DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII

By

Cory Alan Outwater

A THESIS

Submitted to Michigan State University in partial fulfillment of the requirements for the degree of

Plant Pathology – Master of Science

2014

ABSTRACT

DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII

By

Cory Alan Outwater

Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii , is a major disease of tart cherry (Prunus cerasus ) trees, which uncontrolled leads to early defoliation. Pristine, a commonly-utilized fungicide for CLS management in Michigan, is a premix of boscalid, a succinate dehydrogenase inhibitor, and pyraclostrobin, a quinone outside inhibitor. Reduced efficacy of Pristine for CLS control observed in field trials and commercial orchards highlighted the importance of resistance monitoring. A total of

1,288 isolates from commercial orchards and 111 isolates from non-treated trees were collected in 2010 and 2011 and assayed on boscalid-amended media at concentrations of

-1 0, 0.1, 0.5, 1, 2.5, 5, 10, and 25 µg ml . The minimum inhibitory concentration (MIC) of boscalid was determined after incubation at 23 ºC for 14 days . Isolates from non-

-1 treated trees had MIC values ranging from 0.1 to 0.5 µg ml while isolates from

-1 commercial orchards ranged from 0.1 to > 25 µg ml . Isolates with MIC values of > 25

-1 µg ml were considered resistant and comprised 22% and 35% of isolates in 2010 and

2011 respectively. Sequencing of the SDHB gene of resistant isolates led to the detection of the amino acid mutation H272R known to confer boscalid resistance. The occurrence of the H272R mutation in Michigan populations of B. jaapii is correlated with the reduction in sensitivity to boscalid observed in commercial orchards.

To my grandpa Norman Schaub

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ACKNOWLEDGEMENTS

I would like to thank Dr. George Sundin for giving me the opportunity to work in his laboratory and for his guidance through the process of obaining my master’s degree. Thank you Janette Jacobs for working with me during my undergraduate years and teaching me a wealth of techniques and skills that have proven extremely valuable through the years. Thank you to

Dr. Tyre Proffer for guiding me through the world of fungi and for teaching me all the ins and outs of what has became one of my favorite fungal species, Blumeriella jaapii . Thank you to Kim Lesniak for inspiring me to go after a degree in plant pathology, for being there to mentor me through my graduate years, and finally for being a great friend and and source of support through this process. I would also like to thank Gayle McGhee, Gail

Ehret and the entire Sundin lab for being an amazing group of people and for making this experience one that I will cherish for many years to come.

I would like to thank the Michigan cherry growers and my funding sources including the Michigan Cherry Committee for allowing me to work on a very important disease affecting the Michigan cherry industry.

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TABLE OF CONTENTS

LIST OF TABLES………………………………………….....…vii

LIST OF FIGURES……………………………………………..viii

LITERATURE REVIEW…………………………………………1 Introduction…………………………………………………………...1 Plant Pathogens Affecting the Michigan Tart Cherry Industry………5 Cherry Leaf Spot ( Blumeriella jaapii )………………………………..7 Development and Use of Plant Protection Materials in Fruit Production…………………………………………………………...16 The Development and Registration of Pristine……………………...20 Fungicides in the Age of Resistance………………………………...24 LITERATURE CITED……………………………………………...33

CHAPTER 1: DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII ………………………………………...45 Introduction………………………………………………………….45 Materials and Methods………………………………………………49 Fungicide efficacy trials……………………………………...49 Statistical analysis of field data………………………………50 B. jaapii isolate collection……………………………………50 In vitro sensitivity of B. jaapii to boscalid…………………...52 Amplification and identification of the succinate dehydrogenase subunit b gene ( SDHB ) of B. jaapii …………………………..53 Results……………………………………………………………….55 Efficacy of Pristine for control of cherry leaf spot in fungicide field trials at the NWMHRS 1999–2013……………………..55 Grouping of B. jaapii isolates into boscalid sensitivity groups………………………………………………………...57 Sensitivity of B. jaapii isolates to boscalid (2010)…………...59 Sensitivity of B. jaapii isolates to boscalid (2011)…………...62

v Amplification of the SDHB gene and molecular detection of the H272R mutation………………………………………………65 Discussion…………………………………………………………...69 LITERATURE CITED……………………………………………...76

APPENDIX………………………………………………………85 LITERATURE CITED…………………………………………….108

vi

LIST OF TABLES

Table 1. Codon and amino acid residue in the third cysteine-rich cluster of the SDHB gene of 33 B. jaapii isolates grouped by boscalid sensitivity groups. Amino acid residues were changed from a histidine to arginine for all resistant isolates sequenced………….68

vii

LIST OF FIGURES

Figure 1. Efficacy trial results of Pristine treatments for control of CLS. Pristine was evaluated as a full season treatment from 2001 to 2003 and 2009 to 2011. Pristine was evaluated following two applications of chlorothalonil at petal fall and shuck split in 2005 and 2012. Trials were conducted in an experimental orchard of Prunus cerasus cv . Montmorency located at the Northwest Michigan Horticulture Research Center near Suttons Bay, Michigan. CLS incidence and defoliation was assessed in late August or early September of each year. Defoliation data for 2005 was not determined due to low disease pressure. Leaf spot infection and defoliation is scaled as the disease incidence or defoliation divided by the disease incidence or defoliation on unsprayed controls. Scaled infection is represented by black bars and scaled defoliation is represented by hash marked bars………………………………………………………………………………56

Figure 2. Isolates plated on boscalid amended media at concentrations of 0.1, 0.5, 1, -1 2.5, 5, 10, and 25 µg ml . Each colony on plates within a row represents a separate B. jaapii isolate. There are eight different isolates in the sensitive group, ten isolates in the reduced sensitivity group and ten isolates in the resistant group. Minimum inhibitory concentrations were determined as the lowest concentration that completely inhibits mycelial growth following a 14 day incubation period. -1 a. Minimum inhibitory concentration (0.1 µg ml ) of an isolate classified as sensitive. -1 b. Minimum inhibitory concentration (5 µg ml ) of an isolate classified as reduced sensitive. -1 c. Minimum inhibitory concentration (>25 µg ml ) of an isolate classified as resistant...... 58

Figure 3. Boscalid sensitivity distribution of 2010 non-treated and commercial B. jaapii isolates. Baseline isolates are represented by white bars and commercial isolates are represented by black bars………………………………………………………………60

Figure 4. Distribution of 2010 Orchard or site sensitivity groups. Orchards or sites classified as sensitive had average Minimum Inhibitory Concentrations between 0.1 and -1 0.5 µg ml and are identified by white bars. Orchards or sites classified as reduced -1 sensitivity had average Minimum Inhibitory Concentrations between 1 and 10 µg ml and are identified by hatched bars. Orchards or sites classified as resistant had average

viii -1 Minimum Inhibitory Concentrations between 25 and > 25 µg ml and are identified by black bars………………………………………………………………………………...61

Figure 5. Boscalid sensitivity distribution of 2011 non-treated and commercial B. jaapii isolates. Non-treated isolates are represented by white bars and commercial isolates are represented by black bars………………………………………………………………63

Figure 6. Distribution of 2011 Orchard or site sensitivity groups. Orchards or sites classified as sensitive had average Minimum Inhibitory Concentrations between 0.1 and -1 0.5 µg ml and are identified by white bars. Orchards or sites classified as reduced -1 sensitivity had average Minimum Inhibitory Concentrations between 1 and 10 µg ml and are identified by hatched bars. Orchards or sites classified as resistant had average -1 Minimum Inhibitory Concentrations between 25 and > 25 µg ml and are identified by black bars………………………………………………………………………………...64

Figure 7. PCR amplification of the SDHB gene from 7 B. jaapii isolates utilizing the primer pairs SDHB-FL and SDHB-R, SDHB-FS and SDHB-R, and SDHBFEL and SDHB-R. B. jaapii baseline isolates 10PEBJ-1, 3, 4, 9, 11 and B. jaapii boscalid resistant isolates 10BWPB-21, 22 were used for each primer pair. Lanes 1 and 17, DNA ladder; lanes 2, 10 and 19, water control; lanes 3-9, amplification with primer pair SDHB-FL and SDHB-R; lanes 11-16 and 18, amplification with primer pair SDHB-FS and SDHB-R; lanes 20-26, amplification with primer pair SDHBFEL and SDHB-R………………………………………………………………………………….66

Figure 8. Sequences obtained from the second and third cysteine-rich clusters of the SDHB gene of B. jaapii . Both cysteine-rich clusters (shaded areas) were amplified from sensitive, reduced sensitive and resistant isolates. Amino acids highlighted in red have been previously associated with boscalid resistance. No amino acid mutations were detected from the second cysteine-rich cluster from the three sensitivity groups. An amino acid mutation from histidine to arginine was detected for resistant isolates in the third cysteine rich cluster………………………………………………………………...67

Figure A1. Fluopyram sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites previously classified as boscalid sensitive had an Minimum -1 Inhibitory Concentration of 0.5 µg ml and are identified by white bars. The orchard previously classified as boscalid reduced sensitivity had an average Minimum Inhibitory

ix -1 Concentration of 10 µg ml and are identified by hatched bars. The orchard previously -1 classified as resistant had an average Minimum Inhibitory Concentration of > 25 µg ml and are identified by black bars………………………………………………………….88

Figure A2. Boscalid sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites classified as sensitive had an average Minimum Inhibitory -1 Concentration of 0.1 µg ml and are identified by white bars. The orchard classified as -1 reduced sensitivity had an average Minimum Inhibitory Concentration of 5 µg ml and are identified by hatched bars. The orchard classified as resistant had an average -1 Minimum Inhibitory Concentration of > 25 µg ml and are identified by black bars….89

Figure A3. Fluxapyroxad sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites previously classified as boscalid sensitive had an -1 average Minimum Inhibitory Concentration of 0.1 µg ml and are identified by white bars. The orchard previously classified as boscalid reduced sensitivity had an average -1 Minimum Inhibitory Concentration of 2.5 µg ml and are identified by hatched bars. The orchard previously classified as boscalid resistant had an average Minimum Inhibitory -1 Concentration of 25 µg ml and are identified by black bars…………………………...91

Figure A4. Boscalid sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites classified as sensitive had an average Minimum Inhibitory -1 Concentration of 0.1 µg ml and are identified by white bars. The orchard classified as -1 reduced sensitivity had an average Minimum Inhibitory Concentration of 5 µg ml and are identified by hatched bars. The orchard classified as resistant had an average -1 Minimum Inhibitory Concentration of > 25 µg ml and are identified by black bars……………………………………………………………………………………….92

Figure A5. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars had the -1 highest percentage of isolates in the 2.5 µg ml group. The second sample identified by -1 the hatched bars had the highest percentage of isolates in the 25 µg ml group. The third sample identified by the black bars had the highest percentage of isolates in the > 25 µg -1 -1 ml group. The percent of isolates in the > 25 µg ml MIC group had the most dramatic increase over the sampling period………………………………………………………..95

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Figure A6. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars had the -1 highest percentage of isolates in the 5 µg ml MIC group. The second sample identified -1 by the hatched bars had the highest percentage of isolates in the 2.5 µg ml MIC group. The third sample identified by the black bars had the highest percentage of isolates in the -1 25 and > 25 µg ml MIC groups. During the sampling period the highest percentage of -1 -1 isolates shifted from the 5 µg ml groups to the 25 and > 25 µg ml MIC group……...97

Figure A7. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars had the -1 highest percentage of isolates in the 2.5 µg ml MIC group. The second sample -1 identified by the hatched bars had the highest percentage of isolates in the 2.5 µg ml MIC group. The third sample identified by the black bars had the highest percentage of -1 isolates in the > 25 µg ml MIC group. During the sampling period the highest -1 -1 percentage of isolates shifted from the 2.5 µg ml group to the > 25 µg ml MIC group……………………………………………………………………………………99

Figure A8. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars had the -1 highest percentage of isolates in the 2.5 µg ml MIC group. The second sample -1 identified by the hatched bars had the highest percentage of isolates in the 5 µg ml MIC group. The third sample identified by the black bars had the highest percentage of isolates -1 in the 2.5 µg ml MIC group. During the sampling period the highest percentage of -1 -1 isolates remained in the 2.5 µg ml to 5 µg ml MIC groups. By the third sample 0% of -1 isolates were in the > 25 µg ml MIC group…………………………………………..101

Figure A9. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from

xi each sampling time were plotted. The first sample identified by the white bars had the -1 highest percentage of isolates in the 2.5 µg ml MIC group. The second sample -1 identified by the hatched bars had the highest percentage of isolates in the 2.5 µg ml MIC group. The third sample identified by the black bars had the highest percentage of -1 isolates in the 5 µg ml MIC group. During the sampling period the highest percentage -1 -1 of isolates remained in the 2.5 µg ml to 5 µg ml MIC groups. Throughout the -1 sampling period a small percentage of isolates remained in the > 25 µg ml MIC group……………………………………………………………………………………103

Figure A10. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars had the -1 highest percentage of isolates in the 2.5 µg ml MIC group. The second sample -1 identified by the hatched bars had the highest percentage of isolates in the 5 µg ml MIC group. The third sample identified by the black bars had the highest percentage of isolates -1 in the 5 µg ml MIC group. During the sampling period the highest percentage of -1 -1 isolates remained in the 2.5 µg ml to 5 µg ml MIC groups. In the second and third -1 sampling slightly increased in the > 25 µg ml MIC group…………………………105

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LITERATURE REVIEW

INTRODUCTION

Cherries have been cultured since ancient times when they were a delicacy for the great Roman emperors, Greeks and the Chinese. Cherries were first brought to the

Americas in the 1600s as pits carried over on the boats of early settlers (28). At this time they were not commercially cultivated but were inadvertently introduced to surrounding areas by those early settlers. It was the French that first brought over and cultivated cherries (28). Many of the French settlers had grown cherries back in France and subsequently brought them to the New World. Many of those French settlers established cherry trees in their gardens when they settled along the St. Lawrence River and established major cities like Detroit (28).

Cherries were not always a part of Northwest Michigan. In fact it was long thought that the climate and conditions that existed in the region were not conducive to the production of a variety of fruits including sweet and tart cherries. It wasn’t until 1852 when a Presbyterian minister, Peter Dougherty, decided to give it a try and planted a small orchard of cherries on the Old Mission peninsula near Traverse City (28). To the surprise of the natives of the area the trees not only survived but thrived. Dougherty’s small orchard produced a bumper crop of cherries and eventually piqued the interest of residents in the area. It turns out that this region of Michigan is ideal for cherry production. The waters of Lake Michigan that are in close proximity to this Peninsula provide insulation from the harsh Northern Michigan winters and a cooling breeze during the hot summers. The sandy soils and rolling hills of the regions surrounding the lake

xiii which make traditional agriculture challenging turned out to be a perfect fit for cherry production. The sandy soil provides excellent drainage while the rolling topography provides good protection from potentially damaging spring frost. Farmers in the area took note of how successful Dougherty’s orchard was and many decided to establish orchards of their own. The first commercial orchard was established in 1893 and was followed by a rapid increase in the development of the Northern Michigan cherry industry (28). As the industry became established, many support industries such as canneries, packers, and other fruit related enterprises followed. That very first orchard was the spark that ignited the prosperous cherry industry that exists today in Northern

Michigan.

