Understanding the Fungal Biology and Fungicide Control Of

UNDERSTANDING THE FUNGAL BIOLOGY AND FUNGICIDE CONTROL OF

NEOFABRAEA SPECIES CAUSING BULL’S-EYE ROT OF APPLES

GROWN IN THE US PACIFIC NORTHWEST

CHRISTIAN GRACE AGUILAR

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Plant Pathology

JULY 2017

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of CHRISTIAN

GRACE AGUILAR find it satisfactory and recommend that it be accepted.

______Mark Mazzola, Ph.D., Chair

______Weidong Chen, Ph.D.

______Dennis Johnson, Ph.D.

______Chang-Lin Xiao, Ph.D.

ii ACKNOWLEDGMENTS

There are many individuals who provided physical, financial and/or emotional support to me over the course of my Ph.D. studies. I am immensely grateful to these individuals.

Specifically, I would like to thank Dr. Mark Mazzola for “adopting” me into his lab and mentoring me throughout the past five years of my degree. He has guided me not only in terms of my research but has also helped me mature professionally by giving me the opportunity to write manuscripts, grants and network at local and national conferences. His patience has been unwavering and I am very thankful to have had the opportunity to work under his direction.

Similarly, I would like to thank Dr. Chang-Lin Xiao for providing funding support so that I may conduct my research, and for his active involvement during the planning and execution of my research projects. His insights and criticism have helped to strengthen this work. Additionally, I would like to thank Drs. Weidong Chen and Dennis Johnson for sitting through numerous committee meetings, responding to countless emails, and for reviewing and editing this dissertation.

Next I would like to thank Robin Boal, Chris Sater, Sheila Ivanov, Marcia Walter and

Xiaowen Zhao for helping me conduct my projects and for providing suggestions when brainstorming and troubleshooting were needed. I am also appreciative of the various conversations shared with these individuals, to which the tedium of everyday lab activities became much more tolerable. I would like to extend my gratitude to Drs. Rosa Caiazzo and

Parama Sikdar for helping me navigate through my graduate studies and research work, and also for their kindness and friendship.

Furthermore, I would like to recognize the USDA- Agricultural Research Service, the

Seattle Chapter of the Achievement Rewards for College Scientists (ARCS) foundation, the

iii Washington State University Research Assistantships for Diverse Scholars (RADS) program, the

Mike and Kathy Hambelton Fellowship endowment, and the Washington Tree Fruit Research

Commission for providing funding support of this work. Without the provisions granted by these agencies, this research could not exist.

Lastly, I wish to thank my family for enduring this crazy roller coaster ride with me.

Thank you!!!

iv UNDERSTANDING THE FUNGAL BIOLOGY AND FUNGICIDE CONTROL OF

NEOFABRAEA SPECIES CAUSING BULL’S-EYE ROT OF APPLES

GROWN IN THE US PACIFIC NORTHWEST

Abstract

by Christian Grace Aguilar, Ph.D. Washington State University July 2017

Chair: Mark Mazzola

Neofabraea perennans and Neofabraea kienholzii are two of four fungal organisms causing bull’s-eye rot of apples and other pome fruit grown in the US Pacific Northwest.

Artificial wound inoculations conducted on ‘Fuji’ and ‘Red Delicious’ apple twigs using the aforementioned species demonstrated that both fungi are capable of inducing tree cankers that are similar in appearance. Cankers were largest following inoculations held in October compared to all other inoculation events evaluated. Additionally, artificial inoculations conducted on ‘Fuji’ and ‘Red Delicious’ apple fruit throughout the growing season revealed that fruit infections were more likely to be established during the final weeks approaching commercial harvest. Together, these studies demonstrate that these fungi are capable of surviving in the orchard as mycelium in tree cankers throughout the year, and under favorable conditions, can cause fruit infections throughout the fruit-growing season. These results highlight the importance of canker pruning as a means of reducing the pathogen inoculum load in the orchard.

v Trials were also conducted to evaluate the efficacy of various pre-harvest and postharvest applied fungicides for the control of bull’s-eye rot in ‘Fuji’ apples. Findings from these studies demonstrated that among the materials tested, thiophanate-methyl, thiabendazole and pyrimethanil were the most effective fungicides in suppressing incidence of bull’s-eye rot and were capable of mitigating early, mid and late season fruit infections following a single application near/at the end of the apple-growing season. Although effective in controlling this important postharvest disease of apple, incorporation of these fungicides into spray programs should proceed with caution so as to minimize the risk of fungicide resistance in populations of

Neofabraea spp. as well as other major pathogens in the Pacific Northwest.

vi TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iii

ABSTRACT ...... v

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

CHAPTER ONE – Timing of Perennial Canker Development in Apple Trees Caused by

Neofabraea perennans and Neofabraea kienholzii

ABSTRACT ...... 1

INTRODUCTION ...... 2

MATERIALS AND METHODS ...... 4

RESULTS ...... 9

DISCUSSION ...... 12

LITERATURE CITED ...... 17

CHAPTER TWO – Timing of Apple Fruit Infection by Neofabraea perennans and Neofabraea kienholzii in Relation to Bull’s-eye Rot Development in Stored Apple Fruit

ABSTRACT ...... 31

INTRODUCTION ...... 32

MATERIALS AND METHODS ...... 35

vii RESULTS ...... 39

DISCUSSION ...... 42

LITERATURE CITED ...... 47

CHAPTER THREE – Control of Bull’s-eye Rot Caused by Neofabraea perennans and

Neofabraea kienholzii in Stored Apple Using Pre- and Postharvest Fungicides

ABSTRACT ...... 58

INTRODUCTION ...... 59

MATERIALS AND METHODS ...... 62

RESULTS ...... 67

DISCUSSION ...... 70

LITERATURE CITED ...... 74

viii LIST OF TABLES

CHAPTER ONE

1. Comparisons between inoculation event and its effect on acervuli viability of Neofabraea spp. using Fisher’s exact test ...... 22

CHAPTER TWO

1. Dates of fruit inoculation and harvest for bull’s-eye rot field trials conducted at Washington State University Sunrise Research Orchard, and environmental conditions recorded during each fruit inoculation event ...... 52

CHAPTER THREE

1. Influence of pre-harvest fungicide treatment, inoculation event, Neofabraea species and the interaction of these variables on bull’s-eye rot incidence of ‘Fuji’ apples cultivated during the 2013 and 2014 growing seasons ...... 78

2. Influence of postharvest fungicide treatment, inoculation event, Neofabraea species and the interaction of these variables on bull’s-eye rot incidence of ‘Fuji’ apples cultivated during the 2013 and 2014 fruit-growing seasons ...... 79

ix LIST OF FIGURES

CHAPTER ONE

1. Physiological response of ‘Fuji’ apple twigs to wounding and artificial inoculation during the month of October with a sterile potato dextrose agar (PDA) plug serving as the control treatment ( A), a PDA plug containing germinated spores of Neofabraea kienholzii (B), and a PDA plug containing germinated spores of Neofabraea perennans (C). Arrows indicating the wound inoculation site (WIS) and canker margin (CM) are shown. Twigs were harvested and photos were taken at six months post inoculation ...... 24

2. ‘Fuji’ apple twigs wounded and artificially inoculated with potato dextrose agar plugs containing germinated spores of Neofabraea kienholzii (A) and Neofabraea perennans (B) during the month of April at the Washington State University Tree Fruit Research and Extension Center orchard plot in Wenatchee, WA. Black, bulbous structures emanating from the wound inoculation site of the twig canker represent fruiting bodies developed by the pathogen (indicated with an arrow). Photos were taken at six ( A) and two ( B) months post inoculation...... 25

3. Effect of inoculation date on canker development induced by Neofabraea spp. in artificial inoculations of ‘Red Delicious’ apple twigs. Values depicted are average half-length measurement resulting from inoculations conducted at specific inoculation events during the 2012-2013 ( A), 2013-2014 ( B) and 2014-2015 ( C) annual trials of this study. Measurements reported were taken at six months post inoculation and averaged across 16 replicates. Separate statistical analyses were conducted comparing canker size induced by the two Neofabraea spp. and the control treatment at each inoculation event (asterisk); comparing canker size due to N. perennans inoculation across time (uppercase letters); and comparing canker size induced by N. kienholzii across inoculation event (lowercase letters). Treatment means designated with the same letter of the same case, or same number of asterisks are not significantly different from one another according to the Tukey means separation method ( P ≤ 0.05). Error bars represent the standard error of the mean ...... 26

4. Effect of inoculation event on canker development induced by Neofabraea spp. in artificial inoculations of ‘Fuji’ apple twigs. Values depicted are average half-length canker measurement resulting from inoculations conducted at specific inoculation events during the 2012-2013 ( A), 2013-2014 (B) and 2014-2015 ( C) annual trials of this study. Measurements reported were taken at six months post inoculation and averaged across 16 replicates. Statistical analyses comparing canker size induced by two Neofabraea inoculum sources and a control treatment within each individual inoculation event (asterisk) were conducted. Similarly, separate analyses comparing canker size attributed to N. perennans across inoculation event (uppercase letters), and canker size due to N. kienholzii across inoculation event (lowercase letters) were also conducted. Treatments designated with the same letter of

x the same case, or with the same number of asterisks, indicate no significant difference in half-length canker size ( P ≤ 0.05). Error bars specify the standard error of the mean taken from 16 different twig replicates ...... 28

5. Effect of inoculation event on the average proportion of ‘Fuji’ ( A, B) and ‘Red Delicious’ (C, D) apple twigs producing the asexual fruiting body of Neofabraea perennans and Neofabraea kienholzii following artificial wound inoculations conducted at orchard plots located at the Washington State University Tree Fruit Research and Extension Center ( A), Columbia View ( B, D) and Sunrise ( C) research orchards near Wenatchee, WA. Treatment means designated with the same letter are not significantly different ( P ≤ 0.05). Error bars indicate the standard error of the mean ...... 30

CHAPTER TWO

1. Scatterplot showing the linear relationship between timing of ‘Red Delicious’ ( A, B) and ‘Fuji’ ( C, D) apple fruit inoculated with either Neofabraea perennans (A, C) or Neofabraea kienholzii (B, D) in the orchard and total bull’s-eye rot development obtained by the end of the cold storage season. Harvest date is indicated by day ‘0’, and inoculation date is represented by negative integers. The values shown are the percent of diseased apples out of the total number of apples inoculated for each treatment, averaged across 3 replicates .... 53

2. Average total incidence of bull’s-eye rot on ‘Red Delicious’ apples. Apple fruit were inoculated in the orchard with a spore suspension of Neofabraea perennans or Neofabraea kienholzii at various inoculation timings during the 2012 ( A), 2013 ( B) and 2014 ( C) fruit- growing seasons. Fruit were harvested at commercial maturity, and kept under postharvest storage at 0°C for 10 months. Monthly disease ratings were conducted beginning on the fourth month of storage. Difference in total disease incidence for N. perennans and N. kienholzii at each inoculation timing was compared. Means represented by bars within the same inoculation timing and designated with the same letter are not significantly different ( P ≤ 0.05) ...... 54

3. Average total incidence of bull’s-eye rot on ‘Fuji’ apples. Apple fruit were inoculated in the orchard with a spore suspension of Neofabraea perennans or Neofabraea kienholzii at various inoculation timings during the 2012 ( A), 2013 ( B) and 2014 ( C) fruit-growing seasons. Fruit were harvested at commercial maturity, and kept under postharvest storage at 0°C for 9 months. Monthly disease ratings were conducted beginning on the third month of storage. Difference in total disease incidence for N. perennans and N. kienholzii at each inoculation timing was compared. Means represented by bars within the same inoculation timing and designated with the same letter are not significantly different ( P ≤ 0.05) ...... 56

xi CHAPTER THREE

1. Influence of pre-harvest fungicide application on incidence of bull’s-eye rot detected on fruit inoculated with Neofabraea perennans and Neofabraea kienholzii at 5 and 2 weeks before harvest (wbh) during the 2013 ( A) and 2014 ( B) fruit-growing seasons. The Tukey-Kramer method was utilized to identify significant differences among fungicide treatments. Means designated with the same letter are not significantly different ( P < 0.05). Error bars represent the standard error of the mean ...... 80

2. Influence of postharvest fungicide application on incidence of bull’s-eye rot detected on fruit inoculated with Neofabraea perennans and Neofabraea kienholzii at 5 and 2 weeks before harvest (wbh) during the 2013 ( A) and 2014 ( B) fruit-growing seasons. The Tukey-Kramer method was utilized to identify significant differences among fungicide treatments. Means designated with the same letter are not significantly different ( P < 0.05). Error bars represent the standard error of the mean ...... 81

3. Influence of postharvest fungicide drench and thermofog treatments on average bull’s-eye rot incidence due to N. perennans inoculations at 2 wbh during the 2013 ( A) and 2014 ( B) fruit- growing seasons. The Tukey-Kramer method was used to separate treatment means considered statistically significant. Bars with the same letters are not significantly different (P ≤ 0.05) ...... 82

xii CHAPTER ONE

Timing of Perennial Canker Development in Apple Trees Caused by Neofabraea perennans

and Neofabraea kienholzii

C. G. Aguilar , Department of Plant Pathology, Washington State University, Pullman 99164;

M. Mazzola , United States Department of Agriculture-Agricultural Research Service (USDA-

ARS), Tree Fruit Research Laboratory, 1104 N. Western Ave., Wenatchee, WA 98801; and C.

