University of Florida Thesis Or Dissertation Formatting

FUNGICIDE RESISTANCE PROFILE OF QUIESCENT Botrytis cinerea INFECTIONS ON STRAWBERRY TRANSPLANTS

MICHELLE SOUZA OLIVEIRA

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2015

To my family and my fiancé, Leandro

ACKNOWLEDGMENTS

First I would like to thank God, my creator, who is always with me and strengthen me in my most difficult times. “Fear not, I am with you; be not dismayed; I am your God.

I will strengthen you, and help you, and uphold you with my right hand of justice” (Isaiah

41:10).

I am sincere thankful to Dr. Natalia Peres, my advisor, for giving me the opportunity to develop such interesting and important work, believing in my work and for all her guidance and teachings in plant pathology, research and extension. I would like to gratefully thank my committee members, Dr. Nicholas Dufault and Dr. Philip F.

Harmon, for their support with laboratory space while I was in Gainesville and for all their valuable suggestions to my work. I thank all the lab members and interns of the

Strawberry Pathology lab at Gulf Coast Research and Education Center for their teamwork and the help with some evaluations. I thank my family that besides the physical distance are always present in my life achievements, and I gratefully thank my fiancé Leandro for his support and for all his encouragement, patience and unconditional love. And after all the difficulties I’m proud to say I always believed in my dreams and that’s what made me have my accomplishments. “It’s the possibility of having a dream come true that makes life interesting” (Paulo Coelho)

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 9

ABSTRACT ...... 11

CHAPTER

1 LITERATURE REVIEW ...... 13

Strawberry Production ...... 13 Nursery Transplants...... 15 Botrytis cinerea ...... 18 Epidemiology and Life Cycle ...... 18 Symptoms and Signs ...... 19 Disease Control ...... 20 Chemical Control ...... 21 Fungicide Resistance ...... 22 Objectives ...... 26

2 TRANSPLANTS AS A SOURCE OF INFECTION OF Botrytis cinerea TO STRAWBERRY FIELDS IN FLORIDA ...... 30

Introduction ...... 30 Materials and Methods...... 32 Fungal Isolates ...... 32 Fungicide Sensitivity Tests ...... 33 Results ...... 35 Frequency of Infection ...... 35 Fungicide Sensitivity Tests ...... 36 Discussion ...... 41

3 OPTIMIZATION OF A RESAZURIN-BASED ASSAY TO EVALUATE SENSITIVITY OF Botrytis cinerea TO RESPIRATION-INHIBITOR FUNGICIDES ...... 60

Introduction ...... 60 Materials and Methods...... 62 Fungal Isolates ...... 62 Pathogenicity Tests ...... 63 Effect of Conidial Concentration on Resazurin Reduction ...... 63 Evaluation of the Best Medium for the Reduction of Resazurin...... 64

Time of Evaluation of the Microplates and its Effect on Resazurin Reduction .. 65 Effect of SHAM and DMSO on the Respiration of Botrytis cinerea ...... 65 Resazurin Reduction Calculations ...... 66 Conidial Germination Test ...... 67 Results ...... 68 Pathogenicity Tests ...... 68 Effect of Conidial Concentration on Resazurin Reduction ...... 68 Evaluation of the Best Medium for the Reduction of Resazurin...... 69 Time of Evaluation of the Microplates and its Effect on Resazurin Reduction .. 70 Effect of SHAM and DMSO on the Respiration of Botrytis cinerea ...... 70 Conidial Germination Test ...... 70 Discussion ...... 72

4 CONCLUSIONS ...... 88

LIST OF REFERENCES ...... 90

BIOGRAPHICAL SKETCH ...... 100

LIST OF TABLES

Table page

2-1 Frequency of strawberry transplants from different nurseries infected with Botrytis cinerea in 2011, 2012 and 2013...... 55

2-2 Analysis of variance of the frequency of strawberry transplants infected with Botrytis cinerea by nursery and region in 2011, 2012 and 2013...... 55

2-3 F-statistics and P-values for the fungicide inhibition of the mycelial growth of Botrytis cinerea isolates collected from fourteen nurseries in 2011, 2012, and 2013...... 56

2-4 Frequency of resistance of Botrytis cinerea isolates from nurseries to seven different fungicides tested in the mycelial growth assay...... 57

2-5 F-statistics and P-values for the fungicide inhibition of the conidial germination of Botrytis cinerea isolates collected from fourteen nurseries in 2011, 2012, and 2013...... 58

2-6 Frequency of resistance to seven different fungicides of Botrytis cinerea isolates from nurseries tested in the conidial germination assay...... 59

3-1 Botrytis cinerea isolates from strawberry nursery transplants and their sensitivity to boscalid and pyraclostrobin...... 83

3-2 Molar extinction coefficient for Resazurin (Invitrogen 2009)...... 83

3-3 The EC50 (μg/ml) values of Botrytis cinerea isolates in a resazurin reduction assay and the effect of salicylhydroxamic acid (SHAM) and dimethyl sulfoxide (DMSO) on pyraclostrobin-amended medium (complete medium)...... 83

3-4 The EC50 values (μg/ml) of sensitive Botrytis cinerea isolates in a resazurin- reduction assay and the effect of SHAM and DMSO on a pyraclostrobin- amended medium (HA at pH 6.5)...... 84

3-5 The EC50 values (μg/ml) of Botrytis cinerea isolates on boscalid and pyraclostrobin-amended medium comparing a resazurin reduction assay and the conidial germination test...... 84

3-6 Analysis of variation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in a conidial germination test and a resazurin reduction assay with boscalid- and pyraclostrobin-amended medium...... 85

3-7 Correlation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in a conidial germination test and resazurin reduction assay with boscalid- amended medium...... 85

3-8 Correlation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in conidial germination test and resazurin-reduction assay with pyraclostrobin- amended medium...... 85

3-9 Salicylhydroxamic acid (SHAM) and dimethyl sulfoxide (DMSO) effect on the EC50 values (μg/ml) of Botrytis cinerea isolates in conidial germination assay with pyraclostrobin-amended medium...... 86

3-10 Price, time and space required in three different methodologies used to evaluate sensitivity of eleven Botrytis cinerea isolates to one fungicide in eight fungicide concentrations...... 87

LIST OF FIGURES

Figure page

1-1 Strawberry plug transplant ...... 27

1-2 “Bare-root green-top” strawberry transplants ready to be planted on Florida production field...... 27

1-3 Production of certified strawberry planting stock in California ...... 28

1-4 Development of Botrytis gray mold diseases ...... 29

2-1 Potato dextrose agar (PDA) plates with Botrytis cinerea conidial suspensions and incubated inclined at room temperature for subsequent single-sporing...... 48

2-2 Senescent strawberry leaves incubated over a chicken wire, folded in a wave shape, inside clear plastic boxes for isolation of Botrytis cinerea...... 48

2-3 Botrytis cinerea conidiophores and conidia examined under a stereomicroscope (14x) ...... 48

2-4 Plug (4 mm diameter) taken from a 4-day-old Botrytis cinerea colony and transferred to a control potato dextrose agar plate...... 49

2-5 Measurement of Botrytis cinerea mycelial growth on fungicide-amended medium...... 49

2-6 A 15-cm diameter petri dish control plate with yeast bacto agar (YBA) medium seeded with a Botrytis cinerea conidial suspension...... 49

2-7 Evaluation of conidial germination of Botrytis cinerea under the microscope (100x)...... 50

2-8 Overall resistance frequency (%) of Botrytis cinerea to seven different fungicides isolated from nursery transplants in 2011, 2012, and 2013, and evaluated in mycelial growth assays...... 50

2-9 Overall resistance frequency (%) of Botrytis cinerea to seven different fungicides, isolated from nursery transplants in 2011, 2012, and 2013, and evaluated in conidial growth assays...... 51

2-10 Frequency of resistance of Botrytis cinerea isolates collected from nurseries to four different fungicides, determined by mycelial growth for fenhexamid and conidial germination for the other fungicides...... 52

2-11 Frequency of multi-fungicide resistance of Botrytis cinerea from strawberry transplants collected in 2011-2012 from different nurseries from California, Nova Scotia and Quebec...... 53

2-12 Frequency of multi-fungicide resistance of Botrytis cinerea from strawberry transplants collected in 2013 from different nurseries from California, North Carolina, Nova Scotia, Quebec and Ontario...... 54

3-1 Pink fruit harvested from the field, selected by size, and with the calix removed...... 78

3-2 Strawberry fruit in egg cartons ready to be inoculated. Each fruit was inoculated with one isolate and bottom right fruit was the control, treated with water...... 78

3-3 Resazurin reduction by four conidial concentrations of Botrytis cinerea, from 102 to 105 conidia/ml, on boscalid-amended medium...... 79

3-4 Resazurin reduction of Botrytis cinerea isolates (104 conidia/ml) on four liquid media, Complete Medium (CM), Minimal Medium (MM), HA at pH 5.5 and 6.5, amended with boscalid...... 80

3-5 EC50 (μg/ml) of sensitive Botrytis cinerea isolates in a resazurin assay with pyraclostrobin- and boscalid-amended media evaluated from 18 to 30h after preparation of the plates...... 81

3-6 The effect of salicylhydroxamic acid (SHAM) on pyraclostrobin sensitivity in a resazurin-reduction assay...... 82

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

FUNGICIDE RESISTANCE PROFILE OF QUIESCENT Botrytis cinerea INFECTIONS ON STRAWBERRY TRANSPLANTS

Michelle Souza Oliveira

December 2015

Chair: Natalia A. Peres Major: Plant Pathology

Strawberry transplants, produced in nurseries across Canada, the northern U.S., and California, are shipped annually to other strawberry-growing regions such as

Florida. Plants from the nursery carrying quiescent infections of Botrytis cinerea have been suggested as a potential primary inoculum source for the strawberry production fields. In this study, transplants from 14 nurseries located in Canada, North Carolina and California were investigated for the presence of B. cinerea. Overall, the frequency of infection varied from 22.0 to 36.7% in 2011, from 20.0 to 83.3% in 2012, and from 2.5 to 92.5% in 2013. In total, 416 B. cinerea isolates obtained from the nursery transplants were tested for their sensitivity to fenhexamid, pyrimethanil, pyraclostrobin, boscalid, fluopyram, penthiopyrad, and fludioxonil using discriminatory doses determined previously. The frequency of resistant isolates was very high (above 90%) for pyrimethanil and pyraclostrobin, high (above 60%) for boscalid, and very low (below

0.5%) for fluopyram, penthiopyrad, and fludioxonil. Also, multi-fungicide resistance of up to five fungicides was found from the nurseries. In a separate study, eleven isolates were selected to evaluate their sensitivity to boscalid and pyraclostrobin using an

adapted resazurin-based assay. Four liquid media (Complete Medium, Minimal Medium and HA Medium with pH 5.5 and 6.5), four conidial concentrations (from 102 to 105 conidia/ml), and eight fungicide concentrations (from 0 to 10 µg/ml for boscalid, and from 0 to 50 µg/ml for pyraclostrobin) were tested and evaluated from 16 to 32h after plate preparation. The HA medium at pH 6.5, 104 conidia/ml, and evaluation after 24h were the optimal conditions for resazurin reduction. The EC50 values for the sensitive isolates varied from 0.001 and 0.799 µg/ml in the conidial germination test for both fungicides and were highly correlated with the resazurin-based assay. Resistant isolates had EC50 values higher than 10 µg/ml and 50 µg/ml for boscalid and pyraclostrobin, respectively. The results reinforce the need for an integrated approach between strawberry nurseries and production fields for the management of B. cinerea and suggest that the resazurin assay can be used to evaluate the sensitivity of B. cinerea to respiration-inhibitor fungicides.