Today cherries are grown all along the shoreline of lower Michigan. Michigan is the number one producer of tart cherries in the United States (88). In 2011, the United

States produced 105.1 million kilograms of tart cherries of which Michigan produced

71.4 million kilograms (95). This crop had a farm gate value of approximately 47 million dollars (95). Tart cherries are used in products such as pie filling, juice, dried cherries, and many others. Washington, California, and Oregon are the top producers of sweet cherries while Michigan is usually third or fourth. In 2011, the United States produced

311.3 million kilograms of sweet cherries of which Michigan produced 16.3 million kilograms (95). Michigan sweet cherries are sold in the fresh market but are mainly grown for processing. Michigan sweet cherries are used in products such as maraschino cherries, canned cherries, ice cream toppings and many others.

When the tart cherry industry was in the process of becoming established in

Michigan, production of this little red fruit was a very labor intensive process. Orchards

2 were planted by hand, pesticides were applied by hand from the back of horse drawn wagons, and hectares of cherries had to be picked by hand. Harvest involved a small army of people armed with ladders and cherry lugs laboring for days on end to get the crop in before it rotted on the trees. This process was very intensive which subsequently restricted the amount of cherries that could be produced in the state.

To reduce the amount of labor required to harvest such a delicate crop, many harvesting systems were developed over the years. The first mechanization of the tart cherry harvest involved using a mechanical limb shaker and a canvas catching frame

(125). The limb shaker would grab onto individual limbs and vibrate them until cherries came loose and fell onto the catching frame below. Cherries would then be collected from this catching frame and put into boxes. This mechanical shaker sped up the harvesting process but was still time consuming because each limb of a tree would have to be harvested individually.

Early limb shakers were the inspiration that led to the development of the modern day trunk shakers that are employed today to harvest Michigan’s vast cherry crop.

Instead of shaking individual limbs of a tree these new trunk shakers could shake the entire tree simultaneously (125). Many variations of the trunk shaker exist today. Some systems require up to eight laborers while others can be run by a single person. These trunk shakers employ a long boom that is attached to a hydraulically powered pair of rubber padded clamps that grip the trunk of the tree. An operator engages rotating masses in the shaker apparatus that generates and directs intense vibrations throughout the entire tree. These vibrations cause fruit to detach from the tree and fall on a canvas catching frame below. The canvas containing the harvested cherries would then be

3 retracted back into an apparatus containing a conveyor belt. This conveyor belt when activated would move cherries to a metal tank filled with cold water. Due to the soft nature of tart cherries, they are harvested into cold water to prevent damage that would be caused by the weight of cherries stacked on top of each other. Once a tank is filled with cherries it would be transported back to a cooling pad where cherries would be continuously rinsed with cold water to firm up the cherries and prepare them for the processor.

The application of plant protection materials was also an arduous process that was time consuming and often dangerous. To apply the first fungicides, growers would utilize horse drawn wagons and multiple people would apply sprays to trees without the use of any form of personal protection equipment to protect them from what they were spraying. Covering an entire orchard would be a long process that was fairly ineffective at getting these chemicals on the trees in a fast and efficient manner which is necessary for providing adequate protection for diseases or insects. Eventually, new sprayer technologies were invented and implemented leading to the development of the modern air-blast sprayer that is still used by growers today. The modern day air-blast sprayer utilizes a high powered fan that distributes a fine spray mist from nozzles into the canopy of the tree. The sprayer is pulled behind a tractor with a cab which decreases the risk of exposure to the operator and decreases the time it takes to apply pesticides. This technology greatly increased the size of an orchard that could be planted and maintained by a grower, and increased the efficiency and efficacy of plant protection materials.

4 PLANT PATHOGENS AFFECTING THE MICHIGAN

TART CHERRY INDUSTRY

The Michigan tart cherry industry relies heavily on the over 400 year old

Montmorency cultivar which has been the main variety of tart cherry grown in this state since the inception of the industry. This variety, which has a red skin and clear flesh, is self fertile and provides a heavy and consistent yield for growers. Other varieties of tart cherries such as the English Morello, Surefire, Meteor, Northstar, Mesabi, and Balaton are grown but only comprise a very small percentage of Michigan tart cherries. The

Montmorency variety has been a reliable variety widely excepted by growers but this variety does have a host of downfalls. One of the most challenging problems is its high susceptibility to cherry leaf spot (CLS), a disease that greatly impacts tree health and yield potential. In addition to CLS additional diseases affect the production of cherries in the state of Michigan. These diseases include American Brown Rot, European Brown

Rot, Bacterial canker, Powdery Mildew and Armillaria Root Rot.

Cherry leaf spot is caused by the Ascomycete fungus Blumeriella jaapii and elicits heavy defoliation of the tree before harvest. This defoliation causes a reduction in fruit quality and may affect the ability of the tree to overwinter. Cherry leaf spot is the most important disease affecting tart cherries in Michigan and is responsible for the majority of fungicide applications that are required in tart cherry production. Many of these fungicides are single-site fungicides that are at a high risk of developing resistance.

This problem is exacerbated by the fact that there is no new genetic material that has been introduced into this cultivar since its inception. The lack of genetic resistance to this

5 disease is what drives the heavy reliance of growers on fungicides to provide adequate control throughout the season (34).

American brown rot caused by Monilinia fructicola is a fungal disease that affects the fruit on the tree and after harvest in storage. This disease has the ability to rapidly infect fruit and spread throughout an entire orchard overnight when conditions are favorable. It infects all cherry varieties but is most severe on sweet cherry. This fungus can infect flowers in the spring causing then to wilt, turn brown and be retained on the tree through the summer. Fruit infected with brown rot will exhibit small brown spots, under moist conditions; the fungus will sporulate forming a large mass of grey to tan conidia. This disease has the potential to destroy an entire crop before fruit have been harvested. Due to the potential for great loss, yearly fungicide programs are required to suppress this disease (60).

European brown rot caused by Monilinia laxa is a fungal disease that has the potential to infect and kill the flowers and fruiting spurs of tart cherries. This disease is normally not a significant problem on Montmorency tart cherry cultivars that are grown in Michigan but under certain environmental conditions it has the potential to become a serious problem. During years where environmental conditions are conducive, fungicide sprays during bloom have the ability to suppress this disease (60).

Bacterial canker caused by Pseudomonas syringae is a bacterial disease that has the potential to significantly impact the cherry industry. It mainly infects through flowers and can invade the stems. Whole branches or even trees of highly-susceptible cultivars can be killed if conditions are conducive and the bacteria move into the main trunk. This bacterium can also infect leaves of the trees causing a shot-hole symptom. Depending on

6 the severity of infection, leaves can drop or become yellow which reduces the photosynthetic potential of the tree. Copper can be applied before bloom but is only marginally effective at controlling this disease (60).

Powdery mildew caused by Podosphaera clandestina is a disease that can greatly impact the growth of newly elongating shoots and leaves. It appears as a white powdery growth on the stems and leaves that causes the leaves to roll up and become brittle. This disease can reduce the growth of the tree and limit photosynthetic potential of the tree causing reduced vigor and uneven ripening of fruit. This disease can be managed through yearly applications of fungicides that prevent initial infections (60).

CHERRY LEAF SPOT ( BLUMERIELLA JAAPII )

Cherry leaf spot is the most devastating disease affecting the Michigan tart cherry industry. This fungus has the ability to affect production of both tart and sweet cherry varieties. The causal agent of CLS, Blumeriella jaapii has been known by a variety of different names both for it’s anamorph and teliomorph since its discovery. A paper by

Holb (50) illustrates the numerous names that appear in the literature for this fungus.

This difference in naming is partially due to misplacement but also to the fact that the pathogen was discovered independently in Europe and in the United States. For a fairly long time, the sexual stages of CLS isolates found in Europe were considered to be different from isolates found in the United States. The causal organism of CLS was first described by Karsten in 1884 on Prunus padus leaves (66). From this first description it was named Cylindrosporium padi based on its asexual phase (66). Following its discovery it was determined that this disease was prevalent in Europe on both tart and

7 sweet cherry trees in addition to many wild species (4). Shortly after it was discovered and characterized in Europe this disease was reported in fruit tree nurseries in the United

States. This disease greatly affected the ability of young trees in the nursery in overwintering resulting in great economic losses to the nursery operators (112).

During the 1950s, the systematics that was associated with the naming of this leaf spot fungus was called into question by researchers of the time. The name

Cylindrosporium padi that had been given to this leaf spot fungus was disputed because the features of this fungus did not closely match the features that are usually associated with other fungi that comprise the majority of the species in the Cylindrosporium genus

(25, 49, 20). In Europe during 1962, Arx showed that Cylindrosporium padi was closely related to species in the Phloeosporella genus so the annamorph name associated with

CLS was changed to Phloeosporella padi (7).

In the United States, the sexual stage of Phloeosporella padi was discovered and characterized by Bascombre Higgins in 1914 (99). Higgins showed that in addition to the parasitic stage on the leaves of the tree the fungus overwinters on fallen leaves in a stromatic body and forms an apothecium in the spring that subsequently produces the sexual ascospores. This discovery of the sexual stage resulted in the placement of CLS fungus into three different species in the genus Coccomyces in the Ascomycota group.

At the same time in Europe, the perfect stage of the anamorph known as Cylindrosporium padi was found on Prunus padus by Rehm in 1907 (99). He designated this perfect stage as Pseudopeziza jaapii. The question arose as to whether the perfect stage of

Cylindrosporium padi in Europe was the same as the perfect stage of Phloeosporella padi in the United States. The first comparison of the perfect stage found in the United States,

8 Coccomyces, and the perfect stage found in Europe, Pseudopeziza , revealed that the asci and ascospores of the European perfect stage were slightly different then those found in the American perfect stage (20). In 1961 Arx showed that the perfect stage,

Pseudopeziza jaapii, found in Europe was the same as the perfect stage, Coccomyces hiemalis, found in the United States (7). He went on to find that the two names given to these identical perfect stages could not be used because they were incorrect systematically so the name that was officially proposed and the name that is still in use today is Blumeriella jaapii (Rehm) v. Arx.

Blumeriella jaapii overwinters in fallen leaves on the orchard floor. During the spring the fungus will produce sexual cup like fruiting structures called apothecia which will discharge ascospores into the canopy of the tree during favorable conditions.

Apothecia develop optimally around 16.7 ºC and will discharge their spores during periods of heavy rain. This discharge of primary inoculum coincides with the bloom and petal drop period of tart cherry. Once spores attach to the leaves and germinate, the fungus will infect through the natural openings in leaves called stomates (40).

Macroconidia and microconidia are both produced by this fungus. The production of macroconidia which are the major part of the secondary infection period occurs through harvest and postharvest. Once the production of macroconidia ceases the fungus will produce microconidia which are formed on the same type of conidiophores that are used in the production of the macroconidia (48).

During this period of time when macroconidia production switches to microconidia production, the stroma starts to expand downward through the leaf. Masses of hyphae which later form an outer pseudoparenchyma layer around the apothecia grow

9 around this stroma. The stroma will expand to both sides of the leaves while remaining covered by the pseudoparenchyma layer. It has also been indicated that stalked structures with what appear to be spermatia are formed at this stage and will eventually disappear.

It was not confirmed by Higgins that these structures were spermatia but when he plated them they did not germinate which may indicate that they have a role as spermatia (48).

These spermatia-like structures are produced around the same time the structures that have a trichogyne are produced. The conidiophores that produced the microconidia will actually become sticky and hold the outer epidermis cells of the leaves together which aid in covering the stroma beneath (48).

When temperatures start to rise in the spring, the stroma will become active and start to extend to the lower surface of the leaf. The asci will start to develop during the bud swell stage of the tree from ascogenous hyphae which usually grow from the base of the stroma. The asci are formed in the common crozier hook formation and begin to mature at a very slow rate. When the asci are first formed they are spherical and the newly-formed ascospores become elongated as they mature (15).

As the asci grow in size, they begin to push through the covering that protected them through the winter. At this point the ascospores inside the asci are not fully developed but when they are released they rapidly mature. The ascospores are shot up into the air through an opening in the ascus. This stage is responsible for the primary infection of the newly expanded leaves of the cherry tree.

Symptoms associated with CLS first appear as small purple spots on the upper surface of the leaves. These purple spots that are apparent on the surface of the leaf are the result of the development of conidiophores under the epidermis of the leaf (24). As

10 the disease progresses, the spots will become circular and turn brown in the center.

These spots will enlarge slightly and merge with other spots to form large areas of necrosis. In some cases, the necrotic spots will drop out giving the leaf a shot-hole appearance. During the days following this initial swelling of the leaf epidermis, the fungus will produce white to pink masses of conidia in acervuli on the undersides of the leaves. These acervuli full of conidia will rupture through the epidermis of the leaf and release the conidia in masses called cirrhii. These masses of conidia will then be dispersed throughout the tree following a rain event.

Leaves are most susceptible to infection when they first unroll, but infection under favorable environmental conditions can take place until leaves drop naturally in the fall. The discharge and infection by the spores of this fungus is tightly controlled by temperature and periods of leaf wetness. These conidial masses are the most effective diagnostic features used to distinguish this disease from other cherry diseases such as bacterial canker. Infected leaves will turn yellow, and prematurely abscise leading to severe defoliation of the tree. During epidemic years, lesions can develop on the stem and surface of the fruit. Lesions on the stem of the fruit will affect fruit quality and can cause the fruit to prematurely abscise which leads to a loss in yield.

The acervuli and the conidia they produce are very important in the rapid spread of this disease throughout the tree and orchard. The conidia of the asexual stage of

Blumeriella are elongated colorless and are formed on a disk-shaped stroma. Higgins reported that the conidia that were found on different hosts are fairly similar. They are all fairly long, slender, slightly curved and have from 1 to 3 septations. It is unknown whether these are true septations or just pieces of the outer wall (48).

11 When it is first formed, the thickness of the stroma is only a single cell layer making it very delicate. The conidiophores which bear the conidia will arise on the surface in the middle of this stroma and eventually spread out as the stroma expands beneath the epidermis of the leaf. When the conidia have been produced in a large enough mass, they will push their way up and break through the cuticle of the leaf. On

Prunus serotia, the leaf cuticle is very thick so the conidia can only poke a small hole through this layer. This small hole restricts the flow of the conidia into a long strand that extends out from the surface of the leaf giving it a string like appearance. It is also reported that conidia that have dried are not as viable as conidia that are kept in the original acervulus (48).

The conidia are produced in an acervulus and subsequently released and carried by splashing rain land on the surface of the cherry leaf and begin to germinate. The hyphae of the germinating spore will penetrate through the stomata of the leaf and start to grow intercellularly. According to work by Higgins, this fungus produces a haustorium which enters the host cells through small openings which it creates (48). Once the haustorium enters the cell, it forms a circular structure that contains a nucleus and a large vacuole to drain nutrients from the plant cell. The host cell will lay down a cellulose sheath around the haustorium. This resembles that of the relationship between the haustorium of the Erysiphaceae group of fungi as described by Smith (107). Cells that are penetrated by the haustorium are not immediately killed which is evident by the laying down of the cellulose layer by the host cell. Cell death in this case which is observed on the leaf is thought to be caused by desiccation of the host cell when it comes

12 into contact with the asexual stroma. It is not believed that the symptoms observed from

CLS infection are caused by any toxin produced by the fungus (48).

For rapid development of disease in an orchard to occur, environmental conditions favoring the production of primary inoculum in the spring and secondary inoculum must be present. Leaf wetness, the length of leaf wetness, and temperature are important factors affecting the dissemination and germination of asexual conidia of this pathogen. Knowing the specific wetness periods that are most favorable for infection can help determine when the application of protective fungicide programs would be the most effective. A model has been developed that can be used to predict environmental conditions that would be the most conducive for development of CLS infections in

Montmorency tart cherry orchards. Using the apple scab Mills table model as a reference, a multiple regression equation was developed that took into account temperature and length of wetting period in relation to CLS infection. The model was validated in potted trees as well as in orchards and it was determined that the model accounted for 93% of the variation that was observed with leaf infection (33).

