justification of research: The identification and management of plant diseases has been studied since Phytophthora

infestans was first discovered by Anton deBary to be the cause of potato late blight in 1861. The

discovery of CLCD in Australia in 1981 and the U.S. in 2010 makes this a relatively new and

emerging disease problem on a crop that is considered an important food crop worldwide.

Considering Colletotrichum is one of the most common and destructive plant pathogens affecting

a wide range of cultivated hosts with a global distribution (Baroncelli et al. 2015), there is still

much to learn regarding the species, population biology, symptoms and management of

Colletotrichum on celery. Studying CLCD will not only help us to manage this disease but will

provide valuable information that can be related to other Colletotrichum-host pathosystems. In

addition, as climate change continues to progress, it is expected that many places including the

northeastern U.S. will continue to see warmer temperatures and more extreme weather

conditions. It is likely we will only see an increase in disease problems caused by fungi such as C.

acutatum s.l. which prefers warmer weather with high moisture for optimal disease development

(Wharton and Diéguez-Uribeondo 2004).

10

Research Objectives:

The overall goal of this research was to gain a better understanding of the identification,

population biology and management of this relatively new and emerging disease and to provide

better resources and tools for diagnosticians, researchers, extension educators and growers who

are affected by this host-pathosystem. To achieve this, the main objectives of this dissertation

work were to:

1. Identify species and investigate evolutionary relationships of Colletotrichum isolates causing

leaf curl symptoms on celery using morphological and multilocus sequencing techniques.

2. Investigate population structure and diversity between and among isolates collected from

celery in different locations and between two different hosts, celery and apple.

3. Characterize the symptoms caused by three different species of Colletotrichum on two cultivars

of celery under controlled environmental conditions to elucidate similarities and differences in

symptoms and aggressiveness between the three species studied.

4. Evaluate effectiveness of biorational and conventional fungicides that could be used by

growers to manage CLCD outbreaks in the field.

11

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Fujinaga, M., Yamagishi, N., Ogiso, H., Takeuchi, J., Moriwaki, J. and Sato, T. 2011. First report of celery stunt anthracnose caused by Colletotrichum simmondsii in Japan. J. Gen. Plant Pathol. 77:243-247. Gardes, M., and Bruns, T. D. 1993. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113-188. Glass, N. L., and Donaldson, G. C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61:1323-1330. Guerber, J. C. and Correll, J. C. 1997. The first report of the Teleomorph of Colletotrichum acutatum in the United States. Plant Dis. 81:1334. Guerber, J. C., and Correll, J. C. 2001. Characterization of Glomerella acutata, the Teleomorph of Colletotrichum acutatum. Mycologia 93:216-229. Guerber, J. C., Liu, B., Correll, J. C., and Johnston, P. R. 2003. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia 95:872-895. Heaton, J. B., and Dullahide, S. R. 1993. Control of celery leaf curl disease caused by Colletotrichum acutatum. Australas. Plant Pathol. 22:152-155. Huang, F., Chen, G. Q., Hou, X., Fu, Y. S., Cai, L., Hyde, K. D., Li, H. Y. 2013. Colletotrichum species associated with cultivated citrus in China. Fungal Diversity 61:61-74. Johnston, P. R., and Jones, D. 1997. Relationships among Colletotrichum isolates from fruit-rots assessed using rDNA sequences. Mycologia 89:420-430. Jordan, B. Culbreath, A. K., Brock, J., Tyson, C., and Dutta, B. 2018. First report of leaf curl on celery caused by Colletotrichum acutata sensu lato in Georgia. Plant Dis. 102:1657. Kasson, M. T., Pollok, J. R., Benhase, E. B. and Jelesko, J. G. 2014. First report of seedling blight of eastern poison ivy (Toxicodendron radicans) by Colletotrichum fioriniae in Virginia. Plant Dis. 98:995. Kim, W. G., Moon, Y. G., Cho, W. –D., and Park, S. D. 1999. Anthracnose of safflower caused by Colletotrichum acutatum. Plant Pathol. J. 15:62-67. Kulik, T., Pszczolkowska, A., Olszewski, J., Fordonski, G., Plodzien, K. and Sawicka-Sienkiewicz, E. 2005. Identification of Colletotrichum acutatum from yellow and Andean lupin seeds using PCR assay. Electron. J. Pol. Agric. Univ. 8:02. Online http://www.ejpau.media.pl/volume8/issue1/art-02.html Kulshrestha, D. D. 1976. Colletotrichum acutatum- A new seed borne pathogen of Zinnia. Curr. Sci. 45:64-65. Lardner, R., Johnston, P. R., Plummer, K. M., and Pearson, M. N. 1999. Morphological and molecular analysis of Colletotrichum acutatum sensu lato. Mycol. Res. 103:275-285.

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LoBuglio, K.F., and Pfister, D. H. 2008. A Glomerella species phylogenetically related to Colletotrichum acutatum on Norway maple in Massachusetts. Mycologia 100:710-715. Marcelino, J., Giordano, R., Gouli, S., Gouli, V., Parker, B. L., Skinner, M., et al. 2008. Colletotrichum acutatum var. fioriniae (teleomorph: Glomerella acutata var. fioriniae var. nov.) infection of a scale insect. Mycologia 100:353-374. Marcelino, J. A. P., Gouli, S., Parker, B. L., Skinner, M., Schwarzberg, L., Giordano, R. 2009 Host plant associations of an entomopathogenic variety of the fungus, Colletotrichum acutatum, recovered from the elongate hemlock scale, Fiorinia externa. Journal of Insect Science 9:25. McDonald, M. R. and Vander Kooi, K. 2015. Evaluation of various celery cultivars for susceptibility to celery leaf curl, 2015. Muck Vegetable Cultivar Trial and Research Report. p. 98-99. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html National Agricultural Statistics Service (NASS). 2015. Local Food Marketing Practices Survey. Online https://www.nass.usda.gov/Surveys/Guide_to_NASS_Surveys/Local_Food/index.php National Agricultural Statistics Service (NASS). 2019. Vegetables 2018 Summary March 2019. USDA National Agricultural Statistic Service. Online https://downloads.usda.library.cornell.edu/usda-esmis/files/02870v86p/gm80j322z/5138jn50j/vegean19.pdf [Accessed March 31, 2019]. Navazio, J. 2012. The Organic Seed Grower: A Farmer’s Guide to Vegetable Seed. Production. Chelsea Green Publishing. White River Junction, Vermont. Pavel, J. 2016. The etiology, virulence, and phylogenetics of the celery anthracnose pathogen, Colletotrichum fioriniae (= C. acutatum sensu lato). Thesis. University of Arkansas, Fayetteville, AR. Pennycook, S. R. 2017. Colletotrichum fioriniae comb. & stat. nov., resolving a nomenclatural muddle. Mycotaxon 132:149-152. Persley, D., Cooke, T. and House, S. 2010. Diseases of Vegetable Crops in Australia. CISRO Publishing, Melbourne. Pollok, J. R., Mansfield, M. A., Gugino, B. K., and May, S. R. 2012 First report of leaf curl on celery caused by Colletotrichum acutatum in the United States. Plant Dis. 96:1692 Prasanna, K. 1986. Seed health testing of cowpea with special reference to anthracnose caused by Colletotrichum lindemuthianum. Seed Sci. Technol. 13:821-827. Réblová, M., Miller A. N., Rossman, A. Y., Seifert, K. A., Crous, P. W., Hawksworth, D. L., Abdel-Wahab, M. A., Cannon, P. F., Daranagama, D. A., De Beer, Z. W., Huang, S. K., Hyde, K. D., Jayawardena, R., Jaklitsch, W., Jones, E. B. G., Ju, Y. M., Judith, C., Maharachchikumbura, S. S. N., Pang, K. L., Petrini, L. E., Raja, H. A., Romero, A. I., Shearer, C., Senanayake, I. C., Voglmayr, H., Weir, B. S., Wijayawarden, N. N. 2016. Recommendations for competing sexual-asexually typified generic names in Sordariomycetes (except Diaporthales, Hypocreales, and Magnaporthales). IMA fungus. 7:131-153.

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Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2016. Muck Vegetable Cultivar Trial and Research Report. p. 106-107. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2017. Muck Vegetable Cultivar Trial and Research Report. p. 94-95. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2018. Muck Vegetable Cultivar Trial and Research Report. p. 103-104. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Rodriguez-Salamanca, L. M., Enzenbacher, T. B., Byrne, J. M., Feng, C., Correll, J. C., and Hausbeck, M. K. 2012. First report of Colletotrichum acutatum sensu lato causing leaf curling and petiole anthracnose on celery (Apium graveolens) in Michigan. Plant Dis. 96: 1383. Rodriguez-Salamanca, L. M., Quesada-Ocampo, L. M., Naegele, R. P., and Hausbeck, M. K. 2015. Characterization, virulence, epidemiology, and management of leaf curling and petiole anthracnose in celery. Plant Dis. 99:1832-1840. Rubatzky, V. E., Quiros, C. F., and Simon, P. W. 1999. Carrots and Related Vegetable Umbelliferae. CAB International. New York, NY. Sharma, S., Pethybridge, S. J., Buck, E. M., Hay, F. S. 2019. First report of leaf curl on celery (Apium graveolens var. dulce) caused by Colletotrichum fioriniae in New York. Plant Dis. Online 10.1094/PDIS-03-19-0499-PDN. Simmonds, J. H. 1965. A study of the species of Colletotrichum causing ripe fruit rots in Queensland. Qld. J. Agric. Anim. Sci. 22:437-459. Simmonds, J. H. 1966. Host Index of Plant Diseases in Queensland. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Sato, T., and Moriwaki, J. 2013. Molecular re-identification of strains in NIAS Genebank belonging to phylogenetic groups A2 and A4 of the Colletotrichum acutatum species complex. Microbiol. Cult. Coll. 29:13-23. Sreenivasaprasad, S., Mills, P. R., Meehan, B. M., and Brown, A. E. 1996. Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences. Genome 39:499-512. Sreenivasaprasad, S., and Talhinhas, P. 2005. Genotypic and phenotypic diversity in Colletotrichum acutatum, a cosmopolitan pathogen causing anthracnose on a wide range of hosts. Mol. Plant Pathol. 6:361-378. Taglø, V., Aamot, H. U., Strømeng, G. M., Klemsdal, S. S., and Stensvand, A. 2007. Glomerella acutata on highbush blueberry (Vaccinium corymbosum L.) in Norway. Online. Plant Health Prog. doi:10.1094/PHP-2007-0509-01-RS.

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Takeuchi, J., Horie, H., Kubota, M. 2000. First occurrence of anthracnose of Apium graveolens by Colletotrichum acutatum and aspergillus blight of Ruscus hypoglossum by Aspergillus niger in Japan (abstract in Japanese). Jpn. J. Phytopathol. 66:92. Uematsu S., Kageyama K., Moriwaki J., Sato T. 2012. Colletotrichum carthami comb. nov., an anthracnose pathogen of safflower, garland chrysanthemum and pot marigold, revived by molecular phylogeny with authentic herbarium specimens. J Gen Plant Pathol. 78:316-330. Vock, N. T., ed. 1978. A Handbook of Plant Diseases in Colour, Volume 1. Fruit and Vegetables. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Vock, N. T., ed. 1982. A Handbook of Plant Diseases in Colour, Volume 1. Fruit and Vegetables. Second. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Wharton, P. S. and Diéguez-Uribeondo, J. 2004. The biology of Colletotrichum acutatum. Anales del Jardín Botánico de Madrid. 61:3-22. White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a guide to methods and applications. New York, USA: Academic Press, p.315-322. Whitelaw-Weckert, M. A., Curtin, S. J., Huang, R., Steel, C. C., Blanchard, C. L., and Roffey, P. E. 2007. Phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical Australia. Plant Pathol. 56:448-463. Wright, D. G. and Heaton, J. B. 1991. Susceptibility of celery cultivars to leaf curl cause by Colletotrichum acutatum. Australas. Plant Pathol. 20:155-156. Yamagishi, N., Fujinaga, M., Ishiyama, Y., Ogiso, H., Sato, T., and Tosa, Y. 2015. Life cycle and control of Colletotrichum nymphaeae, the causal agent of celery stunt anthracnose. J Gen Plant Pathol. 81:279-286.

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CHAPTER 2:

Taxonomy and phylogenetic relationships of Colletotrichum species associated with leaf curl

on celery

Abstract:

Celery leaf curl disease (CLCD) has been a problem on celery (Apium graveolens var.

dulce) crops in Australia since it was first observed in 1981. The causal agent was identified

morphologically as Colletotrichum acutatum J. H. Simmonds. Molecular taxonomic studies have

demonstrated that the morphological concept of C. acutatum represents a complex comprising at

least 34 phylogenetically diagnosable species. Beginning in 2000, diseases with symptoms

similar to CLCD were identified in Japan and in the United States (U.S.) and Ontario, Canada.

Two different phylogenetic species of Colletotrichum were identified causing disease in Japan

and it was unknown which species were occurring in North America (N.A.). In Australia, it was

still unknown which species within the C. acutatum species complex were causing CLCD. In this

study, multilocus molecular phylogenetic analysis of 66 isolates obtained from celery found that

all isolates examined from N.A. are C. fioriniae, with one isolate from Australia identified as C.

godetiae. Analysis of limited sequence data derived from a dried culture specimen collected in

Queensland, Australia during a 1990 outbreak of CLCD indicates that it is likely C. fioriniae.

Introduction:

Colletotrichum is one of the most common and devastating plant pathogens causing

disease on a wide range of hosts, and is particularly devastating as a fruit rot on fruit and

vegetable crops including strawberry, apple, peach, grape, pepper and tomato (Chen et al. 2016;

Damm et al. 2012; Diao et al. 2017; Hyde et al. 2009; Kepner and Swett 2018; Munir et al. 2016).

Before 1981, there were only a few reports in the literature of Colletotrichum causing disease on

celery including a C. truncatum-like species in the U.S. (Florida, 1956) and C. orbiculare (1959),

17

and C. acutatum (1964) in Australia (Cox 1956; Simmonds 1966). Symptoms of these diseases

included foliar leaf spots, and in the case of C. orbiculare, petiole lesions. In 1981, a new disease

was reported with different symptoms consisting of curling leaves along with leaf spots and

petiole lesions. This new disease was named celery leaf curl disease (CLCD) and the causal agent

was identified morphologically as C. acutatum (Wright and Heaton 1991).

C. acutatum is currently recognized as a species complex (CASC) with 34 distinct

species presently described (Bragança et al. 2016; Crous et al. 2015; Damm et al. 2012; De Silva

et al. 2017; Huang et al. 2013; Uematsu et al. 2012). Diseases on celery with similar symptoms to

CLCD were observed in Japan in 2000 and 2007 and the causal agents were determined to be C.

fioriniae and C. nymphaeae, both species within the CASC. (Fujinaga et al. 2011; Sato and

Moriwaki 2013; Takeuchi et al. 2000). C. acutatum sensu lato (s.l.) isolates were then found

causing leaf curling symptoms on celery in the U.S. in Pennsylvania (PA), Michigan (MI) and

Georgia (Jordan et al. 2018; Pollock et al. 2012; Rodriguez-Salamanca et al. 2012). Recently,

eighteen U.S. isolates obtained from PA, MI and Virginia were identified as C. fioriniae based on

intron sequences of the glutamine synthase gene (Pavel 2016) and an isolate from New York

(NY) was identified as C. fioriniae using sequences of three loci (ITS, ACT, GAPDH) (Sharma et

al. 2019).

On celery, yield losses of 25 to 50% have been reported in severe CLCD outbreaks in

Australia (Wright and Heaton 1991). Disease incidence of 85% was reported in NY (Sharma et

al. 2019) and 99% disease incidence is reported in a natural epidemic in PA (Chapter 5). Since

the first detection in 2010, outbreaks of this disease have been confirmed in PA every year

through samples submitted to the Penn State Plant Disease Clinic.

Accurate identification of the species causing leaf curl globally is essential for furthering

our understanding of its biology, epidemiology and disease cycle. Species identification will aid

in making better management decisions based on our knowledge of the causative phylogenetic

species. Although more research is needed, likely differences between species include disease

18

severity, cultivar resistance, fungicide resistance, optimal environmental conditions for disease

development and survival of the pathogen on seed or in the environment, as well as the ecology

and endemism of the causative agents. Studying these differences and identifying the pathogens

at a fine phylogenetic level will help to inform important management decisions such as crop

rotation, seed and fungicide treatments and cultivar selection.

The most distinctive morphological feature of fungi in the CASC is the presence of

aseptate, hyaline conidia with apices at one or both ends that terminate acutely. These conidia are

formed in acervuli on the host and also produced on the mycelium in culture (Damm et al. 2012).

Colletotrichum species outside the CASC can form conidia with similar shapes, and CASC

isolates can show different morphology when grown on different media or after repeated

subculturing in the laboratory. Distinguishing between species within the CASC is extremely

difficult using morphological characters alone, and therefore molecular methods are needed to

accurately identify species in this complex.

Molecular methods including random amplified polymorphic DNA markers (RAPDs)

and sequences of loci including internal transcribed spacer 1 of the nuclear ribosomal RNA gene

repeat (ITS-1), an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an

intron of the glutamine synthetase (GS), have been used to define the phylogenetic relationships

between CASC isolates. Between 1996 to 2007 several publications using these molecular and

morphological methods divided the species complex into subclades with varying designations

(Guerber et al. 2003; Johnston and Jones 1997; Lardner et al. 1999; Sreenivasaprasad et al. 1996;

Sreenivasaprasad and Talhinhas 2005; Whitelaw-Weckert et al. 2007). Subclades were still

unresolved within the species complex and in 2012 Damm et al. published a multilocus

phylogenetic analysis evaluating 331 isolates using six loci. This analysis resulted in the division

of the complex into five clades with 29 species, including the description of 19 new species.

Combining molecular methods and morphological analyses is a reliable approach for

accurate identification of species of Colletotrichum (Cannon et al. 2012) The objective of this

19

research was to identify the species of Colletotrichum causing CLCD and to understand the

phylogenetic relationships between these isolates using both morphological and multigene

molecular approaches. Strains were compared to isolates from Japan identified as C. fioriniae and

C. nymphaeae. Also, an unknown strain from Victoria, Australia was identified to species, as well

as a dried herbarium culture from a 1990 epidemic in Queensland, Australia.

Materials and methods:

Collection of isolates: Isolates were obtained from commercial production and home gardens

between 2010 to 2017. An effort was made to collect isolates from all states reporting symptoms

of CLCD. Isolates were also provided by Ms. Gail Ruhl (Purdue University), Dr. Mary Hausbeck

(Michigan State University), Dr. James Correll (University of Arkansas), Mr. Stephen Reynolds

(University of Guelph), Ms. Jill Pollok (Virginia Polytechnic Institute and State University), and

Dr. Jacqueline Edwards (Victorian Plant Pathology Herbarium, La Trobe University, Melbourne,

Australia). Isolates from Japan were obtained from the Genetic Resources Center of the National

Agriculture and Food Research Organization (NARO), Tsukuba, Japan. All isolates were

collected from symptomatic celery (Apium graveolens var. dulce) except for isolate 15-1136

which was collected from symptomatic celeriac plants (Apium graveolens var. rapaceum). Isolate

designations, source information and GenBank accession numbers for sequences produced in this

research are presented in Table 2-1.

For isolations, symptomatic petiole, leaf or crown tissue was surface disinfested in 10%

commercial bleach for 30-60 sec. and small sections (2-3 mm) were plated onto potato dextrose

agar amended with streptomycin sulfate at 100 μg/ml (PDA+). Cultures with conidial

morphology and cultural characteristics of Colletotrichum were selected. Monosporic cultures of

each isolate were grown on PDA+ plates with 1-2 cm2 pieces of autoclaved filter paper (VWR®

grade 413). After colonization by the fungus, the filter paper pieces were lifted from the agar

surface with sterile forceps and placed in sterile petri dishes. After drying in a laminar flow hood,

20

colonized filter paper pieces were placed in 1.7 µl microcentrifuge tubes and stored at -20°C.

Isolates were also stored in sterile water by cutting 6, 1-2 cm2 agar cubes from the margin of

colonies grown on PDA+ and placing them in 7 ml sterile distilled water in screw-cap test tubes.

The test tube caps were sealed with parafilm to prevent water loss and stored at 4°C.

An isolate of Colletotrichum (BRIP 17318) causing CLCD in Australia had previously

been deposited in the Queensland Plant Pathology Herbarium

(BRIP) and had been used in cultivar susceptibility studies

(Wright and Heaton 1991). The isolate deposited in BRIP was

no longer viable, however, a portion of a dried culture

specimen of the isolate was obtained from the herbarium and

used for direct amplification and sequencing (Fig. 2-1.).

DNA extraction and phylogenetic characterization: Mycelium was produced for DNA

extraction by placing agar plugs from water storage in shaking liquid cultures of potato dextrose

broth for 7 to 10 days at room temperature. The mycelium was harvested, frozen (-20°C) and

lyophilized. DNA was extracted from lyophilized mycelium using the DNeasy Plant Mini Kit

(Qiagen, Germantown MD). For the dried culture herbarium isolate (BRIP 17318), DNA was

extracted directly from the material using a Mo Bio PowerSoil DNA isolation kit (Mo Bio

Laboratories, Carlsbad, CA) with an increased vortexing time of 20 min following the addition of

the anionic detergent solution C1 for cell lysis using the Mo Bio Vortex Geneie. Isolates were

analyzed using six genomic regions including the 5.8S nRNA gene with the two flanking internal

transcribed spacers (ITS), an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH),

and partial sequences of the chitin synthase 1 (CHS-1), actin (ACT), β-tubulin (TUB2) and

histone 3 (HIS3) genes. The primer pairs used were ITS-1F (Gardes and Bruns 1993) + ITS-4

(White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), CHS-354R + CHS79F (Carbone and

Kohn 1999), CYLH3F +KYLH3R (Crous et al. 2004), ACT-512F + ACT-783R (Carbone and

Fig. 2-1. Image of herbarium isolate BRIP 17318.

21

Kohn 1999) and BT2Fd + BT4R (Woudenberg et al. 2009) or T1 (O’Donnell and Cigelinik 1997)

+ Bt-2b (Glass and Donaldson 1995). PCR reactions were performed in an ExpressGene Thermal

Cycler (Denville Scientific, Inc., South Plainfield, NJ) in a total volume of 25 µl containing 10-50

ng genomic DNA, 0.4 µM of each primer, 9.5 µl filter sterilized ddH2O, and 2x ChoiceTaq

polymerase buffer (Denville Scientific Inc., South Plainfield, NJ) containing 2.5 U of Blue Taq

DNA polymerase, 3 mM MgCl2 and 1.6 mM dNTP. Conditions for PCR reactions for all regions

were the same as those described in Damm et al. (2012). PCR products were visualized after

amplification on a 1.5% agarose gel using Amresco EZ-Vision dye (Amresco, Solon, OH), and

subjected to bi-directional DNA sequencing at the Penn State Genomics Core Facility, University

Park, PA using an ABI 3730 XL automated DNA sequencer (Applied Biosystems, Waltham,

Massachusetts).