L. Xiao , USDA-ARS, San Joaquin Valley Agricultural Sciences Center, 9611 S. Riverbend

Ave., Parlier, CA 93648

ABSTRACT

Multiple species of closely related fungi belonging to the genera Neofabraea and

Phlyctema have been reported to incite canker diseases of apple trees and a postharvest decay of apple fruit referred to as bull’s-eye rot. Neofabraea kienholzii is a recently identified fungus participating in the bull’s-eye rot disease complex of apple and other pome fruit. In this study, apple twigs inoculated with N. kienholzii were shown to develop symptoms of a canker disease closely resembling perennial canker of pome fruit trees caused by Neofabraea perennans .

Cankers resulting from infection by either Neofabraea spp. were more likely to be induced when twig inoculations were held in October, and to a lesser degree in April, compared to all other inoculation events evaluated in this study. While N. kienholzii tended to induce cankers that were smaller in size compared to N. perennans , both pathogens shared similar seasonal trends in the initiation and development of tree cankers. N. perennans and N. kienholzii were recovered from inoculated twigs 6 months post inoculation regardless of when inoculations were conducted,

1 indicating that both pathogens can survive on diseased twigs year round. Furthermore, acervuli were observed more often following twig inoculations held in September and April compared to the other inoculation events assessed.

INTRODUCTION

Bull’s-eye rot is an economically important postharvest disease affecting pome fruit grown in the Pacific Northwest (PNW) region of the United States. This disease is caused by four closely related fungal species belonging to the family Dermateaceae: Neofabraea kienholzii ,

Neofabraea malicorticis , Neofabraea perennans and Phlyctema vagabunda [previously recognized as Neofabraea vagabunda (Chen et al. 2016)]. The distribution of these pathogens within the PNW is geographically variable, sometimes with a single species dominating a pome fruit production district while being entirely absent from another (Kienholz 1939; Spotts et al.

2009). This irregularity may be due to differences in environmental conditions within pome fruit production districts across the PNW and the adaptive abilities of Neofabraea spp. to thrive in a defined environment.

Symptoms of fruit infection by different Neofabraea spp. are indistinguishable from one another and require both morphological and molecular techniques for diagnosis at the species level (de Jong et al. 2001; Gariépy et al. 2003; Kienholz 1939; Soto-Alvear 2013; Spotts et al.

2009). Characteristic symptoms of fruit infection include the development of firm, circular lesions appearing on the fruit surface. Depending on the fruit cultivar, lesions are dark-brown to golden-tan in color with darker pigmented concentric rings appearing sometimes throughout the lesion or confined to the margin of the lesion (Brooks et al.1921; Spotts et al. 2009; Vico et al.

2016). During advanced stages of this disease, mycelial tufts may be visible at the center of the

2 lesion, and cream-colored spore masses oozing from erumpent acervuli may also be found throughout symptomatic tissue (Spotts et al. 2009).

In addition to causing postharvest fruit decay, some of these fungi are also capable of causing tree canker diseases. Anthracnose canker caused by N. malicorticis is widespread in orchards located in the maritime region of the PNW, west of the Cascade Mountain range. The pathogen causes cankers that may encircle the tree branch or trunk resulting in severe girdling and tree death. The fungus appears to be favored by the wet environment and mild temperatures typical of the area (Kienholz 1939; Zeller and Childs 1925; Spotts 2014). Unlike other

Neofabraea spp., N. malicorticis does not require wounds to penetrate bark tissue (Childs 1929;

Spotts 2014). While anthracnose canker expansion on bark tissue typically occurs within the first year following infection, the fungus may continue to actively sporulate for at least three years thereafter (Pscheidt and Ocamb 2016). Although P. vagabunda has been shown to induce cankers on apple and pear trees during artificial inoculation studies, the fungus only rarely causes stem cankers of pome fruit trees in nature (Henriquez et al. 2006). Instead, the fungus presumably behaves as a saprophyte of dead wood and plant matter in the orchard (Tan and

Burchill 1972). Phlyctema vagabunda has been reported to incite ‘coin canker’ on Fraxinus spp.

(Putnam and Adams 2005), a fruit spot of olive fruit [ Olea europaea (Rooney-Latham et al.

2013)] and branch canker of olive trees (Romero et al. 2016), demonstrating a wider host range than the other fungal organisms participating in the bull’s-eye rot disease complex. Neofabraea perennans and N. kienholzii overlap in geographic distribution and are commonly found in pome fruit orchards located in the drier regions of Washington State, especially central Washington

(Henriquez et al. 2004; Kienholz 1939; Spotts et al. 2009). While N. perennans is known to cause perennial canker of apple trees, the over-wintering and survival capabilities of N.

3 kienholzii has not yet been reported. Neofabraea perennans is incapable of directly penetrating bark tissue (Zeller and Childs 1925). Instead colonization by the fungus occurs via wounds found on pome fruit trees. Winter injury, pruning wounds and repeated feeding by woolly apple aphids

(Eriosoma lanigerum ) exacerbate the disease by providing annual points of re-entry for fungal invasion, allowing the disease to persist in a “perennial” nature (Childs 1929; Grove 1990; Zeller and Childs 1925). It appears that N. kienholzii and N. perennans share a similar geographic distribution within the PNW, however it is unknown whether N. kienholzii is capable of inducing cankers of pome fruit trees. Therefore, the primary objectives of this study were to determine whether N. kienholzii is able to incite canker development upon artificial inoculations of wounded apple twigs and if so, determine whether cankers developed in a manner similar to that described for perennial canker. Specifically, the progress of disease development in response to artificial inoculations with N. kienholzii and N. perennans were compared to identify the most favorable timing for canker initiation and canker expansion on wound-inoculated twigs. In addition, year-round pathogen survival in the woody tissue of apple twigs, and timing of acervuli development was monitored and compared between the two pathogen inoculum sources.

MATERIALS AND METHODS

Experimental design. Canker expansion studies were performed on ‘Fuji’ and ‘Red

Delicious’ apple trees planted in 2007 at the Washington State University Sunrise Research

Orchard (SR) near Palisades, WA (47°25'08"N119°54'52"W). The first inoculation was conducted on April 23, 2012 and continued every 8-9 weeks until February 18, 2015. A single annual trial consisted of inoculations beginning in April and continued until February of the following year. During each inoculation event, sixteen trees of each cultivar were

4 randomly selected. Three one-year-old twigs from each tree were wounded and inoculated with either a sterile agar plug (control), an agar plug containing germinated spores of N. kienholzii , or an agar plug containing germinated spores of N. perennans following a randomized complete block design.

Fruiting body evaluation studies were conducted on ‘Fuji’ apple trees planted in 1993 at the Washington State University Tree Fruit Research and Extension Center orchard (TFREC) in

Wenatchee, WA (47°26'26"N120°21'10"W) and on ‘Fuji’ trees planted in 1999 at the

Washington State University Columbia View Research orchard (CV) in East Wenatchee, WA

(47°33'44"N120°14'55"W). Similar studies using the cultivar ‘Red Delicious’ were conducted on trees growing at SR and CV research orchards. ‘Red Delicious’ trees growing at CV and SR orchards were planted in 2001 and 2007, respectively. Wound inoculations for evaluation of fruiting body development were performed on March 3, April 1, May 1, September 1 and

October 1 of 2014. Twelve trees (two replicates of six trees) of each cultivar at each orchard site were selected for this study, with the exception of ‘Red Delicious’ trees growing at SR for which twenty four trees (two replicates of twelve trees) were used. At the CV and TFREC orchard plots, two replicate twigs from each tree were wound inoculated with either N. perennans or N. kienholzii during each inoculation event following a randomized complete block design. Since trees at the SR location were much younger and smaller than those at the other study sites, only a single twig from each tree was wound inoculated with either pathogen during each inoculation event. Weed and insect management was conducted in each orchard plot according to commercial production criteria typical of central Washington (Bush et al. 2008).

5 Inoculation procedure. Isolates CLX5396 ( N. perennans ) and CLX4426 ( N. kienholzii ) were used for all inoculations conducted during this study. CLX5396 originated from decayed ‘Gala’ apple fruit collected from an orchard in East Wenatchee, WA while

CLX4426 originated from decayed ‘Fuji’ apple fruit from an orchard in Tonasket, WA.

Selection of these isolates was based on species identity using morphological (Verkley 1999;

Dugan et al. 1993; Kienholz 1939) and molecular (Gariépy et al. 2003; Soto-Alvear et al.

2013) diagnostics, and due to the fact that these isolates were obtained from orchards in which infections incited by both pathogens were commonly observed. Mycelia from each isolate were preserved on plugs of potato dextrose agar (Difco TM , Becton, Dickinson and

Company, Sparks, MD; PDA) suspended in a 15% glycerol solution stored at -80°C.

The fungal isolates were reactivated from -80°C stocks onto fresh PDA. After incubation at 20°C for 21 days, 10 ml of sterile water was added to each culture plate and a sterile inoculation loop was lightly brushed over the top of each culture to displace conidia into suspension. The conidial suspension was passed through two layers of sterile cheesecloth to filter out mycelial and agar fragments. The concentration of the conidial suspension was adjusted to 5

× 10 4 spores/ml using a hemocytometer and sterile water. Agar plugs were excised from sterile

PDA plates using a 5 mm diameter cork borer and transferred to a sterile container. Ten µl of conidial suspension was added to the top of each agar plug and subsequently incubated at 20°C without light for 48 hours to allow for conidial germination.

On the day of inoculation, twigs were surface disinfested with 70% ethanol and injured roughly mid-length along current year tissue using a 5 mm diameter cork borer. Injuries were superficial, exposing the inner bark tissue. Inoculum plugs were placed conidia-side down onto the wound so that spores were positioned in direct contact with woody tissue. Small squares of

6 sterile moistened cheesecloth (approximately 25 mm 2) were set on top of inoculum plugs and held in place by wrapping parafilm around the twig. The inoculum plug was removed from the wound site after 21 days.

For twigs used in the canker expansion study, the half-length measurement of resulting cankers was recorded every four weeks post inoculation for six months. The half-length measurement is the distance from the center of the wound inoculation site to the proximal end of the canker. Twigs exhibiting symptoms of dieback developed necrotic tissue extending from the inoculation site to the distal end of the twig, making it very difficult to discern the canker margin. Half-length canker measurements were preferred in order to avoid overestimations of canker length, especially when twig dieback symptoms were also observed. On the sixth month post inoculation, twigs were harvested from each tree and cankers were surface disinfested in a

1% sodium hypochlorite solution for 3 minutes followed by washing with deionized water three times for 1 minute each wash. Seven to ten tissue sub-samples were excised from the margin of twig cankers using a sterile scalpel and incubated at 20°C on PDA for 14 days to visually verify the presence of each Neofabraea fungus. Recovery of N. perennans and N. kienholzii was recorded in effort to assess the survival of Neofabraea spp. from inoculated tissues, and in partial fulfillment of Koch’s postulates.

Twigs from the fruiting body development study were destructively harvested at 2, 4, 6,

8, 10 and 12 months post-inoculation. Twigs were surface disinfested with 70% ethanol and cankers were visually inspected for the presence of acervuli using a dissecting microscope.

Acervuli were excised from cankers and transferred onto PDA, incubated at 20°C for 7-14 days and examined visually to assess acervuli viability and verify the presence of Neofabraea spp. in culture.

7 Statistical analysis. Canker half-length measurements recorded at six months post- inoculation were wound-corrected by subtracting 2.5 mm length from each measurement to account for tissue damage induced by the cork borer during the inoculation process.

Measurements were then log transformed and analyzed using a generalized mixed linear model in SAS PROC MIXED (version 9.4; SAS Institute, Cary, NC). The variables of interest were whether inoculation event and/or treatment (control , N. perennans , N. kienholzii ) had any influence over canker expansion, with tree representing the random effect of the model. The Tukey method was used to differentiate means considered statistically significant from one another ( P ≤ 0.05). When a significant interaction between the two explanatory variables in the model was identified, analyses were split to compare the effects of all three treatment levels at each individual inoculation event, while separate analyses evaluating individual treatment across all inoculation events were also conducted. Fisher’s exact test was used to compare differences in pathogen recovery between the two inoculum sources across each inoculation event using SAS PROC FREQ ( P ≤ 0.05). Data pertaining to pathogen recovery from twigs inoculated in February and April 2014 were unavailable and therefore not used in the conduct of these comparisons. For data obtained in determination of fruiting body development, the relationship between pathogen species, inoculation event and twig harvest period on acervuli development was elucidated using logistic regression in SAS

PROC LOGISTIC ( P ≤ 0.05). Firth’s penalized maximum likelihood approach was applied to counteract possible quasi-separation of datasets due to small sample size. Fisher’s exact test was used to compare differences in acervuli viability between inoculation events for each pathogen inoculum treatment by invoking SAS PROC FREQ ( P ≤ 0.05).

8 RESULTS

Canker description. A slight expansion of wounded tissue was observed for the non- inoculated control twigs particularly during winter inoculation periods when temperatures were at or below freezing. Generally, wounded bark tissue appeared white without any noticeable discoloration or necrosis (Fig. 1A). Canker symptoms for twigs inoculated with N. kienholzii (Fig. 1B) were similar in appearance to those induced by N. perennans (Fig. 1C).

Cankers primarily expanded along the length of the twig, and less across the width of the twig. Expansion was often asymmetrical, with greater canker expansion occurring from the center of the wound inoculation point to the distal end of the twig. Cankers were slightly sunken with a reddish-brown, sometimes orange-black discoloration and occasionally gave the appearance of concentric rings. Canker margins were often well-defined, except when twigs demonstrated dieback symptoms. Occasionally, when canker expansion was severe, the upper bark tissue would become paper-like and gradually peel off. Sometimes tissue would become fibrous and threadlike at the point of wounding. Woolly apple aphids were often found colonizing cankers of both N. perennans and N. kienholzii . Acervuli were visible erupting from wounded tissue at the point of twig inoculation. Acervuli appeared black in color, round/bulbous in texture, and were similar in appearance for both Neofabraea spp.