The model showed that CLS infection is highest when the duration of leaf wetness is around 60 hrs and the temperature is between 16 and 20 ºC. Leaf spot infection is lowest when the leaf wetness duration is between 4 and 12 hrs and the temperature is less than 12 ºC or greater than 24 ºC. The model indicates that as the leaf wetness duration increases leaf infection will increase even at unfavorable temperatures.

It also shows that if the temperature is at that optimum level of 16 to 20 ºC infection will still occur even if the duration of leaf wetness is relatively short. It was concluded that a

13 5 hr wetting duration coupled with a temperature of 20 ºC are the minimum environmental conditions that would lead to a CLS infection period. The development of this model was important to growers for the control of this disease. It provided them with the environmental conditions that would be most conducive to disease development so they could observe upcoming environmental events and apply a material to protect trees from infection.

The infection model previously described provides prediction for CLS infection periods but it is based on continuous wetting periods. In nature wetting periods are not always continuous. For instance it could rain heavily for a few hours which would sufficiently wet the leaves and then the sun could come out and dry the leaves followed by another rain event that could again sufficiently wet the leaves. This leads to the question of whether an interruption of the wetting period has any effect on the infection process of the CLS fungus. This is an important question because if this interruption of the wetting period was found to have an effect on the amount of infection by the CLS pathogen then growers could adjust their reaction to these types of wetting events (35).

It was determined that leaves from trees subjected to an interrupted wetting period had less disease severity than leaves subjected to a continuous wetting period. As the duration of the interrupted wetness period was increased, disease severity was reduced.

If the interruption occurred earlier after the initial wetting period, the disease severity would be less then if the interruption occurred more than 8 hrs after the initial wetting period. This information was integrated into the previous model to better predict the possible infection periods by the CLS fungus (35).

14 In addition to leaf wetness duration and temperature, the age of the leaf and the amount of inoculum available are additional factors that affect the severity of CLS infections. In many pathosystems, the age of the host tissue has a direct effect on the ability of the pathogen to successfully infect and colonize the tissue. Most commonly young tissues of the host plant are the most susceptible because they have not yet acquired the defense compounds or processes that are involved in defense against pathogens. Determining the affect of leaf age on infection by B. jaapii has helped provide growers with the insight on when their crop will be the most susceptible to infection and when they need to act to provide chemical protection. Inoculum concentration can also play a substantial role in the severity of disease. In some plant pathogen interactions, the pathogen is only successful in causing disease when the physical concentration of that organism is at high enough levels to overcome the defense systems established by the plant. Knowing the effect of concentration of B. jaapii on disease severity has helped in establishing the effect of reducing inoculum to a certain level has on the development of disease and subsequent severity. Unfolded leaves are not susceptible to infection by B. jaapii which means leaves first become susceptible to infection when they are unrolled (68). Leaves become less susceptible to infection as their age increases (34). This decrease in susceptibility seems to be expressed the most when the inoculum concentration is at its highest level. Susceptibility of different stages of the host to leaf spot infection was applied to the environmental model to better predict the severity of CLS infections.

The development of models to predict the severity of CLS infection based on temperature, leaf wetness duration, leaf age and inoculum concentration have helped

15 growers to prepare for infection events, however, for these models to be useful to growers they need to be incorporated into the decision making process for the timing of a fungicide application. This issue was addressed by bringing together modeling and the actual timing of fungicide application for effective control of CLS. The previously discussed models produce an environmental favorability index or EFI value which is a number between 0 and 100. Zero being the lowest potential for infection and 100 being the highest potential for infection (32). The use of modeling to predict when an infection event might occur and when to apply a protectant fungicide is effective in reducing the number of applications needed to provide adequate disease control when compared to traditional calendar sprays on a 10 to 11 day spray program. Modeling can be used to more effectively and efficiently utilize fungicides which in turn will reduce the number of applications and the subsequent cost incurred by the grower. The only downside of relying on modeling is that it may not be as useful in extremely wet years because the grower may not have an adequate amount of time to apply the protectant spray before an infection event occurs (32).

DEVELOPMENT AND USE OF PLANT PROTECTION

MATERIALS IN FRUIT PRODUCTION

Fruit production and agriculture in general as it exists in its current form relies heavily on fungicides to produce marketable crops for consumers. Fungal pathogens greatly limit the quality and yields that can be obtained from a particular crop. Many of the commonly grown varieties of tree fruit such as apples, pears, peaches, plums and

16 cherries are highly susceptible to numerous plant pathogens. Due to the perennial nature of tree fruit crops, many of the common practices for disease control that are utilized in row crops do not apply. The full season protection and lack of host resistance in fruit crops leads to one of the highest inputs and reliance of fungicides of any other agricultural crop.

Many of the first fungicides utilized in agricultural came from simply observing a phenomenon that occurred when agricultural crops were exposed to certain

th environmental chemicals. The first known fungicide use occurred in the 17 century and involved using a salt water brine wash followed by an application of lime to control bunt on wheat seeds (92). This practice was developed before growers were even aware that a fungal pathogen caused this disease on the wheat seeds. In France in the late 1800s,

Millardet made the observation that a mixture of copper sulfate and lime known commonly as Bordeaux mixture kept the leaves of grape vines healthy through the growing season (117, 92). The initial use of this mixture that actually turned out to be an effective fungicide was originally used to ward off the public from stealing grapes that were planted close to public roads. When sprayed on grapes, this mixture was turquoise in color and would be visible on the plant and cause the grapes to have a bitter taste when eaten. As an unattended consequence of this grape stealing deterrent, this mixture turned out to be very good at controlling downy mildew which at the time was wreaking havoc on French vineyards.

Word of the efficacy of this mixture at controlling fungal diseases made it to the

United States where it was eventually recommended by universities for the control of many fruit tree diseases including CLS (68). The Bordeaux mixture along with varying

17 formulations of copper and sulfur were the only materials available to early tart cherry growers for disease control. Most of these compounds were hand mixed into various solutions on site by growers. These compounds did provide control for fungal disease but commonly caused phytotoxicity in many cropping systems (53). Phytotoxicity was observed on trees in orchards especially when applied in hot dry weather or at high rates.

The copper compounds were especially effective for CLS control as evident by new formulations that are currently utilized in modern tart cherry CLS management programs

(84). These copper compounds still have a risk of causing phytotoxicity but when applied at recommended rates buy during cool weather this risk can be minimized or avoided completely.

The first broad spectrum organic fungicides were developed and released in the

1940s. These new compounds included the dithiocarbamates (thiram, mancozeb, nabam and maneb) (92). These fungicides provided the first step in the generation of new chemistries solely for the purpose of plant protection. Initially these first fungicides were extremely expensive compared to the traditional copper and Bordeaux mixtures, but since they provided excellent disease control without causing extensive phytotoxicity they were widely accepted and utilized by growers of the time (53).

During the 1950s and 1960s, the cherry industry received some of the most important multi-site fungicides that are still in use today. These groups included the phthalimides (captan), the guanidines (dodine), and the phthalonitriles (chlorothalonil).

These products were protectant fungicides that if applied before infection would provide excellent control of many diseases including CLS. Currently, captan and chlorothalonil can not be solely relied upon to provide season long control for CLS because of

18 restrictions on rates and timings that they can be used during the growing season. The loss of these chemistries as the primary materials for CLS control is due to safety concerns with residues on harvested fruit. They are however still utilized early in the season as is the case with chlorothalanil or as tank mix partners as is the case with captan.

Extensive research in the 1960s and early 1970s gave rise to the benzimidazoles and the carboxanilide class of fungicides. The benzimidazoles included thiabendazole, benomyl and thiophanate methyl while the carboxanilides included carboxin and oxycarboxin (92). These fungicides possessed a unique property in that they could systemically move into plant tissues and exhibit post-infection activity. These fungicides were a revolutionary advancement in plant protection that allowed growers to make fungicide applications at lower rates and use longer spray intervals. This class of fungicides was also unique in that the fungicides were site-specific fungicides meaning that unlike the multi-site inhibitors that targeted multiple processes of the pathogen, single-site inhibitors targeted one specific site or process of the pathogen. This first group of site-specific materials was a dramatic change from the previously developed fungicides and at the time there was no concern that this change would have any impact of the efficacy of these fungicides. In fact site-specific fungicides were preferred because of their efficacy at controlling disease and their relative safety to the consumer of end product. This group of fungicides led to the trend in developing single-site inhibitors that continues today.

In the 1970s and 1980s, the sterol demethylation inhibitors were introduced into agriculture (92). This group is one of the largest groups of fungicides developed in the great era of fungicide development and includes propiconazole, tebuconazole,

19 fenbuconazole and many additional compounds. These compounds are site-specific, systemic and highly effective at controlling many economically important pathogens.

Following their introduction, they were widely utilized by the majority of Michigan tart cherry growers for the control of CLS and brown rot (97). They were great fungicides for the industry because they were highly effective, had a slight curative action and reduced the number of applications needed in a season.

The next important groups of fungicides developed were the QoI or strobilurin class of fungicides in the 1990s and early 2000s. This class of fungicides includes azoxystrobin, kresoxim-methyl, trifloxystrobin, pyraclostrobin, and many others (92).

This group of compounds is site-specific, highly effective, and suitable for use on a diverse range of crops. Upon there registration, they were used extensively in many agricultural crops including fruit.

THE DEVELOPMENT AND REGISTRATION OF

PRISTINE

Pristine (BASF Corporation, Research Triangle Park, NC), which is a premix of boscalid and pyraclostrobin is commonly used in CLS management in Michigan tart cherries. Boscalid, a succinate dehydrogenase inhibitor (SDHI), is part of the carboxamide class of fungicides, which inhibit the succinate dehydrogenase complex

(SDH) of the fungal respiration pathway (106). Pyraclostrobin, a quinone outside

20 inhibitor (QoI), is part of the strobilurin class of fungicides which inhibits fungal respiration at complex III of the respiratory chain (6).

To understand the efficacy of fungicides and their potential for use in cropping systems, they must be rigorously evaluated for their efficacy in controlling various pathogens. In Michigan, new and current fungicides are evaluated for their effectiveness of controlling CLS on mature Montmorency tart cherry trees at the Northwest Michigan

Horticultural Research Center (NWMHRC) located near Suttons Bay, MI. Data from these field trials include the period of time where Pristine was developed and subsequently introduced into CLS management programs in Michigan.

Field trials are designed to closely resemble the practices that growers implement in commercial cherry orchards. A common practice for Michigan tart cherry growers used today is to apply Bravo Weather Stik which contains the multi-site inhibitor fungicide, chlorothalonil, at the phenological stages of petal fall and shuck split. This material is excellent for CLS control and is very important in mitigating resistance in B. jaapii to the site-specific materials that are used at later stages in the season.

Chlorothalonil is the preferred material for CLS control but cannot be used after shuck split due to residue concerns at the time of harvest. Each fungicide treatment is applied to individual trees using a handgun sprayer to ensure complete coverage. Multiple applications are made throughout the season with rating of disease severity occurring at harvest and approximately one month later.

Since CLS affects the photosynthetic capacity of the tree, it is critical that fungicides used in CLS management help trees in retaining a majority of their leaves into

September. The rating at or near harvest is important to establish the potential of the

21 fungicide to control CLS through harvest which will ensure that fruit is at its proper ripeness and quality. The rating following harvest is important to evaluate the ability of the fungicide to protect the leaves into September which will ensure the tree overwinters and sets a healthy crop the following spring (61).

The first occurrence of efficacy trials in Michigan involving an active ingredient in Pristine was in 2000 when a new experimental compound from BASF referred to as

BAS 500 20WG 9.6 oz. was evaluated for its effectiveness in controlling CLS on

Montmorency tart cherry trees (62). The active ingredient of this compound was a new strobilurin known as pyraclostrobin. This compound exhibited very little activity against

CLS and was only slightly significantly different from the control. The efficacy of this compound in controlling CLS was not comparable to the high control afforded by industry standard applications of Bravo Ultrex 82.5% WG (chlorothalanil) or the QoI

Flint (62).

The first efficacy trials of Pristine for control of CLS on Montmorency tart cherry in Michigan occurred in 2001 with the new experimental fungicide from BASF identified as BAS 516 38WG (63). This fungicide would later be developed and marketed as a premix of the SDHI boscalid and the QoI pyraclostrobin called Pristine. Full season applications of the BAS 516 38WG compound provided the best control for both CLS infection and defoliation. This compound outperformed all other fungicides tested including full season applications of Bravo Ultrex 82.5% WG. No significant differences were found between the 10.5 oz and 14.7 oz. rates of BAS 516 38WG for CLS infection of defoliation.

22 In 2002 field trials, BAS 516 38WG again provided excellent control for CLS infection and defoliation (64). In this trial, full-season applications of BAS 516 38WG provided statistically similar CLS control as full-season applications of Bravo Ultrex

82.5% WG, Flint 50WG and Flint 50WG + Captan 50W. The rate of BAS 516 38WG again had no significant impact on CLS infection of defoliation. These initial efficacy trials gave the first indication that Pristine would become a valuable part of CLS management programs in Michigan tart cherry orchards.

The registration and subsequent release of Pristine to Michigan tart cherry growers for the control of CLS occurred in 2003. The formulation that was released to

Michigan growers and also tested in field efficacy trials was Pristine 38WG. In 2003, full-season applications of Pristine provided the best control of both CLS infection and defoliation (114). These Pristine trials were essential in confirming the efficacy of

Pristine over a three year period that included varying levels of CLS infection.

Field trials in 2005 provided the first evaluation of the two separate active ingredients that are found in Pristine, boscalid and pyraclostrobin (115). Bravo Ultrex

82.5WDG applied at bloom and shuck split followed by four applications of Endura

70WG, 70% boscalid, provided the best control for CLS infection. Bravo Ultrex

82.5WDG applied at bloom and shuck split followed by four applications of Cabrio

20EG, 20% pyraclostrobin, or full-season applications of Gem 500SC, 42.6% trifloxystrobin, did not provide significant CLS control.

Cabrio and Gem treatments were similar in the levels of CLS control they provided. Bravo Ultrex 82.5 WDG applied at bloom and shuck split followed by four applications of Pristine provided excellent CLS control. The efficacy of Endura for CLS

23 control was significantly better than Pristine and Cabrio. Both Endura and Pristine were significantly more efficacious for CLS control in this field trial. Field trials conducted in

2006 showed that full-season applications of Endura 70WG provided the best control of

CLS. Full season applications of Gem 4.17SC did not provide adequate CLS control.

This field trial helped to determine the component of the Pristine premix that was the most efficacious for CLS control. These results indicated that the SDHI, boscalid was providing the majority of CLS control in the Pristine premix. This information would become a valuable part of the fungicide resistance story associated with this fungicide.

FUNGICIDES IN THE AGE OF RESISTANCE

Fungicide resistance is a relatively new problem when looking at agricultural practices throughout the existence of the human race. Before the extensive use of fungicides became commonplace, the idea of resistance becoming a problem was not even recognized. Even during the extensive fungicide research and development phase in the 1960s, the idea of the development of fungicide resistance to the researchers and chemical companies was foreign. In 1967, it was actually stated by plant pathologists of the time, Georgopoulos and Zaravovtis, “The reported cases of tolerance to agricultural fungicides are very few and the knowledge accumulated hardly justifies a review” (30,

41). This view that resistance was not or would not be a problem would eventually disappear as many cases of fungicide resistance began to appear for important classes of fungicides.