Geneious Biologics version R9 software (Biomatters, Inc., Newark, NJ) was used to edit

and assemble the consensus contigs from the forward and reverse sequences. Sequences were

aligned using the Clustal W algorithm and MEGA 7.0 software and edited manually prior to the

addition of CASC sequences from previous studies. A six-locus dataset was constructed including

sequences from GenBank of type or ex-type isolates for all known species within the

Colletotrichum acutatum species complex (N=34), as well as, additional sequences of C.

fioriniae, C. godetiae, and C. nymphaeae for comparison (Table 2-1) (Bragança et al. 2016;

Crous et al. 2015; Damm et al. 2012; De Silva et al. 2017; Huang et al. 2013; Uematsu et al.

2012). A maximum parsimony (MP) analysis was performed on the six-locus dataset, as well as,

for each locus dataset separately with PAUP* (Phylogenetic Analysis Using Parsimony) v.

4.0a164 (Swofford 2003) using C. orchidophilum CBS 632.80 as the outgroup. Identical taxa

were collapsed within the dataset before performing the analysis. Trees were inferred using the

heuristic search option with Tree Bisection Reconnection (TBR) branch swapping and 100

random sequence additions. Alignment gaps were treated as missing and zero length branches

were collapsed. No more than 10 trees of a score greater than or equal to 10 were saved in each

22

replicate. Maximum parsimony bootstrapping was conducted using the fast-stepwise addition

algorithm and 10,000 replications (Hillis and Bull 1993). Descriptive tree statistics were

calculated, including tree length (TL), consistency index (CI), retention index (RI), rescaled

consistency index (RC), and homoplasy index (HI) for the bootstrap consensus tree. Maximum

likelihood (ML) analyses were performed on the six-locus dataset with identical taxa merged

using the CIPRES web portal with GARLI v.2.01 using the general time reversible model with a

class of invariable sites and gamma distributed rate heterogeneity with 1,000 bootstrap replicates,

10,000 generations without improving topology and 0.025 score improvement threshold (Miller

et al 2010, Zwickl 2006, http://www.phylo.org/portal2/).

Morphological characterization: Nine isolates, representing three species, were evaluated for

their morphological characteristics. Each isolate was grown on two plates of the nutrient-poor

medium Spezieller Nährstoffarmer Agar (SNA) with pieces of autoclaved filter paper (1 cm2,

VWR grade 413) placed on the agar surface to enhance sporulation (Nirenberg 1976). A 6 mm

cork borer plug of each isolate was transferred onto two 100 mm SNA plates and incubated at

25°C with a 12 hr photoperiod for 10 days. Growth rate measurements were taken at 10 days by

measuring two perpendicular diameters per plate using a General Ultratech digital caliper

(General Tools & Instruments LLC, Secaucus, NJ). An Olympus SZ60 dissecting microscope,

Olympus CKX41 inverted microscope and Olympus BX51 light microscope with differential

interference contrast were used for examining the cultures. Structures were mounted in clear

lactic acid and photographed and measured with an Olympus DP26 camera and cellSans

Dimension 1.11 software (Olympus, Tokyo, Japan). Appressoria on hyphae were observed on the

reverse side of SNA plates. Hyphae width and conidia and appressoria dimensions were observed

with 30 measurements per structure and 95% confidence intervals were determined for conidia

and appressoria measurements. Only conidia from acervular conidiomata were used for this study

23

as variations in acervular and hyphal conidia have been observed in previous studies (Cannon et

al. 2000).

Results:

Phylogenetic characterization of Colletotrichum isolates: MP and ML multigene analyses were

conducted on data sets both with and without the herbarium specimen BRIP 17318 isolate data.

In the multilocus MP analysis including herbarium specimen BRIP 17318, 2,223 characters were

processed, of which 279 characters were parsimony-informative, 246 parsimony-uninformative

and 1,698 constant, with 731 most parsimonious trees retained (length = 992 steps, CI = 0.636, RI

= 0.893, RC = 0.568, HI = 0.364). In the multilocus MP analysis without BRIP 17318, 2,223

characters were processed, of which 279 characters were parsimony-informative, 244 parsimony-

uninformative and 1,700 constant, with 750 most parsimonious trees retained (length = 978 steps,

CI = 0.642, RI = 0.895, RC = 0.574, HI = 0.358) (Fig. 2-2). The ML analysis showed tree

topologies with no highly supported conflicting branches compared to those obtained with

parsimony (Fig. 2-2).

Phylogenetic analysis placed all celery isolates from the U.S. within C. fioriniae, with

100% MP and ML bootstrap support. Most of these isolates (60/62) aligned with C. fioriniae

subgroup 1. Two U.S. isolates (15-1327 and 15-706), as well as the Japanese isolate

(MAFF242591), were included in C. fioriniae subgroup 2. Only the Japanese isolate

MAFF242590 was identified as C. nymphaeae. The isolate from Victoria, Australia, VPRI 41701,

was identified as C. godetiae and was placed with other C. godetiae species from Europe, the

U.S., and South Africa and not with the subclade of C. godetiae isolates from Latin America.

High quality sequences from the Queensland, Australia herbarium specimen (BRIP

17318) were obtained only for the ITS, GAPDH, and HIS3 genes. A lower quality sequence was

obtained from the ACT gene and no sequence data were recovered for the CHS-1 or TUB2 genes.

In separate analyses including these sequences, the individual gene MP analyses showed

24

differences in tree topology for the BRIP 17318 isolate, which aligned with C. fioriniae isolates

for the three high-quality gene sequences, and C. godetiae isolates for the ACT gene. This finding

is consistent with this isolate being a hybrid between the two species; however, due to the low

sequence quality and lack of additional sequence data there is not enough information to

determine if this was the case. The phylogenetic position of this isolate inferred from these

incomplete data is indicated in the tree presented in Fig. 2-2.

Morphological characterization: Morphological character measurements (Table 2-2) and

observations were similar to those for other described isolates of the three species examined

(Damm et al. 2012). Isolates 10-788 and VP1 had slightly smaller conidia (11.6-16.7 x 3.1-5.9

μm) than the other C. fioriniae isolates (11.8-17.9 x 3.9-6.1 μm). Isolate 15-706 had larger

appressoria than the other C. fioriniae isolates. No setae were found in any of the cultures. The

average growth for the C. fioriniae isolates was between 61.3-66.7 mm in diameter over ten days,

which was 10.2-11.3 mm more than the growth of the C. godetiae isolate (51.1-55.4 mm) and

9.7-12.8 mm less than the C. nymphaeae isolate (74.1-76.4 mm). Micrographs of conidiomata,

appressoria and conidia are presented in Fig. 2-3.

Discussion:

Three species within the CASC were identified in association with leaf curl symptoms on

celery using morphological and molecular criteria. Previous studies of U.S. celery isolates used

two or three loci for identification of species (Pavel 2016; Sharma et al. 2019). This multilocus

phylogenetic analysis based on six loci provides a more thorough examination of isolates and

details on their genetic diversity, taking advantage of the more comprehensive Damm et al (2012)

study. All U.S. celery-associated isolates were identified as C. fioriniae, and this is the first report

of this species associated with CLCD in six new states (CT, IN, NJ, MA, DE, and VT). C.

fioriniae has a broad host range and causes fruit rot on important cultivated crops including

25

grapes, peaches, blueberries, strawberries and apples (Chen et al. 2016; Damm et al. 2012;

Kepner and Swett 2018; Munir et al. 2016).

Most of the isolates in this study belong to C. fioriniae subgroup 1 and three belong to

subgroup 2. Recent research on C. fioriniae isolates causing fruit rot on peach found variations in

susceptibility to demethylation inhibitor fungicides (DMI) between isolates from subgroup 1 and

2, with subgroup 2 isolates being less sensitive (Chen et al. 2016). Identifying differences such as

fungicide sensitivity and other biological factors between celery isolates belonging to different

subgroups could help inform management decisions based on the dominant isolates found in

specific growing regions.

The herbarium isolate BRIP 17318 was collected during an outbreak of leaf curl disease

causing severe yield losses in Queensland, Australia in 1990. The isolate was no longer viable;

however, DNA was recovered from the dried culture specimen. Three of the loci sequenced (ITS,

GAPDH, HIS3) show strong support for inclusion within C. fioriniae. A fourth gene sequence,

ACT, had reduced quality, but was found to match closely to those of the species C. godetiae

which suggests the possibility that the isolate was a hybrid. Evidence of hybridization in C.

fioriniae has been reported in Damm et al. (2012) where an isolate associated with terminal crook

disease of pine (CBS 797.72) identified as C. acutatum f. sp. pinea was found to have three genes

sharing high sequence identity with C. acutatum s. str. (ACT, HIS3, CHS-1) and three with C.

fioriniae (ITS, GAPDH, TUB2). Damm et al (2012) also reported findings that the in vitro studies

establishing the holotype of Glomerella acutata, the sexual stage of C. acutatum, were the result

of an interspecific hybridization between a C. acutatum and a C. fioriniae isolate (Guerber and

Correll 2001).

Phylogenetic analyses strongly supported placement of the CLCD isolate VPRI41701

from Victoria, Australia within C. godetiae, which is the first association of this species with leaf

curl disease. Colletotrichum godetiae was first described from the seed of a flowering plant

known by the common name “Farewell to Spring” (Clarkia hybrida syn. Godetia hybrida)

26

indicating potential to be a seedborne pathogen (Neergaard 1943). Colletotrichum godetiae

causes fruit rot and anthracnose on several hosts including apple, avocado, grape, strawberry and

olive and has been reported mostly in Europe, South Africa and Latin America (Damm et al.

2012). The isolate VPRI 41701 groups with isolates from Europe and South Africa, as well as, an

isolate from the U.S. (CBS 796.72) used for biocontrol against the weed northern jointvetch

(Aeschynomene virginica) in the 1970s (Daniel et al. 1973). The association of this species with

leaf curl is consistent with the hypothesis that the Queensland, Australia isolate BRIP 17318 is a

hybrid between C. fioriniae and C. godetiae.

Further taxonomic and phylogenetic analysis is needed on more isolates from celery in

Australia to determine the species diversity occurring there. Since C. nymphaeae is seedborne and

both C. fioriniae and C. godetiae have the potential to be seedborne there is a need to further

monitor disease outbreaks to identify introductions of these and other species into new growing

areas. If C. fioriniae is seedborne, this could be one explanation for the sudden appearance of

CLCD in North America. Since C. fioriniae has a broad host range it is also possible that disease

inoculum is originating on other hosts and celery could be a source for disease inoculum for other

crops. It will be important to study differences in the biology of these distinct species, as well as,

between C. fioriniae subgroups 1 and 2 to better understand their life cycles and determine

optimal management strategies.

27

Table 2-1. Isolates used in phylogenetic analysis of Colletotrichum isolates from celery.

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2

C. abscissum COAD 1877, VIC 42850* Citrus sinensis Brazil KP843126 KP843129 KP843132 KP843138 KP843141 KP843135

C. acerbum CBS 128530, ICMP 12921, PRJ 1199.3*

Malus domestica New Zealand JQ948459 JQ948790 JQ949120 JQ949450 JQ949780 JQ950110

C. acutatum CBS 112996, ATCC 56816, STE-U 5292*

Carica papaya Australia JQ005776 JQ948677 JQ005797 JQ005818 JQ005839 JQ005860

C. australe CBS 116478, HKUCC 2616* Trachycarpuse fortunei South Africa JQ948455 JQ948786 JQ949116 JQ949446 JQ949776 JQ950106

C. brisbanense CBS 292.67, DPI 11711* Capsicum annum Australia JQ948291 JQ948621 JQ948952 JQ949282 JQ949612 JQ949942

C. cairnsense BRIP 63642, CBS 140847*

Capsicum annuum Australia KU923672 KU923704 KU923710 KU923722 KU923716 KU923688

C. carthami SAPA100011* Carthamus Japan AB696998 ----- ----- ------ ------ AB696992 C. chrysanthemi

CBS 126518, PD 84/520

Carthamus sp. Netherlands JQ948271 JQ948601 JQ948932 JQ949262 JQ949592 JQ949922

C. citri CBS 134233, ZJUC41* Citrus aurantifolia China KC293581 KC293741 KY856138 KY856309 KC293621 KC293661

C. cosmi CBS 853.73, PD 88/642* Cosmos sp Netherlands JQ948274 JQ948604 JQ948935 JQ949265 JQ949595 JQ949925

C. costaricense CBS 330.75* Coffea arabica Costa Rica JQ948180 JQ948510 JQ948841 JQ949171 JQ949501 JQ949831

C. cuscutae IMI 304802, CPC 18873*

Cuscuta sp. Dominica JQ948195 JQ948525 JQ948856 JQ949186 JQ949516 JQ949846

C. fioriniae 10-788, SM01 Apium graveolens var. dulce ‘Tango’ PA, USA MK628930 MK645647 MK680535 MK680731 MK680600 MK680666

11-946 Apium graveolens var. dulce ‘Tango’ PA, USA MK628933 MK645650 MK680538 MK680735 MK680603 MK680669

11-1392 Apium graveolens var. dulce ‘Tango’ CT, USA MK628931 MK645648 MK680536 MK680732 MK680601 MK680667

11-1393 Apium graveolens var. dulce ‘Tango’ CT, USA MK628932 MK645649 MK680537 MK680733 MK680602 MK680668

12-1092 Apium graveolens var. dulce

PA, USA

MK628934 MK645651 MK680539 MK680735 MK680604 MK680670

12-1227 Apium graveolens var. dulce ‘Early Dell’ PA, USA MK628935 MK645652 MK680540 MK680736 MK680605 MK680671

12-1265 Apium graveolens var. dulce ‘Tall Utah 52-70’ PA, USA MK628936 MK645653 MK680541 MK680737 MK680606 MK680672

12-1575 Apium graveolens var. dulce ‘Tall Utah’ PA, USA MK628937 MK645654 MK680542 MK680738 MK680607 MK680673

28

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2 C. fioriniae (cont.)

13-993 Apium graveolens var. dulce ‘Tango’ PA, USA MK628943 MK645660 MK680548 MK680744 MK680613 MK680679

13-1032 Apium graveolens var. dulce PA, USA MK628938 MK645655 MK680543 MK680739 MK680608 MK680674

13-1051 Apium graveolens var. dulce IN, USA MK628939 MK645656 MK680544 MK680740 MK680609 MK680675

13-1176 Apium graveolens var. dulce PA, USA MK628940 MK645657 MK680545 MK680741 MK680610 MK680676

13-1329 Apium graveolens var. dulce PA, USA MK628941 MK645658 MK680546 MK680742 MK680611 MK680677

13-1347 Apium graveolens var. dulce PA, USA MK628942 MK645659 MK680547 MK680743 MK680612 MK680678

14-071391-0002 Apium graveolens var. dulce

Ontario, Canada

MK628944 MK645661 MK680549 MK680745 MK680614 MK680680

14-854 Apium graveolens var. dulce PA, USA MK628950 MK645667 MK680555 MK680751 MK680620 MK680686

14-1194 Apium graveolens var. dulce ‘Tango’ PA, USA MK628945 MK645662 MK680550 MK680746 MK680615 MK680681

14-1340 Apium graveolens var. dulce ‘Tango’ PA, USA MK628946 MK645663 MK680551 MK680747 MK680616 MK680682

14-1363 Apium graveolens var. dulce ‘Tango’ PA, USA MK628947 MK645664 MK680552 MK680748 MK680617 MK680683

14-1389 Apium graveolens var. dulce ‘Tango’ PA, USA MK628948 MK645665 MK680553 MK680749 MK680618 MK680684

14-1866 Apium graveolens var. dulce PA, USA MK628949 MK645666 MK680554 MK680750 MK680619 MK680685

15-658 Apium graveolens var. dulce ‘Tango’ PA, USA MK628963 MK645680 MK680568 MK680764 MK680633 MK680699

15-663 Apium graveolens var. dulce ‘Tango’ PA, USA MK628964 MK645681 MK680569 MK680765 MK680634 MK680700

15-706 Apium graveolens var. dulce ‘Tango’ NJ, USA MK628965 MK645682 MK680570 MK680766 MK680635 MK680701

15-754 Apium graveolens var. dulce ‘Tango’ PA, USA MK628966 MK645683 MK680571 MK680767 MK680636 MK680702

15-801-1.08 Apium graveolens var. dulce ‘CR-1’

PA, USA MK628967 MK645684 MK680572 MK680768 MK680637 MK680703

15-829 Apium graveolens var. dulce ‘Tango’ PA, USA MK628968 MK645685 MK680573 MK680769 MK680638 MK680704

29

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2 C. fioriniae (cont.)

15-898 Apium graveolens var. dulce ‘Tango’ PA, USA MK628969 MK645686 MK680574 MK680770 MK680639 MK680705

15-899 Apium graveolens var. dulce ‘Tango’ PA, USA MK628970 MK645687 MK680575 MK680771 MK680640 MK680706

15-900 Apium graveolens var. dulce ‘Tango’ PA, USA MK628971 MK645688 MK680576 MK680772 MK680641 MK680707

15-1156 Apium graveolens var. rapaceum ‘Brilliant’ PA, USA MK628951 MK645668 MK680556 MK680752 MK680621 MK680687

15-1158 Apium graveolens var. dulce ‘Merengo’

PA, USA

MK628952 MK645669 MK680557 MK680753 MK680622 MK680688

15-1164 Apium graveolens var. dulce ‘Tango’ PA, USA MK628953 MK645670 MK680558 MK680754 MK680623 MK680689

15-1172 Apium graveolens var. dulce ‘Tango’ PA, USA MK628954 MK645671 MK680559 MK680755 MK680624 MK680690

15-1173 Apium graveolens var. dulce ‘Tall Utah’ PA, USA MK628955 MK645672 MK680560 MK680756 MK680625 MK680691

15-1268 Apium graveolens var. dulce ‘Penn Crisp’ PA, USA MK628956 MK645673 MK680561 MK680757 MK680626 MK680692

15-1276 Apium graveolens var. dulce NY, USA MK628957 MK645674 MK680562 MK680758 MK680627 MK680693

15-1312 Apium graveolens var. dulce PA, USA MK628958 MK645675 MK680563 MK680759 MK680628 MK680694

15-1327 Apium graveolens var. dulce ‘Tango’ PA, USA MK628959 MK645676 MK680564 MK680760 MK680629 MK680695

15-1404 Apium graveolens var. dulce ‘Tall Utah’ PA, USA MK628960 MK645677 MK680565 MK680761 MK680630 MK680696

15-1450 Apium graveolens var. dulce PA, USA MK628961 MK645678 MK680566 MK680762 MK680631 MK680697

15-1553 Apium graveolens var. dulce ‘Tango’ PA, USA MK628962 MK645679 MK680567 MK680763 MK680632 MK680698

16-490 Apium graveolens var. dulce MD, USA MK628979 MK645696 MK680584 MK680780 MK680649 MK680715

16-555 Apium graveolens var. dulce VA, USA MK628980 MK645697 MK680585 MK680781 MK680650 MK680716

16-623 Apium graveolens var. dulce DE, USA MK628981 MK645698 MK680586 MK680782 MK680651 MK680717

16-704 Apium graveolens var. dulce ‘Tango’ PA, USA MK628982 MK645699 MK680587 MK680783 MK680652 MK680718

30

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2 C. fioriniae (cont.)

16-809 Apium graveolens var. dulce ‘Tango’ PA, USA MK628983 MK645700 MK680588 MK680784 MK680653 MK680719

16-874 Apium graveolens var. dulce PA, USA MK628984 MK645701 MK680589 MK680785 MK680654 MK680720

16-1076 Apium graveolens var. dulce MD, USA MK628974 MK645691 MK680579 MK680775 MK680644 MK680710

16-1143 Apium graveolens var. dulce VT, USA MK628975 MK645692 MK680580 MK680776 MK680645 MK680711

16-1365 Apium graveolens var. dulce VT, USA MK628976 MK645693 MK680581 MK680777 MK680646 MK680712

16-301-1.01 Apium graveolens var. dulce ‘Tango’ PA, USA MK628977 MK645694 MK680582 MK680778 MK680647 MK680713

16-401-1.15 Apium graveolens var. dulce ‘Tango’ PA, USA MK628978 MK645695 MK680583 MK680779 MK680648 MK680714

16-010-1 Apium graveolens var. dulce

Ontario, Canada MK628972 MK645689 MK680577 MK680773 MK680642 MK680708

16-09-C-04 Apium graveolens var. dulce ‘Victoria F1’ PA, USA MK628973 MK645690 MK680578 MK680774 MK680643 MK680709

17-926 Apium graveolens var. dulce MA, USA MK628987 MK645704 MK680592 MK680788 MK680657 MK680723

17-1551 Apium graveolens var. dulce PA, USA MK628985 MK645702 MK680590 MK680786 MK680655 MK680721

17-61F Apium graveolens var. dulce ‘Tango’ PA, USA MK628986 MK645703 MK680591 MK680787 MK680656 MK680722

Col-10-4 Apium graveolens var. dulce MI, USA MK628991 MK645708 MK680595 MK680792 MK680661 MK680726

MH01 Apium graveolens var. dulce ‘Tango’ VA, USA MK628992 MK645709 MK680596 MK680793 MK680662 MK680727

MH02 Apium graveolens var. dulce ‘Tango’ VA, USA MK628993 MK645710 MK680597 MK680794 MK680663 MK680728

VP1 Apium graveolens var. dulce ‘Duda C-1’ MI, USA MK628994 MK645711 MK680598 MK680795 MK680664 MK680729

BRIP 17318 Apium graveolens var. dulce Australia MK628990 MK645707 ----- MK680791 MK680660 -----

MAFF242591 Apium graveolens var. dulce Japan MK628989 MK645706 MK680594 MK680790 MK680659 MK680725

ATCC 12097, CPC19392

Rhododendron sp. USA JQ948307 JQ948637 JQ948968 JQ949298 JQ949628 JQ949958

31

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2 C. fioriniae (cont.)