(Fig. 2 A and B).

Timing of canker induction and expansion. A significant interaction between treatment type and inoculation event was detected for both ‘Fuji’ and ‘Red Delicious’ apple cultivars used during the three annual trials of this study ( P < 0.0001). In the 2012-13 trial

(Fig. 3A), ‘Red Delicious’ twigs inoculated with N. perennans in October demonstrated

9 cankers that were significantly larger than those induced during any of the other inoculation events ( P ≤ 0.0027). The second largest cankers formed in response to inoculations conducted in April ( P ≤ 0.0003). When N. kienholzii was used as the inoculum source, cankers initiated in April were significantly larger in size than cankers formed at any other inoculation timing (P ≤ 0.0014) except October ( P = 0.7847). Overall, a noticeable reduction in canker size was observed across all inoculation timings for ‘Red Delicious’ twigs inoculated in 2013-14 (Fig. 3B) compared to the 2012-13 trial. For either pathogen, canker expansion was greater when twigs were inoculated in October compared to cankers initiated in response to inoculations conducted in June ( P ≤ 0.002) or August ( P < 0.0001). During

2014-15 (Fig. 3C), both N. perennans (P < 0.0001) and N. kienholzii (P < 0.0001) induced the largest cankers when twigs were inoculated in October compared to all other inoculation timings.

For ‘Fuji’ twigs inoculated with either N. perennans or N. kienholzii (P < 0.0001) in

2012-13 (Fig. 4A), the greatest canker expansion occurred in response to inoculations completed in October compared to all other inoculation timings. Similar to ‘Red Delicious’, disease severity noted for ‘Fuji’ twigs inoculated during the second year (2013-14) of this study was comparably lower than that obtained during the initial year (2012-2013) of the study (Fig. 4B). Canker expansion due to N. perennans was greatest in response to inoculations held in October compared to all other timings ( P < 0.0001). However, when N. kienholzii was used as the inoculum source, December ( P ≤ 0.0004) and October ( P ≤

0.0107) inoculations resulted in cankers that were significantly larger in size than those initiated in April, June or August. During 2014-15 (Fig. 4C), both N. perennans and N.

10 kienholzii produced the largest cankers when inoculations were conducted in October compared to all other inoculation timings ( P < 0.0001).

Both N. perennans and N. kienholzii were successfully recovered from inoculated twigs harvested at 6 months post inoculation regardless of the time period inoculations were conducted. While the proportion of twigs from which Neofabraea spp. was re-isolated varied across inoculation event, no observable trends in pathogen recovery could be discerned. In general, the pathogen was recovered from at least 50% of the total number of twigs inoculated during each inoculation event tested. Exceptions to this include ‘Red Delicious’ twigs inoculated with N. perennans in April of 2012, December of 2013, June of 2014 and

August of 2014 for which recovery was only at 31.3%, 25%, 25% and 37.5%, respectively.

For N. perennans inoculations conducted on ‘Fuji’, recovery of the pathogen was obtained from only 39.6%, 31.3% and 37.5% of twigs inoculated in December 2013, June 2014 and

February 2015, respectively. Recovery of N. kienholzii was below 50% of ‘Fuji’ twigs inoculated in February 2015 (22.9%). A significant difference in the relative recovery of the two pathogens was detected for inoculations conducted in December 2013 during which N. kienholzii was re-isolated at a significantly greater frequency than N. perennans (P = 0.0002).

Pathogen recovery did not appear to be related to canker expansion. This was evidenced in

2012 when both N. perennans and N. kienholzii were recovered from 100% of twigs inoculated in August, even though average canker size for twigs inoculated in August was very small (less than 3 mm).

Development of acervuli on inoculated apple twigs. Inoculation date was the only significant explanatory variable influencing Neofabraea fruiting body development on ‘Fuji’

11 twigs at TFREC (P = 0.0029; Fig. 5A) and CV ( P = 0.0038; Fig. 5B), and ‘Red Delicious’ twigs at SR ( P = 0.0241; Fig. 5C) orchard plots. No significant explanatory variables for acervuli development were identified for ‘Red Delicious’ twigs inoculated at CV orchard (Fig. 5D). At

TFREC, significantly fewer ‘Fuji’ twigs developed acervuli when inoculations were held in

March ( P < 0.0001), May ( P = 0.0004) and October ( P = 0.0071). At CV, significantly fewer

‘Fuji’ twigs developed acervuli when inoculations were held in March ( P < 0.0001), September

(P = 0.0004) and October ( P < 0.0001) compared to the other inoculation events. At SR, significantly fewer ‘Red Delicious’ twigs developed acervuli when inoculations were held in

March ( P < 0.0001), May ( P < 0.0001) and October ( P = 0.0299) compared to the other inoculation events. No significant difference in acervuli viability was detected between any of the inoculation events for either pathogen inoculum source at any of the orchard plots (Table 1).

However, since twigs were surface disinfested with ethanol prior to microscopic examination and incubation of acervuli on PDA, data collected during the acervuli viability tests are likely an underestimation of true viability experienced under natural conditions.

DISCUSSION

In this study, inoculations of artificially wounded ‘Red Delicious’ and ‘Fuji’ apple twigs with N. kienholzii was shown to induce a canker disease that was similar in behavior and appearance to perennial canker of apple trees caused by N. perennans . Cankers induced by either pathogen were more likely to arise from inoculations conducted in October compared to all other inoculation timings tested in this study. Expansion of cankers initiated in October was often rapid, causing maximum damage to host tissue. Inoculations conducted during the summer

(June, August) were less likely to yield twig cankers that extended in size beyond the wound

12 inoculation site. These results are consistent with the findings of Shear and Cooley (1933), Grove et al. (1992) and Henriquez et al. (2006) in which apple and pear trees inoculated with

Neofabraea spp. demonstrated a higher propensity for canker development in response to inoculations conducted during autumn. These previous studies (Grove et al. 1992; Henriquez et al. 2006) also reported winter as an important period for canker development; however twigs inoculated in December rarely developed obvious cankers in the current study. Rather, twigs demonstrating symptoms of dieback were often observed in response to December inoculations.

While perennial canker can also lead to dieback of infected twigs and branches, these symptoms observed in association with December inoculations may have developed as a synergistic response between wounding and winter injury. This interaction can help explain why wound expansion was observed for ‘Fuji’ twigs inoculated with the control treatment during December

2013, however does not completely explain discrepancies in canker expansion between the three treatment for twigs inoculated in December of 2014. In the current study, half-length canker size was used as an indicator of fungal activity in host tissue. This unit of measurement ignores fungal activity occurring on the distal end of infected twigs, and might explain the discrepancy between our findings and those of previous studies (Grove et al. 1992; Henriquez et al. 2006).

Sizeable cankers were also noted on twigs inoculated in mid-February and April, albeit not as large as those produced in response to October inoculations. Increased activity of these fungi in cankers of pome fruit trees during autumn appears to correspond well with previous reports of higher fruit infection rates observed near the end of harvest as opposed to other months during the fruit-growing season (Aguilar et al. 2017; Henriquez et al. 2008) and with observations made by Miller (1932) who demonstrated Neofabraea spp. are capable of growing on apples and artificial media at a temperature range between 0-20°C, but grow best as temperatures approach

13 20°C. Cankers initiated in spring might also relate to the condition of a weakened host predisposed to infection by winter stress, or in response to nutrient availability in woody tissues following a break from winter dormancy.

During periods favorable to canker development, twig cankers incited by N. kienholzii were on average comparatively smaller than those initiated by N. perennans . This suggests N. kienholzii may not be as aggressive a pathogen of bark tissue as N. perennans . This characteristic could be responsible for the generally lower frequency of incidence in which N. kienholzii has been reported as a causal agent of bull’s eye rot in the PNW relative to other species of

Neofabraea and related fungi (Spotts et al. 2009). However, inoculation studies using N. malicorticis and N. perennans in bark tissue of apple trees by Kienholz (1939) revealed considerable variation in canker development between strains of a single species. Given that pathogenicity is a multifaceted and dynamic process, and that only a single isolate of N. kienholzii was examined in the current study, potential still exists for N. kienholzii to emerge as an important causal agent of bull’s-eye rot and canker of pome fruit trees in the PNW. Since its original identification as a putative Neofabraea spp. among isolates collected from Nova Scotia and Portugal (de Jong et al. 2001), N. kienholzii has been reported in plant materials collected from Australia (Cunnington 2004), the PNW region of the United States (Henriquez et al. 2004;

Spotts et al 2009), Poland (Michalecka et a.l 2015), the Czech Republic (Pešicová et al. 2016) and the Netherlands (Wenneker et al. 2017). Whether these reports indicate current international spread of the pathogen, or merely reflect improvements in species identification through molecular methods is difficult to determine. Genetic analysis of N. kienholzii populations throughout pome fruit production regions could help elucidate the level of diversity existing

14 within this species, and could assist in the formulation of predictions as to the threat this fungus poses to apple production.

In addition to canker expansion, autumn and spring were equally favorable periods for asexual fruiting body development of N. kienholzii and N. perennans . While conidia production was not monitored over the course of this study, previous reports noted abundant acervuli production in cankers of Neofabraea spp. from October through February (Henriquez et al.

2006), with greatest sporulation occurring near the end of summer (Henriquez et al. 2008) and lasting until winter (Grove et al. 1992). Canker sporulation following periods of heavy rainfall and humidity encourages pathogen dispersal (Edney 1956). When this type of atmospheric activity overlaps with fruit development and maturity, fruit infection is highly likely. The fact that Neofabraea spp. can produce acervuli regardless of when twig inoculation occurred, and the fact that the pathogen could be recovered from inoculated twigs sixth months post inoculation indicates that these fungi are very resilient to different environmental conditions experienced in the orchard. Once established in an orchard, the overwintering potential and chance for inoculum spread seem high.

Current recommendations for control of perennial canker concentrate on improvements to the implementation of cultural orchard management practices. This involves canker removal through tree pruning, protecting trees so that wounding and injuries are kept to a minimum, control strategies that help mitigate aphid populations, and use of orchard sanitation practices that minimize the inoculum load of Neofabraea spp. in the orchard (Grove et al. 1992). In the

PNW, apple trees are actively pruned near the end of winter/beginning of spring for horticultural purposes. Since Neofabraea spp. are least active during periods of high temperature and low moisture (i.e. during the summer), from a biological stand-point it might also make sense to

15 scout and prune out branch cankers in the summer as well. This could help prevent additional spread of the fungus from cankers onto developing fruit while at the same time minimizing the potential for infection through newly created pruning wounds. Proper pruning techniques should be employed at all times so that damage to branch collars are avoided. This will stimulate pruning wounds to heal in a healthy manner, making the wound less vulnerable to further infection (Shigo 1984). Over the course of this study, woolly apple aphids were observed colonizing necrotic bark tissue of both perennial canker and cankers induced by N. kienholzii .

Aphids have been shown to induce feeding galls on tree bark tissue which rupture in response to freezing temperatures (Grove et al. 1992), providing points of entry that can be exploited by N. perennans . As aphids were also noted colonizing cankers induced by N. kienholzii , it is highly likely that activities of this insect can exacerbate the disease cycle of N. kienholzii . Further studies documenting the relationship between woolly apple aphids and N. kienholzii cankers are required to address these premises.

16 LITERATURE CITED

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Childs, L. 1929. The relation of woolly apple aphids to perennial canker infection with other notes on the disease. Oregon Agric. Exp. Sta., Sta. Bull. 243. 31 pp.

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de Jong, S. N., Levesque, C. A., Verkley, G. J. M., Abelin, E. C. A., Rahe, J. E., and Braun, P. G. 2001. Phylogenetic relationships among Neofabraea species causing tree cankers and bull’s eye rot of apples based on DNA sequencing of ITS nuclear rDNA, mitochondrial rDNA, and the β- tubulin gene. Mycol. Res. 105:658-669.

17 Dugan, F., Grove, G. G., and Rogers, J. D. 1993. Comparative studies of Cryptosporiopsis curvispora and C. perennans . I. Morphology and pathogenic behavior. Mycologia 85: 551-564.

Edney, K. L. 1956. The rotting of apples by Gloeosporium perennans Zeller & Childs. Ann. Appl. Biol. 44: 113-128.

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Grove, G. G. 1990. Anthracnose and perennial canker. Pages 36-38 in: Compendium of apple and pear diseases. A. L. Jones and H. S. Aldwinckle, eds. American Phytopathological Society, St. Paul, MN.

Grove, G. G., Dugan, F. M., and Boal, R. J. 1992. Perennial canker of apple: seasonal host susceptibility, and perennation of Cryptosporiopsis perennans in infected fruit in eastern Washington. Plant Dis. 76: 1109-1114.

Henriquez, J. L., Sugar, D., and Spotts, R. A. 2004. Etiology of bull’s eye rot of pear caused by Neofabraea spp. in Oregon, Washington, and California. Plant Dis. 88: 1134-1138.

Henriquez, J. L., Sugar, D., and Spotts, R. A. 2006. Induction of cankers on pear tree branches by Neofabraea alba and N. perennans , and fungicide effects on conidial production on cankers. Plant Dis. 90: 481-486.

Henriquez, J. L., Sugar, D., and Spotts, R. A. 2008. Effects of environmental factors and cultural practices on bull’s eye rot of pear. Plant Dis. 92:421-424.