24 One of the influential changes that occurred in fungicide development was the discovery of the single-site inhibitory compounds. Before this discovery, the dominant fungicides developed and used were multi-site inhibitors. These were good chemistries but with many them the mode of action was not completely understood and they were only effective as protectants. They did not have any back action or curative properties and generally did not persist on the crop for a long period of time. The single-site fungicides possessed all of the good qualities of the multi-site inhibitors along with systemic activity, curative properties and a long persistence on the crop. Additionally, the mode of action of these single-site inhibitors was known and was generally specific to the group of pathogens it was designed to control. These were great qualities but with everything in life, it came with a cost.

Having a fungicide with a specific mode of action that only targets one site or process in the pathogen becomes detrimental after continued use in the pathogen population. Targeting a single site or process in pathogen greatly predisposes the population to resistance to that particular fungicide. To become resistant to a single-site fungicide usually only requires a single mutation in the gene that encodes for the target site of the fungicide. Mutations in fungi occur naturally at a rate of 1:100 million and even at this low rate given a high selection pressure for a certain mutation, which is the case with single-site fungicides, it becomes highly probable that a mutation conferring resistance will arise in a population (21).

The first case of resistance to fungicides in the United States that resulted in a loss of desired control of postharvest citrus rot caused by Penicillium spp. and occurred with biphenyl (30). In fruit production, the first case of field resistance occurred with dodine,

25 a guanidine fungicide and the apple scab pathogen, Venturia inaequalis (119). In the years to follow, the number of reported cases of fungicide resistance causing control loses dramatically increased. Resistance to the benzimidazoles fungicides occurred first with

Cercospora arachidicola , the causal agent of peanut leaf spot (31). Resistance to this class of fungicides sparked great concern among growers and the scientific community.

Resistance to the benzimidazoles was of particular concern because it was the first case of resistance to site-specific materials, and this class of fungicides had been widely adapted by growers in many agricultural crops (30).

The early carboximides, carboxin and oxycarboxin, were highly effective at controlling basidiomycete pathogens that caused bunt and smut as well as Rhizoctonia solani (108). Carboxin is a first generation SDHI that was first registered in the United

States in 1968 (21). It was mainly used to control seed and seedling diseases of various field crops. Resistance to this compound first occurred in Ustilago maydis, the causal agent of smut (104, 23, 42, 43, 72).

To determine the resistance mechanism, the iron-sulfur protein subunit of the succinate dehydrogense gene was amplified which revealed the presence of three cysteine-rich regions. A single point mutation was detected in the third cysteine-rich cluster. This single point mutation from a histidine residue to a leucine residue occurred in carboxin resistant strains of Ustilago maydis (23).

Upon alignment and comparison of this region with the same region in other organisms, it was confirmed that this was a highly conserved region across numerous fungal species. This high level of conservation indicated that mutations in this region were not a common occurrence. Using site-directed mutagenesis, it was found that the

26 point mutation causing a change in the amino acid residue from a histidine to leucine in the third cysteine-rich cluster conferred resistance to carboxin (23). This mutation occurred in an important cluster that is associated with the S3 iron-redox center which plays a large role in the binding of fungicides. The cluster where the mutation occurred is thought to play an important role in the mitochondrial respiration pathway due to the high conservation of this region among Eukaryotes. This early work with the carboxamide fungicides established a foundation for future resistance work with a newer

SDHI compound, boscalid.

Following an ever increasing rate a resistance being reported to many classes of fungicides, chemical companies marketing new fungicide products wanted to be proactive in preserving the efficacy of their active ingredients. Premix fungicide products where developed to help delay or prevent resistance from occurring. These products contained a predetermined amount of fungicide with two different modes of actions. The idea behind these premixes is that having two fungicides that each target a different protein of the fungus would simultaneously control any isolates that obtained a mutation to resistance to either of the two fungicides. Pristine was one of these products that was marketed as a premix to protect the boscalid component from selection for resistance. Premixes represented a step by the fungicide industry to prevent the development of resistance but this theory that premixes would not select for resistance was short lived.

Resistance to the SDHI class of fungicides has been well documented in numerous agriculturally important pathogens. Boscalid, developed after carboxin, is active against numerous fungal species occurring in various cropping systems (111). The

27 efficacy of boscalid against numerous plant pathogens led to its widespread use in many fungal management programs. Due to its site-specific mode of action, boscalid has a high potential to select for resistance among pathogen populations (29). Resistance to boscalid has occurred in populations of Alternaria alternata (13, 2, 9, 2), Corynespora cassiicola (55, 91, 90), Botrytis cinerea (127, 75, 77, ), Didymella bryoniae (67, 10),

Podosphaera xanthii (55), Monilinia fructicola (27) and Sclerotinia sclerotiorum (44).

The first case of boscalid field resistance occurred with Alternaria alternata on pistachio reported in 2008 (14). Pristine was introduced into California pistachio orchards and provided excellent control of alternaria late blight. It was useful in controlling this disease where resistance to the strobilurin class of fungicides had been observed. Two years after the registration and use of Pristine, growers reported a reduction in its efficacy. To understand what was occurring in these fungal populations, researchers conducted a survey that involved sampling and in vitro fungicide sensitivity testing of A. alternate isolates from orchards where Pristine had been used two years as well as orchards that had not seen applications of this product. In this case, isolates resistant to both boscalid and pyraclostrobin were found in California populations of A. alternata (14). This was the first study that indicated fungal populations had the ability to develop resistance to both the boscalid and strobilurin components of Pristine.

After establishing that an agriculturally important fungal pathogen has acquired resistance, the next step would be to determine the mode of resistance that allows pathogens to become tolerant to boscalid. The mode of resistance to the early carboxins was already established for pathogens including Ustilago maydis (23). There existed the

28 possibility that since boscalid has the same mode of action as the early carboxamides, the molecular mode of resistance could be similar.

To establish this relationship between the carboxamides and boscalid, the target site of boscalid needed to be studied and characterized. The SDH complex, also referred to as complex II or succinate:ubiquinone oxioreductase (SQR), is a functional part of the aerobic respiratory chain of fungal mitochondria (51, 25). Complex II consist of four subunits, SDHA, SDHB, SDHC and SDHD. These subunits are arranged in two main domains, the membrane-peripheral domain which consists of the SDHA and SDHB subunits and the membrane-anchor domain which consists of the SDHC and SDHD subunits (11). The SDHA and SDHB subunits form the soluble part of the complex and catalyze oxidation of succinate to fumarate. SDHB is an iron-sulfur protein (Ip) which contains three iron-sulfur clusters [2Fe-2S], [4Fe-4S] and [3Fe-4S] (9, 16). These iron- sulfur clusters are important in the transfer of electrons from flavin adenine dinucleotide to the membrane quinone (113, 2, 10).

Resistance work with the SDHI class of fungicides has identified a point mutation that has been associated with high levels of resistance to boscalid. This mutation results in an amino acid change that occurs in the third cysteine-rich region of the SDHB gene of the SDH complex of the fungal respiration chain. A point mutation in this region from a highly conserved histidine to either arginine or tyrosine in the third cysteine-rich cluster of the SDHB gene has been associated with boscalid resistance in several fungal genera

(127, 55, 102, 9, 11 ). This point mutation has only been found in the SDHB gene of isolates that have been exposed to boscalid. The SDHB gene is highly conserved across

29 many species of fungi indicating its importance in the fitness of these varied organisms

(2).

The SDHB gene encodes an iron-sulfur cluster protein that is thought to be important in docking of important molecules associated with the respiration chain as well as the docking of the boscalid molecule. It has been hypothesized that a point mutation in the gene encoding this docking site may cause a conformation change in the binding pocket that would no longer facilitate the binding of the boscalid molecule but that has not changed to the extent that that it can no longer perform its normal function of binding molecules necessary for fungal respiration (9).

Further studies of the sequences of the SDHB gene in various fungi have revealed an additional mutation within this region that has been connected to boscalid resistance.

This mutation was located at a proline residue in the second cysteine-rich region of the

SDHB subunit. This mutation results in an amino acid change from a proline to either a leucine or tyrosine and this mutation was discovered in both laboratory mutants and field isolates of Botrytis cinerea (111, 11). This proline residue is located in a region of the

SDHB subunit that is in close proximity to the ubiquinone-binding site and is part of the

Q-site that comes into contact with ubiquinone (77). This mutation is thought to confer resistance due to its importance and proximity to the ubiquinone binding pocket.

Complex II consists of four subunits (SDHA, SDHB, SDHC and SDHD), which leads to the possibility of the presence of mutations occurring in subunits other than

SDHB that could play a role in conferring boscalid resistance. In previous studies, it was reported that carboxin resistance was conferred by mutations in the SDHC and SDHD subunits of the SDH complex (52, 82, 9, 10). The SDHC and SDHD subunits are

30 hydrophobic membrane-spanning subunits that anchor the flavoprotein and the iron- sulfur protein to the inner mitochondrial membrane (3, 47, 10). The sequencing of these two subunits in boscalid resistant isolates of Alternaria alternata revealed one mutation in the SDHC subunit and two mutations in the SDHD subunit. A mutation from a highly conserved histidine residue at position 89 to an arginine was detected for the SDHC subunit while a mutation from a histidine residue at position 133 to arginine and a mutation from aspartate residue at position 123 to glutamic acid were detected for the

SDHD subunit (10). The isolates in this study that contained the SDHC or SDHD mutations did not contain mutations in the SDHB subunit (10). This suggests that resistance to boscalid can arise if mutations occur in any one of these subunits.

Recently, newer generation SDHIs have been developed that possess the same mode of action as boscalid. These include fluopyram, fluxapyroxad and penthiopyrad.

Due to the presence of resistance in numerous fungal pathogens to boscalid and the older carboxins, there is concern that cross resistance to these newer generations of SDHIs could occur. As these compounds become available to growers and used to control pathogens that have been previously categorized as boscalid resistant, it becomes very important to understand any cross-resistance relationships that may occur.

There have been reports that isolates of Didymella bryoniae and Alternaria alternata resistant to boscalid and that carry mutations in the SDH gene are also resistant to penthiopyrad (9, 12). In populations of A. alternate, the majority of boscalid-resistant and penthiopyrad-resistant isolates tested in vitro on fluopyram were greatly inhibited (9).

Isolates possessing the histidine to tyrosine mutation in the SDHB subunit that were highly resistant to boscalid were not resistant to fluopyram (9). An absence of a cross-

31 resistance to fluopyram has been shown in the case of D. bryoniae, Corynespora cassidicola, Botrytis cinerea and Podosphaera xanthii (55, 121, 9, 12). The difference in cross-resistance relationships between penthiopyrad and fluopyram may suggest the differential binding or binding affinity of these two compounds in the SDH complex. It is possible that a different mutation in one of the SDH genes may confer resistance to fluopyram but currently none have been identified. This suggest that fluopyram may play an important role in managing boscalid-resistant populations, but careful monitoring and resistance avoidance strategies must be implemented to preserve the effectiveness of this fungicide. Cross-resistance data is currently not available for the SDHI fluxpyroxad.

It appears that boscalid is becoming another causality of the striking ability of fungal pathogen populations to evolve resistance and continue to thrive on agriculturally important crops. In some cases, resistance to this highly effective mode of action was reported in as little as two years after its introduction. Even premixes of fungicides such as Pristine do not have the ability to prevent the development of resistance especially when they are used in populations where resistance to the other premix partner is present.

The recent development of the newer SDHI fungicides highlights the importance of understanding how populations of boscalid-resistant isolates develop and persist in field populations repeatedly exposed to these fungicides. The continued trend of the development of single-site fungicides suggest that the problem of fungicide resistance will be an important issue affecting growers for the foreseeable future.

32

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44 CHAPTER 1

DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII

INTRODUCTION

Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii (Rehm) v. Arx, is a major disease of tart ( Prunus cerasus ) cherry trees in Michigan. Leaf spot infection can lead to early defoliation if left untreated which can result in poor fruit quality as trees that are severely defoliated before harvest will produce undersized and poorly ripened fruit.

The formation of flower buds and subsequent fruit set will be greatly reduced the next season following a severe defoliation event due to CLS. Trees that are severely defoliated early in the summer become less cold hardy and are susceptible to winter injury which can result in tree death. Depending on the severity and timing of defoliation, entire orchard blocks can be killed.

CLS affects all commercially grown cultivars of sweet and tart cherries but is most severe on tart cherries. The predominant tart cherry cultivar grown in Michigan is

Montmorency This cultivar requires yearly chemical control programs that consist of six to eight applications of fungicides. A commonly-utilized site specific fungicide for CLS management in Michigan is Pristine (BASF Corporation, Research Triangle Park, NC), a premix of boscalid and pyraclostrobin. Boscalid, a succinate dehydrogenase inhibitor

(SDHI), is part of the carboxamide class of fungicides, which inhibit the succinate dehydrogenase complex (SDH) of the fungal respiration pathway (1). Pyraclostrobin , a quinone outside inhibitor (QoI), is part of the strobilurin class of fungicides which

45 inhibits fungal respiration at complex III of the respiratory chain (4). These site-specific materials used for CLS control have a high risk of developing resistance within fungal populations.

The SDH complex, also referred to as complex II or succinate:ubiquinone oxioreductase (SQR), is a functional part of the aerobic respiratory chain of fungal mitochondria (31, 17). Complex II consist of four subunits, SDHA, SDHB, SDHC and

SDHD. These subunits are arranged in two main domains, the membrane-peripheral domain which consists of the SDHA and SDHB subunits and the membrane-anchor domain which consists of the SDHC and SDHD subunits (8). The SDHA and SDHB subunits form the soluble part of the complex and catalyze oxidation of succinate to fumarate. SDHB is an iron-sulfur protein (Ip) which contains three iron-sulfur clusters

[2Fe-2S], [4Fe-4S] and [3Fe-4S] (6, 11). These iron-sulfur clusters are important in the transfer of electrons between flavin adenine dinucleotide and the membrane quinone (80,

1, 7).

Resistance to the SDHI class of fungicides has been well documented in numerous agriculturally important pathogens. The first case of resistance occurred with

Ustilago maydis and the SDHI, carboxin (75, 16, 25, 26, 47). Carboxin is an SDHI fungicide that inhibits fungal respiration at complex II and is mainly active against the

Basidiomycete group of fungi (5). Boscalid, developed after carboxin, is active against numerous fungal species occurring in various cropping systems (79). The efficacy of boscalid against numerous plant pathogens led to its widespread use in many fungal management programs. Due to its site-specific mode of action, boscalid has a high potential to select for resistance among pathogen populations (20). Resistance to boscalid

46 has occurred in populations of Alternaria alternata (10, 1, 6, 8), Corynespora cassiicola

(34, 65, 67), Botrytis cinerea (90, 91, 89, 60), Didymella bryoniae (45, 7), Podosphaera xanthii (34), Monilinia fructicola (19) and Sclerotinia sclerotiorum (27).

Previous resistance work with the SDHI class of fungicides has identified a point mutation that has been associated with high levels of resistance to boscalid. This mutation results in an amino acid change occurs in the third cysteine-rich region of the SDHB gene of the SDH complex of the fungal respiration chain. A point mutation in this region from a highly conserved histidine to either arginine or tyrosine at position 272 in the SDHB gene has been associated with boscalid resistance (90, 19, 73, 79, 71, 6 ). This point mutation has only been found in the SDHB gene of isolates that have been exposed to boscalid. The SDHB gene is highly conserved across many species of fungi (1) indicating its importance in the fitness of these varied organisms. This gene encodes an iron-sulfur cluster protein that is thought to be important in docking of important molecules associated with the respiratory chain as well as the docking of the boscalid molecule. It has been hypothesized that a point mutation in the gene encoding this docking site may cause a conformation change in the binding pocket that would no longer facilitate the binding of the boscalid molecule, but that has not changed to the extent that that it can no longer perform its normal function of binding molecules necessary for fungal respiration

(6).