CHAPTER 1 LITERATURE REVIEW

Strawberry Production

Cultivated strawberry (Fragaria x ananassa Duch.) is a perennial herbaceous plant of the Rosaceae family that is a hybrid created in Europe from Fragaria virginiana

Duch. and F. chiloensis (L.) Duch, native from North and South America (Santos et al.

2012), respectively, and brought to the USA after 1850 (Maas 1998).

Strawberry is a crop grown worldwide in diverse environments. Production is determined by temperature and photoperiod; however, the heterozygosis of Fragaria spp. allowed the successful development of a large number of existing cultivars with different characteristics (Maas 1998).

World production of strawberries reached 7.7 million tons in 2013. The world’s largest producer, with approximately 3 million tons in 2013, is China, followed by the

US, Spain and Japan (FAOSTAT 2015). During the same period, the U.S. was responsible for the production of 1.36 thousand tons of strawberries, an increase of

16% since 2008 (FAOSTAT 2015), with a planted area of 24,000 hectares and an average yield of 65,000 kg per hectare (Agricultural Statistics 2014).

Florida produces more strawberries than any other state in the southeastern U.S.

The state is the second largest producer in the country, preceded only by California. In

2013, Florida was responsible for 6.23% of the total production of the country, having a planted area of 3,440 hectares with a yield of approximately 28,000 kg per hectare, amounting to US$ 267 million dollars (Agricultural Statistics 2014).

Even though strawberry is a perennial crop, it is grown as an annual in Florida, being planted every year in the production fields. Production in the state is concentrated

in Hillsborough County, around the Plant City area that is known as “the winter strawberry capital of the world” (Brown 2003). The main cultivars currently planted in

Florida are ‘Strawberry Festival’, ‘Florida Radiance’, ‘WinterstarTM’ (‘FL 05-107’), and

‘SensationTM’ (‘Florida127’), all developed at the University of Florida (GCREC)

(Whitaker et al. 2015).

Florida production starts with soil fumigation, using chemicals to suppress nematodes, weeds and soil-borne pathogens (Gilreath et al. 2006). The strawberry production system consists of raised beds covered with black plastic mulch, spaced 1.2 to 1.5 m apart, with staggered rows of holes in the top where the transplants are placed, spaced 30 to 40 cm apart and 30 to 35 cm between rows constituting 39,500 to 54,400 plants per hectare (Cordova et al. 2014; Whitaker et al. 2015). It is important to acquire healthy transplants with good vigor to start production. Transplants are obtained from nurseries in Canada and the northern U.S. Planting starts in mid-September through mid-October either with plug transplants (Figure 1-1), that require less overhead irrigation to establish and start the production earlier in the season; or mainly with bare- root green-top transplants (Figure 1-2), bearing leaves to improve plant adaptation and establishment in Florida weather (Hochmuth et al. 2006). After planting of bare-root transplants, overhead irrigation is used for 7 to 12 days to facilitate plant establishment; additional water and fertilizer is provided by drip irrigation (Santos et al. 2012). The first fruit harvest starts 40 to 60 days after planting and is continuous two to three times per week, with an average of 50 harvests per season in the same area. Production costs are US$35,610 per hectare, and yield ranges between 27,000 and 54,000 kg per

hectare (Brown 2003; Santos et al. 2012). The season usually ends in March, whenever the market is no longer profitable for Florida growers.

Nursery Transplants

Vegetative propagation of strawberry plants preserves the genetic characteristics of the original cultivar (Strand 2008) and is dependent on environmental factors. The longer the photoperiod and the higher the light intensity, the more runners that are produced. Runners are two-node horizontal stems that originate from buds at the leaf axils and form, at the distal node, new strawberry plants called “daughters” that will later produce their own runners (Darnell 2003). One mother plant is able to produce 100 or more daughter plants that survive independently two to three weeks after their production (Strand 2008). The transplants used in strawberry fruiting fields are usually the third generation of the transplants produced in nurseries from mother plants grown in northern latitudes above the 42nd parallel (i.e. Ontario, Quebec, and Nova Scotia) or in higher altitudes in North Carolina, which favors flower initiation in the crown (Brown

2003).

Strawberry nursery production starts with virus-free plants produced by micro- propagation of tissue culture known as foundation plants. At first, they are grown in growth chambers at 37oC for three weeks, then transferred to a mist chamber, and then to a sterile soil in a screen house to protect against insect vectors. In this first stage, a single meristem plant will produce 100 to 1500 clones that are planted in the field.

These foundation plants generate 200 to 300 other plants that are called registered, free of viruses and other diseases, and sold to other nurseries to initiate their production. In high-elevation nurseries, each registered mother plant will produce 20 to 30 daughter plants that are sold to fruit growers or certified for their cleanliness if sold to other

nurseries (Figure 1-3). These plants receive a chilling period of 200 to 400 h below 7oC in the field and initiate flower production in the buds which leads to acceleration of production in the fruiting fields (Strand 2008).

The transplants are planted in the strawberry nurseries in April or May (spring) in rows spaced 0.3 to 0.46 m between plants and 1.0 to 1.3 m between rows so the mother plants can produce runners that fill the gaps forming a “matted-row”. The plant density increases from the 7,000 planted to 100,000 after one year. A few days after planting, flowers produced are removed to stimulate runner production (Pritt 2003).

Low-elevation nurseries produce transplants that are harvested in December and

January and are stored for about 7 months at -2oC to be planted at fruiting fields in the summer; whereas high-elevation transplants are harvested in September and immediately planted in the fruiting fields or stored for brief periods at 1oC. Strawberry transplants destined for Florida are harvested with leaves and the soil is removed, denominating them as “bare-root green-top” (Strand 2008). However, if the plants are grown in trays and sold with substrate they are known as “plug” transplants.

Strawberry transplants shipped from other countries and continents, when reached the U.S. borders, are examined visually for disease symptoms if destined for fruit production (Grunwald et al. 2012; Moslonka-Lefebvre et al. 2011;

U.S.Government 2015). The United States Department of Agriculture (USDA) is responsible for inspecting the plants imported into the US; however, plants are mostly examined for the presence of newly introduced and invasive pests (U.S.Government

2008). According to the Plants for Planting Manual from USDA (2013), the quarantine pests are “the pests that are not present in the U.S. or are not widely distributed in the

country”, and “the propagative plant material entering from Canada is subject to inspection at the Canadian border by Custom and Border Protection, not requiring further inspection by the USDA”. The number of plants received by the U.S. daily is enormous; however, of all the strawberry transplants received, only 1% is further tested for the presence of viruses and other diseases. In 2008, the only approved technique for screening of diseases on strawberry transplants was the leaf grafting method, which is time consuming and the symptoms may lead to a misidentification (Strand 2008).

Serological and molecular techniques may be used currently for pathogen identification but they are more challenging. Some assays like ELISA and nested PCR can be used for identification of various pathogens; however, they are not universally accepted in quarantine regulations because they still needs validation and determination of their limitations, especially related to specificity of the tests (Martin et al. 2000).

Transplants may harbor primary inoculum of several pathogens that arrive from nurseries in quiescent infections, which appose serious and important problems for several crops that depend on vegetative propagated plants to begin cultivation, like sugarcane (Soufi and Komor 2014), banana (Mwangi and Nakato 2009), and strawberry

(Rahman et al. 2015), among others. Seedlings or transplants infected with pathogens lead to disease development, dissemination, and, sometimes, complete loss of crop production. In these cases, growers might be forced to replant later in the season which results in significant economic losses due to decreased yield and increased costs. The entire fruit production process depends on acquisition of healthy transplants; which is possible with transplant certification and trustworthy management of diseases by the nurseries.

Botrytis cinerea

B. cinerea Pers. (1974), the anamorph of Botryotinia fuckeliana (de Bary)

Whetzel (1945), is a fungus from the phylum Ascomycota, family Sclerotiniaceae (Kirk

2015), with a necrotrophic or saprophytic life cycle. This fungus can infect more than

400 hosts including cultivated crops (Davis 1997; Maas 1998; Pearson and Goheen

1988) and wild plants and is classified as a non-specific pathogen. Its reproductive structures, that appear gray in mass, are conidia that are hyaline, asexual, aseptate, one-celled, round to ovoid, hydrophobic, and about 8.12 x 11.74 µm. Their septate hyphae produces dark-brown irregularly branched conidiophores (16-30 µm diameter and 1-5 mm tall). The survival structure is a sclerotium about 2-4 x 1-3 mm, dark- colored and discoid and firmly attached to the substrate (Elad et al. 2007; Maas 1998;

Oliveira 2014). The sexual structures, apothecia, when present, are stalked, brown, bowl-shaped, and germinate from the sclerotia, measuring about 4-5 mm long and produce hyaline ascospores that are one-celled, ovoid-ellipsoid, and about 7 x 5.5. µm

(Oliveira 2014; Pearson and Goheen 1988).

Epidemiology and Life Cycle

Sclerotia of B. cinerea germinate from soil and plant debris at 3 to 27oC producing mycelium (Figure 1-4). Conidia are formed on conidiophores and dispersed by wind and water splash onto plant parts. They germinate when conditions are favorable and penetrate directly or enter through natural openings or wounds. Infection of strawberries occurs between temperatures of 15 to 25oC when relative humidity is higher than 90% or leaves are wet for longer than 13h (Bulger et al. 1987; Maas 1998;

MacKenzie and Peres 2012). Breakdown and degradation of the infected cells is observed resulting in a soft rot, and new conidiophores, the sign of gray mold, are

produced. Sclerotia are formed at high temperature and low humidity to survive adverse weather conditions. Disease cycles are repeated when a potential host is present in the area (Agrios 2005; Maas 1998; Oliveira 2014). High temperatures in Florida during the summer (above 27oC) and fumigation of the soils between plantings break the cycle of

B. cinerea in strawberry fields. Studies have shown that the fungus does not survive as sclerotia on plant debris over-summer (data not published) indicating that the initial inoculum of B. cinerea may come with transplants or from other crops in the area.

Weather conditions that favor B. cinerea growth and disease development, temperatures between 15 and 25oC and leaf wetness higher than 6h (Bulger et al.

1987), are found in nurseries located at high altitudes, and disease management actions are required to avoid losses (Strand 2008). Favorable conditions also occur during flowering and fruiting periods in Florida strawberry fields, increasing the chances of infection and disease development.