18 Kienholz, J. R. 1939. Comparative study of apple anthracnose and perennial canker fungi. J. Agric. Res. 59: 635-665.

Michalecka, M., Bryk, H., Poniatowska, A., and Puławska. 2015. Identification of Neofabraea species causing bull’s eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR. Plant Pathol. 65: 643-654.

Miller, E. V. 1932. Some physiological studies of Gloeosporium perennans and Neofabraea malicorticis . J. Agric. Res. 45: 65-77.

Pešicová, K., Kolařík, M., Hortová, B., and Novotný, D. 2016. Diversity and identification of Neofabraea species causing bull’s eye rot in the Czech Republic. Eur. J. Plant Pathol. DOI: 10.1007/s10658-016-1036-1.

Pscheidt, J. W., and Ocamb, C. M. 2016. Apple ( Malus spp.) anthracnose (bull’s-eye rot). Pacific Northwest plant disease management handbook. Oregon State University. Retrieved from https://pnwhandbooks.org/plantdisease/host-disease/apple-malus-spp-anthracnose-bulls-eye-rot.

Putnam, M. L., and Adams, G. C. 2005. Phlyctema vagabunda causes coin canker of ash (Fraxinus spp.) in North America. Plant Dis. 87: 773.

Rooney-Latham, S., Gallegos, L. L., Vossen, P. M., and Gubler, W. D. 2013. First report of Neofabraea alba causing fruit spot on olive in North America. Plant Dis. 97: 1,384.

Romero, J., Raya, M. C., Roca, L. F., Moral, J., and Trapero, A. 2016. First report of Neofabraea vagabunda causing branch cankers on olives in Spain. Plant Dis. 100: 527.

Shear, E. V., and Cooley, J. S. 1933. Relation of growth cycle and nutrition to perennial apple canker infection (abstract). Phytopath. 23:33.

19 Shigo, A. 1984. Compartmentalization: a conceptual framework for understanding how trees grow and defend themselves. Annu. Rev. Phytopathol. 22:189-214.

Spotts, R. A., Seifert, K. A., Wallis, K. M., Sugar, D., Xiao, C. L., Serdani, M., and Henriquez, J. L. 2009. Description of Cryptosporiopsis kienholzii and species profiles of Neofabraea in major pome fruit-growing districts in the Pacific Northwest USA. Mycol. Res. 113:1301-1311.

Spotts, R. A., “Bull’s-eye rot.” In Compendium of apple and pear diseases and pests , second edition, edited by T. B. Sutton, H. S. Aldwinckle, A. M. Agnello, and J. F. Walgenbach, 78-79. MN: APS Press, 2014.

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Tan, A. M., and Burchill, R. T. 1972. The infection and perennation of the bitter rot fungus, Gloeosporium album , on apple leaves. Ann. Appl. Biol. 70: 199-206.

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20 Zeller, S. M. and Childs, L. 1925. Perennial canker of apple trees (a preliminary report). Oregon Agric. Exp. Sta., Sta. Bull. 217. 17 pp.

21 Table 1 . Comparisons between inoculation event and its effect on acervuli viability of Neofabraea spp. using Fisher’s exact test. Orchard plot, cultivar Inoculum Inoculation events Table compared* probability Tree Fruit Research Neofabraea April May 0.4835 Center, ‘Fuji’ perennans April September 0.2349 April October 0.2838 May September 0.3973 May October 0.4242 September October 0.3522 Neofabraea March April 0.2308 kienholzii March May 0.5000 March September 0.3750 March October 0.6000 April May 0.8462 April September 0.3576 April October 0.2176 May September 0.7500 May October 0.6000 September October 0.3818 Columbia View, ‘Fuji’ N. perennans March April 1.0000 March May 0.5455 March September 0.7143 March October 0.6000 April May 0.0779 April September 0.2941 April October 0.2000 May September 0.4396 May October 0.5035 September October 0.5357 N. kienholzii April May 1.0000 April September 0.0833 April October 1.0000 May September 0.1111 May October 1.0000 September October 0.3333 Columbia View, ‘Red N. perennans March April 0.2500 Delicious’ March May 0.5000 March September 0.6667 April May 0.3182 April September 0.0528 May September 0.2885 N. kienholzii March April 0.6667 March May 0.8333 March September 0.7500 March October 0.5000

22 Orchard plot, cultivar Inoculum Inoculation events Table compared* probability April May 0.4762 April September 0.5000 April October 0.6667 May September 0.4773 May October 0.3333 September October 0.3750 Sunrise, ‘Red N. perennans March April 0.7500 Delicious’ March May 0.6667 March September 0.6923 March October 1.0000 April May 0.5000 April September 0.3831 April October 0.1750 May September 0.4945 May October 0.1818 September October 0.0827 N. kienholzii March April 0.2857 March May 0.5000 March September 0.5714 March October 0.6000 April May 0.8571 April September 0.2424 April October 0.3000 May September 0.5714 May October 0.6000 September October 0.4762 * Comparisons were only made between inoculation events in which acervuli were produced. Acervuli were extracted from twigs and cultured onto potato dextrose agar. If Neofabraea spp. were found growing from acervuli on the agar medium, viability was confirmed. The proportion of twigs from each inoculation event that produced viable acervuli (out of total twigs producing acervuli) was pooled across harvest period within a given inoculation event prior to analysis.

Figure 1. Physiological response of ‘Fuji’ apple twigs to wounding and artificial inoculation during the month of October with a sterile potato dextrose agar (PDA) plug serving as the control treatment ( A), a PDA plug containing germinated spores of Neofabraea kienholzii (B), and a PDA plug containing germinated spores of Neofabraea perennans (C). Arrows indicating the wound inoculation site (WIS) and canker margin (CM) are shown. Twigs were harvested and photos were taken at six months post inoculation.

Figure 2. ‘Fuji’ apple twigs wounded and artificially inoculated with potato dextrose agar plugs containing germinated spores of Neofabraea kienholzii (A) and Neofabraea perennans (B) during the month of April at the Washington State University Tree Fruit Research and Extension Center orchard plot in Wenatchee, WA. Black, bulbous structures emanating from the wound inoculation site of the twig canker represent fruiting bodies developed by the pathogen (indicated with an arrow). Photos were taken at six ( A) and two (B) months post inoculation.

Figure 3. Effect of inoculation date on canker development induced by Neofabraea spp. in artificial inoculations of ‘Red Delicious’ apple twigs. Values depicted are average half-length measurement resulting from inoculations conducted at specific inoculation events during the 2012-2013 ( A), 2013-2014 ( B) and 2014-2015 ( C) annual trials of this study. Measurements reported were taken at six months post inoculation and averaged across 16 replicates. Separate statistical analyses were conducted comparing canker size induced by the two Neofabraea spp.

26 and the control treatment at each inoculation event (asterisk); comparing canker size due to N. perennans inoculation across time (uppercase letters); and comparing canker size induced by N. kienholzii across inoculation event (lowercase letters). Treatment means designated with the same letter of the same case, or same number of asterisks are not significantly different from one another according to the Tukey means separation method ( P ≤ 0.05). Error bars represent the standard error of the mean.

Figure 4. Effect of inoculation event on canker development induced by Neofabraea spp. in artificial inoculations of ‘Fuji’ apple twigs. Values depicted are average half-length canker measurement resulting from inoculations conducted at specific inoculation events during the 2012-2013 ( A), 2013-2014 ( B) and 2014-2015 ( C) annual trials of this study. Measurements reported were taken at six months post inoculation and averaged across 16 replicates. Statistical analyses comparing canker size induced by two Neofabraea inoculum sources and a control

28 treatment within each individual inoculation event (asterisk) were conducted. Similarly, separate analyses comparing canker size attributed to N. perennans across inoculation event (uppercase letters), and canker size due to N. kienholzii across inoculation event (lowercase letters) were also conducted. Treatments designated with the same letter of the same case, or with the same number of asterisks, indicate no significant difference in half-length canker size ( P ≤ 0.05). Error bars specify the standard error of the mean taken from 16 different twig replicates.

Figure 5. Effect of inoculation event on the average proportion of ‘Fuji’ ( A, B) and ‘Red Delicious’ ( C, D) apple twigs producing the asexual fruiting body of Neofabraea perennans and Neofabraea kienholzii following artificial wound inoculations conducted at orchard plots located at the Washington State University Tree Fruit Research and Extension Center ( A), Columbia View ( B, D) and Sunrise ( C) research orchards near Wenatchee, WA. Treatment means designated with the same letter are not significantly different ( P ≤ 0.05). Error bars indicate the standard error of the mean.

30 CHAPTER TWO

Timing of Apple Fruit Infection by Neofabraea perennans and Neofabraea kienholzii in

Relation to Bull’s-eye Rot Development in Stored Apple Fruit

C. G. Aguilar , Department of Plant Pathology, Washington State University, Pullman 99164;

M. Mazzola , United States Department of Agriculture-Agricultural Research Service (USDA-

ARS), Tree Fruit Research Laboratory, 1104 N. Western Ave., Wenatchee, WA 98801; and C.

L. Xiao , USDA-ARS, San Joaquin Valley Agricultural Sciences Center, 9611 S. Riverbend

Ave., Parlier, CA 93648

ABSTRACT

Bull’s-eye rot is a postharvest disease of pome fruit in the U.S. Pacific Northwest. The disease is caused by the fungi Neofabraea kienholzii, Neofabraea malicorticis, Neofabraea perennans and Neofabraea vagabunda . Fruit infection by these pathogens is initiated in the orchard during the fruit-growing season but remains latent at harvest. For fruit held in postharvest cold storage, bull’s-eye rot symptom development is slow to progress, requiring at least three months before symptoms are first visible. In order to determine the timing of pre- harvest fruit infection in relation to bull’s-eye rot development in cold storage, apple fruit of cultivars ‘Red Delicious’ and ‘Fuji’ were inoculated with a conidial suspension of N. perennans or N. kienholzii at different inoculation timings throughout the fruit-growing seasons of 2012-

2014. Fruit were harvested and stored at 0°C for up to 10 months during which time disease incidence was recorded periodically. Results from this study demonstrate that apple fruit

31 infection by either pathogen may occur at any point during the growing season. However, infections occurring over the eight week period immediately prior to harvest yield a higher incidence of bull’s-eye rot in stored fruit compared to infections initiated earlier in the growing season.

INTRODUCTION

Postharvest fruit rot diseases pose a major limitation to the goal of retaining high quality, marketable fruit held under prolonged postharvest storage. In the U.S., Washington State is the leading producer of apples, amassing a multibillion dollar industry. Such high revenues are attained in part, due to utilization of controlled atmosphere storage which extend the fruit storage period, retaining fruit quality and thus enabling the availability of this important agricultural commodity throughout the year (Thompson 1998). However, postharvest diseases of apples such as blue mold, gray mold and bull’s-eye rot caused by Penicillium spp., Botrytis cinerea and

Neofabraea spp. respectively, can cause reductions to fruit yields as high as 5-28%, annually

(Spotts et al. 2009; Xiao and Kim 2008). Economic losses resulting from postharvest diseases can be further exacerbated if the pathogen in question presents a quarantine concern for export markets (Xiao et al. 2011). Diseases such as bull’s-eye rot caused by Neofabraea spp.,

Sphaeropsis rot caused by Sphaeropsis pyriputrescens and speck rot caused by Phacidiopycnis washingtonensis are major production issues for pome fruit growers of the U.S. Pacific

Northwest [(PNW); Xiao and Kim 2008; Yigzaw and Bond 2014]. In addition to diminishing the appearance and quality of fruit grown for domestic consumption, the pathogens associated with these diseases have potential to jeopardize access to international markets for PNW fruit growers

(Warner 2014).

32 Successful control of postharvest diseases requires an understanding of the host infection process and knowledge of the environmental conditions under which infection may occur.

Bull’s-eye rot represents a postharvest disease that arises from infection originating in the orchard but remains quiescent or latent at harvest (Bompeix 1978). Managing the disease can be challenging due to the absence of symptoms at harvest and slow progression on fruit destined for cold storage.

In the PNW, Neofabraea kienholzii, Neofabraea malicorticis , Neofabraea perennans and

Neofabraea vagabunda are all known to cause bull’s-eye rot on fruit of Malus spp. and Pyrus spp., though the distribution of these fungi within this region is geographically fragmented

(Kienholz 1938; Spotts et al. 2009). With the exception of N. kienholzii, these fungi are known to overwinter in the orchard either on margins of sunken stem or branch cankers, or as saprophytes on dead wood and leaves (Edney 1956; Tan and Burchill 1972). Information pertaining to the overwintering status of N. kienholzii is as of yet unavailable due to its recent discovery and identification as a separate bull’s-eye rot-causing species (de Jong et al. 2001; Spotts et al. 2009).

Additional hosts of these canker inducing pathogens include native PNW crabapple ( Malus fusca ), quince ( Cydonia oblonga ), serviceberry ( Amelanchier spp.), mountain ash ( Sorbus acuparia ), and hawthorn (Crataegus spp.) (Creemers 2014; Kienholz 1939). These alternative hosts may serve as important sources of inoculum when growing adjacent to apple or pear orchards. Since native crabapple are believed to be the original host of apple anthracnose

(Kienholz 1939), exclusion of wild crabapple from established orchards as well as proper eradication of infected plant material from the orchard is important in minimizing inoculum buildup (Childs 1929).