Field trials at the Northwest Michigan Horticultural Research Center in Suttons

Bay, MI have indicated a reduction in the effectiveness of Pristine in controlling CLS.

Pristine was registered commercially for use in tart cherry in MI in 2004 and provided excellent control of CLS. Pristine was introduced at a time when resistance to the

47 demethylation-inhibiting (DMI) fungicides was becoming prevalent in Michigan orchards

(70). The widespread resistance of B. jaapii to DMI fungicides has reduced or eliminated the efficacy of this class of fungicides for the control of this disease in Michigan orchards

(38, 70, 54). The DMIs were a reliable group of fungicides that growers had relied upon for many years to control CLS. The loss of the DMI class of fungicides would have been devastating to the industry due to the lack of a suitable replacement for this important class of fungicides. The release of Pristine which was very effective at CLS control filled this gap in the industry. Due to the high levels of DMI resistant isolates in Michigan cherry orchards and the excellent efficacy, Pristine quickly became an important and integral part of many CLS management programs.

Due to the reduction in the efficacy of Pristine in controlling CLS in research trials and a select number of commercial orchards there was a high probability that certain CLS populations had become resistant to Pristine. Field studies have indicated that the boscalid component of Pristine was the most efficacious at CLS control. This suggests that the strobilurin component of Pristine can not be relied upon to provide control for boscalid resistant isolates in an orchard population of B. jaapii, which will increase the risk of resistance development to the boscalid component of Pristine. Owing to its single site mode of action it was likely that boscalid resistant B. jaapii populations would be selected for in Michigan orchards where Pristine is the dominant fungicide utilized for CLS control. Resistance to boscalid of B. jaapii has the potential to be associated with a point mutation of the highly conserved point mutation in a histidine residue of the SDHB subunit.

48 The current study aims to: (i) evaluate the sensitivity to bosaclid of Michigan B. jaapii populations (ii) characterize mutation(s) in the SDHB subunit of B. jaapii that are correlated to boscalid resistance and (iii) determine the distribution of boscalid resistance in Michigan orchard populations of B. jaapii .

MATERIALS AND METHODS

Fungicide efficacy trials

Yearly spray trial programs at the Northwest Michigan Horticultural Research

Center (NWMHRC) near Traverse City, Michigan test the effectiveness of current commercially utilized fungicides as well as experimental fungicides at varying stages of development. Fungicides were tested either in a standard program that includes a Bravo application at the beginning of the year or full season where the fungicide of interest is used throughout the spray trial. Trials were conducted in a block of Prunus cerasus cv.

Montmorency trees established in 1995. Fungicides used in the spray trials were applied at 10 day intervals beginning at late bloom or early petal fall and continue through harvest. Fungicide sprays were applied to individual trees to the point of runoff using a

-2 -1 portable hand gun sprayer at a rate of 330-386 kgcm and 458.9 L water ha .

Experiments were set up in a randomized complete block design with single tree plots and four replications for each fungicide treatment. CLS ratings were conducted at harvest and approximately 4-6 weeks later. Disease rating involved examination of 20 terminals on each tree for disease incidence and defoliation caused by CLS. For each

49 terminal the total number of nodes is counted followed by the total number of leaves that have apparent CLS symptoms and the total number of leaves that have been dropped from the terminal. Defoliation caused by CLS is defined as the number of leaves present on a given terminal divided by the number of nodes on a terminal multiplied by 100.

Statistical analysis of field data

Treatments were evaluated for their effectiveness of controlling CLS incidence and defoliation on Montmorency tart cherry using a one-way analysis of variance for each of the rating timings. Mean separation using a p-value of 0.05 for each dataset was conducted with Fisher’s protected least significant difference test utilizing the M-STAT-

C 4.0 statistical research program (https://www.msu.edu/freed/mstatc.htm).

B. jaapii isolate collection

Orchards were selected for sampling based on reports of control failure from growers that utilized Pristine in their fungicide programs. Conidia from sporulating acervuli were streaked onto coffee water agar (20% brewed coffee, 2% agar) and incubated at 24 ºC for

24 hours. Single conidia were then selected and transferred to and maintained on MMEA

(2% malt extract, 0.1% yeast extract, and 2% agar) plates. When mono-conidial isolates began to form a noticeable colony small agar plugs were taken and transferred to MMEA slants for long term storage. Once a colony was established on the MMEA slant it would be moved to a refrigerated location that was maintained at a constant 5 ºC.

50 A combined total of 1,288 isolates from commercial orchards and 111 isolates from non-treated trees in 2010 and 2011 were collected and used in the boscalid in vitro assay. From these 1,288 commercial isolates 323 were collected in 2010 and 965 were collected in 2011. Of the 111 non-treated isolates, 44 were collected in 2010 and 67 were collected in 2011.

The group of commercial isolates collected in 2010 consisted of isolates from 20 sites with a history of site specific fungicide use for the control of CLS and 1 site that was certified as organic and most likely had not been exposed to site specific fungicides which would include Pristine. Of the 2010 commercial isolates 20 sites were located in the major tart cherry producing areas in Northwest Michigan and one site was located in the West Central tart cherry production area. The 2010 non-treated isolates were obtained from two sites in Ohio which had a high likelihood of never being exposed to site- specific fungicides including Pristine.

The group of isolates collected in 2011 consisted of isolates from 21 sites with a history of site specific fungicide use. Of these 21 sites, 16 were located in the major tart cherry producing areas in Northwest Michigan, 2 were from research plots at the

Michigan Horticultural Research Station, and 3 sites were from the major tart cherry production areas in Southwest Michigan. The 2011 non-treated isolates were obtained from 3 sites around the Michigan State University Campus in mid-Michigan, 1 site located in Livingston County in mid-Michigan, and 1 site in Ohio. These isolates were obtained from tart cherry, sweet cherry and black cherry ( Prunus serotina Ehrh.) trees that have not been treated with Pristine or other site-specific fungicides.

51

In vitro sensitivity of B. jaapii to boscalid

The in vitro sensitivity of B. jaapii to the boscalid component of Pristine was assessed using a mycelial growth assay. The fungicide used in this study was the commercial formulation of Endura® (BASF) which contains 70% a.i. boscalid. To prepare stock solutions of boscalid, Endura was dissolved in 100% acetone and adjusted

-1 -1 -1 to concentrations of 100 mg ml , 10 mg ml and 5 mg ml . Sterilized MMEA cooled to

40 ºC was amended with these stock solutions of boscalid at concentrations of 0.1, 0.5, 1,

-1 2.5, 5, 10, and 25 µg ml . Control media consisted of MMEA amended with the highest concentration of acetone that was added to the fungicide amended media. Mycelial plugs roughly 1mm in diameter were taken from actively growing cultures using a Pasteur pipette and transferred to the fungicide amended plates. Two replicates of each isolate were used in this study. Isolates were incubated at 23-25 ºC for 14 days at which point the growth of each isolate was evaluated and a Minimum Inhibitory Concentration (MIC) determined. The MIC of an isolate is defined as the minimum concentration of a.i. required to fully inhibit the growth of an isolate. In previous in vitro studies conducted with B. jaapii, MIC’s were used effectively to distinguish different fungicide sensitivity groups within populations (16, 21). Since B. jaapii grows at a very slow rate in culture

MIC’s are more appropriate then using an Effective Concentration 50% (EC 50 ) for determining the effect of a fungicide on mycelial growth. MIC’s are also commonly used in antibiotic studies with bacteria where complete growth inhibition is required.

52 Amplification and identification of the succinate dehydrogenase subunit b gene

(SDHB) of B. jaapii

Five isolates from a non-treated Montmorency tart cherry door yard planting and two isolates from a commercial tart cherry orchard in Michigan were chosen for the initial amplification of the SDHB gene of B. jaapii. Mycelium from each isolate was spread onto MMEA media and incubated at 23-25 ºC until a suitable mass of mycelium had developed to facilitate DNA extraction. Approximately 100 mg of mycelium from each isolate was scraped from the surface of the plate and ground using liquid nitrogen and a bead beater homogenizer (FastPrep-24; MP Biomedicals; Solon, OH). DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the protocol provided by the manufacturer.

At the time of this study there was not a published sequence for the SDHB gene of

B. jaapii . However, published SDHB sequences from Alternaria alternate (84), Botrytis cinerea (48, 45, 75, 86, 18), Didymella bryoniae (8), Corynespora cassiicola (3),

Sclerotinia sclerotiorum (27), Podosphaera xanthii (34), Monilinia fructicola (19) and

Mycosphaerella graminicola (73, 76, 1) were available. Sequences of the SDHB gene of

Alternaria alternata, Botrytis cinerea, Corynespora cassiicola and Mycosphaerella graminicola were aligned and used to design a set of degenerate primers aimed at amplifying the SDHB gene of B. jaapii. A region of the SDHB gene that was highly conserved between the four species was used to design three forward degenerate primers and one reverse primer. The three forward primers were SDHB-FL (5'-

GACYTTCCAYATCTACCGCTG-3'), SDHB-FS (5'-ATYTACCCWCTNCCYCACA-

53 3'), SDHB-FEL (TGYGCHATGAACATYRAYGG) and the reverse primer was SDHB-

R (5'-CCKYGAGCAGTTRAGAATVGTG-3'). Degenerate bases used in the primers are defined as: Y=(C/T), W=(A/T), R=(A/G), K=(G/T), V=(A/C/G), H=(A/C/T) and

N=(A/C/G/T).

PCR amplifications were carried out in a 50 µl volume containing 5µl PCR buffer, 1.5 µl MgCl 2, 0.4 µl dNTP’s, 2 µl of each primer, 0.4 µl of Taq polymerase and 5

µl genomic DNA. PCR amplifications were performed with the following conditions: an initial preheating at 94 ºC for 3 minutes which was followed by 40 cycles of denaturation at 94 ºC for 30 s, annealing at 57 ºC for 30 s and extension at 68 ºC for 1.5 minutes, and a final extension at 72 ºC for 5 minutes. Amplified PCR products were separated on 1.5% agarose gels in TAE buffer and photographed after staining with ethidium bromide.

Amplified PCR products were purified with a QIAquick PCR Purification Kit

(QIAGEN Inc., Valencia, CA) and directly sequenced with each corresponding primer pair at the Genomics Technology Support Facility, Michigan State University. Sequences were analyzed and a blast search was performed to confirm the amplification of the

SDHB gene.

These initial sequences were used to design specific primers to further amplify the region of the SDHB gene that contained areas associated with point mutations conferring resistance to boscalid. The new primer pair consisted of the forward primer SDH-FE (5'-

GACGAGCCGACCTCGAAG-3') and a reverse degenerate primer SDH-RE (5'-

CTTCTTGATCTSSGCRATSGCCA-3'). Amplified PCR products were purified with a

54 QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA) and directly sequenced with the SDH-FE and SDH-RE primers. Sequences were analyzed and a blast search was performed to confirm the amplification of the SDHB subunit.

RESULTS

Efficacy of Pristine for control of cherry leaf spot in fungicide field trials at the

NWMHRS 1999–2013

Efficacy trials involving Pristine were conducted from 2001 to 2003, 2005, and

2009 to 2012 at the NWMHRC (Figure 1). Pristine as the experimental compound BAS

516 38WG provided excellent control of CLS in 2001 and 2003. The first reduction in the efficacy of Pristine was observed in 2005 and 2009 during seasons with heavy disease pressure. Pristine was again effective in controlling CLS in 2010. In 2011 and 2012, field trials evaluating the efficacy of Pristine were conducted in a different block of

Montmorency tart cherries at the NWMHRC. The efficacy of Pristine was reduced in

2011 and control provided by Pristine in 2012 was below commercially acceptable levels.

Levels of control provided by Pristine in 2012 provided the first indication that practical resistance to Pristine may be occurring in this block.

Field trials in 2005 provided the first evaluation of the two separate active ingredients that are found in Pristine, boscalid and pyraclostrobin. Bravo Ultrex

82.5WDG applied at bloom and shuck split followed by four applications of Endura

70WG, (70% boscalid), provided the best control for CLS infection. Bravo Ultrex

82.5WDG applied at bloom and shuck split followed by four applications of Cabrio

55 20EG, (20% pyraclostrobin), did not provide significant CLS control. Bravo Ultrex

82.5WDG applied at bloom and shuck split followed by four applications of Pristine provided acceptable levels of CLS control. The efficacy of Endura for CLS control was significantly better than Pristine and Cabrio. Both Endura and Pristine were significantly more efficacious than Cabrio for CLS control in this field trial.

80

70 60 Scaled 50 Infection 40 Scaled 30 Defoliation 20 Infection/Defoliation 10 Scaled%Cherry Leaf Spot 0

2001 2002 2003 2005 2009 2010 2011 2012 Figure 1. Efficacy trial results of Pristine treatments for control of CLS. Pristine was evaluated as a full season treatment from 2001 to 2003 and 2009 to 2011. Pristine was evaluated following two applications of chlorothalonil at petal fall and shuck split in 2005 and 2012. Trials were conducted in an experimental orchard of Prunus cerasus cv . Montmorency located at the Northwest Michigan Horticulture Research Center near Suttons Bay, Michigan. CLS incidence and defoliation was assessed in late August or early September of each year. Defoliation data for 2005 was not determined due to low disease pressure. Leaf spot infection and defoliation is scaled as the disease incidence or defoliation divided by the disease incidence or defoliation on unsprayed controls. Scaled infection is represented by black bars and scaled defoliation is represented by hash marked bars.

56 Grouping of B. jaapii isolates into boscalid sensitivity groups

To determine the distribution of B. jaapii isolates resistant to boscalid, isolates were classified into groups based on their sensitivity to boscalid in the in vitro assay.

Isolates were organized into three sensitivity groups based on MICs determined from the in vitro assay (Figure 2). Isolates classified as sensitive had MIC values between 0.1 and

-1 -1 0.5 g ml . Isolates with MIC values between 1 and 25 g ml were considered to have

-1 reduced sensitivity. Isolates with MIC values greater than 25 g ml were considered resistant. Based on these sensitivity groups, frequency distributions were established for all isolates examined in the populations. The MIC values could then be compared across populations to determine if a certain population as a whole had begun shifting toward resistance.

The B. jaapii populations within each orchard or site were organized into three different categories based on average MIC values obtained in the in vitro assay. An

-1 orchard or site was termed sensitive if the average MIC was between 0.1 and 0.5 g ml ,

-1 reduced sensitive if the average MIC was between 1 and 10 g ml , or resistant if the

-1 average MIC was ≥ 25 g ml . This categorization allowed for the assessment of the differences in MIC distributions of orchards across Michigan.

57

Sensitive Isolates Control 0.1 g/ml 0.5 g/ml 1 g/ml 2.5 g/ml 5 g/ml 10 g/ml 25 g/ml a

Reduced Sensitivity b

Resistant Isolates c

Figure 2. Isolates plated on boscalid amended media at concentrations of 0.1, 0.5, 1, 2.5, 5, 10, -1 and 25 µg ml . Each colony on plates within a row represents a separate B. jaapii isolate. There are eight different isolates in the sensitive group, ten isolates in the reduced sensitivity group and ten isolates in the resistant group. Minimum inhibitory concentrations were determined as the lowest concentration that completely inhibits mycelial growth following a 14 day incubation period. -1 a. Minimum inhibitory concentration (0.1 µg ml ) of an isolate classified as sensitive. -1 b. Minimum inhibitory concentration (5 µg ml ) of an isolate classified as reduced sensitive. -1 c. Minimum inhibitory concentration (>25 µg ml ) of an isolate classified as resistant.