ATCC 28992, CPC 19391

Malus domestica USA JQ948297 JQ948627 JQ948958 JQ949288 JQ949618 JQ949948

CBS 127601, BRIP 28761a

Mangifera indica Australia JQ948311 JQ948641 JQ948972 JQ949302 JQ949632 JQ949962

CBS 127611, DAOM 213703, CF-132

Fragaria x ananassa USA JQ948328 JQ948658 JQ948989 JQ949319 JQ949648 JQ949979

CBS 127614, DAOM 213712 Fragaria x ananassa USA JQ948329 JQ948659 JQ948990 JQ949320 JQ949650 JQ949980

CBS 128517, ARSEF 10222, ERL 1257, EHS 58*

Fiorinia externa USA JQ948292 JQ948622 JQ948953 JQ949283 JQ949613 JQ949943

CBS 129916, CPC 16823

Vaccinium sp. (blueberry) USA JQ948317 JQ948647 JQ948978 JQ949308 JQ949638 JQ949968

CBS 129930, 2.7.3(T1326, ICMP 17941

Malus domestica New Zealand JQ948304 JQ948634 JQ948965 JQ949295 JQ949625 JQ949955

CBS 129931, 1.4.51a(T1166) Malus domestica USA JQ948294 JQ948624 JQ948955 JQ949285 JQ949615 JQ949945

CBS 129938, APPY3 Malus domestica USA JQ948296 JQ948626 JQ948957 JQ949287 JQ949617 JQ949947

CBS 129947, CR46, RB022

Vitis vinifera Portugal JQ948343 JQ948673 JQ949004 JQ949334 JQ949664 JQ949994

CBS 293.67, DPI 13120 Persea americana Australia JQ948310 JQ948640 JQ948971 JQ949301 JQ949631 JQ949961

IMI 363003, CPC 18928 Camellia reticulata China JQ948339 JQ948669 JQ949000 JQ949330 JQ949660 JQ949990

IMI 384569, CPC 18938 Kalmia sp. USA JQ948340 JQ948670 JQ949001 JQ949331 JQ949661 JQ949991

IMI 504882, PJ7 Fragaria x ananassa New Zealand KT153562 KT153552 KT153547 KT153557 KT153542 KT153567

C. godetiae CBS 126520, PD 87/383

Parthenocissus sp. Netherlands JQ948426 JQ948757 JQ949087 JQ949417 JQ949747 JQ950077

CBS 127561, CPC 16426

Ugni molinae Chile JQ948442 JQ948773 JQ949103 JQ949433 JA949763 JQ950093

CBS 129809, T.A.1 Solanum betaceum Colombia JQ948439 JQ948770 JQ949100 JQ949430 JQ949760 JQ950090

CBS 129912, CPC 15125 Podocarpus sp. South Africa JQ948435 JQ948766 JQ949096 JQ949426 JQ949756 JQ950086

CBS 129917, CPC 16002

Schinus molle Mexico JQ948441 JQ948772 JQ949102 JQ949432 JQ949762 JQ950092

CBS 130251, OL 10, IMI 398854

Olea europaea Italy JQ948413 JQ948744 JQ949074 JQ949404 JQ949734 JQ950064

CBS 131332, Agrimonia eupatoria Austria JQ948429 JQ948760 JQ949090 JQ949420 JQ949750 JQ950080

32

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2 C. godetiae (cont.)

CBS 133.44* Clarkia hybrida ‘Kelvon Glory’, seed

Denmark JQ948402 JQ948733 JQ949063 JQ949393 JQ949723 JQ950053

CBS 193.32 Olea europaea Greece JQ948415 JQ948746 JQ949076 JQ949406 JQ949736 JQ950066 CBS 796.72 Aeschynomene virginica USA JQ948407 JQ948738 JQ949068 JQ949398 JQ949728 JQ950058

IMI 351589, CPC 18921

Fragaria x ananassa Ireland JQ948423 JQ948754 JQ949084 JQ949414 JQ949744 JQ950074

VPRI 41701, BRIP 54824

Apium graveolens var. dulce Australia MK628995 MK645712 MK680599 MK680796 MK680665 MK680730

C. guajavae IMI 350839, CPC 18893*

Psidium guajava, fruit India JQ948270 JQ948600 JQ948931 JQ949261 JQ949591 JQ949921

C. indonesiense

CBS 127551, CPC 14986*

Eucalyptus sp. Indonesia JQ948288 JQ948618 JQ948949 JQ949279 JQ949609 JQ949939

C. johnstonii CBS 128532, ICMP 12926, PRJ 1139.3*

Solanum lycopersicum New Zealand JQ948444 JQ948775 JQ949105 JQ949435 JQ949765 JQ950095

C. kinghornii CBS 198.35* Phormium sp. UK JQ948454 JQ948785 JQ949115 JQ949445 JQ949775 JQ950105 C. lacticiphilum

CBS 112989, IMI 383015, STE-U 5303* Hevea brasiliensis India JQ948289 JQ948619 JQ948950 JQ949280 JQ949610 JQ949940

C. limetticola CBS 114.14* Citrus aurantifolia FL, USA JQ948193 JQ948523 JQ948854 JQ949184 JQ949514 JQ949844

C. lupini CBS 109225, BBA 70884*

Lupinus albus Ukraine JQ948155 JQ948485 JQ948816 JQ949146 JQ949476 JQ949806

C. melonis CBS 159.84* Cucumis melo Brazil JQ948194 JQ948524 JQ948855 JQ949185 JQ949515 JQ949845

C. nymphaeae MAFF242590 Apium graveolens var. dulce ‘Cornell 619’ Japan MK628988 MK645705 MK680593 MK680789 MK680658 MK680724

CBS 112202 Fragaria sp. Spain JQ948234 JQ948564 JQ948895 JQ949225 JQ949555 JQ949885

CBS 126366, PD 92/785 Fragaria x ananassa USA JQ948255 JQ948585 JQ948916 JQ949246 JQ949576 JQ949906

CBS 173.51 Mahonia aquifolium Italy JQ948200 JQ948530 JQ948861 JQ949191 JQ949521 JQ949851 CBS 515.78* Nymphaea alba Netherlands JQ948197 JQ948527 JQ948858 JQ949188 JQ949518 JQ949848 CBS 526.77 Nymphaea alba Netherlands JQ948199 JQ948529 JQ948860 JQ949190 JQ949520 JQ949850

IMI 360386, CPC 18925

Pelargonium graveolens India JQ948206 JQ948536 JQ948867 JQ949197 JQ949527 JQ949857

C. orchidophilum CBS 632.80* Dendrobium sp. USA JQ948151 JQ948481 JQ948812 JQ949142 JQ949472 JQ949802

C. paranaense CBS 134729, Col 19, CPC 20901*

Malus Brazil KC204992 KC205026 KC205043 KC205004 KC205077 KC205060

C. paxtonii IMI 165753, CPC18868*

Musa sp. Saint Lucia JQ948285 JQ948615 JQ948946 JQ949276 JQ949606 JQ949936

C. phormii CBS 118194, AR 3546* Phormium sp. Germany JQ948446 JQ948777 JQ949107 JQ949437 JQ949767 JQ950097

33

Species Accession No.a Host/Substrate Location GenBank Accession Numberb

ITS GAPDH CHS-1 HIS3 ACT TUB2

C. pyricola CBS 128531, ICMP 12924, PRJ 977.1*

Pyrus communis New Zealand JQ948445 JQ948776 JQ949106 JQ949436 JQ949766 JQ950096

C. rhombiforme

CBS 129953, PT250, RB011*

Olea europaea Portugal JQ948457 JQ948788 JQ949118 JQ949448 JQ949778 JQ950108

C. salicis CBS 607.94* Salix sp. Netherlands JQ948460 JQ948791 JQ949121 JQ949451 JQ949781 JQ950111

C. scovillei CBS 126529, PD 94/921-3, BBA 70349* Capsicum sp. Indonesia JQ948267 JQ948597 JQ948928 JQ949258 JQ949588 JQ949918

C. simmondsii CBS 122122, BRIP 28519* Carica papaya Australia JQ948276 JQ948606 JQ948937 JQ949267 JQ949597 JQ949927

C. sloanei IMI 364297, CPC 18929*

Theobroma cacao Malaysia JQ948287 JQ948617 JQ948948 JQ949278 JQ949608 JQ949938

C. tamarilloi CBS 129814, T.A.6* Solanum betaceum Colombia JQ948184 JQ948514 JQ948845 JQ949175 JQ949505 JQ949835

C. walleri CBS 125472, BMT(HL)19*

Coffea sp., leaf tissue Vietnam JQ948275 JQ948605 JQ948936 JQ949266 JQ949596 JQ949926

a ATCC: American Type Culture Collection, Manassas, VA; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; IMI: Culture collection of CABI Europe UK Centre, Egham, UK; MAFF: MAFF Genebank Project, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan; BRIP: Plant Pathology Herbarium, Department of Employment, Economic, Development and Innovation, Queensland, Australia; ICMP: International Collection of Microorganisms from Plants; Auckland, New Zealand; SAPA: Hokkaido University Museum, Hokkaido, Japan STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; PD: Plantenziektenkundige Dienst Wageningen, Nederland; VIC: Universidade Federal de Viçosa, Minas Gerais, Brazil; VPRI: Victorian State Government, Victoria, Australia.

bAccession numbers beginning with “MK” were generated for this study.* ex-holotype or ex-epitype cultures.

34

Table 2-2. Measurements of conidia, appressoria and hyphae of Colletotrichum isolates studied and measurement of growth after 10 days on SNA media.

Species Accession No.

Conidia on SNA hyphae width (µm)

Appressoria on SNA Growth on SNA (mm)

length x width (µm)a length x width (µm) mean + stdev

L/W ratio

length x width (µm) mean + stdev

C. fioriniae 10-788 (11.6-)13.5-14.3(-16.7) × (3.1-)4.7-5.1(-5.6)

13.9 ± 1.1 × 4.9 ± 0.6 2.8 1.9-5.8 (7.2-)8.7-9.7(-12.2) × (4.5-)6.3-6.7(-7.8)

68.5-70.8

13-1051 (11.8-)14.0-15.0(-17.6) × (4.1-)4.6-4.9(-5.6)

14.5 ± 1.4 × 4.8 ± 0.4 3.0 1.9-6.3 (6.2-)8.5-9.7(-12.8) × (3.4-)4.5-5.1(-6.5)

60.5-67.6

15-706 (14.3-)15.5-16.1(-17.5) × (4.6-)5.4-5.6(-6.0)

15.8 ± 0.8 × 5.5 ± 0.3 2.9 1.6-4.8 (7.0-)9.4-10.8(-15-8) × (5.0-)6.1-6.7(-8.9)

57.8-64.1

15-1327 (10.0-)13.9-15.0(-17.1) × (4.1-)4.8-5.2(-6.1)

14.4 ± 1.5 × 5.0 ± 0.5 2.9 1.8-5.2 (6.0-)8.4-9.3(-10.8) × (5.4-)6.1-6.4(-7.0)

60.3-64.7

16-490 (12.2-)14.7-15.7(-17.8) × (4.1-)4.8-5.0(5.4)

15.2 ± 1.3 × 4.9 ± 0.4 3.1 1.8-7.0 (6.2-)8.2-9.6(-13.31) × (5.2-)6.0-6.4(-7.2)

61.3-65.3

16-1365 (12.5-)14.6-15.4(-17.9) × (3.9-)4.8-5.1(-5.8)

15.0 ± 1.2 × 5.0 ± 0.5 3.0 2.1-3.7 (5.8-)8.4-9.4(-12.9) × (5.0-)5.7-6.1(-7.0)

61.4-69.5

VP1 (12.2-)13.5-14.2(-16.2) × (4.2-)5.0-5.3(5.9)

13.8 ± 1.0 × 5.1 ± 0.4 2.7 1.6-4.3 (7.1-)8.8-9.9(-12.9) × (4.7-)5.9-6.4(-7.4)

59.3-64.8

C. godetiae VPRI 41701 (11.7-)14.3-15.3(-17.9) × (3.9-)4.8-5.1(-5.8)

14.8 ± 1.3 × 4.9 ± 0.5 3.0 1.5-5.3 (8.2-)11.6-12.9(-16.5) × (5.1-)6.0-6.6(-9.3)

51.1-55.4

C. nymphaeae MAFF242590 (12.2-)13.5-14.5(-17.7) × (4.1-)4.8-5.1(-5.5)

14.0 ± 1.4 × 5.0 ± 0.4 2.8 1.9-5.2 (4.1-)4.8-5.1(5.5) × (4.2-)5.4-5.9(7.3)

74.1-76.4

a (min-) 95% confidence interval (-max)

35

Fig. 2-2. One of the 750 most parsimonious trees obtained from analysis of the combined ITS, GAPDH, CHS-1, ACT, HIS3 and TUB2 sequences alignment showing phylogenetic relationships of Colletotrichum isolates from celery in the CASC. Clades containing celery isolates (taxon labels in bold) are indicated by blocks of different colors. The phylogenetic placement of isolate BRIP 17318 based on ITS, GAPDH, CHS-1, and HIS3 indicated with an arrow. Numbers above branches indicate node support for MP (bold) and ML bootstrap analysis. C. orchidophilum CBS 632.80 was used as the outgroup. 129 identical Apium isolates: 10-788, 11-1392, 12-1265, 13-1051, 13-1329, 13-993, 14-071391-0002, 14-1363, 15-1164, 15-1268, 15-1276, 15-1312, 15-1450, 15-1553, 15-663, 15-754, 15-801-1.08, 15-829, 16-09- C-04, 16-1076, 16-1143, 16-301, 16-401-1.15, 16-555, 16-809, Col 10-4, MH01, MH02. 227 identical Apium isolates: VP1, 11-1393, 11-946, 12-1092, 12-1227, 13-1032, 13-1176, 13-1347, 14-1194, 14-1340, 14-1389, 14-1866, 14-854, 15-1156, 15-1158, 15-1172, 15-658, 15-898, 15-899, 15-900, 16-1365, 16-490, 16-623, 16-874, 17-1551, 17-926, 17-61F. *Ex-holotype or ex-epitype strains. ** Celery strains from outside N.A.

36

Fig. 2-3. Micrographs of conidiomata, appressoria and conidia from celery isolates A. Colletotrichum fioriniae (strain 16-1365), B. Colletotrichum fioriniae (strain 15-1327), C. Colletotrichum nymphaeae (strain MAFF 242590), D. Colletotrichum godetiae (strain VPRI 41701).

37

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Miller, M.A. Pfeiffer W., Schwartz, T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA:1-8. Munir, M., Amsden, B., Dixon, E., Vaillancourt, L., and Gauthier, N. A. W. 2016. Characterization of Colletotrichum species causing bitter rot of apple in Kentucky orchards. Plant Dis. 100:2194-2203.

Neergaard, P. 1943. 8. Aarsberetning fra J. E. Ohlens Enkes Plantepatologiske Laboratorium 1. April 1942-31. Marts 1943. J. D. Qvist & Komp. Bogtrykkeri Akts., København, Danmark. Nirenberg, H. I. 1976. Untersuchungen über die morpholigische und biologische Differenzierung in der Fusarium Sektion Liseola. Mitteilungen aus der Biologischen Bundesanstalt Für Land- und Forstwirtschaft (Berlin-Dahlem). 169: 1-117. O’Donnell, K., and Cigelinik, E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol. Phylogenet. Evol. 7:103-116. Pavel, J. 2016. The etiology, virulence, and phylogenetics of the celery anthracnose pathogen, Colletotrichum fioriniae (= C. acutatum sensu lato). Thesis. University of Arkansas, Fayetteville, AR. Pollok, J. R., Mansfield, M. A., Gugino, B. K., and May, S. R. 2012 First report of leaf curl on celery caused by Colletotrichum acutatum in the United States. Plant Dis. 96:1692 Rodriguez-Salamanca, L. M., Enzenbacher, T. B., Byrne, J. M., Feng, C., Correll, J. C., and Hausbeck, M. K. 2012. First report of Colletotrichum acutatum sensu lato causing leaf curling and petiole anthracnose on celery (Apium graveolens) in Michigan. Plant Dis. 96: 1383. Sato, T., and Moriwaki, J. 2013. Molecular re-identification of strains in NIAS Genebank belonging to phylogenetic groups A2 and A4 of the Colletotrichum acutatum species complex. Microbiol. Cult. Coll. 29:13-23. Sharma, S., Pethybridge, S. J., Buck, E. M., Hay, F. S. 2019. First report of leaf curl on celery (Apium graveolens var. dulce) caused by Colletotrichum fioriniae in New York. Plant Dis. Online 10.1094/PDIS-03-19-0499-PDN. Simmonds, J. H. 1966. Host Index of Plant Diseases in Queensland. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Sreenivasaprasad, S., Mills, P. R., Meehan, B. M., and Brown, A. E. 1996. Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences. Genome 39:499-512. Sreenivasaprasad, S., and Talhinhas, P. 2005. Genotypic and phenotypic diversity in Colletotrichum acutatum, a cosmopolitan pathogen causing anthracnose on a wide range of hosts. Mol. Plant Pathol. 6:361-378. Swofford, D. L. (2003) PAUP: Phylogenetic Analysis Using Parsimony, Version 4.0a164 Computer program distributed by the Illinois Natural History Survey, Champaign, Illinois.

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CHAPTER 3:

Population genetics analysis of Colletotrichum fioriniae isolates causing celery leaf curl

disease in North America and comparison with C. fioriniae isolates from apple

Abstract:

Celery leaf curl disease (CLCD) is a relatively new problem in North America (N.A.) and

many questions remain unanswered regarding the disease cycle and biology of the causal

pathogens. The main species identified causing CLCD in N.A., Colletotrichum fioriniae, has a

broad host range and can survive asymptomatically as an endophyte on many host species.

Population genetics tools were used to gain a better understanding of the genetic diversity and

structure of celery-associated C. fioriniae populations and compare them to isolates from apple.

Linkage disequilibrium analysis of five microsatellite loci revealed a clonal population structure

among celery isolates and a random mating structure between apple isolates suggesting

differences in the disease cycles between these two pathosystems. High genetic diversity was

found in the celery and apple populations and an analysis of molecular variance (AMOVA)

showed differentiation between Pennsylvania (PA) and Michigan (MI) celery populations

collected during the same years, and between celery and apple populations collected from

Pennsylvania fields in the same year. Further comparisons using discriminant analysis of

principal components (DAPC) showed distinctions between these population sets, suggesting

different sources of inoculum for these populations.

Introduction:

CLCD was first described in Australia in 1981 and then appeared abruptly in the

United States (U.S.) in 2010 (Pollok et al. 2012). This disease is caused by species in the

Colletotrichum acutatum species complex (CASC) and the only species identified thus far

causing CLCD in N.A. is C. fioriniae (Chapter 2). There are still many unknown aspects of the C.

fioriniae life cycle and the CLCD disease cycle including sources of inoculum (seed, debris,

42

alternative hosts), overwinter survival and sexual reproduction. Studying the genetic diversity and

structure of C. fioriniae populations can provide further insight into this pathogen and inform

future studies that will help to answer these questions.

Population genetic analyses of plant pathogens helps to address important aspects of

pathogen biology including questions regarding sources and type of inoculum for disease

epidemics and whether populations from different geographic locations are differentiated from

each other by identifying population structure (Grünwald et al. 2017a). Understanding these

biological differences can help inform management strategies. For example, if a pathogen is

seedborne, practices like seed heat treatment and improved seed testing focusing on reducing

such inoculum can be incorporated into the management program (Milgroom and Peever 2003).

If we understand a pathogen’s population structure associated to different hosts, we can evaluate

whether adjusting crop rotations and controlling weeds will provide efficient disease

management. These genetic tools can also help predict how pathogen populations may change

and evolve in the future allowing the anticipation of populations developing fungicide resistance

or overcoming host resistance genes (Kolmer 2013; Li et al. 2014). Population biology combined

with epidemiological studies aids in understanding what important factors may influence

population changes and spread (Milgroom and Peever 2003).

Colletotrichum fioriniae has a broad host range and affects many important crops,

especially fruit, causing fruit rot on grape, peach, blueberry, strawberry and apple (Chen et al.

2016; Damm et al. 2012; Kepner and Swett 2018; Munir et al. 2016). C. fioriniae has also been

found causing anthracnose on garlic leaves, cherry tomato fruit in the U.S. and pepper fruit in

China (Chechi et al. 2019; Diao et al. 2017; Hay et al. 2018). Species in the CASC are known to

survive as epiphytes and endophytes on many plants and C. fioriniae was found as an endophyte

on many hosts in northeastern U.S. forests including sugar maple, barberry, tulip poplar, and

hemlock (Marcelino et al 2009; Wharton and Diéguez-Uribeondo 2004). Additionally, C.

fioriniae was identified as a pathogen on poison ivy (Kasson et al. 2014). These cultivated and

43

forest hosts could serve as possible sources of inoculum for CLCD epidemics in PA where celery

is grown on farms with diversified production that includes small fruit and vegetable crops. In PA

and other eastern states, farms are often located among or near the mixed deciduous forests which

cover a large portion of the region (Widmann 2016). In laboratory studies, Colletotrichum

isolates from other hosts have been shown to cause CLCD symptoms on celery and celery

isolates can also cause disease symptoms on other hosts indicating the potential for alternate hosts

to serve as inoculum sources (Davis 1992; Pavel 2016).

Seeds are another important source of inoculum in many disease systems including

downy mildew on basil (Peronospora belbahrii) and soybean (Peronospora manshurica) and late

blight (Septoria apiicola) and early blight (Cercospora apii) on celery (Davis and Raid 2002;

Mancini et al. 2016; Wyenandt et al. 2015). Fungi in the CASC have also been found in seed,

including safflower, zinnia, cowpea and lupin (Kim et al. 1999; Kulik et al. 2005; Kulshretha

1976; Prasanna 1986). Research in Japan found C. nymphaeae, which causes celery stunt

anthracnose, in celery seed (Yamagishi et al. 2015). Also, the type isolate for C. godetiae, the

species associated with CLCD in Australia in Chapter 2, was originally isolated from seed of the

flowering plant Clarkia (Damm et al. 2012). Celery is a biennial plant and requires cold

vernalization for flower and seed production to occur (Rubatzky et al. 1999). Commercial seed

production for celery occurs in locations with cool winters that do not get below freezing such as

California, the south of France, and Italy (Navazio 2012; Rubatzky et al. 1999).

The survival of Colletotrichum in soil or on plant debris in the soil is another potential

source of inoculum for CLCD epidemics. Soil debris survival has been studied in several C.

acutatum sensu lato (s.l.) pathosystems. Research in New Zealand evaluating the survival of C.

acutatum f. sp. pinea, which causes terminal crook disease on pine, found that the pathogen could

be recovered from inoculated pieces of pine seedlings after 8.5 months in the soil with a re-

isolation rate of 68% (Nair et al. 1983). The researchers were still able to recover the pathogen

from debris after 25 months in the soil. Eastburn and Gubler (1990) found that the pathogen

44

causing strawberry anthracnose could survive for up to nine months in buried strawberry tissue in

California. Norman and Strandberg (1997) were able to detect C. acutatum s.l. from leatherleaf

fern for up to 12 months in infested Florida soil. Research in Israel found that C. acutatum s.l.

could be recovered after five months from artificially inoculated mummified strawberry fruit

buried in the soil (Freeman et al. 2002). In contrast, a study on blueberry in Canada found that C.

acutatum s.l. did not survive on inoculated mummified berries after four months on the soil

surface (Verma et al. 2006).

In warmer climates, C. acutatum s.l. does not require survival structures to endure the

winter months, however in colder climates, like the northeastern U.S., survival as mycelium or

appressoria on different parts of hosts such as twigs, buds and crown tissue is suspected (Wharton

and Diéguez-Uribeondo 2004). Perithecia or sexual structures could also act as overwintering

survival structures although the sexual stage of C. acutatum s.l. is rarely observed and poorly

understood (De Silva et al. 2017). There are only a few reports of the sexual stage of C. acutatum

s.l. occurring in nature with one report on blueberry fruit in Norway and one report on maple

leaves from Massachusetts (LoBuglio and Pfister 2008; Talgø et al. 2007). Mating experiments in

vitro have shown that C. fioriniae is homothallic and may also be heterothallic as the sexual stage

was observed when self-fertile isolates from the scale insect Fiorinia externa and tulip poplar

(Lioriodendron tulipifera) were crossed with each other (Marcelino et al. 2008). Examining the

genetic structure of populations of C. fioriniae to determine if they are clonally- or sexually-

reproducing would shed more light on the life cycle of this pathogen.