Symptoms and Signs

Botrytis spp. cause diseases on several crops; they are commonly known as gray mold, fruit rot (Davis 1997; Maas 1998), blossom-end rot (Yukita 2005), blossom blight (Horst 1983), bunch rot (Ciliberti et al. 2015), damping-off (Konstantinou et al.

2014), neck rot (Park et al. 1995), and bulb rot (Sumner et al. 1994). Flowers and inflorescences are very susceptible to B. cinerea due to their high pollen content, which stimulate conidial germination, and symptoms start with necrotic brown spots and vary from blight to entire rot and abscission (Ciliberti et al. 2015; Oliveira 2014). On strawberry fruit, B. cinerea infection usually starts at any fruit stage under the calyx as a small, round, and brownish lesion that develops a soft rot where the fungus produces a gray mass of conidia, typical of the pathogen. Infected fruit eventually become covered

in spores and mummified. Losses can be increased with frost injuries in the fruit and flowers (Maas 1998; Oliveira 2014; Pearson and Goheen 1988). Older and senescent leaves can also be infected with B. cinerea, showing water soaked lesions that change from brown to gray with the progress of the disease; however, the gray mass of spores are not always found on leaves (Davis 1997; Oliveira 2014; Shim et al. 2014). The signs of B. cinerea are the presence of conidiophores, conidia, and sclerotia on the infected tissue. In strawberries, the disease caused by B. cinerea is called gray mold or

Botrytis fruit rot. The importance of this pathogen is demonstrated by the losses of up to

50% (Cordova et al. 2014; Ellis and Grove 1982; Jarvis 1962) caused in most of the production cycle, including 15% losses pre-harvest even if controlled (Legard et al.

2001), or during transport and in the market (Mertely et al. 2002).

Disease Control

The first step for the management of gray mold or Botrytis fruit rot (BFR) is the acquisition of healthy or resistant transplants. Cultivars vary in their susceptibility to gray mold but no cultivar resistant is available. Of all the cultivars commercially planted in

Florida, two are moderately resistant to BFR: ‘Strawberry Festival’ and ‘Florida Elyana’; the first is susceptible to Colletotrichum crown rot, and the second is a minor cultivar which is susceptible to rain damage (Whitaker et al. 2015). Even with the planting of moderately resistant cultivars, it is recommended that an initial treatment of fungicides before planting be applied to transplants to protect them and delay infection (Maas

1998).

Cultural control alternatives are also adopted by the strawberry growers. Use of plastic mulch and drip irrigation avoid contact of the fruit with the soil and water splash between fruit. The improvement of air circulation and reduction of moist conditions can

be obtained with the raised bed design and reduced plant density (Strand 2008).

Harvesting all the ripe berries, removing diseased and unmarketable fruit, and senescent leaves from the plant also reduces gray mold severity (Legard et al. 2003).

Post-harvest management includes careful handling, cooling the fruit to 1oC soon after harvest and increasing the carbon dioxide concentration during strawberry transport

(Strand 2008). However, none of the control measures cited above are as effective as chemical control.

Chemical Control

Traditionally, fungicides have been considered the standard control method for gray mold on strawberries. Single-site fungicides are those that inhibit a single target in the fungal cell and multi-site are those with multiple targets (Deising et al. 2008). In the

United States, “seven groups of single-site fungicides are currently available for the control of gray mold on strawberry”, including anilino-pyrimidines , hydroxyanilides, phenylpyrroles, dicarboximides, methyl benzimidazole carbamates, quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs) (Amiri et al. 2013; Li et al. 2014b). The recommendation for growers in Florida is to spray during flowering and peak fruit production, due to the susceptibility of flowers to infections of B. cinerea

(Mertely et al. 2002). A common practice is to apply fungicides weekly on a calendar basis (Cordova et al. 2014) or following the Strawberry Advisory System (SAS) (Pavan et al. 2011; Pavan et al. 2012). SAS is a web-based tool that monitors the weather and provides fungicide spray recommendations. It usually requires about half the number of sprays throughout the season to manage the disease compared to the calendar-based system (Cordova et al. 2014; MacKenzie and Peres 2012).

Fungicide Resistance

Botrytis cinerea is considered a high-risk pathogen for development of fungicide resistance due to prolific conidial production, high genetic variability, wide host range, polycyclic nature, and the large number (16 to 22) of fungicide sprays used for its control (Fernandez-Ortuno et al. 2012; Leroux et al. 2002; Veloukas et al. 2014). Gray mold management relies on fungicide applications to maintain a profitable crop production for the grower. However, the more intense the chemical control is, the higher the selection pressure for B. cinerea isolates resistant to fungicides.

With the widespread use of fungicides and high number of sprays to control gray mold, resistance selection pressure increases and the chemicals available no longer control this disease, decreasing strawberry yield. Furthermore, fungicide manufacturers are required to search for new compounds and develop new products that may cost approximately US$256 million per fungicide and take about 10 years for registration

(Oliver and Hewitt 2014). A report by the United Nations Environmental Program

(UNEP) in 1979 suggested that “pesticide resistance ranked as one of the top four environmental problems of the world” (Pimentel 2008).

The Fungicide Resistance Action Committee (FRAC) is a specialist, technical group that classifies fungicides into numbered groups by arranging them based on their mode of action, target site of action, resistance risk, and chemical group (Wedge et al.

2007). FRAC provides guidelines for fungicide resistance management to increase the effectiveness of fungicides and limit crop losses when resistance occurs (FRAC 2015).

Of the fungicides used to control B. cinerea on strawberries in Florida, two are multi- sites, captan (M4) and thiram (M3), and several are single-sites included in the FRAC groups 7, 9, 11, 12, and 17.

The succinate dehydrogenase inhibitors (SDHIs) have a broad-spectrum of action, mainly inhibiting conidial germination and germ tube elongation (Veloukas et al.

2011) by blocking fungal respiration on the mitochondrial complex II. Resistance of B. cinerea to boscalid, a SDHI, was reported soon after its first use in the field and is related to a mutation in the SdhB gene at position 272, with a substitution of histidine

(H) to tyrosine (Y) or leucine (L) (Chatzidimopoulos et al. 2014; Laleve et al. 2014), and has been reported by several authors (Angelini et al. 2010; Fernandez-Ortuno et al.

2012; Leroux et al. 2010; Veloukas et al. 2011; Yin et al. 2011). Penthiopyrad, another

SDHI, has the same mode of action; however, because of its differences in structure and binding location, and to its more recent development (2008) and registration (2012) for use on Florida strawberries, resistance to penthiopyrad is less common (Amiri et al.

2014). Furthermore, fluopyram, another SDHI expected to be registered soon to control

B. cinerea on strawberry, has been evaluated for its resistance risk (Amiri et al. 2014), since it is effective against Mycosphaerella graminicola (Fraaije et al. 2012),

Corynespora cassiicola and Podosphaera xanthii (Ishii et al. 2011). Boscalid (pyridine carboxamides), fluopyram (pyridinyl-ethyl-benzamides) and penthiopyrad (pyrazole-4- carboxamides) are classified in group 7 (FRAC) with a medium to high resistance risk

(FRAC 2015).

Pristine® (BASF Corporation, Research Triangle Park, NC) is a fungicide labeled for use on strawberry crops that contains boscalid and pyraclostrobin. The quinone outside inhibitor (QoI), pyraclostrobin, inhibits respiration by targeting the cytochrome b

(cytb) gene (FRAC 11). It is slightly effective against B. cinerea and is recommended only for suppression of the pathogen. Pyraclostrobin is classified as a high resistance

risk fungicide and a single mutation in the mitochondrial cytb gene can lead to resistance. A substitution of glycine (G) with alanine (A) at the gene position 143 is the most common mutation that confers Botrytis resistance to pyraclostrobin (Samuel et al.

2011; Veloukas et al. 2014); however, a second substitution from phenylalanine (F) to leucine (L) at position 129 confers moderate resistance of other pathogens to QoI fungicides (Pasche et al. 2004; Patel et al. 2012). Sierotzki et al. (2007) found a third substitution of glycine (G) to arginine (R) at position 137 attributing the same low resistance to QoI fungicides on Pyrenophora triticirepentis. A relationship between SDHI and QoI resistance is observed where Botrytis isolates have multiple mutations conferring resistance to both groups (Bardas et al. 2010).

Other groups of fungicides that are used to control B. cinerea on strawberries include anilino-pyrimidines (AP), phenylpyrroles (PP), and the sterol biosynthesis inhibitors (SBI). Pyrimethanil (FRAC 9) and cyprodinil are examples of the AP group that act by inhibiting methionine biosynthesis and have medium risk of resistance; which has been studied but resistance is not understood (Fernandez-Ortuno et al. 2013). The

PP fungicides block the osmotic signal transduction by inhibiting MAP-kinase. This group has low-to-medium risk of resistance and is represented by fludioxonil, which provides very good control of Botrytis. However, the product registered to control gray mold on strawberries that contains fludioxonil (FRAC 12), also contains cyprodinil

(FRAC 9) (Switch® Syngenta Crop Protection, Greensboro, NC), the active ingredient to which resistance has been previously established (Fernandez-Ortuno et al. 2013).

Another group with low-to-medium risk of resistance is the sterol biosynthesis inhibitor

(SBI) group, represented by the hydroxyanilide fenhexamid (FRAC 17) that targets the

3-keto reductase enzyme responsible for inhibition of C4-demethylation. Resistance to fenhexamid is associated with mutations in the erg27 gene at codon 412 (phenylalanine to serine) (Fillinger et al. 2008) and the recently found mutations at the codons 170

(glycine to arginine) and 210 (alanine to glycine) (Amiri and Peres 2014).

Methods to evaluate fungicide sensitivity

There are several methods to evaluate the sensitivity of B. cinerea to fungicides.

The most commonly used assays involve amended agar and evaluation of the growth of the fungus, either by measuring the mycelial growth or by estimating conidial germination and comparing them with a control on non-amended agar. Both of these methods use either a discriminatory dose to determine fungicide resistance or a range of concentrations to determine the effective concentration to inhibit 50% of the fungal growth (EC50). However, they are time and space consuming, the measurements of conidial germination can be subjective, and mycelial tests can only be used with fungicides that have its mode of action towards mycelial growth inhibition.

A new method that has been adapted to test fungicide sensitivity of different pathogens such as Monilinia fructicola (Cox et al. 2009), Verticillium dahliae

(Rampersad 2011), and Alternaria alternata (Vega et al. 2012) is the resazurin- reduction assay, which is a colorimetric assay that uses a non-cytotoxic dye to measure the activity of the cells. Alamar blue® (Life Technologies, Grand Island, NY) is the commercial formulation of the resazurin dye, which in the presence of living cells, is absorbed by the mitochondria and reduced from non-fluorescent blue to a fluorescent pink compound (resorufin). A colorless compound (hydroresorufin) can be formed with elevated number of cells per well, with a long contact between the cells and the dye, or on a low pH environment. This reaction is evaluated fluorometrically or through light

absorbance in a spectrophotometer. This adapted sensitivity assay uses fungicides that inhibit the respiration of the fungi, acting at the same site of reduction of the dye.