58 Sensitivity of B. jaapii isolates to boscalid (2010)

A total of 44 B. jaapii isolates from two non-treated sites and 323 isolates from 21 commercial orchards were assayed for their sensitivity to boscalid. The sensitivity of

-1 -1 isolates to boscalid (determined as MIC) ranged from 0.1 µg ml to > 25 µg ml (Figure

3). There was a large separation of the MIC distribution of non-treated isolates from the

MIC distribution of isolates from commercial orchards. Of the 44 isolates from sites with no known applications of Pristine, 41 isolates were inhibited by a concentration of 0.1 µg

-1 -1 ml and 3 isolates were inhibited by a concentration of 0.5 ml . Of the 323 isolates from commercial orchards with known Pristine applications, 0% of isolates were inhibited by a

-1 concentration of 0.1 µg ml and only 2.2% of isolates where inhibited by a concentration

-1 of 0.5 µg ml . The majority of isolates (75.5 %) were inhibited at a concentration

-1 -1 between 1 µg ml and 25 µg ml while 22.3% of isolates were not inhibited by the

-1 highest concentration of boscalid tested (25 µg ml ).

59

100 90 80 Non-treated 70 Isolates 60

50 40 30 Comercial 20 Isolates Frequency Distribution % Frequency Distribution 10 0

0.1 0.5 1 2.5 5 10 25 >25

Minimum Inhibitory Concentration µg/ml Figure 3. Boscalid sensitivity distribution of 2010 non-treated and commercial B. jaapii isolates. Baseline isolates are represented by white bars and commercial isolates are represented by black bars.

The 2010 B. jaapii populations within each orchard or site were organized into three different categories based on average MIC values obtained in the in vitro assay

(Figure 4). The two non-treated sites were classified as being sensitive to boscalid with

-1 isolate MIC values that were between 0.1 and 0.5 g ml . Of the 21 commercial orchards none were classified as being sensitive. The reduced sensitivity group was comprised of 18 commercial orchards with isolate MIC values that ranged from 0.5 g

-1 -1 ml to > 25 g ml . Most isolates (92.4%) in this reduced sensitivity group had MIC

-1 values in the 1.0 to 10 g ml range, and 2.9% of isolates in this group had MIC values >

60 -1 25 g ml . The resistant group consisted of 3 commercial orchards with isolate MIC

-1 -1 values that ranged from 2.5 g ml to greater than 25 g ml . The majority of isolates

-1 (94.1%) in this resistant group had MIC values of 25 g ml or greater. The least sensitive orchard had a total of 95.8% of isolates that were classified as not being

-1 inhibited at 25 g ml .

100

90 2010 Sensitive 80

70 60 2010 Reduced 50 Sensitivity 40 30 20 2010 Resistant % Frequency Distribution 10 0 0.1 0.5 1 2.5 5 10 25 >25

Minimum Inhibitory Concentration µg/ml

Figure 4. Distribution of 2010 Orchard or site sensitivity groups. Orchards or sites

classified as sensitive had average Minimum Inhibitory Concentrations between 0.1 -1 and 0.5 µg ml and are identified by white bars. Orchards or sites classified as reduced sensitivity had average Minimum Inhibitory Concentrations between 1 and 10 -1 µg ml and are identified by hatched bars. Orchards or sites classified as resistant had -1 average Minimum Inhibitory Concentrations between 25 and > 25 µg ml and are

identified by black bars.

61 Sensitivity of B. jaapii isolates to boscalid (2011)

A total of 67 B. jaapii isolates from five non-treated sites, 96 isolates from field trials at the NWMHRS, and 865 isolates from 20 commercial orchards were assayed for their sensitivity to boscalid (Figure 5). The sensitivity of isolates to boscalid (determined

-1 -1 as MIC) ranged from 0.1 µg ml to > 25 µg ml . There was a large separation of the

MIC distribution of non-treated isolates from the MIC distribution of isolates from orchards with a history of Pristine applications. Of the 67 isolates from sites with no

- known applications of Pristine all isolates were inhibited by a concentration of 0.1 µg ml

1 . Of the 961 isolates from sites with known Pristine applications 0.1% of isolates were

-1 inhibited by a concentration of 0.1 µg ml and 0.3% of isolates where inhibited by a

-1 concentration of 0.5 µg ml . The majority of isolates (65.0 %) were inhibited by a

-1 -1 concentration between 1 µg ml and 25 µg ml while 34.6% of isolates were not

-1 inhibited by the highest concentration of boscalid tested (25 µg ml ).

62

100 90 80 Non-treated 70 Isolates 60 50 40 30 Comercial 20 Isolates Frequency Distribution % Frequency Distribution 10 0 0.1 0.5 1 2.5 5 10 25 >25

Minimum Inhibitory Concentration µg/ml

Figure 5. Boscalid sensitivity distribution of 2011 non-treated and commercial B. jaapii isolates. Non-treated isolates are represented by white bars and

commercial isolates are represented by black bars.

The B. jaapii populations within each orchard or site were organized into three different categories based on average MIC values obtained in the in vitro assay (Figure

6). The five non-treated sites were classified as being sensitive to boscalid with isolate

-1 MIC values that were all 0.1 g ml . Of the 20 commercial orchards or 2 sites from

NWMHRC field trials none were classified as being sensitive. The reduced sensitivity group was made up of 12 commercial orchards and 1 field trial site with isolate MIC

-1 -1 values that ranged from 0.1 g ml to greater than 25 g ml . Most isolates (91.4%) in

-1 this reduced sensitivity group had MIC values in the 1.0 to 10 g ml range, and 6.6% of

-1 isolates in this group had MIC values > 25 g ml . The resistant group was made up of 8

63 commercial orchards and 1 field trial site with isolate MIC values that ranged from 0.5 g

-1 -1 ml to > 25 g ml . The majority of isolates (87.0 %) in this resistant group had MIC

-1 values ≥ 25 g ml . The least sensitive orchard had a total of 86.0% of isolates that were

-1 classified as not being inhibited at 25 g ml .

100 90 2011 Sensitive 80

70 60 2011 Reduced 50 Sensitivity 40

30 20 2011 Resistant Frequency Distribution % Frequency Distribution 10

0 0.1 0.5 1 2.5 5 10 25 >25

Minimum Inhibitory Concentration µg/ml

Figure 6. Distribution of 2011 Orchard or site sensitivity groups. Orchards or sites classified as sensitive had average Minimum Inhibitory Concentrations between 0.1 -1 and 0.5 µg ml and are identified by white bars. Orchards or sites classified as reduced sensitivity had average Minimum Inhibitory Concentrations between 1 and -1 10 µg ml and are identified by hatched bars. Orchards or sites classified as resistant had average Minimum Inhibitory Concentrations between 25 and > 25 µg -1 ml and are identified by black bars.

64 Amplification of the SDHB gene and molecular detection of the H272R mutation

A total of seven isolates were examined using each of the three degenerate primer pairs. Primer pair SDHB-FL and SDHB-R amplified a 650 bp fragment from seven isolates, primer pair SDHB-FS and SDHB-R amplified a 350 bp fragment from six isolates and primer pair SDHB-FEL and SDHB-R amplified a 400 bp fragment from six isolates (Figure 7). A partial nucleotide sequence of the SDHB gene was obtained from sequencing using the described degenerate primers. A search utilizing the BLASTx algorithm revealed homologies to the SDHB subunit of other filamentous fungi. Deduced amino acid sequences revealed three cysteine rich clusters that are known to contain regions with point mutations associated with resistance to boscalid.

The primer pair SDH-FE and SDH-RE amplified a 700 bp fragment and revealed a partial SdhB sequence that encompassed the region of the SDHB subunit containing an amino acid codon change from CAC to CAG leading to an amino acid change from histidine to arginine in the third cysteine rich region.

65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19 20 21 22 23 24 25 26

Figure 7. PCR amplification of the SDHB gene from 7 B. jaapii isolates utilizing the primer pairs SDHB-FL and SDHB-R, SDHB-FS and SDHB-R, and SDHBFEL and SDHB-R. B. jaapii baseline isolates 10PEBJ-1, 3, 4, 9, 11 and B. jaapii boscalid resistant isolates 10BWPB-21, 22 were used for each primer pair. Lanes 1 and 17, DNA ladder; lanes 2, 10 and 19, water control; lanes 3-9, amplification with primer pair SDHB-FL and SDHB-R; lanes 11-16 and 18, amplification with primer pair SDHB-FS and SDHB-R; lanes 20-26, amplification with primer pair SDHBFEL and SDHB-R.

The partial nucleotide sequences from 13 boscalid sensitive, 10 boscalid reduced sensitivity and 10 boscalid resistant isolates were aligned and compared. Analysis of the nucleotide sequences revealed a change of the CAC codon encoding for a histidine

66 residue in the third cysteine rich cluster to CAG encoding for an arginine residue (Figure

8). Of the partial SDHB sequences from 33 isolates, the 10 boscalid resistant isolates

possessed the CAC to CAG mutation. No mutations were detected from the 13 boscalid

sensitive and boscalid reduced sensitivity isolates (Table 1).

Second cysteine-rich cluste r

Sensitive (ECILCACCSTSC PSYWWNSEEYLGPAVLMQSYRWLAD) Reduced Sensitive (ECILCACCSTSC PSYWWNSEEYLGPAVLMQSYRWLAD)

Resistan t (ECILCACCSTSC PSYWWNSEEYLGPAVLMQSYRWLAD)

Third cysteine-rich cluster

Sensitive (SRDQKKEERKAALDNSMSVYRC HTILNCSRTCPKGLNP) Reduced Sensitive (SRDQKKEERKAALDNSMSVYRC HTILNCSRTCPKGLNP) Resistan t (SRDQKKEERKAALDNSMSVYRC RTILNCSRTCPKGLNP)

Figure 8. Sequences obtained from the second and third cysteine-rich clusters of the SDHB gene of B. jaapii . Both cysteine-rich clusters (shaded areas) were amplified from sensitive, reduced sensitive and resistant isolates. Amino acids highlighted in red have been previously associated with boscalid resistance. No amino acid mutations were detected from the second cysteine-rich cluster from the three sensitivity groups. An amino acid mutation from histidine to arginine was detected for resistant isolates in the third cysteine rich cluster.

67

Isolate Boscalid Phenotype Codon in SDHB Sequence Amino Acid Residue 11PEBJ-1 S CAC H (Histidine) 11PEBJ-3 S CAC H 11PEBJ-4 S CAC H 11PEBJ-9 S CAC H 11PEBJ-11 S CAC H 11LIBJMR-1 S CAC H 11LIBJMR-3 S CAC H 11LIBJMR-4 S CAC H 11LIBJMR-5 S CAC H 11LIBJMR-6 S CAC H 11LIBJMR-7 S CAC H 11LIBJMR-8 S CAC H 11LIBJMR-9 S CAC H 10BLGO-2 RS CAC H 10BOGW-24 RS CAC H 10BOW-1 RS CAC H 10BWPB-24 RS CAC H 10BWPB-29 RS CAC H 10BOW-1 RS CAC H 10BOW-3 RS CAC H 10BOW-4 RS CAC H 10BOW-9 RS CAC H 10BOW-12 RS CAC H 10BWPB-21 R CGC R (Arginine) 10BWPB-22 R CGC R 10BWPB-25 R CGC R 10BWPB-27 R CGC R 10BWPB-30 R CGC R 10BOW-5 R CGC R 10BOW-14 R CGC R 10BOW-15 R CGC R 10BOW-16 R CGC R 10BOW-17 R CGC R Table 1. Codon and amino acid residue in the third cysteine-rich cluster of the SDHB gene of 33 B. jaapii isolates grouped by boscalid sensitivity groups. Amino acid residues were changed from a histidine to arginine for all resistant isolates sequenced.

68 DISCUSSION

The management of CLS in commercial tart cherry orchards in Michigan relies upon numerous applications of site specific fungicides due to the high susceptibility of the dominate tart cherry cultivar, Montmorency to CLS. This trend is expected to continue as the Montmorency cultivar is likely to remain the standard used by the industry in the near future. Due to its high sensitivity to CLS, fungicides will continue to be an essential part of any tart cherry management program in Michigan.

The number of effective fungicides available to growers for the control of CLS has decreased over the years. Newer fungicides have been released but at a much slower rate and the higher prices associated with these newer products will limit there use by growers. This limitation on the number of available modes of action greatly increases the risk of developing resistance to many of these commonly used fungicides which include

Pristine. To prolong the effectiveness of these fungicides strict resistance monitoring utilizing in vitro assays as well as molecular techniques need to be implemented. The development of these assays will help to identify where resistance populations exists and give growers in season information for CLS management.

Fungicide trials conducted at the NWMHRC since the introduction of Pristine in

Michigan indicated a reduction in its efficacy for CLS control 10 years after it was first evaluated (Figure 1). Practical resistance has been observed in these field trials and is suspected in select commercial orchards that contained a high percentage of resistant isolates as confirmed by the in vitro boscalid sensitivity assay. Practical resistance is said to occur when the frequency of resistant isolates is high enough in any given population

69 to result in disease control that is not commercially adequate (49). In the 2012 the levels of CLS infection and defoliation was at a level that was not commercially acceptable. If an entire commercial orchard had this level of defoliation and infection the quality of the crop would be severely impacted and there would be a high probability that some of those trees would not survive the winter. The presence of practical resistance in a field trial plot that is constantly rotated with different classes of fungicides indicates the intense selection pressure Pristine has on B. jaapii populations.

The current study has established a baseline sensitivity of non-treated CLS isolates as well as a sensitivity profile of Michigan commercial orchard CLS isolates to the SDHI, boscalid. Non-treated B. jaapii isolates collected from abandoned orchards and homeowner plantings where Pristine had never been applied were extremely sensitive to boscalid. The absence of reduced sensitivity or resistant isolates in non-treated populations of B. jaapii may indicate that B. jaapii populations are naturally sensitive to boscalid and that isolates resistant to boscalid did not exist in these populations before the introduction of Pristine. The baseline sensitivity provides an indication of the natural sensitivity of B. jaapii to boscalid which allows for the determination of the levels of boscalid sensitivity that constitutes boscalid resistance. The population distributions from these non-treated isolates have helped to evaluate the existence of boscalid resistant populations of B. jaapii in commercial orchards.

Population distributions comparing the sensitivity of B. jaapii to boscalid in orchards where Pristine had been used extensively to non-treated B. jaapii populations show a dramatic shift of orchard populations to boscalid resistance. Orchards classified as resistant did not contain a high proportion of isolates classified as being reduced in

70 bosciald sensitivity indicating that the majority the B. jaapii population in those orchards would no longer be affected by the boscalid component of Pristine. Field trials have indicated that the strobilurin component of pristine (pyraclostrobin) is not highly effective at controlling CLS. Therefore it is likely that the continued use of Pristine in these orchards will continue to select for boscalid resistance. If the proportion of resistant

B. jaapii isolates in the orchard population is substantially higher than the proportion of

B. jaapii isolates sensitive to boscalid practical resistance may occur.

Orchards classified as being reduced sensitive had a wide range of sensitivities to boscalid but did not contain a high percentage of sensitive isolates. These orchards did however contain a small percentage of boscalid resistant B. jaapii isolates. This may indicate that B. jaapii isolates in these orchards have shifted but not to the extent that is evident in populations of orchards classified as resistant. Orchards with a reduced sensitivity profile do not contain populations with a high percentage of boscalid resistant isolates and do not appear to be experiencing practical resistance to Pristine. This lack of a high percentage of boscalid resistant isolates and the lack of practical resistance may be due to the different fungicide rotations that are utilized in these orchards. If Pristine is used during the season followed by a different mode of action, boscalid resistant isolates that were selected for by the Pristine application could be controlled by these different modes of action which would subsequently reduce the frequency of boscalid resistant isolates in that population. Although B. jaapii populations in these reduced sensitive orchards appear to be controlled by Pristine the existence of boscalid resistant isolates in these populations have the potential to quickly and dramatically change the distribution of those orchard populations following additional Pristine applications.