Previous studies have evaluated populations of Colletotrichum from celery for genetic

diversity using intersimple-sequence repeat (ISSR) and vegetative compatibility grouping (VCG)

methods. Analysis utilizing ISSRs showed low levels of polymorphism in banding patterns

among celery Colletotrichum isolates, suggesting low population diversity and high clonality

(Rodriguez-Salamanca et al. 2015). This study only identified isolates as belonging to the C.

acutatum species complex and ISSR markers often lack species specificity (Milgroom 2015). In a

45

study by Pavel (2016), VCG diversity was examined in 18 isolates from celery collected from MI

(14), Virginia (VA) (3) and PA (1) and identified them as C. fioriniae based on the sequence of

the intron of the glutamine synthase gene. The isolates belonged to six different VCGs with four

of the six observed among the MI isolates and two additional VCGs found among the PA and VA

isolates. None of the VCGs were found in multiple states indicating possible differentiation of

populations between locations.

Genetic markers that identify short, tandemly repeated motifs called microsatellites (2 to

6 bp) or minisatellites (10 to 60 bp) are frequently used for population genetic studies due to their

high level of polymorphism and the assumption that mutations in these regions are selectively

neutral (Ellegren 2004; Milgroom 2015; Schlötterer 2004). There are many tools available for

analyzing genetic marker data collected from populations of microorganisms, and clonal

populations are especially challenging to evaluate since they violate some common assumptions

made during population data analysis (Grünwald et al. 2017a). Resources specifically developed

for analysis of clonal populations were used in this study to better assess differences in clonal

populations that do not conform with the typical population models for which many analyses

were developed (Grünwald et al. 2017b).

Bitter rot disease on apple fruit is caused by several Colletotrichum species with C.

fioriniae being one of the most common species identified in PA in recent years (Munir et al.

2016; Mr. Phillip Martin, personal communication). Pennsylvania isolates of C. fioriniae from

apple were used in this study to compare these populations to celery isolates. The objective was

to gain insight into the structure, genetic diversity and potential origins of primary inoculum for

CLCD in populations of the principal causal species in N.A., C. fioriniae. A total of 121 isolates

were analyzed from celery and apple to characterize the genetic diversity between populations

from different locations and different hosts and compare the structure of populations by location

and between isolates from celery and apple in Pennsylvania.

46

Materials and methods:

Collection of isolates and DNA extraction: Sixty celery isolates collected in 2010 to 2017 from

twelve U.S. states and Ontario, Canada identified in Chapter 2 as C. fioriniae were used for the

analyses in this chapter. An additional 30 celery isolates were obtained from Dr. Mary Hausbeck

(Michigan State University, 18 isolates), Dr. James Correll (University of Arkansas, 10 isolates)

and Mr. Stephen Reynolds (University of Guelph, 2 isolates). Sixteen isolates were collected

from celery ‘Tango’ seedlings that developed disease naturally in the field at the Russell E.

Larson Agricultural Research Center in Pennsylvania Furnace, PA. In addition, 15 isolates

associated with bitter rot of apple also collected at the Penn State Larson Agricultural Research

Center in Centre County, PA and the Penn State Fruit Research and Extension Centre in Adams

County, PA, were obtained from Mr. Phillip Martin (The Pennsylvania State University) for a

total of 121 isolates. Isolate storage and DNA extraction were as described in Chapter 2, page 20.

The isolates were divided into five populations for analytical purposes (Table 3-2). The

PA celery (Celery_PA; N=43) population included isolates collected across eight years (2010 to

2017) from 21 different counties. The Celery_Other (N=18) population included isolates

collected from ten additional U.S. states and Ontario, Canada between 2010 to 2017. The

Celery_Seedling (N=16) population consisted of isolates collected from naturally infected celery

seedlings in Centre County, PA. These isolates were analyzed as a separate population from the

other PA isolates. Isolates were also obtained from CLCD in Michigan in 2010, 2011, and 2014

(Celery_MI; N=29). Finally, isolates were collected from Centre County and Adams County, PA

in the winter of 2017 (Apple_PA; N=15).

Identification of isolates: The 61 CLCD isolates were previously identified as C. fioriniae

(Chapter 2). Eight of the additional isolates (VP1, VP2, VP3, VP4, VP9, VP10, VP13, VP16)

were also molecularly identified as C. fioriniae by Pavel (2016). The remaining 52 isolates all

had morphological characteristics of C. fioriniae and to verify species identification an intron of

47

the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified using the primer

pair GDF1 + GDR1 (Guerber et al. 2003). PCR reactions were performed in an ExpressGene

Thermal Cycler (Denville Scientific, Inc., South Plainfield, NJ) in a total volume of 25 µl

containing 10-50 ng genomic DNA, 0.4 µM of each primer, 9.5 µl filter sterilized ddH2O, and 2x

ChoiceTaq polymerase buffer (Denville Scientific Inc., South Plainfield, NJ) containing 2.5 U of

Blue Taq DNA polymerase, 3 mM MgCl2 and 1.6 mM dNTP. Conditions for PCR included an

initial denaturation step of 5 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 52°C and

30 s at 72°C, and a final denaturation step of 7 min at 72°C. PCR products were visualized after

amplification on a 1.5% agarose gel using Amresco EZ-Vision dye (Amresco, Solon, OH).

Geneious Biologics version R9 software (Biomatters, Inc., Newark, NJ) was used to edit

and assemble the consensus contigs from the forward and reverse sequences. Sequences were

aligned using the Clustal W algorithm and MEGA 7.0 software. A dataset was constructed

including sequences from GenBank of type or ex-type isolates for known species within the

CASC (N=33), (Damm et al. 2012, Crous et al. 2015, De Silva et al. 2017, Uematsu et al. 2012,

Huang et al. 2013, Bragança et al. 2016). The software PAUP* (Phylogenetic Analysis Using

Parsimony) v. 4.0a165 (Swofford 2003) was used to collapse multilocus genotypes within the

dataset. A Maximum likelihood (ML) analysis was performed on the dataset using the CIPRES

web portal with GARLI v.2.01 using the general time reversible model with a class of invariable

sites and gamma distributed rate heterogeneity with 1,000 bootstrap replicates, 10,000

generations without improving topology and 0.025 score improvement threshold (Miller et al

2010, Zwickl 2006, http://www.phylo.org/portal2/).

Microsatellite sequencing and genotyping: Isolates were genotyped using five microsatellite

primer pairs developed previously (Pun et al. 2015) (Table 3-1). PCR reactions and product

visualization were performed as described above with conditions for PCR including an initial

denaturation step of 10 min at 96°C, followed by 34 cycles of 30 s at 96°C, 60 s at 56°C and 60 s

48

at 72°C. Amplicons were subjected to DNA sequencing at the Penn State Genomics Core

Facility, University Park, PA using an ABI 3730 XL automated DNA sequencer (Applied

Biosystems, Waltham, Massachusetts). Geneious Biologics version R9 software (Biomatters,

Inc., Newark, NJ) was used to manually edit and trim sequences at conserved regions outside of

the repeat motif (Table 3-1). Sequences were aligned using the Clustal W algorithm and MEGA

7.0 software and alleles were scored and recorded manually in a spreadsheet with different scores

assigned to alleles with the same length having at least 1 base pair difference.

Data analysis: All data analyses were done using the software RStudio running R (version 3.5.2).

A genotype accumulation curve was generated using the package poppr version 2.8.1 to

determine if adding more loci to the analyses of the populations collected would potentially

increase the number of haplotypes identified (Arnaud-Hanod et al. 2007; Kamvar et al. 2015).

Within-locus allele diversity was assessed using Simpson’s index (l) and allelic evenness (E5)

(Simpson 1949; Pielou, 1975; Grünwald et al., 2003). Simpson’s index considers the number of

alleles or multilocus geneotypes (MLGs), as well as, the relative abundance of each allele/MLG

and E5 estimates evenness giving a ratio of the number of abundant genotypes to rare genotypes.

Genotypic diversity within populations was assessed by calculating Stoddart and Taylor’s

index (G), Shannon-Weiner index (H), Simpson’s index (l) and evenness (E5) (Shannon and

Weaver 1949; Stoddart and Taylor 1988). When all genotypes are equally abundant, then the

values of G will be the number of MLGs and the value of H will be the natural log of the number

of MLGs. Both measurements combine richness and evenness when calculating genotypic

diversity.

Two tests of linkage disequilibrium, the index of association (IA) and rBarD ( ̅rd), were

used to assess if populations were clonal or randomly mating (Agapow and Burt 2001; Brown et

49

al. 1980; Smith et al. 1993). These two analyses were performed on the full data set, as well as,

on clone corrected data where each MLG in a population was represented only once.

For further analytical purposes, populations were defined as follows to assess diversity

between isolates collected during the same time period 1) Celery_Seedling population and

Apple_PA population in Pennsylvania (collected in 2017) and 2) Celery_PA isolates and

Celery_MI isolates collected from 2010 to 2014. Pairwise genetic diversity was assessed with an

analysis of molecular variance (AMOVA) using GenAlEx version 6.51b2 software using a binary

distance measure. The discriminant analysis of principal components (DAPC) test infers clusters

of genetically related individuals to determine if groups of individuals are genetically distinct

from one another. DAPC was done on the collections defined for the AMOVA analysis using

adegenet in R to identify differentiation between the PA celery/apple populations and celery

PA/MI populations. This method optimizes variance between groups and minimizes variance

within groups to better analyze genetic diversity between groups (Jombart et al. 2010).

Results:

Identification of isolates: The maximum likelihood analysis of the 254 character data set of the

52 additional isolates not previously identified in Chapter 2 revealed that all were placed within

C. fioriniae (Fig. 3-1). All of these isolates grouped with C. fioriniae type specimen CBS 128517

which groups with the first subclade within this species for this locus (subgroup 1). The other

isolates used in this study were also placed within C. fioriniae subgroup 1 (Fig. 2-2). The two

isolates from Chapter 2 that were placed in C. fioriniae subgroup 2 were not used in this analysis

since they are phylogenetically distinct from the subgroup 1 isolates. Placement of isolates in C.

fioriniae has been shown to be supported by the GAPDH sequence alone (Damm et al. 2012).

Microsatellite sequence generation and analysis: Five microsatellite locus sequencing from

121 isolates yielded 595 sequences, with only ten (1.68%) yielding no data due to failed PCR. No

50

sequences were missing from the M16, M22 or M23 loci, and no sequences were missing from

the Celery_PA population. The ten missing sequences were from the two minisatellite markers

(M14 and M18), four of them coming from the Celery_Other population. Allele diversity

analyses showed allele numbers between 4 to 20 for the five loci studied. Allele diversity (l) and

evenness (E5) were moderate to high (l, 0.43 - 0.79; E5, 0.55-0.94) indicating that there was no

one dominant allele at any of the loci used making them good candidates for evaluating genetic

diversity in the populations studied (Table 3-1).

Among the 121 isolates in this study, there were 66 multilocus genotypes identified. The

most abundant genotype (MLG.40) was observed 13 times and was found in all populations

except the seedling population (Fig. 3-2). The genotype accumulation curve showed no clear

plateau for the five loci studied (Fig. 3-3) indicating that more MLGs would likely be obtained by

adding more loci to the analysis of isolates studied. The population diversity results showed that

the PA celery population was the most diverse, showing the highest Shannon-Weiner index value

(H = 3.27) and a G (24.01) value close to the number of MLGs observed (29) (Table 3-3). Both

the Simpson’s index (l = 0.96) and evenness (E5 = 0.91) values for the PA celery population

were close to 1 indicating high diversity and a high number of unique genotypes. The

Celery_Other population had the next highest diversity with H (2.71), G (13.5), l (0.93), and E5

(0.89). The Celery_Seedling population had the lowest diversity compared to the other

populations (H = 1.77, l=0.81). The Celery_MI population had the lowest E5 (0.70) indicating a

higher number of a single genotype (eight isolates of MLG.40, Fig. 3-2).

Populations that are freely recombining show IA and ̅rd values equal to zero. Tests of

linkage disequilibrium showed that all celery isolates combined, and each celery population

analyzed separately had IA and ̅rd that were greater than the expected value of 0 and showed

significant disequilibrium (P = 0.001). The Apple_PA population did not show disequilibrium

from the expected values (P = 0.208). Analysis of the clone corrected data showed the same

51

results with all celery populations showing linkage disequilibrium (P = 0.003-0.001) and the

apple population showing no disequilibrium (P = 0.497) (Table 3-3 and Fig. 3-4).

To test for differentiation between populations an analysis of molecular variance

(AMOVA) was conducted to compare the Celery_Seedling and Apple_PA populations (both

collected in 2017), as well as, to compare the Celery_PA 2010 to 2014 isolates and the

Celery_MI isolates, also collected between 2010 to 2014. Results showed a significant difference

between the Celery_Seedling and Apple_PA populations (PhiPT = 0.068, P = 0.046) and

between the Celery_PA and Celery_MI populations (PhiPT = 0.057, P = 0.025) (Table 3-4). The

within group variance was high for both analyses with 93% for the celery-apple comparison and

94% for the celery PA-MI comparison, therefore a DAPC analysis was done. The calculations for

this analysis minimizes the within group variance and emphasizes between group variance to test

for clustering of isolates to the defined populations.

The DAPC analysis used the first 14 and 23 principal components for the

Celery_Seedling/Apple_PA and Celery_PA/Celery_MI comparisons respectively, representing

100% of the total variance for the populations in both comparisons. The proportions for

successful reassignment to the identified groups were high with Celery_Seedling (100%),

Apple_PA (93%), Celery_PA (82%) and Celery_MI (100%), indicating successful reassignment

of individuals to their original clusters and suggesting differentiation between these groups.

Clusters were visualized by producing density plots of the retained discriminant function and a

histogram of the membership probabilities of the isolates to the four populations (Fig. 3-5).

Discussion:

This study compared the genetic diversity and structure of celery populations of C.

fioriniae in N. A. along with a PA apple population of C. fioriniae. The results reveal that the

celery populations show significant linkage disequilibrium even after clone correction, suggesting

52

these populations are dominated by clonal reproduction, while the apple population shows no

linkage disequilibrium, suggesting sexual reproduction. This indicates that the celery populations

may not consist of sexually-derived isolates, in contrast to the apple population. One possible

explanation for this is that the celery populations are seedborne with a small number of

individuals infecting seed (bottleneck or founder population) and then multiplying asexually on

the seedlings and plants resulting in a field scale outbreak of the disease (Chapter 5, page 91).

Other possible causes for linkage disequilibrium that could explain these results, including

selection and gene flow, which would permit non-seedborne sources as the major sources of

inoculum cannot be ruled out (Milgroom 1996).

The sexual stage of C. fioriniae has not been observed in apple production but the linkage

disequilibrium data indicate populations may be randomly mating in the local environment,

possibly on the dormant trees, in apple debris on the orchard floor, or in soil or nearby plants with

wind borne ascospores initiating new infections in the spring. This possibility helps increase our

understanding of the Colletotrichum-apple disease cycle. The potential increased exchange of

genetic information could inform management recommendations as there would be a greater

possibility for the development of resistance to fungicides and greater chance of overcoming

cultivar resistance. It is not common for apples and celery to be produced on the same farms in

PA, however, many tree fruit and vegetable farms are concentrated in the southeastern and

southcentral parts of the state where disease distribution between these two crops via windborne

ascospores could be possible.

High genetic diversity was observed in isolates collected from celery in PA and other

states over several years. The lowest diversity was found in the Celery_Seedling population

collected from a single outbreak in one experimental field in 2017. The high diversity in the PA

population is not unexpected since these isolates were collected from different cultivars across

several years and across 21 PA counties. The low diversity in the Celery_Seedling population

supports the hypothesis that a smaller number of individuals were brought in on seed and then

53

reproduced clonally on the seedlings. Seedborne populations can also exhibit high genetic

diversity which has been observed in wheat seed infected with Phaeosphaeria nodorum (Bennett

et al. 2005).

While it appears that seed may be a source of inoculum for initiating CLCD disease

epidemics more research is needed to determine if this is the main source throughout the growing

season. It is possible that populations from soil debris, other cultivated hosts or endophytes

surviving on other plants could be contributing to these epidemics. The tools developed in this

study could be used to investigate this further by studying populations of isolates collected from

seeds or seedlings before transplanting and then comparing them to populations collected

throughout the growing season from field plants grown from the same seed lots.

The AMOVA results showed differentiation between the populations analyzed and high

within population variation. The DAPC analysis also showed clear clustering of the

Celery_Seedling vs. Apple_PA, and Celery_PA vs. Celery_MI populations. There was high

support for these clusters indicating differentiation between these populations. If the pathogen

inoculum was coming from the environment or other hosts such as apples, we would not expect

to see this clustering. If the pathogen is seedborne then we may expect to see similarities in the

PA and MI populations collected during the same years unless the cultivars grown at these

locations came from seed produced in different locations. The originating host cultivars are not

known for all of the Celery_PA (2010 to 2014) and Celery_MI isolates (7 Celery_PA and 7

Celery_MI were missing cultivar data). However, among the remaining isolates with known

cultivars, there were none shared between the populations (data not shown). This difference

could account for the differentiation between these two geographic regions even if the pathogen is

primarily seedborne since the seed for the cultivars may have been grown at different locations.

This is the first study to assess genetic structure and diversity in celery and apple

populations of C. fioriniae using microsatellite markers. This analysis shows that these tools can

be effectively used to characterize populations of this pathogen. The results suggest that

54

seedborne inoculum is a potential source of CLCD epidemics and the search for management

practices for susceptible cultivars should be focused on preventing the introduction of inoculum

through seed treatments, improved seed testing, early fungicide treatments and management in

celery seed crops. The population genetics methods used in this study could be used to further

investigate if seedborne inoculum is the main source for CLCD epidemics throughout the

growing season and to explore if these populations can overwinter in soil or nearby hosts and

cause disease in nearby celery crops the following year.

55

Table 3-1. Microsatellite characteristics and allelic diversity for loci used in this study. Microsatellite primers developed by Pun et al. 2015.

Locus Primersa Trimming sequencesb

Repeat motif Number of alleles

λc E5

M14 Fwd*-5’-TTGTGCTGTTACCCTCGACC-3’ Rev-5’-TTGGTTGACACACGAGACCC-3’

5’-AGTATAT-3’ 5’-TTTACT-3’

(RRGRBDVHRMDD) 20 0.79 0.58

M16 Fwd*-5’-GACGCGACGCCCTCTTTATA-3’ Rev-5’-TTTGTATGCCTGGCTCTGGG-3’

5’-GTCAAG-3’ 5’-CCCCAG-3’

(GTT) 10 0.65 0.55

M18 Fwd-5’-CGGGTGGAAGCAACTTGTTG-3’ Rev*-5’-AGAGGCCAGGCGATATATAAGT-3’

5’-GGTTTGG-3’ 5’-GTATTT-3’

(ATTACCTTT) 10 0.78 0.78

M22 Fwd -5’-GCGGAGGTTTGGAGGATCAA-3’ Rev*-5’-CCCCCGGGAATATCATCAGC-3’

5’-GGAGCCC-3’ 5’-CTCAACA-3’

(CTT) 4 0.66 0.94

M23 Fwd*-5’-AGCTTACCGAACGAATGCCA-3’ Rev-5’-AGCACGTTTACGGACACCAT-3’

5’-TGTAAGC-3’ 5’-CTTGCT-3’

(TGY) 4 0.43 0.77

mean 9.6 0.66 0.72

aForward and reverse primer sequences. *Direction sequenced. bTrimming sequences for the beginning (top) and end (bottom) of the region amplified by the primers for each locus. cλ = Simpson’s index, E5 = evenness.

56

Table 3-2. Isolate designation, Genbank accession numbers, year, host and location of isolation for Colletotrichum fiorinae isolates used in this study.

No. Isolate Genbank GAPDH accession No.

Year Population Location

1 F1 MN011888 2017 Apple_PA Pennsylvania

2 F22 MN011889 2017 Apple_PA Pennsylvania

3 F35 MN011890 2017 Apple_PA Pennsylvania

4 F44 MN011891 2017 Apple_PA Pennsylvania

5 F55 MN011892 2017 Apple_PA Pennsylvania

6 F64 MN011893 2017 Apple_PA Pennsylvania

7 F74 MN011894 2017 Apple_PA Pennsylvania

8 F75 MN011895 2017 Apple_PA Pennsylvania

9 F79 MN011896 2017 Apple_PA Pennsylvania

10 F9 MN011897 2017 Apple_PA Pennsylvania

11 RS1 MN011918 2017 Apple_PA Pennsylvania

12 RS2 MN011919 2017 Apple_PA Pennsylvania

13 RS6 MN011920 2017 Apple_PA Pennsylvania

14 RS7 MN011921 2017 Apple_PA Pennsylvania

15 RS8 MN011922 2017 Apple_PA Pennsylvania

16 C44-2 MN011917 2011 Celery_MI Michigan

17 C44-5 MN011907 2011 Celery_MI Michigan

18 C45-4 MN011904 2011 Celery_MI Michigan

19 C46-6 MN011914 2011 Celery_MI Michigan

20 C46-8 MN011903 2011 Celery_MI Michigan

21 C47-3 MN011910 2011 Celery_MI Michigan

22 C49B-57 MN011909 2011 Celery_MI Michigan

23 C49-C-34 MN011915 2011 Celery_MI Michigan

24 C50-1-2 MN011901 2011 Celery_MI Michigan

25 C50-4-12 MN011911 2011 Celery_MI Michigan

26 C50-5-6 MN011913 2011 Celery_MI Michigan

57

No. Isolate Genbank GAPDH accession No.

Year Population Location

27 C51-6-4 MN011912 2011 Celery_MI Michigan

28 C53-C-22 MN011916 2011 Celery_MI Michigan

29 C53D28 MN011908 2011 Celery_MI Michigan

30 C54-5-10 MN011902 2011 Celery_MI Michigan

31 C54-5-6 MN011906 2011 Celery_MI Michigan

32 C55-54 MN011905 2011 Celery_MI Michigan

33 Col_10-4 MK645708 2010 Celery_MI Michigan

34 Col_10-5 (Pavel 2016) 2010 Celery_MI Michigan

35 Col_10-6 (Pavel 2016) 2010 Celery_MI Michigan

36 L49B-48 MN011900 2011 Celery_MI Michigan

37 VP1 MK645711 2014 Celery_MI Michigan

38 VP10 (Pavel 2016) 2014 Celery_MI Michigan

39 VP13 (Pavel 2016) 2014 Celery_MI Michigan

40 VP16 (Pavel 2016) 2014 Celery_MI Michigan

41 VP2 (Pavel 2016) 2014 Celery_MI Michigan

42 VP3 (Pavel 2016) 2014 Celery_MI Michigan

43 VP4 (Pavel 2016) 2014 Celery_MI Michigan

44 VP9 (Pavel 2016) 2014 Celery_MI Michigan

45 11-1392 MK645648 2011 Celery_Other Connecticut

46 11-1393 MK645649 2011 Celery_Other Connecticut

47 16-623 MK645698 2016 Celery_Other Delaware

48 13-1051 MK645656 2013 Celery_Other Indiana

49 16-1076 MK645691 2016 Celery_Other Maryland

50 16-490 MK645696 2016 Celery_Other Maryland

51 17-926 MK645704 2017 Celery_Other Massachusetts

52 15-1276 MK645674 2015 Celery_Other New York

53 14-071391-0002 MK645661 2014 Celery_Other Ontario, Canada

56 14-072199-0002 MN011899 2014 Celery_Other Ontario, Canada

58

No. Isolate Genbank GAPDH accession No.