Pelloux-Prayer et al. (1998) studied the conidial germination of B. cinerea using

Alamar blue as a quantitative indicator evaluated spectro-fluorometrically; however, the method has never been used before to evaluate fungicide sensitivity to this pathogen.

Due to the rapid sporulation and polycyclic cycle nature of B. cinerea on strawberry, rapid detection of fungicide sensitivity is desirable. The resazurin reduction occurs by the “removal of oxygen and replacement by hydrogen, and the dye is intermediate between final reduction of O2 and cytochrome oxidase” (Invitrogen 2009); the respiration-inhibitor fungicides have activity at the cytochrome site, indicating the potential of resazurin use to evaluate fungicide sensitivity. The results of resazurin reduction can be obtained in 24h; the dye is non-toxic to the users and to the environment; and the cost of a resazurin reaction is one-third of the mycelial growth assay (Cox et al. 2009).

Objectives

B. cinerea isolates collected in Florida strawberry fields were found by Amiri et al.

(2013) to have multifungicide resistance to several chemicals sprayed by the growers.

However, the initial inoculum and over summering of the pathogen in strawberry fields were uncertain. Our hypothesis was that the initial B. cinerea inoculum comes with transplants from the nurseries and that the inoculum was already resistant to some of the fungicides used in the production fields. Thus, the objectives of this project were: i) to evaluate strawberry transplants arriving from nurseries from Canada and northern

U.S. for possible quiescent infections of B. cinerea; ii) to determine the fungicide sensitivity profile of the B. cinerea population introduced from nursery transplants to

most fungicides used to control gray mold in Florida; iii) to optimize a resazurin-based assay to evaluate sensitivity of B. cinerea to the respiration-inhibitor fungicides boscalid and pyraclostrobin, and determine the effective fungicide dose to provide 50% control of the fungus (EC50) in comparison with the traditional conidial germination assay.

Figure 1-1. Strawberry plug transplant (Source: Charles O'Dell. The strawberry: a book for growers, others. Gainesville: N. F. Childers Publications, 2003).

Figure 1-2. “Bare-root green-top” strawberry transplants ready to be planted on Florida production field. October 19, 2011. Gulf Coast Research and Education Center. Courtesy of Michelle Souza Oliveira.

Figure 1-3. Production of certified strawberry planting stock in California (Source: Larry L. Strand. Integrated pest management for strawberries. Oakland: University of California, 2008).

Figure 1-4. Development of Botrytis gray mold diseases (Source: George N. Agrios. Plant Pathology. Amsterdam: Elsevier Academic Press, 2005).

CHAPTER 2 TRANSPLANTS AS A SOURCE OF INFECTION OF Botrytis cinerea TO STRAWBERRY FIELDS IN FLORIDA

Introduction

Strawberry transplants are produced in nurseries across Canada, the northern

U.S., and California and shipped annually to other strawberry-growing regions. Florida is the second largest producer of strawberries in the U.S. with 28,000 kg/ha of fruit harvested in approximately 3,500 hectares during 2013/2014 season (Agricultural

Statistics 2014).

Every year, the strawberry production is affected by diseases. Gray mold, caused by Botrytis cinerea, is an important disease of strawberry that infects flowers and fruit, causing pre- and postharvest decay. The symptoms on fruit begin with a small, firm, light-brown lesion that enlarges quickly and becomes covered with a gray fuzzy mass of spores (Maas 1998). Quiescent infections of B. cinerea on transplants have been suggested as a potential source of primary inoculum in strawberry-producing regions

(Amiri et al. 2013; Fernandez-Ortuno et al. 2012; Mercier et al. 2010). Moreover, if the transplants come infected from the nurseries, the management of the disease has to start early in the season.

Gray mold in Florida fields is controlled primarily by fungicide applications. The current management program includes weekly applications of multi- and single-site fungicides as rotations or tank mixtures, with sprays of the multi-sites captan and thiram throughout the season, and application of single-sites during flowering periods when weather conditions are favorable according to the Strawberry Advisory System (Amiri and Peres 2014; MacKenzie and Peres 2012; Mertely et al. 2002; Mertely and Peres

2009). Five groups of fungicides with different mode of actions are labeled to control B.

cinerea in Florida, including SDHIs (succinate dehydrogenase inhibitors), QoIs (quinone outside inhibitors), AP (anilino-pyrimidines), PP (phenylPyrroles), and hydroxyanilides, a sterol biosynthesis inhibitor (Amiri et al. 2013; FRAC 2015).

Some of the fungicides sprayed by the growers are the same as those used in nurseries to produce transplants that are disease-free. However, recommendations ensure the use of broad-spectrum fungicides to avoid an increase in resistance. In

North Carolina, a certified nursery plant production industry was created to assure the health of the transplants (Rahman et al. 2015). Despite of the visual evaluation of certified plants, they can still carry quiescent infections of B. cinerea as well as other pathogens.

Due to the high number of fungicide applications (16 to 22) for gray mold management, and to the high genetic variability, prolific asexual spore production, and polycyclic life style of the pathogen, the risk of resistance selection is high (Fernandez-

Ortuno et al. 2012). Resistance of B. cinerea to several fungicides has already been reported from Germany (Grabke and Stammler 2015), Greece (Myresiotis et al. 2007),

France (Leroux et al. 2010), Chile (Esterio et al. 2011), and several US States (Amiri et al. 2013; Fernandez-Ortuno et al. 2014; Kim and Xiao 2010; Mercier et al. 2010), but none of these previous works described the origin of the resistance.

Amiri et al. (2014) evaluated several B. cinerea fitness factors and indicated that the pathogen does not survive throughout the summer in Florida. Thus, our hypothesis was that the resistant inoculum may arrive with infected transplants. Therefore, the objectives of this study were: i) to evaluate transplants from various nurseries in U.S. and Canada for quiescent infections of B. cinerea; and ii) to determine the fungicide

sensitivity profile of this introduced population to select fungicidal chemistries commonly used to control gray mold in Florida.

Materials and Methods

Fungal Isolates

In 2011 and 2012, ten to twenty strawberry transplants were sampled from six different nurseries, two from Nova Scotia, one from Ontario and one from Quebec in

Canada, one from North Carolina and one from California in the US. The transplants were sampled from the coolers where the growers keep the plants prior to planting. One leaf was removed from each transplant and surface-disinfested in the laboratory with

0.05% sodium hypochlorite for two minutes, then rinsed twice with sterile deionized water. The leaves were incubated in sealed 15 x 25 cm plastic bags for 7 days at 23°C with a damp cotton ball to maintain moisture before examination under a stereomicroscope (14X) for the presence of B. cinerea. Each B. cinerea isolate recovered from the leaves was transferred to a 6-cm diameter petri dish containing malt yeast agar (MYA; 20g malt extract, 5g yeast extract, 12g agar).

In total, 30 and 56 B. cinerea colonies were single-spored from 2011 and 2012, respectively, using an adapted protocol of Gronover et al. (2001), where conidia were harvested from fully sporulated B. cinerea colonies and suspended in 1 ml of deionized water. The conidial suspension was spread on potato dextrose agar (PDA) with a sterile glass rod, the plates were incubated inclined, to avoid overlap of germinating conidia, at room temperature (~23oC) (Figure 2-1), and after 24h, a single germinated conidium was transferred to MYA plate.

In the 2013-2014 season, forty transplants were sampled from 13 different nurseries three from North Carolina, one from California, four from Nova Scotia, two

from Ontario, and three from Quebec. The transplants were collected from the coolers where the growers keep the transplants prior to planting. One leaf from each of the forty nursery-transplants was disinfested in 0.02% sodium hypochlorite for 2 minutes and rinsed twice with sterile deionized water upon arrival in Florida. The leaves were incubated over a chicken wire, folded in a wave shape, inside clear plastic boxes (31.5 x

25 x 10 cm) with 250 ml of deionized water at the bottom to maintain 99-100% humidity

(Figure 2-2). The same 250 ml water was collected from the boxes to guarantee the humidity of the chamber. After 7 days of incubation at room temperature (~23°C), the leaves were examined under a stereomicroscope (14X) for the presence of B. cinerea

(Figure 2-3) and 473 isolates were transferred to malt yeast agar medium (MYA).

Approximately 20 isolates per cultivar/nursery combination were selected for testing of fungicide sensitivity, a total of 330 isolates. All 416 isolates from the three years were preserved at -80oC as a conidial suspension in 1 ml of 20% glycerol. The percentage of infected plants was calculated and data were transformed using arcsine of square root to be analyzed using SAS 9.4 software (Statistical Analysis System, Cary, NC, USA) following a general linear model for analysis of variance to compare quiescent infections among nurseries.

Fungicide Sensitivity Tests

Mycelial growth

All 86 isolates from 2011 and 2012 and the 330 isolates from 2013 were grown on MYA for 4 days. One plug (4 mm diameter) was taken with a cork borer (Figure 2-4) from an actively growing colony and placed upside down on a 6-cm diameter petri dish containing potato dextrose agar (PDA; Becton Dickinson, Sparks, MD) amended with

500, 10, 500, 10, 10 µg/ml of boscalid (Endura®, BASF, Research Triangle Park, NC),

penthiopyrad (Fontelis®, DuPont, Wilmington, DE), pyraclostrobin (Cabrio® EG, BASF,

Research Triangle Park, NC) plus SHAM (Acros Organics, New Jersey, USA) at 100

µg/ml, fluopyram (Luna® Privilege, Bayer CropScience, Research Triangle Park, NC) and fenhexamid (Elevate® 50 WDG, Arysta LifeScience, San Francisco, CA), respectively, or on CzapeK-Dox agar (CZA) (Myresiotis et al. 2007) amended with 5

µg/ml of pyrimethanil (Scala™ SC, Bayer CropScience, Research Triangle Park, NC) or

CZA amended with 5 g/liter of yeast extract and 0.3 µg/ml fludioxonil (Medallion®,

Syngenta, Greensboro, NC); the appropriate volume of the fungicide was added before dispensing the medium in the plates. Discriminatory doses of the fungicides tested were previously determined by Amiri et al. (2013). Cultures were incubated at 23°C for 2 days, hyphal growth measured with a caliper (Figure 2-5) from the edge of the plug to the edge of the colony and the percent inhibition calculated relative to the PDA/CZA control. One plate was used per isolate/fungicide combination. The test was repeated twice. Data were transformed using arcsine of square root and resistance frequencies between nurseries were analyzed using general linear model to perform analysis of variance on SAS 9.4 statistical software.

Conidial germination

The 416 isolates from 2011, 2012 and 2013 were tested for fungicide sensitivity in a conidial germination test. Isolates were grown initially on MYA for 5 to 6 days until full sporulation and a suspension of 2.5x105 conidia/ml was prepared for each isolate.

Each 15-cm diameter petri dish was divided into thirty 1.5 x 2.0 cm rectangles to guide conidial placement and the subsequent measurements. Two 7-µl drops of the spore suspension were placed diagonally in each square on the plate (Figure 2-6) containing yeast bacto agar (YBA; 10g bacto peptone, 20g sodium acetate, 10g yeast extract, 15g

agar) or malt extract agar (MEA; 10g malt extract, 15g agar) amended with fungicides.