71 Resistance to boscalid of B. jaapii isolates has been correlated with a single point mutation in the third cysteine-rich cluster of the SDHB subunit of the succinate dehydrogenase gene. The amino acid change from a histidine to an arginine was only detected in B. jaapii isolates that were classified as resistant in the in vitro boscalid sensitivity assay. This mutation was not detected in B. jaapii isolates that were classified as sensitive or reduced sensitive in the in vitro boscalid sensitivity assay. The detection of this mutation only in B. jaapii isolates classified as boscalid resistant provides validation to the results and sensitivity groupings obtained in the in vitro boscalid sensitivity assay.

Boscalid resistance has also been associated with point mutations in additional regions of the SDHB gene. In the second cysteine-rich region of the SDHB gene an amino acid change in a proline residue that is replaced by phenylalanine or leucine has been associated with boscalid resistance (6). The current study compared sequences of the second cysteine-rich cluster of B. jaapii isolates from each sensitivity group to detect any potential mutations. Amino acid changes to this proline residue were not detected in any of the three sensitivity groups indicating that the histidine amino acid change in B. jaapii isolates is the major mutation responsible for conferring boscalid resistance.

In the current study the histidine to arginine mutation was detected in boscalid resistant isolates but additional mutations in the SDHC and SDHD subunits are also known to confer varying levels of resistance to boscalid (6, 7). Mutations in these regions could explain the group of commercial B. jaapii isolates that were characterized as having a reduced sensitivity to boscalid but did not contain the histidine to arginine amino acid change. To locate additional mutations in the SDH gene of B. jaapii

72 amplification and subsequent sequencing of the SDHA SDHC and SDHD subunits will be conducted.

It is important to evaluate and characterize boscalid resistance in Michigan populations of B. jaapii due to the lack of novel fungicide compounds that can be utilized to control CLS. Growers can no longer utilize the same fungicide every year and expect the same levels of control even if that fungicide is a premix such as Pristine. The DMI class of fungicides that were once an important tool in CLS management provide a great example of the reliance of the tart cherry industry on a continual stream of new fungicides to manage changing B. jaapii populations. When the DMIs started to decline in effectiveness for CLS control in the majority of Michigan orchards there was a new fungicide, Pristine, to fill in the gap left by the loss of DMIs. Currently Pristine is in the same decline as the DMIs but the replacement fungicides, fluxapyroxad and fluopyram, do not possess a novel mode of action. These new generation SDHIs utilize the same mode of action as boscalid. The new SDHIs fluopyram and fluxapyroxad have been developed and registered in Michigan for CLS control and due to their single-site mode of action it is likely that these compounds are at a high risk of selecting for resistance in

B. jaapii populations. Currently there have not been claims of cross resistance between boscalid and these newer generation SDHIs in other fungal species (34, 86, 9). To date cross resistance between boscalid, fluopyram and fluxapyroxad has not been assessed for

2010 and 2011 populations of B. jaapii . The current study will provide a collection of B. jaapii isolates that have not been exposed to these new SDHI compounds. This collection of isolates with varying levels of boscalid sensitivity will be useful for the evaluation of cross resistance to the new generation of SDHIs. These new SDHI fungicides may play a

73 crucial role in controlling CLS populations that are resistant to boscalid in Michigan but cross resistance relationships need to be evaluated for these compounds before they are extensively used as a replacement for Pristine.

To avoid the complete loss of Pristine as a control option for CLS and putting these new SDHIs at risk for resistance it is important to identify orchards where boscalid resistance exist and limit the use of Pristine in those particular orchards. This study was able to provide information on which orchards contained high levels of B. jaapii isolates resistant to boscalid. Since different orchards have varying populations of B. jaapii with different boscalid sensitivities it is important to identify orchards that are at a high risk for the occurrence of practical resistance to Pristine and tailor a management program that fits that particular population. In orchards where a high percentage of resistant isolates exist Pristine would not be recommended for CLS control.

In orchards where resistance to boscalid has led to the reduction in efficacy of

Pristine for CLS control additional classes of fungicides such as copper, dodine, strobilurins and the SDHIs, fluopyram and fluxapyroxad, can be tank mixed with multisite inhibitors such as captan. CLS management programs utilizing the multi-site inhibitor, chlorothalanil, which is highly effective in CLS control could be implemented if regulations regarding its use in tart cherries are loosened. Currently chlorothalanil can only be used up to the phonological stage of shuck split in tart cherry due to concerns with residues on harvested fruit. In recent years there has been a push to allow chlorothalanil to be used past shuck split since harvesting of tart cherries occurs in tanks of water and these tanks are rinsed for a lengthy period of time before delivery to the processor limiting potential residues on the fruit. A section 24(c) special local need

74 registration was issued by the U.S. EPA for use of Bravo Weather Stik (chlorothalanil) on mechanically harvested tart cherries that are used for processing (61). This local need registration is only a temporary solution that can only be utilized in Michigan but there is the possibility that the label will be permanently changed allowing growers to utilize this effective fungicide for CLS control. The availability of a multisite inhibitor that can be used full season along with monitoring of Michigan B. jaapii populations will greatly help to reduce boscalid resistant isolates in orchards and may allow Pristine to again be part of a fungicide rotation for CLS control.

Resistance to boscalid in Michigan commercial orchard populations of B. jaapii has been detected and associated with a point mutation in the SDHB gene. This survey shows that boscalid resistance is widespread across the tart cherry producing regions of

Michigan but this resistance has not led to practical field resistance to Pristine in all areas where Pristine is utilized in CLS management programs. Pristine can still be utilized for

CLS control in Michigan but careful considerations made by growers when determining when are where it is appropriate to utilize this product. The presence of boscalid resistant

B. jaapii isolates in a majority of Michigan tart cherry orchards indicate that the potential for developing practical resistance is high and has the potential to occur rapidly with continued use of Pristine. Additional surveys will be required to determine if boscalid resistant populations of B. jaapii will continue to persist in orchards and what affect these populations will have on the effectiveness of the new SDHIs in controlling CLS.

75

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76. Skinner, W., Bailey, A., Renwick, A., Keon, J., Gurr, S., and Hargreaves, J. 1998. A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola . Curr. Genet. 34:393-398.

77. Spiegel, J., and Stammler, G. 2006. Baseline sensitivity of Monilinia laxa and M. fructigena to pyraclostrobin and boscalid. J. Plant Dis. Protect. 113:199-206.

78. Stammler, G., and Speakman, J. 2006. Microtiter method to test the sensitivity of Botrytis cinerea to boscalid. J. Phytopathol . 154:508-510.

79. Stammler, G., Brix, H. D., Nave, B., Gold, R., and Schoefl, U. 2008. Studies on the biological performance of boscalid and its mode of action. Pages 45-51 in: Modern Fungicides and Antifungal Compounds V. H. W. Dehne, H. B. Deising, U. Gisi, P. E. Russell, and H. Lyr, eds. DPG, Braunschweig, Germany.

80. Sun, F., Huo, X., and Zhai, Y. 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121:1043–57.

81. Sundin, G. W., Ehret, G. R., Nugent, J. E., Klein, W. M., and Anderson, M. D. 2003. Evaluation of new fungicides on Montmorency tart cherries for leaf spot control, 2003. Fungicide and Nematicide Tests 59:STF001.

83 82. Sundin, G. W., Ehret, G. R., Proffer, T. J., Nugent, J. E., Klein, W. M., Anderson, M. D., and Rothwell, N. L. 2005. Evaluation of new fungicides rotations for resistance management on Montmorency tart cherries in NW Michigan, 2005. Fungicide and Nematicide Tests 61:STF004.

83. Sundin, G. W., Ehret, G. R., Rothwell, N. L., Klein, W. M., Lizotte, E. M., and Anderson, M. D. 2011. Control of cherry leaf spot, powdery mildew, and brown rot with new fungicides in NW Michigan, 2010. Plant Dis. Manag. Rep 5:STF010.

84. Szeto, S. S. W., Reinke, S. N., Sykes, B. D., and Lemire, B. D. 2007. Ubiquinonebinding site mutations in the Saccharomyces cerevisiae succinate dehydrogenase generate superoxide and lead to the accumulation of succinate. Journal of Biological Chemistry 37(Suppl. 282):27518–26.

85. Ulrich, J. T., and Mathre, D. E. 1972. Mode of action of oxathiin systemic fungicides. V. Effect on electron transport system of Ustilago maydis and Saccharomyces cerevisiae . J. Bacteriol. 110:628-632.

86. Veloukas, T., and Karaoglanidis, G. S. 2012. Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity. Pest Manage. Sci. 68:858-864.

87. Veloukas, T., Leroch, M., Hahn, M., and Karaoglanidis, G. S. 2011. Detection and molecular characterization of boscalid-resistant Botrytis cinerea isolates from strawberry. Plant Dis. 95:1302-1307.

88. Wharton, P. S., Iezzoni, A. and Jones, A. L. Screening Cherry Germ Plasm for Resistance to Leaf Spot. Plant Disease 87:471-77.

89. Yanase, Y., Yoshikawa, Y., Kishi, J. and Katsuta, H. 2007. The history of complex II inhibitors and the discovery of penthiopyrad. Pesticide Chemistry . Crop Protection, Public Health, Environmental Safety. 295–303.

90. Yin, Y. N., Kim, Y. K., and Xiao, C. L. 2011. Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple. Phytopathology 101:986-995.

91. Zhang, C. Q., Yuan, S. K., Sun, H. Y., Qi, Z. Q., Zhou, M. G., and Zhu, G. N. 2007. Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid. Plant Pathol. 56:646-653.

84

APPENDIX

85 INTRODUCTION

The new SDHIs fluopyram and fluxapyroxad have been developed and registered in Michigan for CLS control. Due to their single-site mode of action, it is likely that these compounds are at a high risk of selecting for resistance in B. jaapii populations. Currently there have not been claims of cross resistance between boscalid and these newer generation SDHIs in other fungal species (1, 2, 3). To date cross resistance between boscalid, fluopyram and fluxapyroxad has not been assessed for 2011 populations of B. jaapii . The current study will evaluate the sensitivity of B. jaapii isolates to these new

SDHI compounds. An additional objective of this study is to evaluate the change in population distributions of boscalid resistant isolates throughout the growing season when treated with different SDHI fungicides in field trials at the Northwest Michigan

Horticultural Research Center. These new SDHI fungicides may play a crucial role in controlling CLS populations that are resistant to boscalid in Michigan but cross resistance relationships need to be evaluated for these compounds before they are extensively used as a replacement for Pristine.

B. jaapii fluopyram and fluxapyroxad sensitivity

A collection of isolates from non-boscalid treated, reduced boscalid sensitivity, and boscalid resistant orchards were chosen for the fluopyram and fluxapyroxad cross- resistance assay. Eight isolates from a non-boscalid treated site, 11LIBJ-MR, 20 isolates from orchards with a reduced sensitivity to boscalid, 11BL-CBC, and 50 isolates from boscalid resistant orchards were used in the fluopyram sensitivity assay. Nine isolates

86 from a non-boscalid treated site, 10PEBJ, 29 isolates from orchards with a reduced sensitivity to boscalid, 11BA-S, and 49 isolates from boscalid resistant orchards were used in the fluxapyroxad sensitivity assay.

The in vitro sensitivity of B. jaapii to the boscalid, fluopyram and fluxapyroxad was assessed using a mycelial growth assay. The fungicides used in this study were the technical formulations of fluopyram (Bayer) and fluxapyroxad (BASF). To prepare stock solutions of fluopyram and fluxapyroxad were dissolved in 100% acetone and adjusted to

-1 -1 -1 concentrations of 100 mg ml , 10 mg ml and 5 mg ml . Sterilized MMEA cooled to

40 ºC was amended with these stock solutions of boscalid at concentrations of 0.1, 0.5, 1,

-1 2.5, 5, 10, and 25 µg ml . Control media consisted of MMEA amended with the highest concentration of acetone that was added to the fungicide amended media. Mycelial plugs roughly 1mm in diameter were taken from actively growing cultures using a Pasteur pipette and transferred to the fungicide amended plates. Two replicates of each isolate were used in this study. Isolates were incubated at 23-25 ºC for 14 days at which point the growth of each isolate was evaluated and a Minimum Inhibitory Concentration (MIC) determined. The MIC of an isolate is defined as the minimum concentration of a.i. required to fully inhibit the growth of an isolate. The frequency distribution of MIC from each orchard assayed for fluopyram and fluxapyroxad sensitivity were compared to MIC frequency distributions of those same orchards from the boscalid sensitivity assay.

Results from the fluopyram study indicate that that there is a reduction in the sensitivity of B. jaapii isolates to fluopyram from the commercial orchards when compared to the sensitivity of non-treated isolates. Of the isolates from the boscalid

87 -1 resistant orchard 48% were not inhibited at > 25 µg ml of fluopyram (Figure 1). For the orchard that exhibited a reduction in sensitivity to boscalid 10% of isolates were not

-1 inhibited at > 25 µg ml of fluopyram (Figure 1). In comparisons between boscalid sensitivities and fluopyram sensitivities of non-treated isolates it appears that B. jaapii is less sensitive to fluopyram in vitro (Figure 1, 2).

100 90

80 11LIBJ-MR 70 60

50 11BL-CBC 40 30 20 Frequency Distribution % Frequency Distribution 10 11BO-GW 0 0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration

(Fluopyram)ug/ml

Figure A1. Fluopyram sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites previously classified as boscalid sensitive had an Minimum -1 Inhibitory Concentration of 0.5 µg ml and are identified by white bars. The orchard previously classified as boscalid reduced sensitivity had an average Minimum Inhibitory -1 Concentration of 10 µg ml and are identified by hatched bars. The orchard previously -1 classified as resistant had an average Minimum Inhibitory Concentration of > 25 µg ml and are identified by black bars.

88

100 90

80 11LIBJ-MR 70

60 50 40 11BL-CBC 30

20 Frequency Distribution % Frequency Distribution 10 11BO-GW 0 0.1 0.5 1 2.5 5 10 25 > 25

Minimum Inhibitory Concentration (Boscalid)µg/ml

Figure A2. Boscalid sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites classified as sensitive had an average Minimum Inhibitory -1 Concentration of 0.1 µg ml and are identified by white bars. The orchard classified -1 as reduced sensitivity had an average Minimum Inhibitory Concentration of 5 µg ml and are identified by hatched bars. The orchard classified as resistant had an average -1 Minimum Inhibitory Concentration of > 25 µg ml and are identified by black bars.

89 Initial results from the fluopyram sensitivity assay suggest that B. jaapii populations are inherently less sensitive to the rates of fluopyram used in this study when compared to boscalid sensitivities at the same rates. It has been shown with boscalid resistant isolates of Alternaria alternata that the amino acid mutation in the SDHB subunit from a histidine to a arginine conferring resistance to boscalid may also confer low levels of resistance to fluopyram while the histidine to tyrosine mutation in the same

SDHB location only confers high levels of boscalid resistance and no cross-resistance to fluopyram. This may be what is occurring in this situation as the only amino change detected in boscalid resistant isolates is the histidine to arginine mutation. Sequencing of the entire SDH gene for isolates that in this study appear resistant to both fluopyram and boscalid will provide insight into this potential cross-resistance situation.