33 In general, conidia released from acervuli of Neofabraea spp. on cankers or other overwintering sources become rain/irrigation-splash dispersed onto neighboring twigs and developing fruit during the apple-growing season (Gariépy et al. 2005; Sharples 1959; Wilkinson

1944). Germinating conidia infect fruit by hyphal penetration of epidermal tissue preferentially through open lenticels on the apple surface (Wilkinson 1944), however infections of the stem bowl or calyx region of fruit is also possible (Spotts et al. 2009). Though infection is asymptomatic at harvest, over time fruit held at cold storage begin to develop tan colored lesions with alternating concentric dark brown rings visible on the fruit skin. During advanced stages of bull’s-eye decay, white to cream-colored mycelium producing acervuli with cream-colored spore masses can be visible growing from these target-like lesions.

Current recommendations for managing bull’s-eye rot rely primarily on pruning cankers from infected trees, or applying pre- and/or postharvest fungicides to fruit (Creemers 2014;

Spotts 2014). It is speculated that Neofabraea spp. causing bull’s-eye rot are capable of sporulating from cankers and other overwintering sources throughout the entire apple fruit- growing season. Given the presence of appropriate environmental conditions, it is plausible fruit infection may occur at any time during the fruit maturation process (Grove et al. 1992).To assist in the development of effective fungicide spray schedules which minimize application periods, the current study was conducted to identify the timing under which these fungi infect fruit in the orchard in relation to bull’s-eye rot development of fruit kept in cold storage. The specific objectives of this study were to (i) determine the timing of apple fruit infection by N. perennans and N. kienholzii in the orchard, in relation to development of bull’s-eye rot during postharvest cold storage, and to (ii) compare the relative capacity of N. kienholzii and N. perennans to incite

34 disease on apple fruit at varying stages of fruit maturity as indicated by differences in disease incidence resulting from artificial fruit inoculations.

MATERIALS AND METHODS

Isolate selection. Fruit inoculations in the orchard were conducted with N. perennans isolate CLX5396 and N. kienholzii isolate CLX4426. Isolate CLX5396 was originally isolated from decayed ‘Gala’ fruit harvested from an orchard located in East Wenatchee,

Washington, while isolate CLX4426 originated from decayed ‘Fuji’ fruit grown in Tonasket,

WA. The isolates were obtained from locations where fruit infection by both species was prevalent. These isolates were previously identified to species using molecular and morphological diagnostic techniques (Dugan et al. 1993; Gariépy et al. 2003; Spotts et al.

2009). Isolate CLX4426 was one of ten isolates utilized to formally describe N. kienholzii as a separate species capable of inciting bull’s-eye rot (Spotts et al. 2009). Fungal isolates were maintained on potato dextrose agar (Difco TM , Becton, Dickinson and Company, Sparks, MD;

PDA) plugs in 15% glycerol and stored at -80°C for long term preservation.

Orchard location and Inoculation timing. Fruit inoculations were conducted in the field on ‘Fuji’ apple trees growing in a 0.49 hectare block and ‘Red Delicious’ apples growing in a 0.6 hectare block both planted in 2007 at the Washington State University

Sunrise Research Orchard northwest of Palisades, WA (47°25'08"N119°54'52"W).

Experiments were conducted for three consecutive growing seasons beginning in 2012. Fruit were inoculated on a monthly basis beginning in May at petal fall and continuing until

35 harvest, roughly on a five to six week interval (Table 1). Under-tree irrigation, pruning, thinning and pest control were conducted following regional commercial production standards (Bush et al. 2008). Pest control entailed the application of multiple insecticides and herbicides throughout the growing season. Fungicides were not applied to any trees used over the course of this experiment.

Inoculum preparation. Conidial suspensions of N. kienholzii and N. perennans were prepared from 3 week old PDA cultures grown in the dark at 20°C. Cultures were flooded with 10 ml of sterile distilled water and agitated using a sterile inoculation loop to dislodge conidia into the liquid. The resulting spore suspension was then filtered through two layers of sterile cheesecloth to remove mycelial and agar fragments and collected into a sterile

Erlenmeyer flask. A hemocytometer was used to determine spore concentration of the suspension and sterile distilled water was used to adjust the suspension to a concentration of

5 x 10 5 spores/ml. This spore concentration was selected based on similar fruit inoculation studies done with postharvest decay pathogens prevalent in the PNW (Kim et al. 2014;

Sikdar et al. 2014). The suspension was transferred into a plastic spray bottle and kept in a cooler filled with ice until use in inoculations conducted later the same day. Sterile distilled water applied to fruit using plastic spray bottles was used as the control inoculation treatment.

Inoculation procedure. Fruit inoculations in the orchard were conducted in the evenings approximately 3 hours prior to sunset in order to minimize spore exposure to direct

36 sunlight and extreme heat. Trees were selected and assigned to each treatment following a randomized complete block design with a total of three replicates per treatment and 40 fruit per replicate. Depending on fruit set, one to three trees were used to attain a sufficient number of fruit necessary to represent a single replicate. Fruit were sprayed to run-off with inoculum of either N. kienholzii , N. perennans or sterile distilled water as the control using a hand sprayer. Inoculated fruit were immediately covered with a moistened white plastic trash bag of varying sizes (15, 30 and 49 liters) depending on the length of the tree branch fruit were growing on. Bags were sealed using clothespins in order to maintain high humidity to facilitate the establishment of infection. A WatchDog data logger (Spectrum Technologies,

Inc., Aurora, IL) was placed into one of the bags during each inoculation date to record temperature and relative humidity experienced during each incubation period (Table 1).

Plastic bags were removed from the fruit approximately 15 hours post-inoculation, and fruit remained on the tree until harvest. Fruit were harvested at commercial maturity, and harvest was conducted in the morning on the dates indicated on Table 1. After harvest, fruit were transported to Washington State University Tree Fruit Research and Extension Center in

Wenatchee, WA for visual inspection. Fruit were manually sorted to remove any apples with obvious injuries or abnormalities. Fruit were then packed onto fiberboard fruit trays and placed inside cardboard boxes lined with polyethylene bags. Packed fruit were stored in regular atmosphere (RA) at 0°C for up to ten months (about four months longer than commercial RA standards).

Disease evaluations. Due to the differential fruit maturation timing common to the two cultivars used in this study, ‘Red Delicious’ apples were harvested one month prior to

37 ‘Fuji’ fruit. Thus visual disease evaluations were initiated after four months of cold storage for ‘Red Delicious’ and after three months for ‘Fuji’ apples. Any fruit exhibiting symptoms resembling bull’s-eye rot or with unidentifiable lesions were removed from their boxes, and information pertaining to treatment was recorded. Tissue was excised from the margin of lesions and placed onto PDA for isolation of potential causal fungi. Cultures were incubated at 20°C for a period of 14 days. Resulting colony morphology and growth characteristics of fungi emerging from fruit tissue in media were used to verify pathogen presence (Dugan et al. 1993; Verkley 1999). Disease evaluations continued throughout the cold storage season on a monthly basis for seven months (ten months total storage for ‘Red Delicious’ apples, and nine months total storage for ‘Fuji’).

Statistical Analysis. To determine the relationship between total bull’s-eye rot appearing during postharvest cold storage and the timing of apple fruit inoculation with either N. perennans or N. kienholzii in the orchard, the incidence of diseased fruit from each replicate of each treatment was arcsine square root transformed and subjected to a linear regression analysis using SAS PROC REG (version 9.4; SAS Institute, Cary, NC) with date of fruit harvest indicated by time ‘0’ and date of fruit inoculation prior to harvest designated with negative values. To assess whether differences in the relative capacity of these two fungi to incite disease exists, disease incidence for the three treatments was analyzed within a single month following a generalized linear model using SAS PROC GENMOD. The standard addition transformation was applied to all incidence values to account for an excess of zeros observed in the control treatment. Means separation was computed following the

Tukey method ( P ≤ 0.05).

38 RESULTS

In general, average relative humidity during the fruit inoculation incubation period ranged from 80-99% with the exception of inoculations conducted in June 2012 when average relative humidity was recorded at 61%. Typically, the highest temperature during the post-inoculation incubation period was recorded in July (approximately 35°C) with the exception of ‘Red Delicious’ fruit inoculations completed in 2014 when the highest recorded temperature during incubation was experienced in September (34.9°C), and ‘Fuji’ inoculations in 2014 when the highest incubation temperature occurred in June (35.5°).

Temperature trends were usually cooler in May, September and October, and warmest in July

(Table 1).

Symptomatic fruit were recovered during each disease rating, demonstrating that when held in cold storage at 0°C, bull’s-eye rot symptoms can manifest as early as three months into the postharvest season, and as late as ten months. Bull’s-eye rot lesions were commonly observed at fruit lenticels, however occasionally lesions originating at the calyx and stem bowl region of fruit were also recorded. It should also be noted that bull’s-eye rot was observed and the pathogen recovered from fruit treated with the water control representing multiple inoculation timings. Disease incidence for the control was limited, ranging from 0-2.5% with only one occurrence where fruit rot reached 5.0% (‘Fuji’ apples inoculated in June 2013). These infections may represent natural sources of inoculum present in the orchard. No attempts were made to identify which Neofabraea spp. induced these infections.

With the exception of ‘Red Delicious’ apples inoculated with N. kienholzii during May of

2013, postharvest development of bull’s-eye rot was confirmed from both apple cultivars representing each inoculation timing tested in this study. This demonstrates that regardless of

39 apple fruit physiology, fruit infection by Neofabraea spp. can be initiated throughout the entire fruit-growing season given favorable conditions for infection are met. In general, bull’s-eye rot incidence increased gradually when fruit were inoculated early in the growing season, declined in July, and then continued to increase for the remainder of the fruit-growing season. This could suggest inhibition of fungal growth when temperatures approach of exceed 30°C. Total fruit infection by either pathogen was consistently greatest when inoculations took place at the end of the growing season compared to early or mid-season infections. Excluding ‘Red Delicious’ apples inoculated with N. kienholzii in 2014, the relationship between total postharvest bull’s-eye rot resulting from N. perennans or N. kienholzii inoculations increased significantly ( P ≤ 0.04) in a linear manner as the timing of fruit inoculation approached harvest (Fig. 1).

Disease incidence for ‘Red Delicious’ apples inoculated with either Neofabraea spp. was significantly different from the control treatment at each inoculation timing tested during the

2012 fruit-growing season (Fig. 2A). When inoculations were held in September 2012, N. perennans induced significantly greater disease than N. kienholzii (P < 0.0001). However, no significant difference in disease incidence between the two pathogens was detected at any other inoculation timing tested during that year. In 2013, significantly more disease was attributed to

N. perennans compared to N. kienholzii or the control except for inoculations conducted in July, during which disease incidence was low and not significantly different between the three treatments (Fig. 2B). Additionally, no disease was observed from fruit inoculated with N. kienholzii during the May inoculation timing. For inoculations conducted during the 2014 fruit- growing season (Fig. 2C), disease incidence was significantly greater for N. perennans compared to N. kienholzii during the months of July, August and September 2014. In general, disease

40 incidence on the fruit inoculated with N. kienholzii in 2014 was very low, and only significantly different from the control treatment during the September inoculation timing.

When inoculations were conducted on ‘Fuji’ apples during the 2012 fruit-growing season

(Fig. 3A), disease incidence for N. perennans and N. kienholzii was significantly greater than the control during each inoculation timing except in July when disease levels were low for both pathogens. Additionally, significantly more fruit were infected when N. perennans was used as the inoculum source compared to N. kienholzii for inoculations conducted in September, while the opposite was true for inoculations completed in October. No detectable difference in disease incidence could be discerned between the two pathogens at any other inoculation timing during that year. In 2013 (Fig. 3B), N. perennans incited significantly more disease than N. kienholzii when inoculations were conducted in May, June and September, whereas inoculations held in

July and October resulted in significantly more N. kienholzii infections. No significant difference in disease incidence between the two pathogens was detected for fruit inoculated in August.

During the 2014 fruit-growing season (Fig. 3C), N. perennans caused more disease than N. kienholzii at each inoculation period except June when no significant difference in disease was detected between the two pathogens ( P = 0.3271), and except in October when N. kienholzii induced significantly more fruit infections. Due to low disease incidence by N. kienholzii , no significant difference between the control and N. kienholzii treatment could be detected during the May ( P = 0.0674) and July ( P = 0.2467) inoculations.

41 DISCUSSION

Results from these studies demonstrate that both ‘Red Delicious’ and ‘Fuji’ apple cultivars are susceptible to fruit infection by the bull’s-eye rot-causing pathogens throughout the growing season, but are most susceptible to infection during the final weeks prior to fruit harvest.

These results are consistent with the disease cycles of other major pome fruit postharvest pathogens resident to the Pacific Northwest such as Phacidiopycnis washingtonensis (Sikdar et al. 2014) and Sphaeropsis pyriputrescens (Kim et al. 2014). In artificial inoculations, infection by N. perennans and N. kienholzii increased gradually over the course of the growing season with the exception of a sharp decline occurring during the July inoculation time-point (for trials conducted in 2012 and 2013). This was followed by a sharp increase in disease incidence for fruit inoculated during the final two to three inoculation timings. Although there were some fluctuations between the infection capabilities of the two bull’s-eye rot pathogens, overall N. perennans and N. kienholzii were capable of inducing disease at comparable levels. This indicates that although only recently identified as a component of the pathogen complex inciting bull’s-eye rot of apple (de Jong et al. 2001), N. kienholzii appears to be well adapted to the conditions encountered in the major pome-fruit-growing regions of central Washington (Gariépy et al. 2005; Spotts et al. 2009). Additionally, while bull’s-eye rot symptoms were not visible at harvest, typical bull’s-eye lesions were observed as early as three months into the cold storage season and were initiated by as late as the tenth month of storage (data not shown).