YBA was amended with 2 or 5 µg/ml of boscalid; 2 or 5 µg/ml of fluopyram; or 1 or 5

µg/ml of penthiopyrad; and MEA was amended with 50 µg/ml of pyraclostrobin plus

SHAM (100 µg/ml); 1 or 50 µg/ml of fenhexamid; 0.1 or 10 µg/ml fludioxonil; or 1 or 25

µg/ml pyrimethanil. Discriminatory doses of the fungicides tested were previously determined by Amiri et al. (2013). Cultures were incubated at 24°C for 18 to 24h and conidial germination evaluated microscopically. One-hundred conidia were counted for each B. cinerea isolate and conidia that had germ tubes equal to or larger than the conidial diameter were recorded as germinated (Figure 2-7). The percent inhibition was calculated relative to the control on a non-amended YBA or MEA plate. One plate was used per isolate/fungicide combination. The test was repeated once or twice.

Resistance frequencies between nursery data were transformed using arcsine of square root; an analysis of variance using the general linear model was performed using SAS

9.4 statistical software to identify significant factors.

Results

Frequency of Infection

The frequency of plants infected with Botrytis cinerea in 2011 was 22.0 and

28.6% from the two Nova Scotia nurseries and 36.7% from the Quebec nursery (Table

2-1). In 2012, the percentage of infection was 55.0, 56.7, 83.3, 20.0 and 20.0% from

California, Nova Scotia, Quebec, Ontario, and North Carolina nurseries, respectively.

The same nursery from California (Cr) had an increase of infection of 39.8% (from 55.0 to 76.9%) in 2013 but the number of plants tested was five times higher that year. The same nursery from Nova Scotia (G) had an increase in infection of 98.2% (from 28.6 to

56.7%) from 2011 to 2012 and 31.0% (from 56.7 to 74.3%) from 2012 to 2013. From

Quebec, the same nursery (L) showed an increase of 127.0% (from 36.7 to 83.3%) from

2011 to 2012 and a decrease of 7.6% (from 83.3 to 77.0%) from 2012 to 2013. The frequency of infection for 2013 was 76.9% from the California nursery (Cr); 75.0, 92.5,

2.5, and 74.3% from the Nova Scotia nurseries (HB, CK, CM, and G); 22.9, 49.3, and

88.6% from North Carolina (B, NC, and Tr); 70.0, 58.5, and 77.0% from Quebec (Pe,

PL, and L); and 92.5 and 64.1% from the Ontario nurseries (EG and T). Overall, the frequency of infection in 2013 was higher than in 2011 and 2012. In 2013, there was one nursery (CM) with 2.5% infection, the lowest observed from all nurseries sampled

(Table 2-1). There was no significant difference in the percentage of plants infected by region (p=0.845) or by nursery (p=0.528) (Table 2-2).

Fungicide Sensitivity Tests

Mycelial growth

Percent of mycelial growth inhibition was evaluated three times for all fungicides tested (Table 2-3). Resistance frequencies across experiment repetition were significantly different for all the fungicides tested but fenhexamid (p=0.4). The two similar experiments for boscalid and the three experiments for fluopyram were statistically different among them (p≤0.05; data not shown).

The nursery factor was significant (p≤0.01) for all fungicides tested (Table 2-3).

Most nurseries had more than 50% isolates resistant to pyraclostrobin and boscalid, but one nursery from Nova Scotia (HB) had 20% resistance to pyraclostrobin and 25% resistance to boscalid (Table 2-4); the same nursery also had 40.0% resistance to pyrimethanil and no resistance to fenhexamid. Almost all nurseries had more than

50.0% of the isolates resistant to pyrimethanil, and four nurseries had resistance of

30.0% (CK), 40.0% (HB), 44.4% (Tr) and 45.0% (Pe) in 2013. Most of the isolates were

sensitive to fluopyram and fludioxonil showing that these fungicides are still effective for

B. cinerea. Resistance to penthiopyrad was below 20.0% in 13 nurseries; there were only two nurseries from 2011 (G) and 2012 (L) that had more than 20.0% resistant isolates. Six nurseries had no resistance to fenhexamid and two nurseries had 50.0% of isolates resistant, all the others had resistance frequencies in this range (from 0.0% to

50.0%). Nurseries from Quebec had the highest frequencies of isolates resistant to fenhexamid (Table 2-4).

Comparing the isolates from 2011, 2012 and 2013, the overall frequency of resistance was almost 100.0% for pyraclostrobin and boscalid, higher than 60.0% for pyrimethanil, about 20.0% for fenhexamid and penthiopyrad, and not detected or below

9.1% for fludioxonil and fluopyram, respectively (Figure 2-8). The percentage of resistant isolates remained the same or very close for pyraclostrobin, boscalid, fluopyram, penthiopyrad and fludioxonil; increased by 52.8% for fenhexamid and 36.8% for pyrimethanil from 2011 to 2012; and decreased by 37.9% for fenhexamid, 37.0% for penthiopyrad and 30.2% for pyrimethanil from 2012 to 2013 (Figure 2-8).

There was one nursery from Nova Scotia (G) and one from Quebec (L) evaluated in all three years (Figure 2-4). For the Nova Scotia nursery (G) there was an increase of

12.4%, 20.5%, and 5.5% resistance to pyrimethanil (from 66.7 to 75.0%), pyraclostrobin

(from 77.8 to 93.8%) and boscalid (from 88.9 to 93.8%), respectively; and a reduction of

100.0% resistance to fenhexamid (from 11.1 to 0.0%) and penthiopyrad (from 22.2 to

0.0%) from 2011 to 2012. The same nursery showed a decrease of 14.5% resistance to pyrimethanil (from 75.0 to 64.1%) with an increase of 10.3%, 6.6%, 6.6%, and 15.4% resistance to fenhexamid (from 0.0 to 10.3%), pyraclostrobin (from 93.8 to 100.0%),

boscalid (from 93.8 to 100.0%), and penthiopyrad (from 0.0 to 15.4%), respectively from

2012 to 2013. The nursery from Quebec (L) had an increased resistance of 134.0% for fenhexamid (from 21.4 to 50.0%), 48.5% for pyrimethanil (from 64.3 to 95.5%), and

90.9% for penthiopyrad (from 14.3 to 27.3%) from 2011 to 2012; in the following year, there was a decrease in the resistance to all three fungicides, 36.8% reduction for fenhexamid (from 50.0 to 31.6%), and 42.1% for pyrimethanil (from 95.5 to 55.3%) and penthiopyrad (from 27.3 to 15.8%). All the other fungicides remained consistent from year to year for those two nurseries. One nursery from North Carolina (Tr) and one from

Ontario (T) were evaluated in 2012 and 2013; however, no comparison was made because of their low number of isolates in 2012.

Conidial germination

Three experiments were done in which all the isolates were tested for the fungicide inhibition of conidial germination. The experiment repetitions were significantly different (p≤0.01) for all fungicides tested but pyraclostrobin (p=0.07) (Table 2-5).

Fungicide concentration was a significant factor (p≤0.01) in inhibition of conidial germination for all isolates tested, with higher inhibition at the highest concentration tested for each fungicide (Table 2-5). Pyraclostrobin was only tested at a high concentration (50 µg/ml) to determine highly resistant isolates. When testing for nursery effect, there was significant difference (p≤0.01) in frequency of conidial germination inhibition for all fungicides tested.

Pyrimethanil, pyraclostrobin and boscalid were the fungicides with the highest frequencies of resistance in the conidial germination test (Table 2-6). One nursery from

Nova Scotia (HB) had isolates significantly more sensitive to both pyraclostrobin

(20.0%) and boscalid (25.0%) with similar results observed in both mycelial growth and

conidial germination tests (Tables 2-4 and 2-6). Almost all isolates were sensitive to fluopyram, penthiopyrad and fludioxonil; one single isolate from a Quebec nursery (L) and one isolate from an Ontario nursery (EG) was resistant to fluopyram and penthiopyrad, respectively. The frequency of resistance to fenhexamid varied among nurseries, with the lowest 6.3% (G), the highest 97.4% (L), and no correlation between nurseries from the same region (Table 2-6). Of all isolates tested, 29 (7.0%) were resistant to pyraclostrobin but sensitive to boscalid and two (0.5%) were resistant to boscalid but sensitive to pyraclostrobin.

The overall frequency of resistant isolates was above 90.0% for pyrimethanil and pyraclostrobin, above 60.0% for boscalid, and below 0.5% for fluopyram, penthiopyrad, and fludioxonil (Figure 2-9). An increase of resistance to boscalid was observed from year to year, 10.9% from 2011 to 2012, and 18.8% from 2012 to 2013. Of the nurseries evaluated in all three years, L and G, in contrast to the mycelial growth test, had mostly increases in resistance (Table 2-6). The only decreases in resistance observed were in the Nova Scotia nursery (G), from 2011 to 2012, with a decrease of 43.6% to fenhexamid (from 11.1 to 6.3%) and 6.2% to pyrimethanil (from 100.0 to 93.8%). This nursery had similar resistance frequency to fluopyram, penthiopyrad and fludioxonil.

The nursery from Quebec (L) showed similar resistance frequencies for pyrimethanil, pyraclostrobin, penthiopyrad and fludioxonil, and increases of resistance of 2.6%, 9.0%, and 42.8% to fluopyram, boscalid and fenhexamid, respectively.

The frequency of resistant isolates is also presented by fungicide and by year

(Figure 2-10). Data for fenhexamid is from mycelial growth test since results for this fungicide were similar for all three experiments (Table 2-3). Fluopyram, penthiopyrad,

and fludioxonil were not included because the frequency of resistance was zero for almost all nurseries.

Resistance to fenhexamid was observed in all nurseries (Figure 2-10). For the nurseries sampled in 2013, when more isolates were collected, Nova Scotia had lower frequency of resistant isolates (all below 10.0%) than Quebec nurseries (around 40.0% for the three nurseries). A pattern was observed for distribution of isolates resistant to pyrimethanil, pyraclostrobin, and boscalid, where a single nursery from Nova Scotia

(HB) had the lowest frequency for all three fungicides (85.0%, 20.0% and 25.0%). B. cinerea isolates had the highest resistance frequency to pyraclostrobin (100.0% in 12 of the nurseries tested) in 2013 when compared to the other fungicides in the same year.

In 2012, resistance frequencies were 100.0% for pyraclostrobin and pyrimethanil in three nurseries tested; and, in 2011, 100.0% resistance to pyrimethanil was found in three nurseries, while 100.0% resistance to pyraclostrobin was found in two nurseries.

The frequency of resistance to boscalid increased from year to year with the highest frequencies in 2013 independent of nursery region.