Results from the fluxapyroxad study shows that that there was not a dramatic reduction in the sensitivity of B. jaapii isolates to fluxapyroxad from the commercial orchards when compared to the sensitivity of non-treated isolates. Of the isolates from the

-1 boscalid resistant orchard only 8% were not inhibited at > 25 µg ml of fluxapyroxad

(Figure 3). For the orchard that exhibited a reduction in sensitivity to boscalid, 0% of

-1 isolates were not inhibited at > 25 µg ml of fluxapyroxad (Figure 3). However when looking at the MIC distributions of the boscalid resistant orchard 0% of isolates are

-1 inhibited at a concentration of ≤ 2.5 µg ml of fluxapyroxad (Figure 3). In comparisons between boscalid sensitivities and fluxapyroxad sensitivities of non-treated isolates it appears that non-treated B. jaapii populations are equally sensitive to fluxapyroxad in vitro (Figure 3, 4).

90

100 90 80 10PEBJ 70 60 50 11BA-S 40 30 11BO-W 20

Frequency Distribution % Frequency Distribution 10 0 0.1 0.5 1 2.5 5 10 25 > 25

Minimum Inhibitory Concentration (Fluxapyroxad)ug/ml

Figure A3. Fluxapyroxad sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites previously classified as boscalid sensitive had an average -1 Minimum Inhibitory Concentration of 0.1 µg ml and are identified by white bars. The orchard previously classified as boscalid reduced sensitivity had an average Minimum -1 Inhibitory Concentration of 2.5 µg ml and are identified by hatched bars. The orchard previously classified as boscalid resistant had an average Minimum Inhibitory -1 Concentration of 25 µg ml and are identified by black bars.

91

100 90

80 70 10PEBJ 60 50 11BA-S 40 30

20 11BO-W

Frequency Distribution % Frequency Distribution 10

0 0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration

(Boscalid)ug/ml

Figure A4. Boscalid sensitivity distribution of non-treated and commercial B. jaapii isolates. Non-treated sites classified as sensitive had an average Minimum Inhibitory -1 Concentration of 0.1 µg ml and are identified by white bars. The orchard classified as -1 reduced sensitivity had an average Minimum Inhibitory Concentration of 5 µg ml and are identified by hatched bars. The orchard classified as resistant had an average -1 Minimum Inhibitory Concentration of > 25 µg ml and are identified by black bars.

These initial results suggest that there might be cross-resistance between boscalid and fluxapyroxad but the percentage of isolates resistance to both of these materials is relatively low in orchard populations. These sensitivity tests were conducted with isolates collected in 2010 and 2011 and would not have been exposed to fluxapyroxad since it was not released in Michigan until 2013. The appearance of isolates with a reduction in sensitivity to fluxapyroxad before it had been used in commercial orchards may suggest that a low level of cross resistant isolates exist in these orchards. If cross-resistance does

92 exist applications of fluxapyroxad will quickly select for isolates resistant to this product and lead to the increase of the proportion of fluxapyroxad resistant isolates in these orchards. It will be important to monitor these sites and also establish the molecular mode of resistance to fluxapyroxad to better understand how B. jaapii populations will evolve to this new SDHI.

Season long sensitivity of B. jaapii to boscalid

Yearly spray trial programs at the Northwest Michigan Horticultural Research

Center (NWMHRC) near Traverse City, Michigan test the effectiveness of current commercially utilized fungicides as well as experimental fungicides at varying stages of development. Fungicides were tested either in a standard program that includes a Bravo application at the beginning of the year or full season where the fungicide of interest is used throughout the spray trial. Experiments were set up in a randomized complete block design with single tree plots and four replications for each fungicide treatment. CLS ratings were conducted at harvest and approximately 4-6 weeks later. Disease rating involved examination of 20 terminals on each tree for disease incidence and defoliation caused by CLS. In 2012 fungicides evaluated in the research plot included Merivon, Luna

Sensation, Pristine, Bravo, and Gem. Merivon is a premix of the SDHI, fluxapyroxad, and a strobilurin, Luna Sensation is a premix of the SDHI, fluopyram, and a strobilurin,

Pristine is a premix of the SDHI, boscalid, and a strobilurin, Bravo is a multisite inhibitor, chlorothalanil, and Gem is a strobilurin.

This study involved sampling B. jaapii populations from the different fungicide treatments at three different time points during the tart cherry growing season. The

93 objective of this study was to determine if applications of these different SDHI fungicides would change the distribution of boscalid resistant B. jaapii isolates across the three sampling dates. Six B. jaapii infected leaf samples from each of the four replications of

th rd each treatment were collected on June 29 , August 1st and August 23 . Isolates originating from a single spore obtained from sampled leaves were evaluated for their sensitivity to boscalid. The distributions of minimum inhibitory concentration proportions were plotted for each of the fungicides tested from the three sampling times.

For the Merivon treatment the first sample had 10% of isolates that were not

-1 inhibited at 25 µg ml , the second sample had 33% of isolates that were not inhibited at

-1 -1 25 µg ml and the third sample had 79% of isolates that were not inhibited at 25 µg ml

(Figure 5). Results suggest the Merivon treatment selected for boscalid resistant isolates in these populations. The highest percentage of isolates in the first sampling had a

-1 boscalid MIC of 2.5 µg ml while in the last sampling the highest percentage of isolates

-1 had a boscalid MIC > 25 µg ml (Figure 5.) These results indicate that Merivon was selecting for boscalid resistant CLS isolates and controlling boscalid sensitive isolates even without the application of Pristine.

94

100

90 80 June 29 70

60 50 August 1

40

30 August 23 20 Frequency Distribution % Frequency Distribution 10 0

0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration µg/ml

Figure A5. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified -1 by the white bars had the highest percentage of isolates in the 2.5 µg ml group. The second sample identified by the hatched bars had the highest percentage of -1 isolates in the 25 µg ml group. The third sample identified by the black bars had -1 the highest percentage of isolates in the > 25 µg ml group. The percent of -1 isolates in the > 25 µg ml MIC group had the most dramatic increase over the

sampling period.

For the Luna Sensation treatment the first sample had 25% of isolates that were

-1 not inhibited at 25 µg ml , the second sample had 8% of isolates that were not inhibited

95 -1 at 25 µg ml and the third sample had 33% of isolates that were not inhibited at 25 µg

-1 ml (Figure 6). Results suggest the Luna Sensation treatment may only be moderately selecting for boscalid resistant isolates in these populations. The highest percentage of

-1 isolates in the first sampling had a boscalid MIC of 5 µg ml while in the last sampling

-1 the highest percentage of isolates had a boscalid MIC of 25 and > 25 µg ml (Figure 6).

These results indicate that Luna Sensation is selecting for boscalid resistant CLS isolates but at a much less dramatic rate than Merivon.

96

100

90 80 June 29

70

60

50 August 1 40

30

Frequency Distribution % Frequency Distribution 20 August 23 10

0 0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration µg/ml

Figure A6. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars -1 had the highest percentage of isolates in the 5 µg ml MIC group. The second sample identified by the hatched bars had the highest percentage of isolates in the -1 2.5 µg ml MIC group. The third sample identified by the black bars had the highest -1 percentage of isolates in the 25 and > 25 µg ml MIC groups. During the sampling -1 period the highest percentage of isolates shifted from the 5 µg ml groups to the 25 -1 and > 25 µg ml MIC group.

For the Pristine treatment the first sample had about 8% of isolates that were not

-1 inhibited at 25 µg ml , the second sample had 25% of isolates that were not inhibited at

-1 -1 25 µg ml and the third sample had 54% of isolates that were not inhibited at 25 µg ml

97 (Figure 7). Results suggest the Pristine treatment selected for boscalid resistant isolates in these populations. The highest percentage of isolates in the first sampling had a boscalid

-1 MIC of 2.5 µg ml while in the last sampling the highest percentage of isolates had a

-1 boscalid MIC > 25 µg ml (Figure 7). These results indicate that Pristine is selecting for boscalid resistant CLS isolates and controlling boscalid sensitive isolates. This distribution indicates the rapid selection pressure that can occur if Pristine is used in B. jaapii populations that have a small percentage of boscalid resistant isolates present in the population.

98

100

90 80 June 29 70

60 50 August 1

40

30

Frequency Distribution % Frequency Distribution 20 August 23 10

0 0.1 0.5 1 2.5 5 10 25 > 25

Minimum Inhibitory Concentration µg/ml Figure A7. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified -1 by the white bars had the highest percentage of isolates in the 2.5 µg ml MIC group. The second sample identified by the hatched bars had the highest -1 percentage of isolates in the 2.5 µg ml MIC group. The third sample identified -1 by the black bars had the highest percentage of isolates in the > 25 µg ml MIC group. During the sampling period the highest percentage of isolates shifted from -1 -1 the 2.5 µg ml group to the > 25 µg ml MIC group.

99 For the non-treatment control the first sample had about 17% of isolates that were

-1 not inhibited at 25 µg ml , the second sample had 0% of isolates that were not inhibited

-1 - at 25 µg ml and the third sample had 0% of isolates that were not inhibited at 25 µg ml

1 (Figure 8). Results suggest the without the selection pressure of a fungicide treatment, boscalid resistant isolates will not be selected for in these populations. The highest percentage of isolates in the first sampling and the third sampling had boscalid MIC

-1 values of 2.5 µg ml (Figure 8). These results indicate that under low selection pressure the percentage of boscalid resistance can actually decrease during the growing season.

100

100 90

80 June 29

70 60

50 August 1

40 30

Frequency Distribution % Frequency Distribution 20 August 23 10

0 0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration µg/ml

Figure A8. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars -1 had the highest percentage of isolates in the 2.5 µg ml MIC group. The second sample identified by the hatched bars had the highest percentage of isolates in the 5 -1 µg ml MIC group. The third sample identified by the black bars had the highest -1 percentage of isolates in the 2.5 µg ml MIC group. During the sampling period the -1 -1 highest percentage of isolates remained in the 2.5 µg ml to 5 µg ml MIC groups. -1 By the third sample 0% of isolates were in the > 25 µg ml MIC group.

For the Bravo treatment the first sample had about 8% of isolates that were not

-1 inhibited at 25 µg ml , the second sample had 4% of isolates that were not inhibited at

-1 -1 25 µg ml and the third sample had 12% of isolates that were not inhibited at 25 µg ml

101 (Figure 9). Results suggest the Bravo treatment did not heavily select for boscalid resistant isolates in these populations. The highest percentage of isolates in the first

-1 sampling had a boscalid MIC of 2.5 µg ml while in the last sampling the highest

-1 percentage of isolates had a boscalid MIC 5 µg ml (Figure 9). These results indicate that Bravo was able to maintain relatively the same distribution of boscalid sensitivities throughout the three sampling times.

102

100

90 June 29 80 70

60

50 August 1 40 30

Frequency Distribution % Frequency Distribution 20 August 23 10

0 0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration µg/ml Figure A9. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by the white bars had the highest percentage of isolates in the -1 2.5 µg ml MIC group. The second sample identified by the hatched bars had -1 the highest percentage of isolates in the 2.5 µg ml MIC group. The third sample identified by the black bars had the highest percentage of isolates in the 5 -1 µg ml MIC group. During the sampling period the highest percentage of -1 -1 isolates remained in the 2.5 µg ml to 5 µg ml MIC groups. Throughout the -1 sampling period a small percentage of isolates remained in the > 25 µg ml MIC group.

For the Gem treatment the first sample had 0% of isolates that were not inhibited

-1 -1 at 25 µg ml , the second sample had 4% of isolates that were not inhibited at 25 µg ml

103 -1 and the third sample had 12% of isolates that were not inhibited at 25 µg ml (Figure

10). Results suggest the Gem treatment did not have a high selection for boscalid resistant isolates in these populations. The highest percentage of isolates in the first

-1 sampling had a boscalid MIC of 2.5 µg ml while in the last sampling the highest

-1 percentage of isolates had a boscalid 5 µg ml (Figure 10). These results indicate that

Gem like the Bravo treatment does not have a large on the distribution of boscalid resistant isolates in a population.

104

100 90

80 June 29

70 60 50 August 1

40 30

Frequency Distribution % Frequency Distribution 20 August 23

10

0 0.1 0.5 1 2.5 5 10 25 > 25 Minimum Inhibitory Concentration µg/ml

Figure A10. Boscalid sensitivity distribution of B. jaapii isolates from three sampling dates in 2012, (June 29, August 1, and August 23), from a fungicide field trial plot at the NWMHRC. The frequency distributions of Minimum Inhibitory Concentrations from each sampling time were plotted. The first sample identified by -1 the white bars had the highest percentage of isolates in the 2.5 µg ml MIC group. The second sample identified by the hatched bars had the highest percentage of -1 isolates in the 5 µg ml MIC group. The third sample identified by the black bars -1 had the highest percentage of isolates in the 5 µg ml MIC group. During the -1 sampling period the highest percentage of isolates remained in the 2.5 µg ml to 5 -1 µg ml MIC groups. In the second and third sampling slightly increased in the > 25 -1 µg ml MIC group.

105 The results from this study help provide an understanding of the population structure of B. jaapii under the selection pressure of varying fungicides. The Pristine and

Merivon treatments had the greatest increase in the proportion of isolates that where not

-1 inhibited by the > 25 µg ml concentration of boscalid suggesting that the SDHI component in these two fungicides was selecting for boscalid resistant isolates. This selection for boscalid resistant isolates is understandable for the Pristine treatments as boscalid is the SDHI component of that fungicide but for Merivon the SDHI component is fluxapyroxad. Fluxapyroxad should not select for boscalid resistance unless there is some kind of cross-resistance relationship with boscalid. Luna Sensation did have some selection for boscalid resistance but this selection was more variable throughout the sampling period and was not as dramatic as the Merivon and Pristine treatments. The untreated control, Bravo, and Gem treatments did not select for boscalid resistance. For the untreated control there was a small percentage of the B. jaapii population that was not

-1 inhibited at the 25 µg ml level but this population decreased or was outweighed by boscalid sensitive isolates. The Bravo and Gem treatments did not select for high levels of boscalid resistant isolates and was able to maintain the majority of the population in

-1 the 2.5 to 5 µg ml boscalid sensitivity range throughout the sampling period. This suggests that when Pristine or another SDHI containing fungicide is not used during the season the frequency of boscalid resistant isolates may go down. If isolates that are sensitive to boscalid remain in the orchard their proportion in the overall population may increase when SDHIs are not used. Sensitivity to fluxapyroxad and fluopyram for this

106 study will be determined and should allow for the evaluation of the selection pressure of these products for resistance in B. jaapii populations.

107

LITERATURE CITED

108 LITERATURE CITED

1. Avenot, H. F., Thomas, A., Gitaitis, R. D., Langston, D. B., Jr., and Stevenson, K. L. 2012. Molecular characterization of boscalid and penthiopyrad resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram. Pest Manage. Sci. 68:645-651.

2. Ishii, H., Miyamoto, T., Ushio, S., and Kakishima, M, 2011. Lack of cross resistance to a novel succinate dehydrogenase inhibitor, fluopyram, in highly boscalid-resistant isolates of Corynespora cassiicola and Podosphaera xanthii . Pest Manage. Sci. 67:474-482.

3. Veloukas, T., and Karaoglanidis, G. S. 2012. Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity. Pest Manage. Sci. 68:858-864.

109