Most fungal postharvest pathogens gain entry into their hosts either through wounds, direct penetration of intact tissue, or colonization of natural openings such as lenticels, stems and pedicels (Prusky and Lichter 2007). Neofabraea species causing bull’s-eye rot primarily enter their hosts via open lenticels appearing on the surface of fruit skin (Edney 1956). While lenticel

42 number is static throughout maturity, lenticels can enlarge due to lengthening of micro-cracks within the cuticle in response to fruit growth (Harker and Ferguson 1988). One explanation as to the propensity for bull’s-eye rot pathogens to cause greater disease near the end of a growing season could be due to changes in the availability of these natural openings with response to fruit maturity. Over time, mineral content and other environmental factors may influence the breakdown of lenticels (Turketti et al. 2012). Changes to the structural integrity of these lenticels throughout development may facilitate their accessibility by pathogen propagules. The importance of structural resistance to infection by lenticel-colonizing fungi was demonstrated by

Guan et al. (2015) who showed that apple ring rot caused by Botryosphaeria dothidea was more severe in cultivars exhibiting a higher number of natural openings and a thinner layer of cuticular wax on the fruit surface. As apple fruit approach maturity, spores of N. perennans and N. kienholzii are likely better able to exploit the vulnerabilities of a weakened cuticle and thusly can invade lenticels of their host to a much higher success rate.

Successful invasion of a host is influenced by the metabolic processes occurring in both host and pathogen at the time of infection. Processes occurring during the early development of fruitlets may vary drastically with those occurring near the completion of fruit maturation. For example, levels of total phenolic compounds isolated from apple fruit flesh and skin were found to decrease significantly with fruit maturity, and these compounds were found to be inhibitory to

Botryosphaeria ribis infection of immature apples (Hwang 1983). Similarly, increased production of the antifungal diene, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene in unripe avocado is inhibitory to colonization by the postharvest pathogen, Colletotrichum gloeosporioides (Prusky 1996; Prusky et al. 1983; Prusky and Lichter 2007; Wang et al. 2004).

However as fruit ripen, the diene becomes metabolized by avocado lipoxygenase (Karni et al.

43 1989; Prusky and Licher 2007) leaving fruit vulnerable to cell wall degrading enzymes produced by C. gloeosporioides , namely pectate lyase and polygalacturonase (Prusky and Licher 2007;

Wattad et al. 1994). Additionally, secretion of proteases by Nectria galligena was shown to behave as an elicitor for benzoic acid production in immature apple fruit, restricting colonization of host tissue by the pathogen. However, during ripening pH and sugar content within fruit increase, creating an environment favoring fungal degradation of benzoic acid (Brown and

Swinburne 1973). Protease secretion and variations to benzoic acid concentrations over the course of fruit ripening have been shown to play important roles in eliciting infections by quiescent pathogens such as Nectria galligena and Neofabraea species (Swinburn 1975). Still, the metabolic processes conferring resistance to pathogen invasion in apple fruit may be far more dynamic that initially perceived. In a study conducted by Janisiewicz and co-authors (2016), production of reactive oxygen species (ROS) in accession lines of wild Malus sieversii shown to be resistant to Penicillium expansum declined sharply immediately after fruit were wounded.

Counterintuitively, certain accession lines deemed highly susceptible to P. expansum infection produced high levels of ROS. These results indicate that the metabolic processes historically regarded as necessary for pathogen inhibition may not always behave in a predictable manner and may interact in way that are far more complicated than initially interpreted. Studies looking into the defense response of apples against invasion by Neofabraea species are lacking.

Unlike P. expansum which can produce blue mold symptoms relatively shortly after colonizing wounds on apple fruit, bull’s-eye rot-causing fungi undergo a period of inactivation otherwise known as quiescence shortly after coming into contact with host tissue. During quiescence, fungal growth and disease development is arrested, resuming only after certain physiological and/or biochemical cues in the host have been satisfied (Coats and Johnson 1997).

44 As was demonstrated in this study, the length of quiescence for bull’s-eye rot-causing species can vary tremendously depending upon how early during the growing season fungal spores have made contact with their host. Quiescence in the Neofabraea -apple pathosystem appears to be disrupted shortly after harvest, but the exact timing and cues causing this transition are unknown and should be the subject of future investigations. As climacteric fruit, apples continue to undergo respiration and transpiration after harvest. While in cold storage, these processes continue, albeit at a slower rate depending on storage conditions (Thompson 1998). As time progresses, fruit continue to senesce and ripen causing an accumulation in ethylene production and depletion of stored nutrients and water reserves (Coats and Johnson 1997). Transition out of quiescence for Neofabraea species is likely affected by the availability of nutrients during storage, but also in response to volatile production during ripening.

Fruit infection by N. perennans and N. kienholzii was observed at every inoculation timing tested in this study, suggesting that while temperature and other environmental factors may be important influences of infection, they are not necessarily determinants of infection. It seems likely that the fungi causing bull’s-eye rot are capable of withstanding a wide range of temperatures (Henriquez et al. 2008). Similarly, minimal infection during the July inoculation timing may indicate an aversion for higher temperatures, since average daytime temperatures experienced in central Washington often exceed 30°C during this time. Molecular and physiological processes affecting pathogen and host interactions during the initial phases of host colonization may be more important predictors for successful infection. However, it is important to mention that while temperature and other conditions may not affect the infection process directly, their effect on host and pathogen health can indirectly influence host susceptibility to infection. By enclosing fruit in moistened bags during the inoculation procedure, an optimum

45 microclimate was created on the fruit surface. The 15 hour incubation period during which fruit were exposed to these conditions helped facilitate the establishment of infections. Because

Neofabraea spp. are quiescent pathogens of pome fruit, N. perennans and N. kienholzii must be resilient to withstand a variety of environmental conditions before resuming the infection process after harvest. The intricacies of the environment and its influence over host-pathogen interactions should be explored further to better develop predictive models that can assess the risks associated with difference orchard practices on fruit infection and subsequent postharvest decay in storage.

Because Neofabraea spp. exhibit a higher success in initiating infection near the end of the apple fruit-growing season, application of a systemic fungicide during the final weeks leading up to harvest or after harvest should help minimize the incidence of this disease (Coats and Johnson 1997). By targeting this period, a reduction in frequency of fungicide applications can be attained. Additionally, management practices prior to the commencement of the growing season should be directed toward scouting and removal of tree cankers in the orchard as these are the primary source of pathogen inoculum. Further recommendations to help minimize pathogen inoculum in the field include regular pruning and skirting to increase air circulation within the tree canopy and inhibit optimum conditions for infection. Decaying organic material such as twigs, leaves and fruit should be removed from the orchard floor as these can also support the overwintering of pathogen propagules. Lastly, since spore dispersal of bull’s-eye rot pathogens is largely dependent on transmission via water splash, use of over-head sprinkler systems should be avoided (Henriquez et al. 2008).

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51 Table 1. Dates of fruit inoculation and harvest for bull’s-eye rot field trials conducted at Washington State University Sunrise Research Orchard, and environmental conditions recorded during each fruit inoculation event. Apple Inoculation Average Minimum Maximum Harvest date cultivar event relative temperature temperature humidity (°C) (°C) (%) x Red 17 May 2012 99.4 4.9 22.1 2 October Delicious 20 June 2012 61.3 17.9 28.3 2012 24 July 2012 94.9 16.8 34.9 20 September 92.9 8.6 22.9 2012 14 May 2013 82.6 9.4 30.8 25 September 13 June 2013 92.1 6.9 26.4 2013 18 July 2013 88.7 17.5 36.2 20 August 2013 82.7 13.3 32.4 9 September 2013 91.8 14.8 28.0 19 May 2014 84.3 10.1 30.4 17 September 18 June 2014 84.8 13.3 28.0 2014 24 July 2014 89.1 12.1 34.5 21 August 2014 96.6 15.6 27.2 9 September 2014 95.2 11.3 34.9 Fuji 17 May 2012 99.4 4.9 22.1 25 October 20 June 2012 61.3 17.9 28.3 2012 24 July 2012 94.9 16.8 34.9 20 September 92.9 8.6 22.9 2012 9 October 2012 93.9 3.2 24.4 14 May 2013 82.6 9.4 30.8 22 October 13 June 2013 92.1 6.9 26.4 2013 18 July 2013 88.7 17.5 36.2 22 August 2013 92.9 17.9 27.2 11 September 93.2 18.7 34.5 2013 3 October 2013 92.1 4.5 21.7 20 May 2014 90.2 11.7 34.1 15 October 19 June 2014 83.9 15.2 35.3 2014 24 July 2014 79.9 12.1 29.1 21 August 2014 96.4 15.2 26.0 9 September 2014 98.4 20.2 29.9 2 October 2014 93.2 5.3 17.5 X Relative humidity was averaged over the entire course of incubation lasting approximately 15 hours for each inoculation timing.

Figure 1. Scatterplot showing the linear relationship between timing of ‘Red Delicious’ ( A, B) and ‘Fuji’ ( C, D) apple fruit inoculated with either Neofabraea perennans (A, C) or Neofabraea kienholzii (B, D) in the orchard and total bull’s-eye rot development obtained by the end of the cold storage season. Harvest date is indicated by day ‘0’, and inoculation date is represented by negative integers. The values shown are the percent of diseased apples out of the total number of apples inoculated for each treatment, averaged across 3 replicates.

Figure 2. Average total incidence of bull’s-eye rot on ‘Red Delicious’ apples. Apple fruit were inoculated in the orchard with a spore suspension of Neofabraea perennans or Neofabraea kienholzii at various inoculation timings during the 2012 ( A), 2013 ( B) and 2014 (C) fruit- growing seasons. Fruit were harvested at commercial maturity, and kept under postharvest storage at 0°C for 10 months. Monthly disease ratings were conducted beginning on the fourth month of storage. Difference in total disease incidence for N. perennans and N. kienholzii at each

inoculation timing was compared. Means represented by bars within the same inoculation timing and designated with the same letter are not significantly different ( P ≤ 0.05).

Figure 3. Average total incidence of bull’s-eye rot on ‘Fuji’ apples. Apple fruit were inoculated in the orchard with a spore suspension of Neofabraea perennans or Neofabraea kienholzii at various inoculation timings during the 2012 ( A), 2013 ( B) and 2014 ( C) fruit-growing seasons. Fruit were harvested at commercial maturity, and kept under postharvest storage at 0°C for 9 months. Monthly disease ratings were conducted beginning on the third month of storage. Difference in total disease incidence for N. perennans and N. kienholzii at each inoculation

timing was compared. Means represented by bars within the same inoculation timing and designated with the same letter are not significantly different ( P ≤ 0.05).

CHAPTER THREE

Control of Bull’s-eye Rot Caused by Neofabraea perennans and Neofabraea kienholzii in

Stored Apple Using Pre- and Postharvest Fungicides

C. G. Aguilar , Department of Plant Pathology, Washington State University, Pullman 99164;

M. Mazzola , United States Department of Agriculture-Agricultural Research Service (USDA-

ARS), Tree Fruit Research Laboratory, 1104 N. Western Ave., Wenatchee, WA 98801; and C.

L. Xiao , USDA-ARS, San Joaquin Valley Agricultural Sciences Center, 9611 S. Riverbend

Ave., Parlier, CA 93648

ABSTRACT

Bull’s-eye rot is a major postharvest disease of apple caused by multiple species of fungi belonging to the Neofabraea and Phlyctema genera. Chemical control of these fungi is a crucial component of disease management for conventionally grown apples. The efficacy of several pre- harvest and postharvest applied fungicides were evaluated to identify chemistries that can control bull’s-eye rot incited by Neofabraea perennans and Neofabraea kienholzii on apples. In general, the pre-harvest fungicide thiophanate-methyl was found to be effective at reducing the incidence of disease caused by N. perennans and N. kienholzii . Occasionally, application of zinc also reduced disease on fruit inoculated with N. kienholzii , however disease control using this fungicide was often inconsistent and not as effective as thiophanate-methyl. Two postharvest fungicides, thiabendazole and pyrimethanil also provided disease control that was far superior to

other chemical compounds evaluated in this study. The efficacy of thiabendazole and pyrimethanil was unaffected by application method (fungicide dip compared to thermofog).

Overall, disease incidence was lower for fruit treated with the postharvest chemicals thiabendazole or pyrimethanil, compared to the pre-harvest fungicide thiophanate-methyl.

Despite providing satisfactory control of bull’s-eye rot, integration of these three chemicals into disease management programs should proceed judiciously with consideration of their impact on the development of fungicide resistance and their influence on fungal diversity of other economically important apple postharvest pathogens.

INTRODUCTION

The Pacific Northwest region of the United States is a major area for pome fruit production due to the temperate climate, rich volcanic soils and expansive irrigation network characteristic of the area (Schotzko and Granatstein 2005). For many decades Washington State has remained at the forefront of apple production, accounting for approximately 70% of the total annual U.S. crop (Agriculture Marketing Resource Center 2015). As a multi-billion dollar industry, Washington-grown apples constitute a major component of the agricultural economy for the state. About one third of the total apple crop produced in Washington is exported with major consumers including Mexico, Canada and India (Geisler 2013). Recently, efforts to expand existing trade agreements applicable to tree fruits between the U.S. and Chinese market were curtailed for an extended period due to the detection of quarantine pests. Identification of the postharvest pathogens causing bull’s-eye rot ( Neofabraea spp., Phlyctema sp.), Sphaeropsis rot ( Sphaeropsis spp.) and speck rot ( Phacidiopycnis washingtonensis ) in apple shipments from

Washington State resulted in a temporary suspension of apple shipments to China during the summer of 2012 (Wheat 2013). After two years of deliberations, access to this market was reinstated with the stipulation that Pacific Northwest growers and packers abide by strict phytosanitary guidelines and shipment inspections to prevent exportation of pest contaminated fruit (Heping 2015; Warner 2014).