Multi-fungicide resistance of the B. cinerea isolates from different nurseries was also observed (Figures 2-11 and 2-12). Each pie graph represented one nursery and the wedges were the percentage of fungicide resistant isolates. In 2011-2012, resistance to three or four fungicides was observed in isolates from nurseries in Nova

Scotia and Ontario, and 25.0% of the isolates from a nursery in California had resistance to five fungicides; no fungicide resistance was observed in only one nursery from Nova Scotia (Figure 2-11).

In 2013, resistance to three or four fungicides was the most observed across nurseries (Figure 2-12). Frequency of isolates from California resistant to three and four fungicides was 44.0% and 41.0%, respectively. One nursery from Ontario had 5.0% of the isolates resistant to one fungicide; however, the other nursery from the same region had all of its isolates resistant to at least three fungicides. In Quebec, the majority of isolates were resistant to four fungicides for three of four nurseries, where one of them had 100.0% of its isolates resistant to four fungicides (fenhexamid, pyrimethanil, pyraclostrobin, and boscalid); the remaining nursery had 57.0% and 43.0% of its isolates resistant to three and four fungicides, respectively. Two North Carolina nurseries had more than 50.0% of their isolates resistant to four fungicides; the third nursery had 72.2% of the isolates resistant to three fungicides. The results from Nova

Scotia were noteworthy because of the single nursery with 15.0% sensitive isolates and

15.0% of isolates resistant to only one fungicide (pyrimethanil); this nursery also had

45.0% of isolates resistant to two fungicides (fenhexamid and pyrimethanil), and the remaining isolates were resistant to either three or four, including boscalid and pyraclostrobin. The nurseries that had a percentage of sensitive isolates were different in 2011-2012 and 2013, but were both from Nova Scotia. No resistance to fluopyram or fludioxonil was found across nurseries.

Discussion

The results of this study demonstrate that nursery transplants are an important source of initial inoculum of B. cinerea to Florida strawberry fields which could indicate that B. cinerea is first established from quiescent infections in the transplants. To our knowledge, this is the first indication that fungicide resistant B. cinerea is introduced into

strawberry fields by the transplants. This is an important discovery that affects disease management in strawberry fields and an alert for nurseries and fruit production growers.

Strawberry plant production starts with either plugs or bare-root transplants from other regions, planted in the nursery widely spaced but that form a “matted-row” with a large number of plants per area. The transplants shipped to begin Florida production are the third or fourth generation from the original mother plant; moreover, the transplants are subject to infection of B. cinerea and other pathogens, which requires nursery grower action for control (Rahman et al. 2015). Previous work (Fernandez-

Ortuno et al. 2012; Mercier et al. 2010) found some evidence that the B. cinerea resistant strains could be coming from nursery sources, since they found such strains even in organic fields. This study confirms the presence of B. cinerea on transplants and demonstrates the existence of strains resistant to one or more fungicides from nurseries.

No association between the nursery region and resistance profile was apparent.

This could be related to the fact that many nurseries start their production with plant stock from a few other common nursery sources, mainly other nurseries from California.

Since the same fungicides that control B. cinerea in the field are also used to control other diseases like anthracnose, caused by Colletotrichum spp., and powdery mildew, caused by Podosphaera aphanis, in the nurseries (Mercier et al. 2010; Rahman et al.

2015; Whitaker et al. 2015), the selection pressure for resistant isolates is increased, wherefore the strawberry growers receive plants already infected with resistant pathogens. The weekly fungicide sprays and the continued use of the same group of fungicides for several years, like the QoIs in Florida since 1998 (Amiri et al. 2013), are

other factors that contribute for selection of resistant isolates (Bardas et al. 2010; Kim and Xiao 2010).

B. cinerea populations with resistance to boscalid, a succinate dehydrogenase inhibitor (SDHI), was found in this study at frequency levels about 85.0%, similar to those also reported in Greece (57.0%) (Bardas et al. 2010), France and Germany

(Leroux et al. 2010), as well as in U.S. states such as California (66.0%) (Mercier et al.

2010), South and North Carolina (61.5%) (Fernandez-Ortuno et al. 2012), and Florida

(85.4%) (Amiri et al. 2013). Some other work demonstrated lower resistance frequency than our study, like resistance to boscalid in China (19.0%) (Yin et al. 2011), Germany

(21.5%) (Weber 2011), Greece (35.0-50.0%) (Veloukas et al. 2011) and the US-

Washington state (20.0%) (Kim and Xiao 2010). The increasing resistance to boscalid might be the result of higher number of isolates tested in 2013 or the consistent use of this product.

Pristine (boscalid + pyraclostrobin) has been recommended for the control of B. cinerea on strawberries for 10 years in Florida. Since boscalid and pyraclostrobin are always sprayed together, their resistances are closely related, as demonstrated by some of the work mentioned above (Bardas et al. 2010; Fernandez-Ortuno et al. 2012;

Kim and Xiao 2010; Mercier et al. 2010; Veloukas et al. 2014). However, a small population that is sensitive to boscalid (28.2%) was reported by Fernandez-Ortuno et al.

(2012) that also found 11 isolates resistant to pyraclostrobin but sensitive to boscalid. In this study, less than 15% of the isolates were sensitive to boscalid and 29 isolates of

416 (7.0%) were found resistant to pyraclostrobin but sensitive to boscalid.

The results of this study indicate that the conidial germination assay is more adequate for evaluation of fungal sensitivity to SDHI fungicides due to the similar results in all three experiments. Veloukas and Karaoglanidis (2012) also found that B. cinerea germ tube elongation is more sensitive than mycelial growth on fluopyram-amended medium, a difference that was not noted in this study for fluopyram but was observed with penthiopyrad. Luna® Tranquility (fluopyram + pyrimethanil) and Luna® Sensation

(fluopyram + trifloxystrobin) are two commercial products containing fluopyram that are registered for other crops in Florida but their registration for use on strawberries is still pending. Because of the low resistance frequency found to fluopyram (below 2.7%), it can be considered a prospective alternative for gray mold management, but the risk of increase of B. cinerea isolates resistant to the new SDHIs should be observed carefully.

Another fungicide tested, pyrimethanil, an anilino-pyrimidine, had resistance frequencies as high as boscalid and pyraclostrobin and, for most of the nurseries, more than twice as high as in some previous reports (Esterio et al. 2011; Myresiotis et al.

2007). Due to the high resistance frequency (above 60.0%) to these three active ingredients, Pristine (boscalid + pyraclostrobin) and Scala (pyrimethanil) are no longer recommended for gray mold management on strawberries in Florida (Peres 2015b).

The noteworthy fact that one nursery (HB) showed frequencies of resistance to the three compounds (25.0%, 20.0%, and 85.0%) lower than the other nurseries tested could not be explained. It would be interesting to get spray records from the nurseries tested to compare them and determine the reason for the differential B. cinerea sensitivity in only one nursery (HB). Positive cross-resistance between pyraclostrobin and azoxystrobin, and between boscalid and carboxin has been observed before

(Leroux et al. 2010) but no correlation between boscalid, pyraclostrobin and pyrimethanil has been previously reported. Another anilino-pyrimidine that has been used in strawberry fields since 2004 is cyprodinil, usually applied in a commercial formulation that also contains fludioxonil (Switch® 62.5WG), and it has shown cross resistance with pyrimethanil (Myresiotis et al. 2007). The first report of field resistance of

B. cinerea to cyprodinil was in Switzerland in 1996 (Baroffio et al. 2003), and also has been found on U.S. strawberries (Fernandez-Ortuno et al. 2013; Mercier et al. 2010).

No resistance to fludioxonil was found in either mycelial growth or conidial germination tests. This result agrees with some previous work (Bardas et al. 2010;

Fernandez-Ortuno et al. 2013; Myresiotis et al. 2007) other than the 41.0% resistance found on Germany (Weber 2011) and four resistant isolates from blackberries and strawberries in the US (Li et al. 2014a). Field trials show that Switch® (fludioxonil + cyprodinil) provides best control to B. cinerea in Florida strawberry fields (Cordova et al.

2014). Since resistance to cyprodinil is known to be present (Amiri et al. 2013), the efficacy of Switch® is likely due to fludioxonil.

Resistance to the sterol biosynthesis inhibitor (SBI), fenhexamid, in nursery isolates varied from zero to 50.0%, agreeing with widespread distribution in the

Carolinas (Grabke et al. 2013), with 16.8% resistant isolates recovered. The same range of resistance frequencies was found in B. cinerea in other locations, i.e. 46% from

Chilean vineyards (Esterio et al. 2011), 30.0 to 50.0% from French and German vineyards (Fillinger et al. 2008), 45.0% from Northern German berries (Weber 2011),

30.0% to 36.0% from Florida strawberries (Amiri et al. 2014), 25.0% from California strawberries (Mercier et al. 2010), but no resistance found on Greek kiwifruit (Bardas et

al. 2010). Better management of gray mold was achieved when fenhexamid was applied during the second peak bloom period in Florida strawberry fields (Legard et al.

2005). Since the resistant population to this product is relatively low, it can still be used as an option for control of gray mold in strawberries. It would be interesting to get the spray records from the nurseries to understand whether the differences in resistance patterns are aligned with their management practices. One example is with the use of fenhexamid and pyrimethanil; these products are mainly effective against gray mold, which in theory should not be a major disease problem in plant production fields since flowers and fruit should not be present. However, day neutral cultivars tend to flower continuously and control might be implemented in the nursery if flowers are not removed. Another point is that the baseline sensitivity of nursery isolates before the introduction of these fungicides is not known.

Finding multi-fungicide resistance (MFR) in B. cinerea from the nurseries is a significant challenge for the management of gray mold in the production system, because it reduces the options for controlling the resistant isolates and managing the disease. Resistance to up to five fungicides was present in the isolates tested; however, the highest MFR population found had resistance to three fungicides reinforcing the widespread distribution of MFR found in Florida fields in 2013 (Amiri et al. 2013). B. cinerea populations with 70.0% and 85.0% resistance to at least two fungicides were reported on Florida (Amiri et al. 2013) and California (Mercier et al. 2010) strawberry fields, respectively. The reason for the existing MFR is explained by the existing multiple mutations in genes related to fungicide resistance, like the mutation N230I on sdhB gene that causes moderate resistance to boscalid, fluopyram and fluxapyroxad

(Veloukas et al. 2013), the cytb mutation (G143A) in pyraclostrobin-resistant isolates

(Yin et al. 2012), or the mutation on erg27 gene that promotes resistance to fenhexamid

(Amiri and Peres 2014).

An option to reduce resistance coming from nurseries could be the use of different spray programs in the nurseries than in the production fields, possibly multi-site fungicides, or the registration of products exclusively for nursery use, with different modes of action than the ones used in the commercial farms. Nursery growers should also invest in removing flowers and avoiding fruiting in the field for transplant production to reduce Botrytis inoculum survival and dispersal, while practicing good sanitation to avoid dispersal of other diseases.

Current spray recommendations for the Florida strawberry growers include early treatment of the plants and avoiding the use of Pristine and pyrimethanil. The SDHIs penthiopyrad and fluxapyroxad are recommended in alternation with fenhexamid when conditions are moderate for gray mold development according to the Strawberry

Advisory System (Peres 2015b). Whenever fluopyram is registered for strawberries in

Florida, it will be recommended in rotation with Switch® to avoid rapid resistance selection (Amiri et al. 2014; Peres 2015a).