Year Population Location

55 16-005-2 MN011898 2016 Celery_Other Ontario, Canada

54 16-010-1 MK645689 2016 Celery_Other Ontario, Canada

57 16-1143 MK645692 2016 Celery_Other Vermont

58 16-555 MK645697 2016 Celery_Other Virginia

59 17-137 MN011871 2017 Celery_Other Virginia

60 MH01 MK645709 2010 Celery_Other Virginia

61 MH02 MK645710 2010 Celery_Other Virginia

62 MH03 (Pavel 2016) 2010 Celery_Other Virginia

63 10-788 MK645647 2010 Celery_PA Pennsylvania

64 11-946 MK645650 2011 Celery_PA Pennsylvania

65 12-1092 MK645651 2012 Celery_PA Pennsylvania

66 12-1227 MK645652 2012 Celery_PA Pennsylvania

67 12-1265 MK645653 2012 Celery_PA Pennsylvania

68 12-1575 MK645654 2012 Celery_PA Pennsylvania

69 13-1032 MK645655 2013 Celery_PA Pennsylvania

70 13-1176 MK645657 2013 Celery_PA Pennsylvania

71 13-1329 MK645658 2013 Celery_PA Pennsylvania

72 13-1347 MK645659 2013 Celery_PA Pennsylvania

73 13-993 MK645660 2013 Celery_PA Pennsylvania

74 14-1194 MK645662 2014 Celery_PA Pennsylvania

75 14-1340 MK645663 2014 Celery_PA Pennsylvania

76 14-1363 MK645664 2014 Celery_PA Pennsylvania

77 14-1389 MK645665 2014 Celery_PA Pennsylvania

78 14-1866 MK645666 2014 Celery_PA Pennsylvania

79 14-854 MK645667 2014 Celery_PA Pennsylvania

80 15-1158 MK645669 2015 Celery_PA Pennsylvania

81 15-1164 MK645670 2015 Celery_PA Pennsylvania

82 15-1172 MK645671 2015 Celery_PA Pennsylvania

59

No. Isolate Genbank GAPDH accession No.

Year Population Location

83 15-1173 MK645672 2015 Celery_PA Pennsylvania

84 15-1268 MK645673 2015 Celery_PA Pennsylvania

85 15-1312 MK645675 2015 Celery_PA Pennsylvania

86 15-1404 MK645677 2015 Celery_PA Pennsylvania

87 15-1450 MK645678 2015 Celery_PA Pennsylvania

88 15-1553 MK645679 2015 Celery_PA Pennsylvania

89 15-658 MK645680 2015 Celery_PA Pennsylvania

90 15-663 MK645681 2015 Celery_PA Pennsylvania

91 15-754 MK645683 2015 Celery_PA Pennsylvania

92 15-801-1.08 MK645684 2015 Celery_PA Pennsylvania

93 15-829 MK645685 2015 Celery_PA Pennsylvania

94 15-898 MK645686 2015 Celery_PA Pennsylvania

95 15-899 MK645687 2015 Celery_PA Pennsylvania

96 15-900 MK645688 2015 Celery_PA Pennsylvania

97 16-09-C-04 MK645690 2016 Celery_PA Pennsylvania

98 16-1365 MK645693 2016 Celery_PA Pennsylvania

99 16-301-1.01 MK645694 2016 Celery_PA Pennsylvania

100 16-401-1.15 MK645695 2016 Celery_PA Pennsylvania

101 16-704 MK645699 2016 Celery_PA Pennsylvania

102 16-809 MK645700 2016 Celery_PA Pennsylvania

103 16-874 MK645701 2016 Celery_PA Pennsylvania

104 17-1551 MK645702 2017 Celery_PA Pennsylvania

105 184 MN011872 2017 Celery_PA Pennsylvania

106 17-61F MK645703 2017 Celery_Seedling Pennsylvania

107 17-62F MN011873 2017 Celery_Seedling Pennsylvania

108 17-63F MN011874 2017 Celery_Seedling Pennsylvania

109 17-64F MN011875 2017 Celery_Seedling Pennsylvania

110 17-65F MN011876 2017 Celery_Seedling Pennsylvania

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No. Isolate Genbank GAPDH accession No.

Year Population Location

111 17-66F MN011877 2017 Celery_Seedling Pennsylvania

112 17-67F MN011878 2017 Celery_Seedling Pennsylvania

113 17-68F MN011879 2017 Celery_Seedling Pennsylvania

114 17-70F MN011880 2017 Celery_Seedling Pennsylvania

115 17-71F MN011881 2017 Celery_Seedling Pennsylvania

116 17-72F MN011882 2017 Celery_Seedling Pennsylvania

117 17-77F MN011883 2017 Celery_Seedling Pennsylvania

118 17-80F MN011884 2017 Celery_Seedling Pennsylvania

119 17-81F MN011885 2017 Celery_Seedling Pennsylvania

120 17-84F MN011886 2017 Celery_Seedling Pennsylvania

121 17-88F MN011887 2017 Celery_Seedling Pennsylvania

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Table 3-3. Genotypic diversity of populations used in this study and linkage disequilibrium on complete and clone corrected (CC) data.

Population N (MLG)a eMLG H G λ E5 IA ̅rd P IA CC ̅rd CC P CC ALL Celery 106 (58) --- 3.78 30.70 0.97 0.69 0.460 0.116 0.001 0.329 0.083 0.001 Pennsylvania 43 (29) 13.13 (1.08) 3.27 24.01 0.96 0.91 0.46 0.12 0.001 0.33 0.08 0.001 Michigan 29 (15) 9.78 (1.23) 2.42 8.17 0.88 0.70 0.34 0.08 0.001 0.30 0.08 0.003 Other 18 (16) 13.50 (0.60) 2.71 13.50 0.93 0.89 1.09 0.27 0.001 0.78 0.20 0.001 Seedling 16 (7) 6.81 (0.39) 1.77 5.12 0.81 0.85 1.51 0.38 0.001 1.46 0.37 0.001 Apple 15 (12) 12.00 (0.0) 2.40 9.78 0.90 0.88 0.13 0.03 0.21 -0.004 -0.001 0.497

aN (MLG) = Number of individuals (number of Multilocus genotypes), H = Shannon-Weiner Index, G = Stoddard and Taylor’s Index, λ = Simpson’s Index, E5 = Evenness, IA = index of association, ̅rd = RbarD.

Table 3-4. Analysis of molecular variance (AMOVA) of Celery_Seedling and Celery_Apple and Celery_PA and Celery_MI (2010 to 2014) populations.

Populations analyzed dfa Sum of squares

Mean square

Estimated variance

% Variation Phi Statistic

P-value

Celery_Seedling and Apple_PA Between populations 1 3.241 3.241 0.111 7% 0.068 0.046 Within populations 29 44.275 1.527 1.527 93%

Total 30 47.516 1.637 100% Celery_PA and Celery_MI Between populations 1 3.930 3.930 0.103 6% 0.057 0.025 Within populations 44 75.330 1.712 1.712 94%

Total 45 79.260 1.816 100% adf = degrees of freedom, P-value based on 999 permutations.

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Fig. 3-1. Maximum likelihood tree obtained from analysis of the GAPDH gene sequence alignment showing phylogenetic relationships of Colletotrichum isolates from celery in the C. acutatum species complex. The clade containing the celery isolates is indicated by a yellow color block. Maximum likelihood bootstrap support values above 70% are shown at the nodes. C. orchidophilum CBS 632.80 was used as the outgroup.

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Fig. 3-2. Number of multilocus geneotypes (MLG) found in each population studied showing very little overlap of genotypes between populations except for MLG.40 (*), which appears in each population except the Celery_Seedling population.

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Fig. 3-3. Genotype accumulation curve showing the number of multilocus genotypes (MLGs) observed with up to four loci. The curve does not plateau indicating that adding more loci to the analysis may reveal more MLGs in the populations studied.

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Fig. 3-4. Graphs of linkage disequilibrium analysis showing the distribution of expected ̅rd values assuming no linkage and the observed ̅rd value for the populations (blue dotted line) A. Celery_PA, B. Celery_MI, C. Celery_Seedling, D. Apple_PA. Values show significant linkage disequilibrium for all celery populations and no disequilibrium for the apple population.

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Fig. 3-5. Discriminant analysis of principal components (DAPC) density plots and histograms for comparison of A. Apple_PA and Celery_Seedling populations and B. Celery_MI and Celery_PA (2010 to 2014 isolates). DAPC analysis shows clustering of isolates into their defined populations exhibiting differentiation between the populations.

A B

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CHAPTER 4:

Comparison of symptoms caused by three Colletotrichum species associated with leaf curl on celery

Abstract:

The use of differing names in the literature for Colletotrichum diseases with comparable

symptoms on celery has caused difficulty in understanding and communicating about this

pathosystem. Clarification is needed to understand if distinct symptoms are caused by the

different Colletotrichum species that cause disease on celery. Three species, C. fioriniae, C.

nymphaeae and C. godetiae were evaluated on two celery cultivars under controlled

environmental conditions. All three species were observed causing symptoms of leaf curling, leaf

spots, petiole lesions and crown rot. Smaller plants incubated for 72 hr in high humidity after

inoculation showed more severe symptoms than larger plants incubated for 24 hr in high

humidity. Overall, when inoculated on the smaller plants, the isolates showed increased variation

in symptom severity than inoculations on larger plants with less post inoculation incubation.

Isolates also showed differences in symptom severity between the two cultivars studied. Although

the isolates showed diversity in aggressiveness they all caused the same symptoms concluding

that they should all be considered causal agents of the same disease and not different diseases.

Introduction:

While it is well documented that Colletotrichum spp. can cause significant yield losses in

celery production, the fact that different names are being used for diseases caused by

Colletotrichum species on celery has led to confusion in the literature and resulted in multiple

disease names being used to describe the same or very similar symptoms (Fujinaga et al. 2011;

Pollok et al. 2012; Rodriguez-Salamanca et al. 2012). The earliest reports of Colletotrichum on

celery were in the United States (Florida, Cox 1956) and Australia (Simmonds 1966). Both

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diseases were called anthracnose and the causal agents were identified morphologically as a C.

truncatum-like species (Florida) and C. orbiculare (Australia). Symptoms of these diseases

included foliar leaf spots, and in the case of C. orbiculare, also petiole lesions. There have been

no reports in the scientific literature regarding these diseases since they were first described

except for the mention of C. orbiculare in a vegetable disease handbook called A Handbook of

Plant Diseases in Colour (Vock 1978, 1982).

In 1981 a new disease was reported on celery in Australia displaying different symptoms

consisting of curling leaves along with leaf spots and petiole lesions. This new disease was named

celery leaf curl disease (CLCD) and the causal agent was identified morphologically as C.

acutatum (Wright and Heaton 1991). In 2000 there was a brief report of anthracnose on celery in

Japan caused by C. acutatum (Takeuchi et al. 2000). Fujinaga et al. (2011) further studied this

pathogen and found that it caused leaf curling and leaf spotting symptoms and the fungus was

identified as C. fioriniae using ITS and β-tubulin sequence data. They kept the name anthracnose

for this disease and additionally identified and described another new disease with similar leaf

curling symptoms; however, they chose to call the new disease celery stunt anthracnose. The

stunt anthracnose pathogen was identified as C. simmondsii and was later re-identified as C.

nymphaeae based on a broader multi-gene sequence analysis (Sato and Moriwaki 2013). The

Japanese group chose not to call either disease “celery leaf curl” as they noted some differences

in the symptomology and they did not have an isolate from Australia as a comparison reference.

Colletotrichum was first found causing leaf curling symptoms on celery in the United

States (U.S.) in 2010 and has been observed so far in twelve U.S. states, as well as, Ontario,

Canada (Jordan et al. 2018; Pollock et al. 2012; Rodriguez-Salamanca et al. 2012; Sharma et al.

2019). In Chapter 2 isolates from North America were identified as C. fioriniae and the literature

references a range of common names for this disease including leaf curl, anthracnose and leaf curl

with petiole anthracnose (Jordan et al. 2018; Pollock et al. 2012; Rodriguez-Salamanca et al.

2015; Sharma et al. 2019).

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Colletotrichum has a wide host range and has been reported to infect most host plant

parts including the leaves, petioles, flowers, fruit, stems, crown and roots (Moreira et al. 2019;

Peres et al. 2005). In many host-Colletotrichum pathosystems, including strawberry, pepper,

apple and tomato, multiple species of Colletotrichum have been identified causing similar

symptoms on the same host and are considered causal agents of the same disease on that host

(Baroncelli et al. 2015; Chechi et al. 2019; Diao et al. 2017; Munir et al. 2016). Different

Colletotrichum species are sometimes associated with different symptoms on the same host

species as in the case of strawberry anthracnose where fruit rot and root necrosis are associated

with C. nymphaeae and C. fioriniae, while crown rot is associated with C. gloeosporioides

(Mangandi et al. 2015; Wang et al. 2019).

In Chapter 2, isolates associated with celery leaf curl symptoms from North America,

Australia and Japan were identified as C. fioriniae, C. nymphaeae and C. godetiae; however, it is

unknown if these species are causing the same or different diseases on celery. The primary

symptoms observed with these diseases include downward curling of the leaves (leaf epinasty),

small translucent to yellow leaf spots that can turn into tan to brown necrotic leaf spots, and

sunken brown elongated lesions on petioles and crown rot (Fig. 1-1, page 5).

Research conducted in environmentally controlled growth chambers in Michigan

evaluated the effect of temperature and leaf wetness on CLCD symptom development and

showed that high temperatures and high leaf wetness after inoculation resulted in more disease

(Rodriguez-Salamanca et al. 2015). The highest temperature tested (30°C) showed significantly

more petiole lesion development than the other three temperatures tested (15, 20, 25°C) and

showed significantly more leaf curling than inoculated plants grown at 15 and 20°C. Plants

incubated at 100% relative humidity in a dew chamber for 96 hr after inoculation showed

significantly more lesion development and leaf curling than plants incubated for five other

lengths of time (0, 12, 24, 48, 72 hr).

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The objective of this study was to determine if symptomology could be used to

characterize the diseases caused by these three species of Colletotrichum. All symptom types that

have been reported in association with Colletotrichum diseases on celery were evaluated on two

cultivars of celery grown under controlled environmental conditions. Comparisons were made

between different species and between multiple isolates of one species (C. fioriniae) on ‘Tango’

and ‘Tall Utah 52-70 R Improved’ plants. For simplicity, ‘Tall Utah 52-70 R Improved’ will be

referred to as ‘Tall Utah’ from this point forward.

Materials and methods:

For all experiments, the plants were started by direct seeding into 50-cell trays filled with

Metro-Mix 300 professional growing media (Sun Gro Horticulture, Agawam, MA) and thinned to

one plant per cell. Due to limitations of growth chamber space, experiments were divided into

three groups, A, B and C (Table 4-1). Seedlings were transplanted into classic 100 size round pots

(Nursery Supplies, Inc., Fairless Hills, PA) for experiment groups A and B and seedlings were

transplanted into 4-in. square pots for experiment group C. Plants were watered as needed and

fertilized with Miracle-Gro All Purpose Plant Food, 0.5 tsp/gallon water, 24-8-16 (N-P-K)

(Scott’s Miracle-Gro Products, Port Washington, NY). All experiments were conducted in growth

chambers set at 25°C, 14-hr photoperiod and 70% relative humidity. Pots were placed into

individual plastic containers (5 in. x 5 in. x 4 in.). Each container was then placed into an 8-in.

diameter galvanized wire cage to keep plants separated and prevent cross contamination of the

isolates tested (Fig. 4-1). The plants were bottom watered by placing a 24-in. long piece of 0.25

in. polyvinyl chloride (PVC) pipe in each cage and using a funnel to add water through the pipe

to the bottom of the plastic container.

Plants were inoculated with isolates grown for 11 to 15 days on potato dextrose agar

amended with streptomycin sulfate at 100 μg/ml (PDA+). Inoculum was prepared by flooding

plates with sterile distilled water and scraping plates with a sterile metal spear to dislodge spores.

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Spores were filtered through four layers of cheese cloth and inoculum concentration was adjusted

using a hemocytometer. Plants were placed in plastic bags and inoculum was applied with small

spray bottles adjusted to deliver 6 ml/10 sprays. The plastic bags were closed and left on for 24 hr

post-inoculation for experiment groups A and B and 72 hr for group C to create a high humidity

environment to facilitate infection.

Group A experiments: comparison of C. fioriniae and C. godetiae: Group A experiments

evaluated the symptomology of four isolates of C. fioriniae (10-788, 16-1365, 13-1051, 16-490),

and one isolate of C. godetiae (VPRI 41701) on ‘Tango’ and ‘Tall Utah’. Each experiment was

conducted using 93-day-old ‘Tango’ and ‘Tall Utah’ plants with four replicates per isolate and

cultivar. This experiment was repeated using 110-day-old plants with five replicates per isolate

and cultivar. Inoculum concentration was 1 x 106 conidia/ml and 6 ml/plant of inoculum was

applied as described previously.

Group B experiments: comparison of C. fioriniae and C. nymphaeae: Group B experiments

were conducted to compare one C. fioriniae (16-1365) and one C. nymphaeae (MAFF 242590)

isolate on 125-day-old ‘Tango’ and ‘Tall Utah’ plants with four replicates per isolate and cultivar.

This experiment was repeated using 125-day-old plants with four replicates per isolate and

cultivar. Inoculum concentration was 2 x 105 conidia/ml and 9 ml/plant of inoculum was applied

as described previously.

Group C experiments: comparison of three species with high leaf wetness period: Group C

experiments were conducted to compare each of the three species on smaller plants with a longer

incubation time at high humidity to increase ideal initial infection conditions. Two isolates of C.

fioriniae (16-1365, 13-1051), one of C. nymphaeae (MAFF 242590) and one of C. godetiae

(VPRI 41701) were inoculated onto 92-day-old ‘Tango’ and ‘Tall Utah’ plants with four

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replicates per isolate and cultivar. This experiment was repeated using 92-day-old plants with

four replicates per isolate and cultivar. Although the plants were the same age in days as the

plants used in the group A experiments they were grown in smaller 4-in. square pots and were

shorter (Table 4-1). Inoculum concentration was 4 x 105 conidia/ml and 12 ml/plant of inoculum

was applied as described previously.

Symptom evaluation: Assessments were made to evaluate different types of symptoms including

leaf curling, leaf spotting, petiole lesions, crown rot, and stunting. The incidence of leaf curl was

rated by counting the number of leaves with curling symptoms out of the total number of leaves

for all leaves measuring 6 in. in length or longer. For experiment groups A and B, leaf spot

severity was measured using an ordinal scale where 0 = no spotting, 1 = trace-5%, 2=5-15%,

3=15-30%, 4=20-50%, 5=50-70%, 6=70-100%. For experiment group C, leaf spot incidence was

measured by counting the number of leaflets with spotting out of the total number of leaflets and,

if spots were present, then the longest diameter of two leaf spots per plant were measured. For all

experiments the total number of petiole lesions was counted on all leaves longer than 6 in. Spore-

producing structures (conidiomata) often form on the petiole lesions and small adventitious roots

will also grow on the damaged petiole tissue, therefore, petiole lesions were examined under a

dissecting microscope to check for the presence of conidiomata and adventitious roots. Crown rot

severity was rated using an ordinal scale where 0 = no crown rot, 1 = < 50%, 2 = 50 - 75%, and 3

= 75 - 100%. Stunting was assessed by measuring the height of each plant before inoculation and

again at the end of the experiment and then comparing the difference in the two heights to the

water inoculated controls.

Isolate verification:

To verify the presence of the pathogen in symptomatic plants, isolations were made from

each plant. Plant tissue was surface disinfested with 10% commercial bleach for 30 to 60 sec and

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rinsed in sterile water for 30 to 60 sec. Small sections (2 to 3mm) were plated onto PDA+ with

four pieces per plate and two plates for each plant part (curled leaves, spotted leaves, petioles,

crown, and roots). For asymptomatic plants, isolations were made from leaves, petioles, crown

and roots, following the same methodology as described above. Cultures were identified based on

morphological characteristics according to descriptions of the species in Damm et al. 2012 and

the study done in Chapter 2.

Data analysis: Data were analyzed using PROC MIXED and Tukey’s HSD test (P < 0.05) for

differences in leaf curling incidence, leaf spotting incidence, petiole lesions and stunting using

SAS v.9.4 statistical software (SAS Institute, Cary, NC). Symptoms of leaf severity and crown rot

severity were analyzed using Kruskal Wallis multiple comparison with Minitab v.18 statistical

software (Minitab Inc., State College, PA). Differences in symptom severity between the two

cultivars was tested using the two-sample t-test or Kruskal Wallis test. For all data the residual

normal distributions were tested using normality plots and the Anderson-Darling test or the Ryan-

Joiner test and equal variances were tested using Levene’s test with Minitab v.18 statistical

software. Where data did not meet assumptions for normality or equal variances the square root

transformed data were analyzed and the back-transformed data are presented in the results. Data

for the water inoculated controls are reported in the results but were not included in the data

analyses except for the comparison for plant stunting.

Results:

Each experiment group (A, B, and C) included two independent experiments assessing

each of the two cultivars. Data were analyzed for the symptoms on each cultivar to see if the data

from the two independent experiments could be combined within each group (e. g. could the leaf

curl data from the two experiments in group A be combined for isolate 10-788 on ‘Tango’).

Statistical analysis of the two independent experiments within each group (A, B, and C) showed

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that there were no significant differences between experiments so the same data between

experiments within each group were combined for further analysis. The only data that could not

be combined was the height measurements used to assess stunting for the ‘Tango’ plants in the

group A and C experiments. The measurements were analyzed separately for these experiments.

The results for assessment of the symptoms are presented in Table 4-2 and further described

below.

Group A experiments: comparison of C. fioriniae and C. godetiae: For the group A

experiments which evaluated four C. fioriniae and one C. godetiae isolate, all isolates tested

resulted in the full range of symptoms evaluated on both cultivars except for stunting which was

not observed with any of the isolates inoculated on ‘Tango’ or ‘Tall Utah’. No significant

differences in leaf curling, number of petiole lesions and crown rot severity were observed on

‘Tango’ between the four C. fioriniae isolates or between C. fioriniae and C. godetiae. However,

significantly higher leaf spot severity was observed with isolate 16-1365 (C. fioriniae) compared

to isolate VPRI 41701 (C. godetiae) (P = 0.005). The ‘Tango’ plants showed no stunting

compared to the water inoculated control in both experiments, however, in one of the two

experiments, differences were found between two C. fioriniae isolates with 10-788 showing

significant stunting compared to isolate 16-490 (P = 0.02).

In ‘Tall Utah’ there were no significant differences in crown rot severity, however, unlike

‘Tango’, differences in leaf curl and petiole lesions were observed. Two of the four C. fioriniae

isolates (16-1365 and 16-490) caused significantly more leaf curling than C. godetiae (VPRI

41701) (P = >0.001) and isolate 10-788 had significantly fewer petiole lesions than isolate 16-490

(both C. fioriniae) (P = 0.009).’Tall Utah’ did show significant plant height differences between

the water inoculated control and two C. fioriniae isolates (16-1365 and 10-788) (P = 0.011),

however the inoculated plants were unexpectedly taller than the control plants. No differences in

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stunting were observed between the isolates. While ‘Tango’ showed differences in leaf spot

severity, no differences were observed in ‘Tall Utah’.