Postharvest decay management can be a costly issue for the Washington State apple industry, especially when mitigating diseases of quarantine concern such as bull’s-eye rot.

Phlyctema vagabunda , Neofabraea kienholzii , Neofabraea malicorticis , and Neofabraea perennans are fungal pathogens each responsible for causing bull’s-eye rot of apple and pear.

Though geographically fragmented, these fungi are all present with varying frequency throughout the Pacific Northwest (Gariépy et al. 2005; Spotts et al. 2009). In central and eastern

Washington where a majority of apples are grown, N. perennans is the predominant causal species contributing to bull’s-eye rot outbreaks in the area (Spotts et al. 2009). While N. kienholzii is present throughout the Pacific Northwest with much less frequency than other

Neofabraea species, due to its recent identification as a component of the bull’s-eye rot complex, relatively little is known about this organism (de Jong et al. 2001).

Bull’s-eye rot is characterized by circular lesions appearing on the fruit surface with concentric or “bull’s-eye-like” rings originating at the fruit lenticels (Spotts et al. 2009).

Symptoms develop slowly in cold storage and decayed tissue remains relatively firm throughout the disease process. Although fruit infection is initiated in the orchard, symptoms are quiescent at harvest and generally do not become apparent until at least three months into the postharvest storage season. Year-round survival of Phlyctema and Neofabraea spp. in the orchard is due to the ability of these fungi to persist in cankers produced on pome fruit trees and/or survive on

organic matter in orchard soils (Henriquez et al. 2006). In the primary apple production region of central Washington State, these canker diseases are not as economically important to growers as fruit infections, however it is through this canker association that the fungi are able to disseminate inoculum throughout the growing season, in turn leading to fruit infections

(Kienholz 1939).

Management practices to control Phlyctema and Neofabraea spp. in the orchard include pruning cankers from infected trees to minimize the buildup of inoculum during the fruit- growing season and minimal use of over-tree irrigation systems since the pathogen is spread through water splash dissemination of conidia onto neighboring fruit (Creemers 2014). For conventionally grown fruit, fungicide application is another important component of bull’s-eye rot management. Fungicide application is typically performed either during the fruit-growing season or after harvest prior to cold storage. Despite the importance of bull’s-eye rot as a major postharvest disease, little has been published relating to effective control of the causal fungi through application of chemical fungicides registered for pome fruit production in the U.S.

(Grantina-Ievana 2015; Lopatecki and Burdon 1966; Spotts et al. 2009; Weber and Palm 2010).

Therefore, the primary objectives of this study were to: (i) evaluate the efficacy of various pre- harvest applied fungicides for controlling bull’s-eye rot fruit infections caused by N. perennans and N. kienholzii ; (ii) compare the efficacy of different postharvest fungicides when applied as a drench or thermofogging treatment for controlling bull’s-eye rot fruit infections caused by N. perennans and N. kienholzii ; (iii) and to determine whether fungicide efficacy is affected by the seasonality of fruit infection.

MATERIALS AND METHODS

Orchard study sites. This study was conducted during the 2013 and 2014 fruit-growing seasons on cv. ‘Fuji’ apple trees planted in 2007 on a 0.4 hectare plot at the Washington State

University Sunrise Research Orchard located southhwest of Pallisades, WA

(47°25'08"N119°54'52"W). An under-tree irrigation system was employed, and trees were pruned, thinned and maintained for weed and insect control as per commercial orchard standards for the Pacific Northwest region (Bush et al. 2008).

Inoculum preparation. Neofabraea perennans isolate CLX5396 and N. kienholzii isolate CLX4426 were selected for use in fruit inoculations. Isolate CLX5396 was originally collected in 2007 from decayed ‘Gala’ apple fruit grown in East Wenatchee, WA. Isolate

CLX4426 was initially cultured from decayed ‘Fuji’ apple fruit originating in Tonasket, WA in

2005. The speciation of these two isolates was determined using both morphological (Dugan et al. 1993; Spotts et al. 2009; Verkley 1999) and molecular diagnostic methods (Gariépy et al.

2003; Soto-Alvear 2013). Isolates were revived from 15% glycerol stocks stored at -80°C and cultured on potato dextrose agar (PDA; Difco TM , Becton, Dickinson and Company, Sparks, MD).

Cultures were incubated at 20°C in total darkness for 14 days, transferred to fresh PDA and incubated at 20°C without light for an additional 21-30 days. To prepare conidial suspensions of each isolate, 10mL of sterile distilled water was flushed over the fungal culture and a sterile inoculation loop was applied over the mycelium to detach conidiospores into the solution. In effort to separate conidia from agar debris and mycelial fragments, the suspension was passed through two layers of sterile cheesecloth and collected into a sterile beaker. Spore concentration

of the suspension was adjusted to 5 x 10 5 spores/ml using a hemocytometer. The suspension was transferred to plastic spray bottles and kept on ice for no longer than five hours.

Inoculation procedure. Due to the large volume of fruit used in this study and amount of time needed to properly inoculate fruit, pathogen inoculations were completed on two separate days. As such, fruit were split into two groups – those to be treated with a preharvest applied fungicide and those to be treated with a postharvest applied fungicide. In general, preharvest treated fruit were inoculated one day prior to postharvest treated fruit.

During the 2013 growing season, two inoculation events were used: a mid-season inoculation occurring 5 weeks before harvest (wbh), and a late season inoculation occurring 2 weeks prior to harvest. For the 2014 fruit-growing season, one additional inoculation event occurring 18 wbh was included to represent early season fruit infections. In 2013, mid-season fruit inoculations were conducted on September 18th/19th, and late season inoculations were done on October 8th/9th. For the 2014 season, the early season fruit inoculation event was completed on June 9th/10th, while mid-season and late season inoculations were conducted on

September 10th/11th and September 30th/October 1st, respectively.

Inoculations were conducted within three hours of sunset to prevent excess exposure of the spore suspension to sunlight and to avoid daytime high temperatures. Trees were selected according to a completely randomized design, with a final sample size of twenty fruit per tree (an excess of fruit were inoculated to account for incidental losses experienced throughout the growing season) and a total of four replicate trees for each treatment combination (pathogen by inoculation period by fungicide). Attached fruit were sprayed with a conidiospore suspension of

either N. perennans or N. kienholzii until run-off. After the spore suspension was applied, pre- moistened white plastic trash bags of different sizes (15, 30 and 49 liters depending on tree limb length) were used to cover fruit in order to shield spores from direct sunlight and to maintain a favorable microclimate for spore germination and hyphal penetration (Edney 1958). Information pertaining to temperature and relative humidity during the inoculation process was recorded by placing a WatchDog Data Logger (Spectrum Technologies, Inc., Aurora, IL) inside one of the plastic bags containing inoculated fruit. Bags were removed from trees after a fifteen-hour incubation period and fruit were left attached to trees for the remainder of the growing season.

Fungicide application. Zinc as Ziram 76DF at 3.59 g per liter water (United Phosphorus,

Inc, King of Prussia, PA), thiophanate-methyl as Topsin-M WSB at 0.61 g per liter water

(United Phosphorus Inc.), and a pyraclostrobin plus boscalid mixture as Pristine at 0.55 g per liter of water (BASF Corporation, Research Triangle Park, NC) were independently applied to runoff on fruit surfaces using a powered handgun sprayer at approximately 14 (zinc) and 2

(thiophanate-methyl, pyraclostrobin plus boscalid) days before harvest. During the 2013 trial, pre-harvest fungicide application dates were completed on October 10th for zinc and October

21st for both thiophanate-methyl and pyraclostrobin plus boscalid. In 2014, pre-harvest fungicides were applied on October 3rd for zinc and October 14th for both thiophanate-methyl and pyraclostrobin plus boscalid.

The fungicides fludioxonil as Scholar SC at 1.24 mL per liter water (Syngenta,

Greensboro, NC), thiabendazole as Mertect 340-F at 1.24 mL per liter water (Syngenta), pyrimethanil as Penbotec 400SC at 1.24 mL per liter water (Pace International, Seattle, WA),

and a difenoconazole plus fludioxonil mixture as Academy at 1.24 mL per liter water (Syngenta) were applied to selected fruit as a postharvest drench treatment by dipping the fruit in 40 liters of a fungicide solution for 30 seconds within four hours of harvest. Addtionally, the postharvest fungicides fludioxonil as ecoFOG 80 at 60 g per ton of apples (Pace, International), pyrimethanil as ecoFOG 160 at 40 g per ton of apples (Pace, International), thiabendazole as ecoFOG TBZ at

80 g per ton of apples (Pace, International) and difenoconazole as an experimental formulation at

40 g per ton of apples (Pace, International) were applied as a thermofog treatment by placing fruit in a plastic crate (L60 x W40 x H29 cm) and exposing to each designated chemical for four hours. After fogging, fruit were packed into apple boxes and held in cold storage as described for other treatments below. The difenoconazole plus fludioxonil drench and difenoconazole thermofog treatments were incorporated into this study for only a single year while all other chemical formulations were tested for two growing seasons. All pre-harvest and postharvest treated fruit were compared against control fruit that were pathogen-inoculated yet lacking fungicide treatment (herein referred to as the ‘untreated control’ or ‘control’).

Fruit harvest and storage. In 2013, postharvest fungicide treated fruit were harvested on

October 23rd while pre-harvest fungicide treated fruit were harvested on October 24th. In 2014 postharvest fungicide treated fruit were harvested on October 16th while pre-harvest fungicide treated fruit were harvested on October 17th.

Pre-harvest fungicide treated fruit were sorted immediately after harvest and any fruit with abnormalities or wounds were discarded so that a minimum sample size of twenty fruit per replicate was achieved. Fruit were then transferred to sterilized fiberboard apple trays and placed

inside cardboard apple boxes lined with perforated polyethylene bags. Fruit destined for postharvest treatment were sorted immediately after harvest and treated with postharvest fungicides following sorting. After treating fruit with the assigned drench or thermofog chemical, fruit were packed onto fiberboard trays in cardboard boxes as described above. All fruit were stored at 0°C under regular atmosphere for up to nine months.

Fruit evaluations. Fruit were visually inspected for symptoms of bull’s-eye rot beginning on the third month of cold storage and continuing monthly until the ninth month of storage. Any fruit with visble symptoms of postharvest decay were removed and tissue isolations were conducted from decayed fruit to identify the causal pathogen inducing these symptoms.

Briefly, symptomatic tissue was removed from the margin of lesions using a sterile scalpel and incubated on PDA at 20°C for approximately ten days. Typical morphological characteristics of

N. perennans and N. kienholzii colonies growing in culture were used to identify whether symptomatic fruit were due to bull’s-eye rot (Dugan et al. 1993; Spotts et al. 2009). Incidence of bull’s-eye rot was recorded for each treatment.

Data analysis. Disease incidence data for preharvest and postharvest treatments were analyzed according to a generalized linear model using SAS PROC GENMOD (version 9.4).

The standard addition transformation was utilized (a positive integer of 1 was added to each data-point) in order to account for data sets with an abundance of zero values. Analyses were conducted separately for each fungicide application type (pre-harvest, postharvest) within a single growing season (2013, 2014).The Tukey-Kramer method was utilized to separate

treatment means significantly different from one another ( P ≤ 0.05). Since N. perennans was the sole inoculum source used for the postharvest thermofog application, and only a single inoculation timing was evaluated, a separate analysis following the aforementioned statistical procedure was conducted comparing disease incidence from fruit treated with the thermofog fungicides alongside data from drench treated fruit previously inoculated with N. perennans at two weeks before harvest.

RESULTS

Pre-harvest fungicides. Incidence of bull’s-eye rot during the 2013 growing season was significantly influenced by time of inoculation (i.e. inoculation event; P < 0.0001) as well as a significant interaction between pathogen species and pre-harvest fungicide ( P = 0.0011; Table 1).

Overall, bull’s-eye rot incidence was significantly greater when inoculations were conducted at 2 wbh compared to 5 wbh ( P < 0.0001). Thiophanate-methyl provided superior control of the disease and fruit receiving this treatment had a significantly lower incidence of fruit decay relative to the untreated control, pyraclostrobin plus boscalid or zinc treatments ( P < 0.0001) regardless of which Neofabraea spp. was utilized as the inoculum source (Fig. 1A). Relative to the control, zinc application also reduced bull’s-eye rot caused by N. kienholzii (P = 0.0024), but was not effective at mitigating disease attributed to N. perennans (P = 1.0).

A significant three-way interaction between inoculation event, pathogen species and pre- harvest fungicide treatment ( P = 0.0269) was observed for incidence of bull’s-eye rot during the

2014 trial of this study (Table 1). There was no significant difference in the incidence of bull’s- eye rot among any of the fungicide treatments when either pathogen was inoculated at 18 wbh.

This trend held true for inoculations occurring at 5 wbh as well. However, when inoculations were conducted at 2 wbh, fruit treated with thiophanate-methyl exhibited the lowest incidence of bull’s-eye rot relative to the control ( P < 0.0001; Fig. 1B). Contrary to results obtained from the

2013 trial, zinc reduced fruit decay caused by N. perennans (P = 0.0445), but was ineffective against N. kienholzii (P = 0.9779). When disease levels for the control treatments were compared across inoculation events, there were no significant differences in disease induced by N. kienholzii when inoculations were conducted at 18 wbh compared to 5 ( P = 0.1881) or 2 wbh ( P

= 0.9467), but there was a significant increase in disease at 2 weeks compared to 5 wbh ( P =

0.0002). This suggests that early and late season infection due to N. kienholzii was comparable during this trial. Among control fruit inoculated with N. perennans, only inoculations conducted at 2 wbh demonstrated significantly greater disease incidence than inoculations conducted at 18

(P < 0.0001) or 5 wbh ( P = 0.0015).