Finally, it is important to improve the communication between nursery and fruit production growers to avoid the use of the same fungicides and reduce the frequency of resistance coming with the transplants. Perhaps, the multi-site fungicides should be the mainstream of disease management in nurseries to extend the life of current single-site fungicides available.

Figure 2-1. Potato dextrose agar (PDA) plates with Botrytis cinerea conidial suspensions and incubated inclined at room temperature for subsequent single-sporing. May 12, 2015. Courtesy of Michelle Souza Oliveira.

A B Figure 2-2. Senescent strawberry leaves incubated over a chicken wire, folded in a wave shape, inside clear plastic boxes for isolation of Botrytis cinerea. A) View from the top. B) Lateral view. December 30, 2013. Courtesy of Michelle Souza Oliveira

Figure 2-3. Botrytis cinerea conidiophores and conidia examined under a stereomicroscope (14x). March 26, 2015. Courtesy of Michelle Souza Oliveira.

Figure 2-4. Plug (4 mm diameter) taken from a 4-day-old Botrytis cinerea colony (left) and transferred to a control potato dextrose agar plate (right). March 26, 2013. Courtesy of Michelle Souza Oliveira.

Figure 2-5. Measurement of Botrytis cinerea mycelial growth on fungicide-amended medium. September 6, 2014. Courtesy of Michelle Souza Oliveira.

Figure 2-6. A 15-cm diameter petri dish control plate with yeast bacto agar (YBA) medium seeded with a Botrytis cinerea conidial suspension. February 28, 2014. Courtesy of Michelle Souza Oliveira.

A B Figure 2-7. Evaluation of conidial germination of Botrytis cinerea under the microscope (100x). A) Germinated conidia. B) Non-germinated conidia. February 6, 2014. Courtesy of Michelle Souza Oliveira.

100

40 2011 (n=34) 2012 (n=52)

20 2013 (n=330) Resistant Isolates (%) Isolates Resistant

Figure 2-8. Overall resistance frequency (%) of Botrytis cinerea to seven different fungicides isolated from nursery transplants in 2011, 2012, and 2013, and evaluated in mycelial growth assays. The concentrations of fungicides used were 10 µg/ml for fenhexamid (Elevate® 50WDG), fluopyram (Luna® Privilege), and penthiopyrad (Fontelis®), 500 µg/ml for pyraclostrobin (Cabrio® EG) and boscalid (Endura®), 5 µg/ml for pyrimethanil (ScalaTM SC), and 0.3 µg/ml for fludioxonil (Medallion®). An isolate was considered resistant when mycelial growth was higher than 80% on pyraclostrobin- and boscalid-amended media or higher than 50% on the other fungicide-amended media tested.

100

60 2011 (n=34) 40 2012 (n=52)

20 2013 (n=330) Resistant Isolates (%) Isolates Resistant

Figure 2-9. Overall resistance frequency (%) of Botrytis cinerea to seven different fungicides, isolated from nursery transplants in 2011, 2012, and 2013, and evaluated in conidial growth assays. The concentrations of fungicides used were 5 µg/ml for boscalid (Endura®), fluopyram (Luna® Privilege), and penthiopyrad (Fontelis®), 50 µg/ml for fenhexamid (Elevate® 50 WDG) and pyraclostrobin (Cabrio® EG), 25 µg/ml for pyrimethanil (ScalaTM SC), and 10 µg/ml for fludioxonil (Medallion®). An isolate was considered resistant when conidial germination was higher than 50% on all fungicide-amended media tested.

Fenhexamid - 2011 Fenhexamid - 2012 Fenhexamid - 2013 2 100 100 100 80 80 80 60 60 22 60 20 40 40 40 38 7 11 14 40 9 11 20 20 20 20 39 59 20 18 16 1 20 1 20 8 20

00 00 00

ResistantIsolates (%)

EG HB

G Bl L G Cr L Tr T NC CM

Pyrimethanil - 2011 Pyrimethanil - 2012 Pyrimethanil - 2013 9 11 14 16 11 22 1 39 20 1 59 38 7 20 20 18 8 20 20 40 100 100 100 20 80 80 80 60 60 2 60 40 40 40 20 20 20

00 00 00

ResistantIsolates (%)

EG HB

G Bl L G Cr L Tr T NC CM

Pyraclostrobin 2011 Pyraclostrobin 2012 Pyraclostrobin 2013 11 14 16 11 22 1 39 20 1 59 38 7 20 20 18 8 20 20 40 100 100 100 9 80 80 80 60 60 2 60 40 40 40 20 20 20 20

00 00 00

ResistantIsolates (%)

EG HB

G Bl L G Cr L Tr T NC CM

Boscalid - 2011 Boscalid - 2012 Boscalid - 2013 1 2 1 38 20 20 18 8 20 20 40 22 39 59 100 14 100 100 20 7 80 11 80 11 80 9 16 60 60 60 40 40 40 20 20 20 20

00 00 00

ResistantIsolates (%)

EG HB

G Bl L G Cr L Tr T NC CM

Nova Scotia California Quebec North Carolina Ontario

Figure 2-10. Frequency of resistance of Botrytis cinerea isolates collected from nurseries to four different fungicides, determined by mycelial growth for fenhexamid and conidial germination for the other fungicides. There is a column for each nursery in the graph, with the number of isolates above it and the same pattern for the same region of the nursery. The concentrations of the fungicides tested were 10 µg/ml for fluopyram, 25 µg/ml for pyrimethanil, 50 µg/ml for pyraclostrobin, and 5 µg/ml for boscalid.

Figure 2-11. Frequency of multi-fungicide resistance (color coded) of Botrytis cinerea from strawberry transplants collected in 2011-2012 from different nurseries from California, Nova Scotia and Quebec. The fungicides tested were Fenhexamid, Pyrimethanil, Pyraclostrobin, Boscalid, Fluopyram, Penthiopyrad, and Fludioxonil. (Yellow=sensitive, Red=resistant to one fungicide, Green=resistant to two fungicides, Purple=resistant to three fungicides, Blue=resistant to four fungicides, Orange=resistant to five fungicides).

Figure 2-12. Frequency of multi-fungicide resistance (color coded) of Botrytis cinerea from strawberry transplants collected in 2013 from different nurseries from California, North Carolina, Nova Scotia, Quebec and Ontario. The fungicides tested were Fenhexamid, Pyrimethanil, Pyraclostrobin, Boscalid, Fluopyram, Penthiopyrad, and Fludioxonil. (Yellow=sensitive, Red=resistant to one fungicide, Green=resistant to two fungicides, Purple=resistant to three fungicides, Blue=resistant to four fungicides, Orange=resistant to five fungicides).

Table 2-1. Frequency of strawberry transplants from different nurseries infected with Botrytis cinerea in 2011, 2012 and 2013. Number of plants Year Region Nursery sampled Number of isolates Plants infected (%) 2011 Nova Scotia Bl 50 11 22.0 Nova Scotia G 28 8 28.6 Quebec L 30 11 36.7 2012 California Cr 20 11 55.0 Nova Scotia G 30 17 56.7 Quebec L 30 25 83.3 Ontario T 10 2 20.0 North Carolina Tr 5 1 20.0 2013 California Cr 108 83 76.9 Nova Scotia HB 40 30 75.0 Nova Scotia CK 40 37 92.5 Nova Scotia CM 40 1 2.5 Nova Scotia G 70 52 74.3 North Carolina B 35 8 22.9 North Carolina NC 71 35 49.3 North Carolina Tr 35 31 88.6 Quebec Pe 40 28 70.0 Quebec PL 41 24 58.5 Quebec L 74 57 77.0 Ontario EG 40 37 92.5 Ontario T 78 50 64.1

Table 2-2. Analysis of variance of the frequency of strawberry transplants infected with Botrytis cinerea by nursery and region in 2011, 2012 and 2013. Source of variation Degrees of freedom SS Mean Squares F value P value Regiona 4 0.156 0.039 0.340 0.845 Nurseryb 13 1.287 0.099 1.000 0.528 aFrequency of infection was evaluated from five locations: Nova Scotia, Quebec, Ontario, California, and North Carolina. bFrequency of infection was evaluated from fourteen nurseries: Bl, G, L, Cr, T, Tr, HB, CK, CM, B, NC, Pe, PL, and EG. Note: F-value is the group variance against the hypothesis that the means are the same. P-value is the probability of occurrence of variation in the frequency of infection between regions or nurseries.

Table 2-3. F-statistics and P-values for the fungicide inhibition of the mycelial growth of Botrytis cinerea isolates collected from fourteen nurseries in 2011, 2012, and 2013. Fenhexamid Pyrimethanil Pyraclostrobin Boscalid Penthiopyrad Factor Df F P F P F P F P F P experimenta 2 0.92 0.40 6.28 <0.01 18.68 <0.01 30.68 <0.01 15.62 <0.01 nurseryb 13 32.07 <0.01 4.77 <0.01 21.08 <0.01 11.06 <0.01 4.87 <0.01 exp*nur 26 0.17 1.00 1.14 0.28 2.19 <0.01 1.84 <0.01 0.50 0.99 aThere were three experiments evaluated, where each isolate was tested in a fungicide-amended medium. bFungicide inhibition was evaluated from fourteen nurseries: Bl, G, L, Cr, T, Tr, HB, CK, CM, B, NC, Pe, PL, and EG.

Table 2-4. Frequency of resistance of Botrytis cinerea isolates from nurseries to seven different fungicides tested in the mycelial growth assay. Total Resistant Isolates (%) Year Region Nursery Isolates Fenhexamid Pyrimethanil Pyraclostrobin Boscalid Fluopyram Penthiopyrad Fludioxonil 2011 Nova Scotia G 9 11.1 66.7 77.8 88.9 0.0 22.2 0.0 Nova Scotia Bl 11 18.2 63.6 100.0 100.0 0.0 18.2 0.0 Quebec L 14 21.4 64.3 100.0 100.0 0.0 14.3 0.0 2012 California Cr 11 9.1 100.0 100.0 100.0 9.1 18.2 0.0 Nova Scotia G 16 0.0 75.0 93.8 93.8 0.0 0.0 0.0 Quebec L 22 50.0 95.5 100.0 100.0 0.0 27.3 0.0 North Carolina Tr 1 0.0 100.0 100.0 100.0 0.0 0.0 0.0 Ontario T 2 100.0 50.0 100.0 100.0 0.0 50.0 0.0 2013 Nova Scotia G 39 10.3 64.1 100.0 100.0 0.0 15.4 0.0 Nova Scotia CK 20 0.0 30.0 100.0 100.0 0.0 10.0 0.0 Nova Scotia CM 1 0.0 100.0 100.0 100.0 0.0 0.0 0.0 Nova Scotia HB 20 0.0 40.0 75.0 45.0 0.0 0.0 0.0 California Cr 59 8.5 71.2 98.3 100.0 0.0 16.9 0.0 Quebec L 38 31.6 55.3 100.0 100.0 0.0 15.8 0.0 Quebec LB 7 28.6 85.7 100.0 100.0 0.0 0.0 0.0 Quebec Pe 20 50.0 45.0 100.0 100.0 0.0 15.0 0.0 Quebec PL 20 10.0 75.0 100.0 100.0 0.0 15.0 0.0 North Carolina Tr 18 5.6 44.4 100.0 100.0 0.0 11.1 0.0 North Carolina B 8 0.0 87.5 100.0 100.0 0.0 0.0 0.0 North Carolina NC 20 15.0 65.0 100.0 100.0 0.0 0.0 0.0 Ontario EG 20 5.0 85.0 100.0 100.0 0.0 10.0 0.0 Ontario T 40 37.5 65.0 100.0 100.0 0.0 5.0 0.0 Note: The concentrations of fungicides used were 10 µg/ml for fenhexamid (Elevate® 50WDG), fluopyram (Luna® Privilege), and penthiopyrad (Fontelis®), 500 µg/ml for pyraclostrobin (Cabrio® EG) and boscalid (Endura®), 5 µg/ml for pyrimethanil (ScalaTM SC), and 0.3 µg/ml for fludioxonil (Medallion®). Data on mycelial growth inhibition for similar experiments were used to calculate frequency of resistance to the fungicides. Fluopyram data is from the first experiment, for the other fungicides data is an average of two or three experiments. An isolate was considered resistant when mycelial growth was higher than 80% on pyraclostrobin- and boscalid-amended media or higher than 50% on the other fungicide- amended media tested.