Group B experiments: comparison of C. fioriniae and C. nymphaeae: For the group B

experiments, which was a comparison between one isolate of C. fioriniae (16-1365) and one

isolate of C. nymphaeae (MAFF 242590), both isolates caused the full range of symptom types

except for crown rot which was not observed with the C. nymphaeae isolate on either cultivar or

stunting which was not observed on ‘Tall Utah’. The C. fioriniae isolate caused stunting when

compared with the water inoculated control plants (P = 0.044) on ‘Tango’. There was no

significant difference in symptom severity (leaf curling, number of petiole lesions, leaf spotting

and crown rot) between the two isolates on both cultivars.

Group C experiments: comparison of three species with high leaf wetness period: The group

C experiments compared all three Colletotrichum species (2 isolates of C. fioriniae and one

isolate each of C. nymphaeae and C. godetiae) inoculated on smaller plants which were incubated

at high humidity for 72 hr after inoculation instead of 24 hr. Inoculated plants developed all

symptom types on both cultivars except no stunting was observed on ‘Tall Utah’. On ‘Tango’ the

two C. fioriniae isolates (16-1365 and 13-1051) showed significantly more leaf curling than the

C. nymphaeae isolate (MAFF 242590) (P = >0.001). The C. fioriniae isolate 16-1365 also caused

more curling than the C. godetiae isolate (VPRI 41701). Both C. fioriniae isolates caused

significantly more leaf spotting than isolates of the other two species (P = 0.000). All species did

cause some leaf spots, however there were no significant differences in the leaf spot diameters

with sizes ranging from 3.0 to 4.8 mm (P = 0.409) (data not shown). Both C. fioriniae isolates

also resulted in significantly more petiole lesions than the other two species (P = 0.000). Crown

rot caused by C. fioriniae isolate 13-1051 was significantly more severe than the C. nymphaeae

and C. godetiae isolates (P = 0.008). Stunting was variable on ‘Tango’ between the two Group C

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experiments. In one experiment there were no differences in plant height between the inoculated

and control plants for any of the isolates while in the other experiment, both C. fioriniae isolates

caused significant stunting compared to the other two species and water inoculated controls.

Across all the isolates, there were no significant differences in crown rot severity on ‘Tall

Utah’ and stunting was not observed. In C. fioriniae isolate 13-1051 more leaf curl was observed

than in the other two species. Also, C. fioriniae isolate 16-1365 resulted in more severe curling

than C. nymphaeae (MAFF 242590) and developed a larger number of leaf spots than both the C.

nymphaeae and C. godetiae isolates. In addition, C. fioriniae isolate 13-1051 developed more leaf

spots than the C. nymphaeae isolate. Although all species did show some leaf spots, there were no

significant differences in the leaf spot diameters between species with sizes ranging from 2.2 to

3.0 mm (P = 0.792) (data not shown). The two C. fioriniae isolates (16-1365 and 13-1051)

resulted in a higher number of petiole lesions than the other two species.

Comparison of symptom severity between ‘Tango’ and ‘Tall Utah’ was assessed for each

isolate in the group C experiments using the two-sample t-test for symptoms of leaf curling,

spotting and petiole lesions and the Kruskal Wallis test for crown rot severity. Across all

symptom types, disease severity was higher for all isolates on ‘Tango’ compared to ‘Tall Utah’

demonstrating its increased susceptibility to these three species of Colletotrichum. Significant

differences were found between the two cultivars for all symptoms on plants inoculated with

isolate 16-1365 (C. fioriniae) and all symptoms except for leaf curl for 13-1051 (C. fioriniae). For

the C. godetiae isolate (VPRI 41701), leaf curl and petiole lesions showed significant differences

and leaf spot and crown rot did not. For the C. nymphaeae isolate (MAFF 242590), all symptoms

except for petiole lesions showed differences between the two cultivars. Results for this

comparison are presented in Table 4-3.

Isolate verification: Isolates with morphology consistent with the inoculated species of

Colletotrichum were recovered from all symptomatic plant tissues cultured. No Colletotrichum

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was recovered from the water inoculated control plants. Colletotrichum was also recovered from

asymptomatic parts of inoculated plants including petiole, crown and root tissue.

Discussion:

Diagnosis of fungal plant diseases such as CLCD relies on the ability to match the

symptoms described for the disease with the presence of the causal pathogen. Often the pathogen

is identified morphologically since more accurate molecular methods are too expensive and time

consuming to use in many clinical settings or have not been developed yet. There are several

examples of species within the same genera causing different diseases with different symptoms

on the same host including late blight (Phytophthora infestans) and buckeye rot (Phytophthora

parasitica) on tomato and early blight (Alternaria solani, A. tomatophila) and Alternaria stem

canker (Alternaria alternata f. sp. lycopersici) on tomato (Jones et al. 2014). These different

diseases on tomato not only have different symptoms, but also differ in crop loss severity and

management recommendations. More commonly, however, species from the same genus cause

similar symptoms on the same host and are regarded as the same disease. This is the case with

many diseases caused by Colletotrichum species on other hosts including strawberry, pepper,

tomato and apple (Baroncelli et al. 2015; Chechi et al. 2019; Diao et al. 2017; Munir et al. 2016).

This study examined the symptoms caused by three different species of Colletotrichum

on two cultivars of celery under controlled conditions. The results showed that all three species

caused the same types of symptoms on the cultivars studied, although severity varied between the

Colletotrichum species and between the two cultivars for some of the symptom types. The

difference between cultivars is not surprising because differences have been observed in other

celery cultivars infected with CLCD (Renynolds et al. 2016, 2017, 2018). Although no

completely resistant cultivars have been identified some host resistance has been observed and

‘Tall Utah’ proved to be less susceptible to all of the species in this study.

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Differences in species severity were observed in the group A experiments where ‘Tango’

plants inoculated with C. fioriniae isolate 16-1365 showed significantly more leaf spotting than

did plants inoculated with the C. godetiae isolate. The C. fioriniae isolates, 16-1365 and 16-490,

caused significantly more leaf curl on ‘Tall Utah’ than the C. godetiae isolate. In the group C

experiments C. fioriniae (16-1365 and 13-1051) caused significantly more leaf curl, spotting,

petiole lesions and stunting on ‘Tango’ compared to C. nymphaeae and C. godetiae isolates.

These differences indicate variations in aggressiveness between species rather than differences in

symptomology which demonstrates that these three Colletotrichum species, C. fioriniae, C.

godetiae, and C. nymphaeae, cause the same disease on celery rather than different diseases as

observed in the other host-pathosystems previously described.

The variations in symptom incidence and severity between the two cultivars and between

the experiments on smaller plants with longer post-inoculation incubation compared to the larger

plants highlights the range of disease symptoms in this pathosystem and does not indicate that

these are different diseases. Furthermore, the variation in stunting observed between the two

independent experiments in Groups A and C on ‘Tango’ shows the variability of this symptom

which likely depends on the extent of crown rot affecting the growing point of the plant. The

unpredictability of this symptom was also highlighted in the Group A experiments on ‘Tall Utah’

where the C. fioriniae plants inoculated with isolates 16-1365 and 10-788 were significantly taller

than the water inoculated control.

I propose the name “celery leaf curl disease” or simply “leaf curl” for the common name

of this disease caused so far by three species in the C. acutatum species complex (C. fioriniae, C.

godetiae, and C. nymphaeae). This is the oldest name associated with this disease in the literature

(Wright and Heaton 1991) and it adequately describes the most noticeable symptom caused by

these pathogens. Using the same common name in publications and other communications will

also help to effectively educate growers and home gardeners about this disease, as well as

communicate with other researchers and extension educators.

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The results of this study show that C. godetiae and C. nymphaeae cause the same symptoms

as C. fioriniae, which is the only species that has been found causing CLCD in North America

thus far. Additionally, C. godetiae and C. nymphaeae isolates used in this study were found to

cause less severe symptoms than C. fioriniae in some of the experiments conducted. This

suggests that losses due to these species would be similar to current losses experienced with

CLCD caused by C. fioriniae. More research is needed to determine if there are species

differences regarding fungicide effectiveness, cultivar susceptibility, and biological factors such

as winter survival and modes of transmission. For example, C. nymphaeae has been shown to be

seedborne (Yamagishi et al. 2015) and it is suspected that C. fioriniae is seedborne as well

(Appendix).

In depth studies of the different symptoms caused by pathogens is essential for furthering

our ability to accurately diagnose and manage emerging disease problems like CLCD. Extensive

assessment of symptoms is particularly valuable for locations where more advanced identification

methods such as serology or molecular identification are not available or are too costly for

producers. Availability of information is expanding globally with increased access to web-based

resources. Detailed and specific information on symptoms can assist diagnosticians, extension

specialists, growers and home owners in making rapid and accurate identification of important

plant diseases.

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Fig. 4-1. Wire cages used to separate plants and prevent cross contamination of isolates. Each plant was placed in a plastic container and placed inside a wire cage. A 1/4 in. diameter PVC pipe was placed next to each pot in the plastic container and was used to add water to the bottom of the containers to prevent splashing of conidia. Table 4-1. Division of experiments into three groups (A-C) to compare species on larger plants (A and B) and smaller plants (C). Treatments were replicated within each experiment and each experiment was repeated once. Experiment

Group Isolates tested

(origin of isolate) Species Size of plants (in.)

(Age in days) Tango Tall Utah

A

10-788 (Pennsylvania) C. fioriniae

15.2 / 21.8 (93 / 110)

21.3 / 24.3 (93 / 110)

16-1365 (Pennsylvania) C. fioriniae 13-1051 (Indiana) C. fioriniae 16-490 (Maryland) C. fioriniae VPRI 41701 (Australia) C. godetiae

B 16-1365 (Pennsylvania) C. fioriniae 20.2 / 19.5

(125) 21.8 / 22.0

(125) MAFF 242590 (Japan) C. nymphaeae

C

16-1365 (Pennsylvania) C. fioriniae

12.3 / 10.4 (92)

14.7 / 11.0 (92)

13-1051 (Indiana) C. fioriniae VPRI 41701 (Australia) C. godetiae MAFF 242590 (Japan) C. nymphaeae

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Table 4-2. Assessment of symptoms on ‘Tango’ and ‘Tall Utah’ caused by three species of Colletotrichum (C. fioriniae, C. godetiae, and C. nymphaeae) evaluated in three groups of experiments (A, B, and C).

Group A experiments Tangoz Tall Utah

Species Isolate Leaf Curl Leaf Spoty Petiole Lesion

s

Crown Rot

Stuntingx Leaf Curl Leaf Spot Petiole

Lesions Crown

Rot Stunting 1 2

C. fioriniae 16-1365 31.0 1.3 a 2.1 0.2 3.0 ab 9.3 24.3 ab 1.0 2.1 bc 0.3 3.9 a 13-1051 26.2 1.0 ab 0.4 0.1 5.3 ab 11.6 15.8 bc 1.0 3.2 bc 0.1 2.1 ab 10-788 33.4 1.0 ab 3.5 0.3 1.8 b 9.7 0.5 c 1.0 0.6 c 0.1 4.0 a 16-490 45.7 1.0 ab 1.7 0.3 7.0 a 7.7 19.5 ab 1.0 4.5 ab 0.4 2.5 ab

C. godetiae VPRI 41701 30.5 0.7 b 1.2 0.3 5.4 ab 10.5 1.9 c 1.0 1.1 bc 0.1 2.1 ab Water Control 0.0 0.7 0.1 0.0 3.4 ab 11.6 0.0 0.6 0.2 0.0 1.3 b P = value 0.32 0.005 0.17 0.78 0.02 0.19 >0.001 1.0 0.009 0.875 0.011

Group B experiments C. fioriniae 16-1365 40.7 0.9 3.6 0.25 3.7 b 7.1 0.6 0.4 0.1 5.3 C. nymphaeae MAFF 242590 24.0 1.0 2.1 0.0 4.7 ab 9.7 0.8 0.1 0.0 5.9 Water Control 1.0 1.0 0.0 0.0 6.5 a 0.0 0.9 0.0 0.0 4.6 P = value 0.255 0.537 0.143 0.143 0.044 0.567 0.602 0.506 0.317 0.448

Group C experiments 1 2 C. fioriniae 16-1365 91.0 a 85.9 a 34.8 a 1.4 ab -0.4 b 2.0 47.4 ab 54.8 a 13.4 a 0.1 3.6 13-1051 84.6 ab 70.5 a 30.3 a 1.8 a -0.3 b 1.3 64.1 a 40.1 ab 15.0 a 0.3 3.4 C. godetiae VPRI 41701 67.8 bc 24.0 b 6.6 b 0.6 b 3.3 a 2.2 30.2 b 23.5 bc 1.2 b 0.3 3.6 C. nymphaeae MAFF 242590 57.6 c 29.6 b 6.2 b 0.6 b 3.7 a 2.6 1.6 c 13.1 c 4.7 b 0.1 4.6 Water Control 0.0 0.5 0.0 0.0 5.7 a 2.7 1.4 0.4 0.0 0.0 4.0 P = value >0.001 0.000 0.000 0.008 0.000 0.297 0.000 0.000 0.000 0.898 0.755

zData in columns was pulled across two experiments except for ‘Tango’ stunting data in Group A experiments. Means followed by the same letter within columns within experiment group are not significantly different at P = 0.05.

yLeaf spot symptoms were evaluated using an ordinal scale (0 = no spotting, 1 = trace-5%, 2=5-15%, 3=15-30%, 4=20-50%, 5=50-70%, 6=70-100%) in experiment groups A and B. Leaf spot incidence was evaluated in experiment group C as the number of leaflets per plant with leaf spotting out of the total number of leaflets.

xStunting data were analyzed separately for group A and C experiments due to significant differences between the experiments.

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Table 4-3. Comparison of symptoms between celery ‘Tango’ and ‘Tall Utah’ for each isolate studied in the Group C experiments.

Species Isolate Cultivar Leaf Curl Leaf Spot Petiole Lesions Crown Rot C. fioriniae 16-1365 Tango 91.0 a 85.9 a 34.3 a 1.4 a

Tall Utah 47.4 b 54.8 b 13.5 b 0.1 b P-value 0.000 0.008 0.001 0.004 13-1051 Tango 84.5 70.5 a 30.4 a 1.8 a Tall Utah 64.1 40.1 b 15.0 b 0.3 b P-value 0.059 0.015 0.023 0.001 C. godetiae VPRI 41701 Tango 67.8 a 24.0 6.6 a 0.6 Tall Utah 30.2 b 23.5 1.2 b 0.3 P-value 0.005 0.922 0.004 0.097 C. nymphaeae MAFF 242590 Tango 57.6 a 29.6 a 6.2 0.6 a Tall Utah 1.6 b 13.1 b 4.7 0.1 b P-value 0.000 0.034 0.486 0.046

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Literature Cited: Baroncelli, R., Zapparata, A., Sarrocco, S., Sukno, S. A., Lane, C. R., Thon, M. R., Vannacci, G., Holub, E., Sreenivasaprasad, S. 2015. Molecular diversity of anthracnose pathogen populations associated with UK strawberry production suggests multiple introductions of three different Colletotrichum species. PLoS ONE 10(6): e0129140. doi:10.1371/journal.pone.0129140. Chechi, A., Stahlecker, J., Zhang, M., Luo, C. X., Schnabel, G. First report of Colletotrichum fioriniae and C. nymphaeae causing anthracnose on cherry tomatoes in South Carolina. Plant Dis. Online https://doi.org/10.1094/PDIS-09-18-1696-PDN. Cox, R. S. 1956. Control of diseases in the celery seed bed. Fla. Ag. Exp. Station J. Series. 532:242-244 Damm, U., Cannon, P. F., Woudenberg, J. H. C., and Crous, P. W. 2012. The Colletotrichum acutatum species complex. Stud. Mycol. 73:37-113. Diao, Y. -Z., Zhang, C., Liu, F., Wang, W. -Z., Liu, L., Cai, L., Liu, X. -L. 2017. Colletotrichum species causing anthracnose disease of chili in China. Persoonia 38: 20-37. Fujinaga, M., Yamagishi, N., Ogiso, H., Takeuchi, J., Moriwaki, J. and Sato, T. 2011. First report of celery stunt anthracnose caused by Colletotrichum simmondsii in Japan. J. Gen. Plant Pathol. 77:243-247. Jones, J. B., Zitter, T. A., Momol, T. M., Miller, S. A. eds. 2014. Compendium of Tomato Diseases and Pests, Second Edition. American Phytopathological Society Press. St. Paul, MN. Jordan, B. Culbreath, A. K., Brock, J., Tyson, C., and Dutta, B. 2018. First report of leaf curl on celery caused by Colletotrichum acutata sensu lato in Georgia. Plant Dis. 102:1657. Mangandi, J., Peres, N. A., and Whitaker, V. M. 2015. Identifying resistance to crown rot caused by Colletotrichum gloeosporioides in Strawberry. Plant Dis. 99:954-961. Moreira, R. R., Vandresen, D. P., Glienke, C., and May-De-Mio, L. L. 2019. First report of Colletotrichum nymphaeae causing blossom blight, peduncle and fruit rot on Pyrus pyrifolia in Brazil. Plant Dis. Online. https://doi.org/10.1094/PDIS-12-18-2263-PDN

Munir, M., Amsden, B., Dixon, E., Vaillancourt, L., and Gauthier, N. A. W. 2016. Characterization of Colletotrichum species causing bitter rot of apple in Kentucky orchards. Plant Dis. 100:2194-2203.

Peres, N. A., Timmer, L. W., Adaskaveg, J. E., Correll, J. C. 2005. Lifestyles of Colletotrichum acutatum. Plant Dis. 89:784-796.

Pollok, J. R., Mansfield, M. A., Gugino, B. K., and May, S. R. 2012 First report of leaf curl on celery caused by Colletotrichum acutatum in the United States. Plant Dis. 96:1692. Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2016. Muck Vegetable Cultivar Trial and Research Report. p. 106-107. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html

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Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2017. Muck Vegetable Cultivar Trial and Research Report. p. 94-95. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2018. Muck Vegetable Cultivar Trial and Research Report. p. 103-104. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Rodriguez-Salamanca, L. M., Enzenbacher, T. B., Byrne, J. M., Feng, C., Correll, J. C., and Hausbeck, M. K. 2012. First report of Colletotrichum acutatum sensu lato causing leaf curling and petiole anthracnose on celery (Apium graveolens) in Michigan. Plant Dis. 96: 1383. Rodriguez-Salamanca, L. M., Quesada-Ocampo, L. M., Naegele, R. P., and Hausbeck, M. K. 2015. Characterization, virulence, epidemiology, and management of leaf curling and petiole anthracnose in celery. Plant Dis. 99:1832-1840. Sato, T., and Moriwaki, J. 2013. Molecular re-identification of strains in NIAS Genebank belonging to phylogenetic groups A2 and A4 of the Colletotrichum acutatum species complex. Microbiol. Cult. Coll. 29:13-23. Sharma, S., Pethybridge, S. J., Buck, E. M., Hay, F. S. 2019. First report of leaf curl on celery (Apium graveolens var. dulce) caused by Colletotrichum fioriniae in New York. Plant Dis. Online 10.1094/PDIS-03-19-0499-PDN. Simmonds, J. H. 1966. Host Index of Plant Diseases in Queensland. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Takeuchi, J., Horie, H., Kubota, M. 2000. First occurrence of anthracnose of Apium graveolens by Colletotrichum acutatum and aspergillus blight of Ruscus hypoglossum by Aspergillus niger in Japan (abstract in Japanese). Jpn. J. Phytopathol. 66:92. Vock, N. T., ed. 1978. A Handbook of Plant Diseases in Colour, Volume 1. Fruit and Vegetables. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Vock, N. T., ed. 1982. A Handbook of Plant Diseases in Colour, Volume 1. Fruit and Vegetables. Second. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Wang, N. -Y., Forcelini, B. B. and Peres, N. A. 2019. Anthracnose fruit and root necrosis of strawberry are caused by a dominant species within the Colletotrichum acutatum species complex in the United States. Phytopathology in press. Online https://doi.org/10.1094/PHYTO-12-18-0454-R. Wright, D. G. and Heaton, J. B. 1991. Susceptibility of celery cultivars to leaf curl cause by Colletotrichum acutatum. Australas. Plant Pathol. 20:155-156. Yamagishi, N., Fujinaga, M., Ishiyama, Y., Ogiso, H., Sato, T., and Tosa, Y. 2015. Life cycle and control of Colletotrichum nymphaeae, the causal agent of celery stunt anthracnose. J Gen Plant Pathol. 81:279-286.

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CHAPTER 5

Integrating host resistance with biorational fungicides for management of celery leaf

curl disease

Abstract:

Celery leaf curl disease (CLCD) has led to yield loss on Pennsylvania farms since it was

first discovered in 2010. Pennsylvania has many small diversified farms growing organic produce

and while there are conventional materials available for CLCD management limited research has

been conducted to evaluate products for organic production. Biopesticides are of particular

interest due to their low environmental impact compared to copper-based fungicides. In this study

copper-based as well as biofungicide products are compared to conventional fungicides in three

field trials with cultivar susceptibility being integrated into one of those trials (cultivars ‘Tango’

vs ‘Merengo’). None of the biofungicide materials provided season long control compared to

untreated inoculated plants except for Champ WG combined with Regalia when applied to the

moderately susceptible cultivar Merengo. Accurate timing of fungicide applications is essential

for managing CLCD in both organic and conventional production systems.

Introduction:

CLCD is an emerging disease with the potential to cause significant economic losses in

celery production. Yield losses between 25 to 50% were reported in severe CLCD outbreaks in

Australia (Wright and Heaton 1991) and this chapter reports 99% disease incidence in a natural

epidemic. Australia, Japan, Canada and the United States (U.S.) have reported CLCD and related

diseases caused by species in the Colletotrichum acutatum species complex (Fujinaga et al. 2011;

Jordan et al. 2018; McDonald et al. 2016; Pollock et al. 2012; Rodriguez-Salamanca et al. 2012;

Vock 1982). Isolates from Pennsylvania (PA) and other states in the U.S. were identified as C.

fioriniae (Chapter 2). Sources of inoculum for these disease epidemics is unclear, however,

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seedborne pathogen transmission is suspected to be a significant source and has been shown to

occur in Japan with the species C. nymphaeae (Yamagishi et al. 2015).

Infected plants develop symptoms of leaf epinasty (downward curling of leaves), petiole

lesions with adventitious root formation and eventually crown rot and death of entire plants (Fig.

1-1, page 6). Disease development is favored by warm temperatures (25-30°C) and prolonged

leaf wetness periods (Rodriguez-Salamanca, 2015). All cultivars tested to date are susceptible to

CLCD, however, research has shown some variation in susceptibility indicating that the use of

less susceptible cultivars in combination with fungicides would be beneficial for disease

management (Davis 1992; McDonald and Vander Kooi 2015; Reynolds et al. 2016, 2017, 2018;

Wright and Heaton 1991).