Drench application of postharvest fungicides. A significant three-way interaction between inoculation event, pathogen species and postharvest fungicide ( P < 0.0001) was detected during the 2013 fruit-growing season trial (Table 2). Both pyrimethanil and thiabendazole significantly reduced bull’s-eye rot decay relative to the control for either

Neofabraea spp. when inoculations were conducted at 5 wbh ( P < 0.0001; Fig. 2A). Similarly, when N. kienholzii inoculations were conducted at 2 wbh, both pyrimethanil ( P < 0.0001) and thiabendazole ( P < 0.0001) treatment resulted in significantly fewer decayed fruit relative to the control. However, only thiabendazole effectively reduced bull’s-eye rot incidence for fruit inoculated with N. perennans at 2 wbh ( P = 0.0001). Disease incidence observed in the control treatment was higher when inoculations were conducted at 2 wbh compared to 5 wbh when N.

kienholzii inoculum was used ( P = 0.0002) but was not a significant factor when N. perennans inoculum was used ( P = 1.0).

Three signficant two-way interactions were detected in trials performed during 2014

(Table 2), specifically between inoculation event and pathogen ( P = 0.0012), inoculation event and postharvest fungicide treatment ( P = 0.0038) and pathogen by postharvest fungicide treatment ( P = 0.0337). Fruit inoculated with N. kienholzii exhibited significantly more disease when inoculations occurred at 2 wbh compared to 5 (P < 0.0001) or 18 wbh ( P = 0.0092), but no significant difference in disease was observed between inoculation events at 18 and 5 wbh ( P =

0.3944). When fruit were inoculated with N. perennans , a significant difference in disease incidence was observed between all three inoculation events ( P ≤ 0.0069) with disease incidence being highest when inoculations were conducted at 2 wbh ( P < 0.0001). Only pyrimethanil ( P ≤

0.0002) and thiabendazole ( P < 0.0001) significantly reduced bull’s-eye rot incidence relative to the control when inoculations were conducted at 5 and 2 wbh (Fig. 2B). For fruit inoculated at 18 wbh, only pyrimethanil significantly reduced disease relative to the control ( P = 0.0058). For either Neofabraea spp., pyrimethanil ( P < 0.0001) and thiabendazole ( P < 0.0001) effectively minimized bull’s-eye rot incidence relative to the control and all other postharvest fungicide treatments evaluated in this study ( P ≤ 0.0113).

Thermofogging application of postharvest fungicides. Fog applications of pyrimethanil ( P = 0.0083) and thiabendazole ( P < 0.0001) significantly reduced bull’s-eye rot incidence caused by N. perennans relative to the control for inoculations conducted at 2 wbh

(2013; Fig. 3A). When applied as a fog, pyrimethanil performed far better in controlling bull’s-

eye rot than when applied as a drench ( P = 0.0049). However, application method did not significantly ( P = 0.9979) alter the level of disease control attained in response to thiabendazole postharvest treatment.

During the 2014 trial (Fig. 3B), a significant reduction in bull’s-eye rot relative to the control was observed for fog treatments of difenoconazole ( P = 0.0328), fludioxonil ( P =

0.0043), pyrimethanil ( P < 0.0001) and thiabendazole ( P < 0.0001). When application method was compared for each postharvest fungicide, no significant difference in disease incidence was observed.

DISCUSSION

Postharvest bull’s-eye rot development was induced on fruit artificially inoculated with

Neofabraea spp. during three separate inoculation events representing early, mid and late season fruit infection. In general, disease incidence increased as inoculations were conducted closer to the fruit harvest period, possibly reflecting fluctuations in host susceptibility as a factor of apple fruit maturity (Xu and Robinson 2010). While the efficacy of the different fungicides tested in this study varied slightly depending on the influence of certain experimental variables and the year the study was conducted, in general, when disease pressure was high, thiophanate-methyl was the most effective pre-harvest fungicide for control of bull’s-eye rot. Mean disease incidence for fruit treated with this fungicide ranged from 7.5%-27.5%, compared to 14.9%-88.5% in the pre-harvest untreated controls. Additionally, thiabendazole and pyrimethanil were the most effective postharvest fungicides at minimizing the incidence of bull’s-eye rot. When applied as a drench, disease incidence ranged from 0%-28.0% and 1.0%-61.3% on fruit treated with

thiabendazole and pyrimethanil, respectively, compared to 17.5%-89.0% in the postharvest untreated controls. Fruit treated with pyraclostrobin plus boscalid, fludioxonil, and difenoconazole with or without fludioxonil often possessed the highest disease incidences, occassionally having levels of disease that were greater than the untreated control. Typically disease incidence following zinc application was as high as the control, but on certain rare occassions, bull’s-eye rot was significantly reduced by this pre-harvest fungicide (for example,

N. kienholzii inoculated fruit during the 2013 growing season and fruit inoculated with N. perennans at two wbh during the 2014 growing season). However, the lack of year to year or pathogen to pathogen consistency in these trials demonstrates that zinc may not be suitable for use in the control of bull’s-eye rot.

While thiophanate-methyl and thiabendazole provided the most effective control of bull’s-eye rot, both products belong to the methyl benzimidazole carbamate (MBC) group of fungicides. These chemistries target a single mode of action involved with interference of beta- tubulin assembly during mitosis. As such, these fungicides have been deemed high risk for development of resistance in fungal populations by the Fungicide Resistance Action Committee

(FRAC). In pome fruit production districts of northern Germany where bull’s-eye rot is common, multiple isolates of N. perennans and P. vagabunda were found to demonstrate varying degrees of resistance to thiophanate-methyl (Palm and Weber 2007; Weber and Palm 2010). Judicious application of these fungicides must proceed in the Pacific Northwest so as to ensure their long- term efficacy. To accomplish this, one must ensure that fungicides are applied according to manufacturer recommendations, that label rates are being adhered and that spray coverage is optimal. Furthermore, fungicides sharing the same mode of action should not be applied in the same growing season nor in consecutive growing seasons at a specific orchard site. Alternating

between fungicides targeting different modes of action can help reduce the risk of resistance development. In pome fruit packinghouses of the Pacific Northwest, repeated and excessive use of benzimidazole and anilino-pyrimidine fungicides favored selection of fungicide resistant strains within populations of Penicillium expansum (Caiazzo et al. 2014; Li and Xiao 2008) and

Botrytis cinerea (Zhao et al. 2010). While these fungi exhibit shorter life cycles and higher rates of reproduction and sporulation in comparison to Neofabraea spp., the potential for fungicide resistance in pathogen populations causing bull’s-eye rot exists. Fortunately, implementation of fungicide rotation programs in recent years has helped alleviate selection pressure on resistant strains of Penicillium and Botrytis and presumably could also be applied to minimize the risk of fungicide resistance development in Neofabraea species as well.

During the course of this study, the fungicides Luna Sensation (fluopyram plus trifloxystrobin; Bayer CropScience), Aprovia (benzovindiflupyr; Syngenta), and Inspire Super

(difenoconazole plus cyprodinil; Syngenta) were registered for use in pome fruit production.

Trifloxystrobin and fluopyram belong to the quinone outside inhibitor (QoI) and succinate dehydrogenase inhibitors (SDHI) group of fungicides, respectively. In the present study, the pre- mixture pyraclostrobin plus boscalid was shown to provide inadequate control of bull’s-eye rot.

Pyraclostrobin and boscalid also belong to the QoI and SDHI group of fungicides, respectively.

While it would be unwise to make predictions as to the efficacy of this newly registered fungicide, use of fluopyram plus trifloxystrobin and pyraclostrobin plus boscalid within a single growing season or in consecutive annual rotation should be avoided. Given that little information exists regarding bull’s-eye rot control with these newly registered fungicides, studies evaluating baseline sensitivites of Phlyctema and Neofabraea spp.to these recently registered products

should serve the apple industry well, and improve spray programs currently implemented by pome fruit growers of the Pacific Northwest.

Results from the present study indicate that a single application of thiophanate-methyl during the final weeks prior to harvest, or a single application of thiabendazole or pyrimethanil immediately following harvest, may be sufficient to mitigate early, mid and late season fruit infections. However, since Neofabraea spp. are considered quarantine pests in certain countries abroad, this type of spray program may not be sufficiently stringent for fruit intended for international export. Predictive models identifying key periods during the growing season when pathogen inoculum is accumulating in the orchard would be very beneficial to growers, and could help pin-point the most appropriate time for fungicide application. Additionally, integrated pest management strategies that incorporate fungicide spray programs are highly encouraged in order to minimize disease risk caused by bull’s-eye rot organisms. This entails pruning and destroying twig and branch cankers harboring Neofabraea spp. from fruit trees so as to minimize primary inoculum available in the orchard, practicing proper orchard sanitation techniques that minimize the amount of plant debris left decomposing on the orchard floor (Grove et al. 1992), and reducing the use of overhead sprinklers or evaporative cooling, especially during the final weeks prior to harvest, as this may create a favorable microclimate encouraging infection by

Neofabraea spp. (Henriquez et al. 2008).

Thermofog application of fungicides were shown to provide similar efficacy against bull’s-eye rot compared to drench applications of the same compound. Fog application of postharvest fungicides may be advantageous over conventional drench systems in that less chemical waste is accumulated. However, it is important to note that fungicide distribution using thermofog application can be affected by many factors including air circulation and temperature

within the cold storage room at the time of fungicide application (Delele et al. 2012), and in the past this has been shown to affect fungicide residue levels on fruit (Bertolin et al.1995). While many improvements have been made in recent years to help perfect chemical application via thermofogging, packers should be cognizant of potential issues that can be encountered during the application process should storage conditions in cold rooms not be precisely monitored.

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Table 1 . Influence of pre-harvest fungicide treatment, inoculation event, Neofabraea species and the interaction of these variables on bull’s-eye rot incidence of ‘Fuji’ apples cultivated during the 2013 and 2014 growing seasons. 2013 Trial Bull’s-eye rot incidence Source Chi-sq. Pr > Chi-Sq. Pre-harvest fungicide (F) 283.37 < 0.0001 Inoculation event (I) 71.53 < 0.0001 Neofabraea species (N) 2.55 0.1100 I*F 4.34 0.2269 N*F 16.10 0.0011 I*N 3.52 0.0605 I*N*F 2.70 0.4410

2014 Trial Bull’s-eye rot incidence Source Chi-sq. Pr > Chi-Sq. Pre-harvest fungicide (F) 67.74 < 0.0001 Inoculation event (I) 65.99 < 0.0001 Neofabraea species (N) 7.19 0.0073 I*F 21.78 0.0013 N*F 1.16 0.7622 I*N 5.40 0.0671 I*N*F 14.26 0.0269

Table 2 . Influence of postharvest fungicide treatment, inoculation event, Neofabraea species and the interaction of these variables on bull’s-eye rot incidence of ‘Fuji’ apples cultivated during the 2013 and 2014 fruit-growing seasons. 2013 Trial Bull’s-eye rot incidence Source Chi-sq. Pr > Chi-Sq. Postharvest fungicide (F) 414.45 < 0.0001 Inoculation event (I) 26.90 < 0.0001 Neofabraea species (N) 12.48 0.0004 I*F 2.08 0.5567 N*F 69.87 < 0.0001 I*N 0.01 0.9125 I*N*F 22.87 < 0.0001

2014 Trial Bull’s-eye rot incidence Source Chi-sq. Pr > Chi-Sq. Postharvest fungicide (F) 198.31 < 0.0001 Inoculation event (I) 50.79 < 0.0001 Neofabraea species (N) 36.16 < 0.0001 I*F 22.71 0.0038 N*F 10.43 0.0337 I*N 13.47 0.0012 I*N*F 7.34 0.5008

Figure 1. Influence of pre-harvest fungicide application on incidence of bull’s-eye rot detected on fruit inoculated with Neofabraea perennans and Neofabraea kienholzii at 5 and 2 weeks before harvest (wbh) during the 2013 ( A) and 2014 ( B) fruit-growing seasons. The Tukey- Kramer method was utilized to identify significant differences among fungicide treatments (P. + bos. represents the formulation pyraclostrobin plus boscalid; T. methyl represents the formulation thiophanate-methyl). Means designated with the same letter are not significantly different ( P < 0.05). Error bars represent the standard error of the mean.

Figure 2. Influence of postharvest fungicide application on incidence of bull’s-eye rot detected on fruit inoculated with Neofabraea perennans and Neofabraea kienholzii at 5 and 2 weeks before harvest (wbh) during the 2013 ( A) and 2014 ( B) fruit-growing seasons. The Tukey- Kramer method was utilized to identify significant differences among fungicide treatments (Flu. represents the formulation fludioxonil; Pyri. represents the formulation pyrimithenail; Tbz. represents the formulation thiabendazole; Dif. + flu. represents the formulation difenoconazole plus fludioxonil). Means designated with the same letter are not significantly different ( P < 0.05). Error bars represent the standard error of the mean.

Figure 3. Influence of postharvest fungicide drench and thermofog treatments on average bull’s- eye rot incidence due to N. perennans inoculations at 2 wbh during the 2013 ( A) and 2014 ( B) fruit-growing seasons. The Tukey-Kramer method was used to separate treatment means considered statistically significant (Flu. represents the formulation fludioxonil; Pyri. represents the formulation pyrimethanil; Tbz. represents the formulation thiabendazole). Bars with the same letters are not significantly different ( P ≤ 0.05).