Fungicide research related to CLCD on celery has been limited over the past three

decades and has focused largely on conventional options with a few trials evaluating some

copper-based products that could be used for organic production. Many conventional fungicide

active ingredients reduced CLCD disease incidence and yield loss when applied preventatively

including protectant multisite mode of action fungicides: chlorothalonil (Bravo Weather Stik

and Bravo ZN), copper oxychloride (Cuprox 50 WP), thiram (Thiotox 80% WP) and mancozeb

(Dithane M-45, Manzate Pro-Stick 75DF); single site targeted fungicides: propiconazole (Tilt

2.08EC), tebuconazole (Folicur), azoxystrobin (Quadris 2.08SC), difenoconazole (Inspire 5SC),

pyraclostrobin (Cabrio 3.3EC) and trifloxystrobin (Flint); and combination or premix

fungicides: azoxystrobin/propiconazole (Quilt SC), boscalid/pyraclostrobin (Pristine WG),

fluxapyroxad/pyraclostrobin (Priaxor SC, Merivon SC) and cyprodinil/fludioxonil (Switch)

(Heaton and Dullahide 1993; Raid et al. 2013, 2014; Rodriguez-Salamanca et al. 2015). However,

some of these products have been discontinued (Cuprox 50 WP, Thiotox 80%) or are not

currently labeled for use on celery (Inspire SC, Priaxor SC, Tilt, Folicur, Dithane M-45, Manzate

Pro-Stick, and Flint).

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Fungicide modes of action are divided into groups and given a FRAC code designation

by the fungicide resistance action committee (FRAC 2019). Pathogen populations can quickly

develop resistance to single site mode of action fungicides such as azoxystrobin and

pyraclostrobin (FRAC code 11) and limiting the number of product applications seasonally, as

well as, rotating or combining them with multisite fungicides is recommended. This reduces the

risk of pathogen populations developing resistance and increases the likelihood that these

fungicides will continue to remain effective management options.

Biofungicides are products containing microorganisms (bacteria and fungi) as the active

ingredient while biorational products are non-living and derived from living organisms as is the

case with Regalia, which is an extract of the plant Reynoutria sachalinensis (giant knotweed). For

simplicity, all of these types of products will be referred to as biofungicides from this point

forward. Interest in using biofungicides for organic production and in combination with

conventional materials is growing (Shishkoff and McGrath 2002). Copper-based fungicides are

used in both organic and conventional systems but growing concern over the effects of excess

copper in the environment has led to regulations reducing the use of these products in European

countries (Pena and Anton 2017). The United States Department of Agriculture (USDA) National

Organic Program declared that organically approved copper pesticides used in USDA certified

organic production must be used in a manner that minimizes accumulation of copper in the soil

(USDA 2019). This highlights the need for more research into copper-based alternatives for

organic growers that could also be integrated into conventional production systems.

The objective of this research is to evaluate biofungicide products that are labeled for use

on celery and compare them to commercially available conventional standards. Several products

certified for use in organic production were chosen for evaluation and represent different types of

biological activity including plant defense activators (Regalia SC, Double Nickel 55), pathogen

excluders (Actinovate AG, Double Nickle 55), anti-fungal metabolite producers (Actinovate AG,

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Double Nickel 55, Serenade Max), cellular protein disruptors (Champ WG, Badge X2), and

disinfectant/cell wall oxidizers (Oxidate 2.0).

Three products evaluated consist of bacterial biocontrol organisms including

Streptomyces lydicus (Actinovate AG) and Bacillus amyloliquefaciens (Double Nickel 55 and

Serenade Max) while Regalia SC is an extract of the giant knotweed plant and Oxidate 2.0 is a

hydrogen dioxide-based product. All of these products tested are currently labeled for use on

celery. Activity against other Colletotrichum species has been previously shown in studies with

Streptomyces species and B. amyloliquefaciens (Kim and Chung 2004; Palaniyandi et al 2013;

Yoshida et al 2001).

In addition, several studies in Ontario, Canada showed the cultivar Merengo was among

the least susceptible cultivars evaluated showing only 14.4 and 23.1% disease incidence in 2016

and 2017 compared to the susceptible control TZ9779 which showed 94.4 and 54.4% disease

incidence (Renynolds et al. 2016, 2017, 2018). Therefore, ‘Merengo’ was compared with the

susceptible cultivar Tango together with combinations or rotations of biofungicides for disease

management.

Materials and methods:

Plot layout: Four experiments were conducted during the 2015 through 2018 growing seasons at

the Pennsylvania State University Russell E. Larson Agricultural Research Center in

Pennsylvania Furnace, PA. Fertility in the research fields was adjusted based on soil tests with a

portion broadcasted and incorporated prior to planting and subsequent fertility applied through

fertigation throughout the season. Standard production guidelines from the Mid-Atlantic

Commercial Vegetable Production Recommendations were followed for crop management.

Transplants of cultivars CR-1 (2015), Tango (2016, 2017), or Tango + Merengo (2018) were

planted on 26 May 2015, 15 Jun 2016, 7 Jun 2017 and 30 May 2018. Transplants were obtained

from transplant growers in Michigan (2015) and Pennsylvania (2016). In 2017 and 2018

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transplants were grown onsite in a greenhouse from heat treated seed at the Larson Research

Center. The plots were 15 ft long with double rows on 6 in. high raised beds covered with 1mil.

standard black plastic mulch with one row of drip irrigation below the plastic. Each row

contained 15 plants (2015, 2016) or 14 plants (2017, 2018) with 8 in. spacing between plants for a

total of 28 or 30 plants per plot. There were 5 ft breaks between plots within the row and 5 ft

between row centers. Treatments were replicated 4 times in a randomized complete block design

(2015, 2016, 2017) or split plot design (2018) with cultivar as the main plot and fungicide

treatment as the sub-plot.

Fungicide treatments: In 2015, 2016 and 2018, fungicides were applied using a tractor-

mounted, R&D CO2-powered side boom sprayer calibrated to deliver 28 gal/A at 32 psi at the

tank through 3 TX-18 hollow-cone nozzles. In 2015 fungicide applications were made on 6, 12,

21, 27 Aug and 2, 9, 16, 23 Sep. In 2016 applications were made on 27 Jul and 3, 11, 16, 25 Aug.

In 2018 applications were made on 21, 29 Jun and 5, 12, 19, 26 Jul. Treatments applied in 2015

and 2016 included Bravo Weather Stik 6SC 1.5pt/A (chlorothalonil, FRAC group M5, Syngenta

Crop Protection, LLC), Equus 720SST 2.0pt/A (chlorothalonil, FRAC M5, Amvac Chemical

Corporation), Manzate Pro-Stick 3.0 lb/A (mancozeb, FRAC M3, UPL NA, Inc.), Quadris 2.08

SC 12.0 fl. oz./A (azoxystrobin, FRAC 11, Syngenta Crop Protection, LLC), Cabrio EG 16.0

oz/A (pyraclostrobin, FRAC 11, BASF Ag Products), Regalia SC 4.0 qt/A (Reynoutria

sachalinensis, FRAC P5, Marrone Bio Innovations), Actinovate AG 12.0 oz/A (Streptomyces

lydicus WYEC 108, FRAC BM2, Novozymes BioAg Inc), Double Nickel 55 3.0 lb/A (Bacillus

amyloliquefaciens strain D747, FRAC 44, Certis USA, LLC), Champ WG 2.0 lb/A (copper

hydroxide, FRAC M1, Nufarm Americas Inc.), and Oxidate 2.0 32 fl oz./A (hydrogen dioxide

and peroxyacetic acid, BioSafe Systems). In 2018 the following treatments were applied to both

‘Merengo’ and ‘Tango’ plots: Cabrio EG 16.0 oz/A alternated with Bravo WS 6SC 2.0 pt/A,

Champ WG 2.0 lb/A, Badge X2 3.57 lb/A (copper oxychloride and copper hydroxide, FRAC M1,

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Gowan USA), Champ WG 2.0 lb/A plus Regalia SC 4.0 qt/A, Champ WG 2.0 lb/A alternated

with Actinovate AG 12.0 oz/A, Champ WG 2.0lb/A alternated with Oxidate 2.0 128 fl oz/A, and

Champ WG 2.0 lb/A plus Serenade Max 3.0 lb/A (Bacillus amyloliquefaciens strain QST 713,

FRAC 44, AgraQuest, Inc.).

Inoculum preparation and application: In all years, symptoms resulting from natural inoculum,

likely brought in with seed or transplants, was observed prior to inoculation. Nevertheless, to

promote uniform disease distribution plants were artificially inoculated. In 2015, one row in each

plot was artificially inoculated on 17 Aug and 1 Sep 2015 with Pennsylvania C. fioriniae isolate

(10-788) at concentrations of 6.12 x 105 and 4.12 x 105 spores/ml respectively. On 8 Aug 2016

both rows in each plot were inoculated with Pennsylvania C. fioriniae isolate (16-809) at a

concentration of 7.40 x 104 spores/ml. In 2017, plants became severely infected before fungicides

could be applied. Incidence data was collected and data on this natural epidemic are presented in

the results. No artificial inoculum was applied in 2017. On 9 Jul 2018 both rows in each plot were

inoculated with a combination of two Pennsylvania C. fioriniae isolates (16-704 and 16-809) at a

concentration of 1.0 x 106 spores/ml.

Inoculum was prepared by growing isolates on potato dextrose agar amended with

streptomycin sulfate at 100 μg/ml for 7-15 days. Plates were flooded with sterile distilled water

and scraped with a sterile metal spear to dislodge spores. Spores were filtered through four layers

of cheese cloth and inoculum concentration was adjusted using a hemocytometer. Inoculum was

applied using a Hudson Home & Garden handheld sprayer directed towards the mid-canopy of

the plants. All fungicide trials had inoculated untreated controls and the 2015 and 2018 trials also

had uninoculated untreated controls to monitor for natural inoculum.

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Disease rating: Disease incidence was evaluated in 2015 and 2017 by examining 11-15 plants

(2015) or all 28 plants per plot (2017) for symptoms of leaf epinasty, petiole lesions and crown

rot. In 2016 disease severity was evaluated as a percentage of the plant showing symptoms of leaf

epinasty and crown rot and 13 plants per plot were evaluated on each rating date. In 2018, an

ordinal rating scale was developed where 12 plants per plot were each rated on a 0-5 scale (0=no

symptoms, 1=1-25% curling, 2=25-50% curling, 3=50-75% curling, 4=75-100% curling, 5=75-

100% curling with severe crown rot). Disease ratings were determined on 17, 30 Aug, 7, 14, 20

Sep, and 6 Oct 2015; 8, 20, 27 Aug 2016; 4, 19 July and 3 Aug 2017; and 26 Jun, and 11, 19, 29

Jul 2018. Precipitation totals were 22.45 in. for 26 May - 6 Oct 2015, 17.46 in. for 15 Jun - 27

Aug 2016, 13.15 in. for 7 Jun - 3 Aug 2017, and 13.8 in. for 30 May - 29 Jul 2018.

Data analysis: The 2015 and 2016 data were analyzed using one-way Analysis of Variance

(ANOVA) and Fisher’s Least Significant Difference test (P < 0.05) using Minitab v.18 statistical

software (Minitab Inc., State College, PA). Incidence of plants with CLCD in 2015 was analyzed

with ANOVA using the arcsine-square-root transformed proportion of average incidence from

each plot. In 2016, data from one replication were removed from the analysis due to high levels

of natural infection early in the season before fungicides were applied. In 2016, disease severity

data were averaged for each plot and the area under the disease progress curve (AUDPC) was

also calculated (Shaner and Finney 1977). In 2018, the average disease severity medians and

square root transformed AUDPC data were analyzed using PROC MIXED and partitioned

lsmeans analysis (P < 0.05) using SAS v.9.4 statistical software (SAS Institute, Cary, NC). For all

data the residual normal distributions were tested using normality plots and the Anderson-Darling

test or the Ryan-Joiner test and equal variances were tested using Levene’s test with Minitab v.18

statistical software. Data for the untreated un-inoculated plots was not included in the data

analyses but is reported in the results.

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Results:

2015 Fungicide trial: Despite inoculating the plants twice, disease incidence (DI) was low in

2015 ranging between 13 and 33% in the untreated inoculated control plots. Significant

differences were found between treatments for final disease incidence assessment (Table 5-1).

Based on this incidence data, the conventional fungicides Cabrio EG, Quadris 2.08SC and

Manzate Pro-Stick had a significantly lower percentage of symptomatic plants than the untreated

inoculated control (P = 0.01). Bravo WS 6SC, Equus 720SST and Champ WG reduced disease

slightly, but were not significantly better than the untreated inoculated control. The copper and

biofungicide treatments were less effective (DI 9.1 to 27.3%) than the conventional products (DI

0.9 to 9.4%) and Actinovate AG actually had a higher average incidence (27.3%) than the

untreated inoculated control (22.5%) (Fig. 5-1). No disease developed in the untreated un-

inoculated control plots, however, natural inoculum was observed in some plots prior to

inoculation.

2016 fungicide trial: Disease incidence and severity were higher in 2016 with untreated

inoculated control plots reaching 77.8% severity by the end of the trial. No significant differences

were found between treatments on any of the disease severity rating dates or over the course of

the whole season based on the AUDPC data (Table 5-1). However, in general by both the second

and third ratings compared to the untreated controls the conventional fungicides (Bravo WS 6SC,

Equus 720SST, Manzate Pro-Stick, Quadris 2.08SC, and Cabrio EG) numerically reduced disease

severity to between 27.1 to 34.5% (second rating) and 27 to 36.4% (third rating) compared to the

biofungicide products which only reduced disease severity by 10.2 to 23.0% and 6.0 to 21.6%

respectively (Fig. 5-2, 5-3).

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2017 and 2018 fungicide trials: In 2017 a natural disease epidemic occurred before fungicide

applications could be made however, disease incidence data was collected to evaluate disease

progress during the season. As expected if left unmanaged, the disease progressed during the

season with an incidence of 62.0% on 4 Jul, 90.0% on 19 Jul and 99.1% by 3 Aug. In 2018,

disease incidence and severity were high with a final median disease severity rating for the 12

plants per plot of 5 on a 0-5 scale across all treatment and control plots of susceptible ‘Tango’.

Disease incidence at the end of the season in the inoculated untreated control plots was 95.8% for

‘Merengo’ and 100% for ‘Tango’. There was no cultivar*treatment interaction (Table 5-2),

however, when the cultivars were combined for analysis no differences were observed among the

treatments for disease severity on 19 Jul (P = 0.62) or the end of season AUDPC (P = 0.25).

Therefore, cultivar data were analyzed separately to observe differences between treatments

within each cultivar.

In general, over the course of the season, disease severity was lower for ‘Merengo’

compared to ‘Tango’. Mid-season on 19 Jul, Cabrio EG alternated with Bravo WS 6SC and

Champ WG plus Serenade Max were both significantly better at managing CLCD than Champ

WG alternated with Actinovate on the moderately resistant ‘Merengo’. By the end of the season

based on AUDPC data, Cabrio EG alternated with Bravo WS 6SC and Champ WG plus Regalia

SC significantly reduced disease compared to the untreated inoculated control. On ‘Tango’,

however, Cabrio EG alternated with Bravo WS 6SC and Champ WG alone significantly reduced

disease compared to the untreated inoculated control from mid- through the end of the season.

Natural infection was noted in the field on 15 Jun and the untreated un-inoculated plots showed

overall AUDPC ratings of 0.9 (‘Merengo’) and 47.3 (‘Tango’) indicating the presence of natural

inoculum.

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Discussion:

These trials demonstrate the potential for celery leaf curl disease to cause significant

yield losses, especially for organic celery growers who have more limited management options.

Infected plants with initial leaf curling symptoms are still marketable for fresh and processing

markets as the curled leaves can be removed prior to being sold. However, under conditions of

warm temperatures and high moisture, infected plants quickly develop petiole lesions and crown

rot accompanied by secondary bacterial rot making them completely unmarketable. The natural

field epidemic in 2017 showed rapid disease development with 62% of plants developing

symptoms less than one month after planting and 90% of plants showing symptoms only 15 days

later. Frequent precipitation at the Larson Research Center during these weeks likely created ideal

conditions for disease development and spread with 5.78 in. of precipitation recorded between

planting on 7 Jun and 4 Jul when 62% of plants had developed symptoms.

In the 2015 trial there was very low disease pressure with only 22.5% of plants showing

symptoms in the untreated inoculated control. While an exact reason for this is unclear, it could

have been that unbeknownst to us ‘CR-1’ is less susceptible to CLCD. Unfortunately, ‘CR-1’ is a

proprietary line and is not widely available for Pennsylvania celery growers. Another possible

cause is the low frequency of precipitation events recorded in 2015 between inoculation and the

last date for data collection during the disease epidemic. In 2015, only three precipitation events

were recorded at the Larson Research Center totaling 6.1 in. while in 2016 and 2018, there were

nine (4.17 in.) and eight (7.51 in.) events respectively. Longer periods of leaf wetness have been

shown to increase CLCD disease severity and these rain events likely corresponded to longer leaf

wetness periods (Rodriguez-Salamanca et al. 2015).

In 2016 disease severity was high and throughout the season no differences were

observed between the fungicide treatments and the untreated inoculated control. Two fungicide

treatment applications were made preventatively starting on 27 Jul prior to inoculation on 8 Aug.

However, natural inoculum had already been observed in the field and by 19 Jul 11% of the

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plants in the field were showing symptoms of CLCD. Plants showing symptoms prior to

fungicide applications were not used in the data analysis, however this high percentage of initial

inoculum indicates that there may have been infected asymptomatic plants in the field and

fungicide applications were started too late to effectively prevent disease development. In 2015

and 2018 there was also natural inoculum present before fungicide applications were started with

0.8% incidence in 2015 and 6% incidence in 2018. Plants showing symptoms before fungicide

applications were also excluded from data analysis in these years. These results highlight the need

for early fungicide applications as part of a CLCD management program. Fungicide applications

should start as soon as symptoms are observed or earlier in the case of farms with a history of

CLCD problems or when highly susceptible cultivars are being grown.

Fungicides in the QoI group (Quinone outside Inhibitors, FRAC group 11) provided the

best disease control with Quadris 2.08SC (azoxystrobin) in the 2015 trial and Cabrio EG

(pyraclostrobin) in both 2015 and 2018 (alternated with Bravo WS 6SC) outperforming all the

other products tested. Fungicides in the QoI group are at a high risk for resistance development in

pathogen populations and require limited applications and rotating or combining with other

products to reduce the risk of fungicide resistance developing. Manzate Pro-Stick (mancozeb) is a

broad-spectrum fungicide that provided significant control in the 2015 trial, however this product

is not currently labeled for use on celery. Cabrio EG alternated with the broad-spectrum fungicide

Bravo WS 6SC provided season long control on both ‘Merengo’ and ‘Tango’ in the 2018 trial and

is a recommended option for conventional growers.

This study shows the high potential for the ‘Merengo’ to be used as part of a CLCD

disease management strategy in organic production with the Champ WG plus Regalia SC

treatment significantly reducing disease compared to the inoculated control on ‘Merengo’ in the

2018 trial. Cultivar Merengo is available through several seed companies and also has resistance

to Fusarium yellows. Cultivar Tango, however, is reported to be sweeter and more tender than

‘Merengo’ therefore some growers may be reluctant to switch cultivars. Further research to

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identify resistance in other cultivars would be beneficial for both organic and conventional

producers. More research needs to be done to investigate biorational fungicide options for CLCD

management with a focus on timing of fungicide applications for the best disease control.

Table 5-1. Effect of fungicide treatments on end of the season celery leaf curl disease incidence on celery ‘CR-1’ plants (2015) as well as disease severity on ‘Tango’ plants (2016). Research plots were inoculated with Pennsylvania isolates of C. fioriniae.

Treatment and rate/A

Disease Incidencez

2015

Severity 20 Aug 2016

Severity 27 Aug 2016

AUDPCx 2016

Bravo Weather Stik 6 SC 1.5 pt 9.4 b-ey 41.0 50.8 699.0 Equus 720SST 2.0 pt 7.4 b-e 33.6 41.9 618.0 Manzate Pro-Stick 3.0 lb 4.9 de 35.6 48.0 644.7 Quadris 2.08SC 12.0 fl oz 6.2 cde 36.8 41.4 710.0 Cabrio EG 16.0 oz 0.9 e 34.1 43.3 647.0 Regalia SC 4.0 qt 12.9 a-d 57.6 66.7 1048.0 Actinovate AG 12.0 oz 27.3 a 55.3 71.8 1113.0 Double Nickel 55 3.0 lb 20.2 abc 57.8 68.2 977.0 Champ WG 2.0 lb 9.1 b-e 46.3 56.2 773.0 Oxidate 2.0 32 fl oz 19.8 abc 45.1 57.3 860.0 Untreated Inoculated Control 22.5 ab 68.1 77.8 1228.5 Untreated Un-inoculated Control

0.0 --- --- ---

P-value 0.004 0.143 0.144 0.254

z Back-transformed disease incidence data. Untreated un-inoculated control not used in data analysis

y Means followed by the same letter within columns are not significantly different at P = 0.05 as determined by Fisher’s protected least significant difference test.

x AUDPC = Area under disease progress curve was calculated from 1 to 27 Aug according to the formula : ∑n

i=1[(Ri+1 + Ri)/2] [ti+1 – ti], where R = disease severity rating (% of leaf surface affected) at the ith observation, ti = time (days) since the previous rating at the ith observation, and n = total number of observations.

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Fig. 5-1. Boxplot showing the effect of fungicide treatments on end of the season celery leaf curl disease incidence on celery ‘CR-1’ inoculated with C. fioriniae in the 2015 field trial. Treatments are ordered by means. Light gray boxes are conventional treatments, dark gray boxes are biofungicide treatments and the white box is the inoculated untreated control. Non-inoculated untreated control plots (not shown) had 0% incidence.

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Fig. 5-2. Boxplot showing the effect of fungicide treatments on severity of celery leaf curl disease on 20 Aug 2016 on celery ‘Tango’ inoculated with C. fioriniae in the 2016 field trial. Treatments are ordered by means. Light gray boxes are conventional treatments, dark gray boxes are biofungicide treatments and the white box is the inoculated untreated control. No significant differences were observed between treatments (P = 0.143).

Fig. 5-3. Boxplot showing the effect of fungicide treatments on season-long CLCD severity as calculated using the AUDPC values on celery ‘Tango’ inoculated with C. fioriniae in the 2016 field trial. Treatments are ordered by means. Light gray boxes are conventional treatments, dark gray boxes are copper and biorational treatments and the white box is the inoculated untreated control. No significant differences were observed between treatments (P = 0.254).

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Table 5-2. Effect of fungicide treatments on celery leaf curl disease severity on celery ‘Tango’ and ‘Merengo’ in the 2018 field trial. Research plots were inoculated with Pennsylvania isolates of C. fioriniae.

Treatment and rate/A CLCD severityz

19 Jul 2018 AUDPCy Merengo (Resistant cultivar)x 0.6 b 23.3 b Cabrio EG 16.0 oz alt Bravo Weather Stik 6 SC 2.0 pt

0.1 ab 9.8 c

Champ WG 2.0 lb 0.9 ab 27.4 ab Badge X2 3.57 lb 0.5 ab 24.2 ab Champ WG 2.0 lb + Regalia SC 4.0 qt 0.1 ab 18.2 bc Champ WG 2.0 lb alt Actinovate AG 12.0 oz

1.3 a 31.3 a

Champ WG 2.0 lb alt Oxidate 2.0 128 fl oz

1.0 ab 29.5 ab

Champ WG 2.0 lb + Serenade Max 3.0 lb 0.0 b 19.4 abc Untreated Inoculated Control 1.1 ab 31.4 a Untreated Un-inoculated Control 0.0 0.9 Tango (Susceptible cultivar)x 3.8 a 61.6 a Cabrio EG 16.0 oz alt Bravo Weather Stik 6 SC 2.0 pt

2.0 c 39.8 c

Champ WG 2.0 lb 3.3 b 56.2 bc Badge X2 3.57 lb 3.8 ab 64.8 ab Champ WG 2.0 lb + Regalia SC 4.0 qt 4.0 ab 61.6 ab Champ WG 2.0 lb alt Actinovate AG 12.0 oz

4.1 ab 64.5 ab

Champ WG 2.0 lb alt Oxidate 2.0 128 fl oz

4.8 a 78.4 a

Champ WG 2.0 lb + Serenade Max 3.0 lb 4.0 ab 62.3 ab Untreated Inoculated Control 4.5 a 69.1 ab Untreated Un-inoculated Control 2.9 47.3 P-value (cultivar) 0.0001 0.0001 P-value (treatment) 0.0001 0.0001 P-value (cultivar*treatment) 0.1517 0.7541

zData were analyzed using proc mixed and a partitioned lsmeans analysis (SAS 9.4, SAS Institute, Cary, NC). Means followed by the same letter within columns are not significantly different at P = 0.05. The untreated un-inoculated control was excluded from the analysis.

yAUDPC = Area under disease progress curve was calculated from 11 to 29 Jul according to the formula : ∑n

i=1[(Ri+1 + Ri)/2] [ti+1 – ti], where R = disease severity rating (% of leaf surface affected) at the ith observation, ti = time (days) since the previous rating at the ith observation, and n = total number of observations. Back-transformed AUDPC data presented.

xNumbers in this row are statistical means for all treatments applied to the cultivar. Means followed by the same letter within columns within these rows are not significantly different at P = 0.05

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Fig. 5-4. Boxplot showing the effect of fungicide treatments on severity of celery leaf curl disease on 19 Jul 2018 on celery ‘Merengo’ and ‘Tango’ inoculated with C. fioriniae in the 2018 field trial. Treatments are ordered by medians. Light gray boxes are ‘Merengo’ treatments, dark gray boxes are ‘Tango’ treatments and the white box is the inoculated untreated control. Means followed by the same letter within cultivars are not significantly different at P = 0.05.

Fig. 5-5. Boxplot showing the effect of fungicide treatments on severity (AUDPC) of celery leaf curl disease on celery ‘Merengo’ and ‘Tango’ inoculated with C. fioriniae in the 2018 field trial. Treatments are ordered by medians. Light gray boxes are ‘Merengo’ treatments, dark gray boxes are ‘Tango’ treatments and the white box is the inoculated untreated control. Means followed by the same letter within cultivars are not significantly different at P = 0.05.

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Literature Cited:

Davis, R. D. 1992. Leaf curl disease of celery. Thesis. University of Queensland, Brisbane, Australia. Fujinaga, M., Yamagishi, N., Ogiso, H., Takeuchi, J., Moriwaki, J. and Sato, T. 2011. First report of celery stunt anthracnose caused by Colletotrichum simmondsii in Japan. J. Gen. Plant Pathol. 77:243-247. Fungicide resistance action committee (FRAC). 2019. FRAC Code List 2019: Fungal control agents sorted by cross resistant pattern and mode of action (including FRAC Code numbering). Online http://www.frac.info/docs/default-source/publications/frac-code-list/frac-code-list-2019.pdf?sfvrsn=98ff4b9a_2 Heaton, J. B., and Dullahide, S. R. 1993. Control of celery leaf curl disease caused by Colletotrichum acutatum. Australas. Plant Pathol. 22:152-155. Jordan, B. Culbreath, A. K., Brock, J., Tyson, C., and Dutta, B. 2018. First report of leaf curl on celery caused by Colletotrichum acutata sensu lato in Georgia. Plant Dis. 102:1657. Kim, P. I. and Chung, K.-C. 2004. Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908. FEMS Microbiology Letters 234:177-183. McDonald, M. R. and Vander Kooi, K. 2015. Evaluation of various celery cultivars for susceptibility to celery leaf curl, 2015. Muck Vegetable Cultivar Trial and Research Report. p. 98-99. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html McDonald, M. R., Vander Kooi, K., Celetti, M. 2016. Evaluation of fungicide efficacy and cultivar susceptibility for the management of anthracnose on celery. Poster 242-P. American Phytopathological Society Annual Meeting. July 30-Aug 3, 2016. Tampa, FL. Palaniyandi, S. A., Yang, S. H. and Suh, J.-W. 2013. Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes. Journal of Applied Microbiology. 115:207-217. Peña, N. and Antón, A. 2017. Is copper fungicide that bad? Acta Hortic. 1164 333-337. Pollok, J. R., Mansfield, M. A., Gugino, B. K., and May, S. R. 2012 First report of leaf curl on celery caused by Colletotrichum acutatum in the United States. Plant Dis. 96:1692. Raid, R., Klamer, D., and Klamer, J. 2013. Evaluation of fungicides for management of Colletotrichum acutatum on celery, 2012. Plant Dis. Manag. Rep. 7:V029. Raid, R., Spriensma, D., Klamer, J., and Klamer, B. 2014. Evaluation of fungicides for management of celery anthracnose caused by Colletotrichum acutatum, 2013. Plant Dis. Manag. Rep. 8:V307. Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2016. Muck Vegetable Cultivar Trial and Research

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Report. p. 106-107. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2017. Muck Vegetable Cultivar Trial and Research Report. p. 94-95. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Reynolds, S., Celetti, M. J., Jordan, K., McDonald, M. R., Screening for cultivar resistance to manage leaf curl on celery crops in Ontario, 2018. Muck Vegetable Cultivar Trial and Research Report. p. 103-104. University of Guelph. Office of Research and Department of Plant Agriculture. Online https://www.uoguelph.ca/muckcrop/annualreport.html Rodriguez-Salamanca, L. M., Enzenbacher, T. B., Byrne, J. M., Feng, C., Correll, J. C., and Hausbeck, M. K. 2012. First report of Colletotrichum acutatum sensu lato causing leaf curling and petiole anthracnose on celery (Apium graveolens) in Michigan. Plant Dis. 96: 1383. Rodriguez-Salamanca, L. M., Quesada-Ocampo, L. M., Naegele, R. P., and Hausbeck, M. K. 2015. Characterization, virulence, epidemiology, and management of leaf curling and petiole anthracnose in celery. Plant Dis. 99:1832-1840. Shaner, G. and Finney, R. E. 1977. The effect of nitrogen fertilization on the expression of slow-mildewing resistance in knox wheat. Phytopathology 67:1051-1056. Shishkoff, N. and McGrath, M. T. 2002. AQ10 biofungicide combined with chemical fungicides of AddQ spray adjuvant for control of cucurbit powdery mildew in detached leaf culture. Plant Dis. 86:915-918. USDA. 2019. Electronic code of federal regulations. The National List of Allowed and Prohibited Substances. Online https://www.ecfr.gov/cgi-bin/text-idx?c=ecfr&SID=9874504b6f1025eb0e6b67cadf9d3b40&rgn=div6&view=text&node=7:3.1.1.9.32.7&idno=7#sg7.3.205.g.sg0 Vock, N. T., ed. 1982. A Handbook of Plant Diseases in Colour, Volume 1. Fruit and Vegetables. Second. Queensland Department of Primary Industries. Brisbane, Queensland, Australia. Wright, D. G. and Heaton, J. B. 1991. Susceptibility of celery cultivars to leaf curl cause by Colletotrichum acutatum. Australas. Plant Pathol. 20:155-156. Wyenandt, C. A., Kuhar, T. P., Hamilton, G. C., VanGessel, M. J., Arancibia, R. A. Eds. 2019 Mid-Atlantic Commercial Vegetable Production Recommendations. Online https://njaes.rutgers.edu/pubs/publication.php?pid=e001 Yamagishi, N., Fujinaga, M., Ishiyama, Y., Ogiso, H., Sato, T., and Tosa, Y. 2015. Life cycle and control of Colletotrichum nymphaeae, the causal agent of celery stunt anthracnose. J Gen Plant Pathol. 81:279-286. Yoshida, S., Hiradate, S., Tsukamoto, T., Hatakeda, K., and Shirata, A. 2001. Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves. Phytopathology 91:181-187.

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CHAPTER 6:

Research summary and future directions

The overarching goal of this research was to gain a better understanding of the

Colletotrichum-celery pathosystem and provide information for improved diagnosis and

management of celery leaf curl disease (CLCD). This work will help diagnosticians, extension

educators and researchers to accurately identify CLCD and furthers our understanding of the

disease cycle and biology of the main causal pathogen in North America (N.A.), C. fioriniae.

In Chapter 2 the phylogenetic analysis revealed a new species-host association with the

identification of C. godetiae collected from symptomatic plants in Australia. All isolates collected

from N.A. were identified as C. fioriniae and all but two aligned with the first subgroup within

this clade. In addition, a dried herbarium specimen from an epidemic of CLCD in Australia was

identified as C. fioriniae and may have been a hybrid between C. fioriniae and C. godetiae. The

knowledge that C. fioriniae is the main causal pathogen in N.A. will help to build our

understanding of this disease system. Connections can be made to research on this species from

other pathosystems such as apple, peach and blueberry where C. fioriniae is a major species

found causing fruit rot on these hosts. Sequence data generated from the multilocus analysis of 66

isolates was deposited in GenBank where it can be utilized for further research on these species.

Microsatellite loci and population genetics tools were used to better understand the

biology and relationships of C. fioriniae populations from both celery and apple (Chapter 3). This

study revealed high clonality in celery populations of C. fioriniae, whereas sexual reproduction

was detected in the apple population. Furthermore, populations from these two hosts were found

to be significantly different from each other indicating a separation in the populations between

these two hosts. One possibility for these differences is that inoculum for the CLCD outbreaks is

seedborne. More research is needed to further investigate this hypothesis and determine if seed is

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a major source of inoculum for CLCD epidemics. However, since seed is a possible source,

management recommendations including using heat treated seed, scouting frequently for disease

symptoms and using preventative fungicide applications early in the season should be followed to

reduce losses for potential seedborne inoculum. Research focused on improving seed detection

methods would also help to identify and remove contaminated seed from production systems.

Since Colletotrichum survives as an endophyte it is likely that infected seeds may be originating

from plants with no visible symptoms of CLCD making it difficult to identify sources of

contaminated seed.

An extensive study of the symptomology caused by three species of Colletotrichum (C.

fioriniae, C. godetiae, and C. nymphaeae) on two celery cultivars was conducted (Chapter 4).

This study revealed differences in aggressiveness between isolates and species but no differences

in the types of symptoms that developed, concluding that all three species cause the same disease

with “celery leaf curl disease” proposed as the common name. While leaf curling is the most

characteristic symptom of this disease other symptoms produced include small leaf spots, petiole

lesions, adventitious roots, crown rot and stunting. The development and severity of symptoms

varied depending on environmental conditions, host cultivar, species, and isolate. The cultivar

‘Tall Utah’ was identified as less susceptible than ‘Tango’ and should be investigated further as

an option for CLCD management. This study resolves the confusion regarding differences in

symptomology and indicates that using the same common disease name for all of these species

would allow for clearer communication and more accurate identification of CLCD. Research

evaluating disease symptoms caused by other Colletotrichum species on celery should continue to

be conducted as past studies have found that some species only cause leaf spotting symptoms and

do not cause the other symptoms associated with CLCD.

Fungicide trial research showed no effective organic or biorational products for disease

management on the highly susceptible cultivar Tango (Chapter 5). Additionally, season long

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reduction of CLCD symptoms was only achieved in 2015 under low disease pressure using the

QoI fungicides azoxystrobin (Quadris 2.08SC) or pyraclostrobin (Cabrio EG) or the broad

spectrum Mancozeb Pro-Stick which is not currently labeled for use on celery. None of the

fungicides applied under high disease pressure in 2016 were effective and only Cabrio EG

alternated with Bravo WS 6SC provided season long management on ‘Tango’ in 2018. This

highlights the need for further research on both biorational and conventional fungicides for

CLCD management especially on highly susceptible cultivars.

In the 2018 fungicide trial, ‘Merengo’ showed high possibility for use in organic and

conventional systems where CLCD is a problem. This cultivar showed significantly less disease

for all treatments compared to ‘Tango’ throughout the season. In addition, plants treated with

Champ WG plus Regalia SC showed significantly less disease than the untreated inoculated

control and could be a management option for organic production systems. More research is

needed to further test combinations of resistant cultivars and biorational treatments to identify

options for organic production that not only reduce disease but also reduce environmental impacts

of frequent copper fungicide use.

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APPENDIX

Detection of seedborne Colletotrichum fioriniae through vertical transmission from

inoculated celery plants to seeds

Introduction:

Celery leaf curl disease (CLCD) appeared abruptly in the United States (U.S.) in 2010 on

farms that were growing their own transplants from seed (Pollok et al. 2012). The fact that CLCD

appeared suddenly on farms growing their own transplants indicates that the pathogen may be

seedborne and that seed may be the primary source for inoculum of CLCD epidemics. This

disease is caused by species in the Colletotrichum acutatum species complex and the only species

identified thus far causing CLCD in the U.S. is C. fioriniae (Marcelino & Gouli) Pennycook

(Chapter 2). Fungi in the C. acutatum species complex have been found in seed of several hosts

including safflower, zinnia, cowpea and lupin (Kim et al. 1999; Kulik et al. 2005; Kulshretha

1976; Prasanna 1986). Research in Japan found C. nymphaeae (Pass) Aa, which causes celery

stunt anthracnose, in celery seed by plating seed onto selective culture media (Yamagishi et al.

2015).

Celery is a biennial which requires cold vernalization for flower and seed production to

occur (Rubatzky et al. 1999). Commercial seed production for celery occurs in locations with

cool winters that do not get below freezing such as California, the south of France, and Italy

(Navazio 2012; Rubatzky et al. 1999). Any seedborne inoculum would be coming from locations

where seed is produced and not the local environment. The objective of this research was to

determine if C. fioriniae can be transmitted from inoculated plants into seed produced in the

following year. Demonstration of vertical transmission of the pathogen would support the

hypothesis that C. fioriniae can be distributed in seed.

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Materials and methods:

Transplants of celery ‘CR-1’ (2015) and ‘Tango’ (2016) were planted on 26 May 2015

and 15 Jun 2016 at the Pennsylvania State University Russell E. Larson Agricultural Research

Center in Pennsylvania Furnace, PA. In 2015, plants were artificially inoculated on 17 Aug and 1

Sep with PA C. fioriniae isolate 10-788 at concentrations of 6.12 x 105 and 4.12 x 105 conidia/ml

respectively. In 2016, plants were inoculated on 8 Aug with PA C. fioriniae isolate 16-809 at a

concentration of 7.40 x 104 conidia/ml. In the fall, 5-6 plants were dug up, potted into plastic pots

and stored in a cold room at 4°C for 6-7 months to overwinter the plants and expose them to a

cold vernalization period. Plants were watered periodically to prevent pots from completely

drying out. After cold vernalization, plants were brought outside and allowed to flower and form

seed. Mature seed heads were harvested and allowed to fully dry then were cleaned of large

debris and stored in a refrigerator at 4°C.

Two independent experiments were conducted to assess seed for the presence of

Colletotrichum in the two seed lots collected. Seed lot A consisted of seeds from CR-1 plants

grown and inoculated in 2015 and harvested in 2016. Seed lot B consisted of seeds from ‘Tango’

plants grown and inoculated in 2016 and harvested in 2017. Seeds were plated onto sterile 100

mm petri dishes containing 9.0 cm autoclaved filter paper and approximately 2.5 ml sterile

deionized water. The number of seeds plated were 1,045 (lot A) and 1,104 (lot B) for experiment

#1 and 1,059 (lot A) and 1,178 (lot B) for experiment #2. Plates were closed with parafilm and

incubated in a growth chamber at 25°C with 60% relative humidity and a 12 hr photoperiod.

Additional sterile water was added to the plates if drying out of the filter paper was observed.

Seeds were examined after two and three weeks of incubation to look for the presence of

Colletotrichum on the seedlings.

Seeds with evidence of Colletotrichum on the seed coat or emerging seedling were plated

onto potato dextrose agar amended with streptomycin sulfate at 100 μg/ml (PDA+) (Fig. A-1).

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Cultures were identified after 7-10 days as C. fioriniae based on morphological characteristics of

the cultures as described by Damm et al. 2012 and observed in similar isolates in Chapter 2.

To verify the identity of the isolates, two isolates were saved from each seed lot per

experiment for a total of eight isolates. An agar plug of each culture was grown in shaking liquid

cultures of potato dextrose broth for 7-10 days at room temperature. The mycelium was harvested

and, frozen (-20°C) and DNA was extracted from mycelium using the Mo Bio PowerSoil Kit

(Qiagen, Germantown MD). An intron of the glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) gene was amplified using the primer pair GDF1 + GDR1 (Guerber et al. 2003). PCR

reactions and sequence editing were performed as described in Chapter 2, page 21. Geneious

Biologics version R9 software (Biomatters, Inc., Newark, NJ) was used to edit and assemble the

consensus contigs from the forward and reverse sequences. The four sequences from each seed

lot were aligned with GAPDH sequences from the C. fioriniae ex-type isolate CBS 128517 and

the isolates used to inoculate the plants 10-788 (2015) and 16-809 (2016) using the Clustal W

algorithm in the Geneious Biologics software. The percent pairwise identity was calculated for

the six aligned isolates for each seed lot.

Results:

In experiment #1, Colletotrichum developed on 21% (219/1,045) of lot A seeds and 20%

(222/1,104) of lot B seeds. In experiment #2, Colletotrichum developed on 27% (289/1,059) of

lot A seeds and 24% (282/1,178) of lot B seeds. Acervuli and masses of conidia developed on the

seed coat, as well as, the emerging seedling roots and cotyledons (Fig. A-1). Sequence

comparison of the GAPDH gene showed 99.9% pairwise identity of the four seed isolates from

lot A with C. fioriniae isolates CBS 128517 and 10-788. Comparison of GAPDH sequences from

lot B isolates showed 100% pairwise identity with C. fioriniae isolates CBS 128517 and 16-809.

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Discussion:

This study has demonstrated that C. fioriniae can be vertically transmitted from infected

plants into developing seed the following year. This research provides strong support for the

hypothesis that this pathogen can be distributed in seed and would explain how CLCD appeared

suddenly on farms in the U.S. growing their own transplants from seed. Seedborne inoculum

could be the main source for CLCD epidemics therefore research and management practices

should focus on minimizing entry of the pathogen through seed. Research with C. nymphaeae

showed when artificially infested seeds were treated with hot water at 60°C for 10 to 30 min. or

50 and 55°C for 30 min. the pathogen did not grow when the treated seeds were plated on media.

Seed germination was significantly affected by treatments at 55 and 60°C, therefore treatment at

50°C for 30 min. is recommended (Yamagishi et al. 2015).

In addition to seed treatments, research focusing on improving detection of the pathogen

in seed is warranted to identify and prevent the introduction of infested seeds into the production

system. Since asymptomatic or latent infections may occur in young seedlings grown from

infested seeds, early fungicide applications may be needed to reduce further development and

spread of the disease in young plants. Frequent scouting for disease symptoms will aid in timing

fungicide applications and additional research on fungicide efficacy will help to identify more

products that can be used for CLCD management.

Fig. A-1. Images of C. fioriniae sporulating on celery A) seed coat, B) emerging root, C) emerging cotyledon. Images A and B are from lot A seeds and C is from lot B seeds.

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Literature Cited:

Guerber, J. C., Liu, B., Correll, J. C., and Johnston, P. R. 2003. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia. 95:872-895. Kim, W. G., Moon, Y. G., Cho, W. –D., and Park, S. D. 1999. Anthracnose of safflower caused by Colletotrichum acutatum. Plant Pathol. J. 15:62-67. Kulik, T., Pszczolkowska, A., Olszewski, J., Fordonski, G., Plodzien, K. and Sawicka-Sienkiewicz, E. 2005. Identification of Colletotrichum acutatum from yellow and Andean lupin seeds using PCR assay. Electron. J. Pol. Agric. Univ. 8:02. Available at http://www.ejpau.media.pl/volume8/issue1/art-02.html Kulshrestha, D. D. 1976. Colletotrichum acutatum- A new seed borne pathogen of Zinnia. Curr. Sci. 45:64-65. Navazio, J. 2012. The Organic Seed Grower: A Farmer’s Guide to Vegetable Seed. Production. Chelsea Green Publishing. White River Junction, Vermont. Pollok, J. R., Mansfield, M. A., Gugino, B. K., and May, S. R. 2012 First report of leaf curl on celery caused by Colletotrichum acutatum in the United States. Plant Dis. 96:1692 Prasanna, K. 1986. Seed health testing of cowpea with special reference to anthracnose caused by Colletotrichum lindemuthianum. Seed Sci. Technol. 13:821-827. Rubatzky, V. E., Quiros, C. F., and Simon, P. W. 1999. Carrots and Related Vegetable Umbelliferae. CAB International. New York, NY. Yamagishi, N., Fujinaga, M., Ishiyama, Y., Ogiso, H., Sato, T., and Tosa, Y. 2015. Life cycle and control of Colletotrichum nymphaeae, the causal agent of celery stunt anthracnose. J Gen Plant Pathol 81:279-286.

VITA Sara R. May

srm183@psu.edu

Education August 2019 Ph.D. Plant Pathology The Pennsylvania State University, University Park, PA December 2003 M.Agr. Plant Pathology The Pennsylvania State University, University Park, PA May 2001 B.S. Horticulture, minor in Plant Pathology The Pennsylvania State University, University Park, PA Professional Experience and Research May 2009-Present Coordinator, Plant Disease Clinic, Dept. Plant Pathology and

Environmental Microbiology, Penn State, University Park, PA Jan. 2003-May 2009 Research Technician supervised by Dr. Barbara Christ, Dept.

Plant Pathology, Penn State, University Park, PA May 2001-Dec. 2003 Graduate Research Assistant advised by Dr. Barbara Christ,

Dept. Plant Pathology, Penn State, University Park, PA Jan. 1999-May 2001 Undergraduate Research Assistant advised by Dr. Barbara

Christ, Dept. Plant Pathology, Penn State, University Park, PA May 2000-Aug. 2000 Undergraduate Research Assistant advised by Dr. Walter

Stevenson, Dept. Plant Pathology, University of Wisconsin, Madison, WI

Grants and Awards Dec. 2012 Graduate Student Competitive Grant June 2015 Leonard J. Francl Memorial Endowment June 2016 Larry J. Jordan Memorial Endowment Publications 2012 Pollok, J. R., Mansfield, M. A, Gugino, B. K., May, S. R. 2012.

First report of leaf curl on celery caused by Colletotrichum acutatum in the United States. Plant Disease. 96:1692