Table 2-5. F-statistics and P-values for the fungicide inhibition of the conidial germination of Botrytis cinerea isolates collected from fourteen nurseries in 2011, 2012, and 2013. Fenhexamid Pyrimethanil Pyraclostrobin Boscalid Penthiopyrad Factor Df F P F P F P F P F P Experimenta 2 22.48 <0.01 9.16 <0.01 2.64 0.07 8.04 <0.01 7.81 <0.01 1b 141.85 <0.01 271.92 <0.01 128.63 <0.01 483.61 <0.01 Concentration . . c 13 39.86 <0.01 7.96 <0.01 <0.01 82.33 <0.01 4.90 <0.01 Nursery 95.59 13 5.73 <0.01 1.08 0.36 0.93 0.57 0.58 0.96 exp*nur 0.37 0.83 aThere were three experiments evaluated, where each isolate was tested in a fungicide-amended medium. bPyraclostrobin was tested at only one concentration, then concentration and its interactions could not be evaluated cFungicide inhibition was evaluated in fourteen nurseries: Bl, G, L, Cr, T, Tr, HB, CK, CM, B, NC, Pe, PL, and EG.

Table 2-6. Frequency of resistance to seven different fungicides of Botrytis cinerea isolates from nurseries tested in the conidial germination assay. Total Resistant Isolates (%) Year Region Nursery Isolates Fenhexamid Pyrimethanil Pyraclostrobin Boscalid Fluopyram Penthiopyrad Fludioxonil 2011 Nova Scotia G 9 11.1 100.0 77.8 55.6 0.0 0.0 0.0 Nova Scotia Bl 11 18.2 100.0 100.0 63.6 0.0 0.0 0.0 Quebec L 14 28.6 100.0 100.0 78.6 0.0 0.0 0.0 2012 California Cr 11 27.3 100.0 100.0 63.6 0.0 0.0 0.0 Nova Scotia G 16 6.3 93.8 93.8 62.5 0.0 0.0 0.0 Quebec L 22 68.2 100.0 100.0 86.4 0.0 0.0 0.0 North Carolina Tr 1 0.0 100.0 100.0 100.0 0.0 0.0 0.0 Ontario T 2 50.0 50.0 50.0 100.0 0.0 0.0 0.0 2013 Nova Scotia G 39 17.9 97.4 100.0 87.2 0.0 0.0 0.0 Nova Scotia CK 20 15.0 100.0 100.0 85.0 0.0 0.0 0.0 Nova Scotia CM 1 0.0 100.0 100.0 100.0 0.0 0.0 0.0 Nova Scotia HB 20 55.0 85.0 20.0 25.0 0.0 0.0 0.0 California Cr 59 47.5 100.0 100.0 89.8 0.0 0.0 0.0 Quebec L 38 97.4 100.0 100.0 94.7 2.6 0.0 0.0 Quebec LB 7 71.4 100.0 100.0 85.7 0.0 0.0 0.0 Quebec Pe 20 75.0 95.0 100.0 95.0 0.0 0.0 0.0 Quebec PL 20 45.0 100.0 100.0 100.0 0.0 0.0 0.0 North Carolina Tr 18 83.3 100.0 100.0 100.0 0.0 0.0 0.0 North Carolina B 8 37.5 100.0 100.0 100.0 0.0 0.0 0.0 North Carolina NC 20 75.0 100.0 100.0 95.0 0.0 0.0 0.0 Ontario EG 20 80.0 100.0 100.0 95.0 0.0 5.0 0.0 Ontario T 40 57.5 100.0 100.0 97.5 0.0 0.0 0.0 Note: The concentrations of fungicides used were 5 µg/ml for boscalid (Endura®), fluopyram (Luna® Privilege), and penthiopyrad (Fontelis®), 50 µg/ml for fenhexamid (Elevate® 50 WDG) and pyraclostrobin (Cabrio® EG), 25 µg/ml for pyrimethanil (ScalaTM SC), and 10 µg/ml for fludioxonil (Medallion®). Data on conidial germination inhibition experiments that were similar were used to calculate frequency of resistance to the fungicides. Presented data was an average of two or three experiments for all fungicides tested. An isolate was considered resistant when conidial germination was higher than 50% on all fungicide-amended media tested.

CHAPTER 3 OPTIMIZATION OF A RESAZURIN-BASED ASSAY TO EVALUATE SENSITIVITY OF Botrytis cinerea TO RESPIRATION-INHIBITOR FUNGICIDES

Introduction

Botrytis cinerea is a non-host specific pathogen that can cause several diseases, including gray mold or Botrytis fruit rot (BFR) in strawberries. The main control for gray mold relies on the use of fungicides. Throughout the strawberry season in Florida, fungicides are applied from the beginning of flowering in October-November to the end of the season in March-April, a total of 16 to 22 sprays (Amiri et al. 2013; Mertely et al.

2002). Several groups of single-site fungicides are available for gray mold management including: succinate dehydrogenase inhibitors (SDHI), quinone outside inhibitors (QoI), anilino-pyrimidines (AP), phenylpyrroles (PP), and sterol biosynthesis inhibitors (SBI).

Among these, the respiration-inhibitors boscalid (SDHI) and pyraclostrobin (QoI) are frequently used since the introduction of the pre-mixture product Pristine (BASF,

Research Triangle, NC) in 2004 (Amiri et al. 2013). Boscalid, a pyrimidine-carboxamide, works by inhibiting the complex II of respiration (FRAC 2015), whereas pyraclostrobin, a methoxy-carbamate, inhibits complex III or cytochrome bc1 at the Qo site (Bartlett et al.

2001) both interfering with the mitochondrial electron transport chain of the fungus and stopping energy production for the fungal cell.

Resistance of B. cinerea to fungicides is rapidly selected due to its abundant conidia production, high genetic variability, and the large number of fungicide sprays used to control it (16 to 22). Resistance of B. cinerea to boscalid and pyraclostrobin has already been reported in Florida (Amiri et al. 2013), California (Mercier et al. 2010),

North and South Carolina (Fernandez-Ortuno et al. 2012), Washington (Kim and Xiao

2010), and several countries (Leroux et al. 2010; Myresiotis et al. 2008). It is likely that

researchers have only identified a small proportion of the B. cinerea resistant populations within strawberry production regions. Further testing is needed to understand the true impact of these populations on gray mold management.

A common assay used to determine respiration-inhibitor fungicides sensitivity is conidial germination agar plate test because these fungicides interfere primarily at this fungal life stage (Russell 2002). This test basically evaluates the inhibition of the conidial growth and development in a medium amended with fungicide. However, it is time consuming, expensive, requires laboratory space, and measurements could vary depending on the person evaluating. Thus, there is a need for assay methods that can process a high throughput of B. cinerea isolates giving a faster resistance profile while spending less time and money.

Resazurin is a non-toxic dye that is used for determination of cell viability by measuring quantitatively the proliferation of the cells. Some fungicide sensitivity tests started to use the resazurin dye as an indicator of fungal growth in the presence of fungicide. The results were successful because the methodology is inexpensive, requires little space, is reliable, and gives quick results (Cox et al. 2009). The resazurin reduction occurs in the mitochondria and is evaluated fluorometrically or by absorbance readings (Vega et al. 2012), which can be made in as little as 60 min depending on the microorganism tested, e.g. Staphylococcus spp. (Pettit et al. 2005), and it can extend until fluorescence is no longer observed.

When optimizing a resazurin assay for fungal sensitivity to respiration-inhibitor fungicides, some factors to be considered are sporulation capacity of the specimen, spore density, type of medium and fungicide used, spectrophotometrical reading point,

frequency of evaluation, reagent concentrations, and relative-reduction calculations necessary to adjust the analysis.

Our hypothesis was that the resazurin-reduction assay used for other pathogens could work for determining fungicide sensitivity of B. cinerea to respiration-inhibitor fungicides. Thus, the objectives of this study were: i) to optimize a resazurin-reduction assay to evaluate the sensitivity of B. cinerea to the respiration-inhibitor fungicides boscalid and pyraclostrobin; and ii) to determine the effective fungicide dose that reduces fungal growth by 50% (EC50) and compare with the traditional conidial germination assay.

Materials and Methods

Fungal Isolates

Eleven single spore isolates of B. cinerea, with different sensitivities previously accessed through conidial germination inhibition (Table 3-1), were tested for fungicide sensitivity in a resazurin-reduction based assay optimized from the procedure of Vega, et al. (2012). The isolates were collected from strawberry nursery transplants in 2013, by placing the leaves over a wire rack in a humid chamber for a week. B. cinerea isolates were then transferred into a malt yeast agar (MYA) for single spore and posterior conidial production. The isolates were stored at -80oC in a suspension of 1 ml of 20% glycerol. In preparation for testing, B. cinerea colonies were incubated at room temperature (~23oC) for 7 days for full sporulation, the 6-cm diameter plates were flooded with 7 ml of deionized water, the conidia were harvested by rubbing the mycelial surface with a glass rod, and the suspension was filtered through two layers of sterile cheese cloth into a 15-ml Eppendorf tube. The solution was stored in a refrigerator, at -

4oC, for up to 10 days, and it did not germinate.

Pathogenicity Tests

To determine whether the isolates from senescent leaves were infective on fruit, the eleven isolates were inoculated on ‘Winterstar’ strawberry fruit following the method of Forcelini (2013) for the detached fruit assay. Immature pink strawberry fruit were harvested arbitrarily from the field. The fruit were selected by size in the laboratory and the calyx removed (Figure 3-1). Fruit with an average of 2 to 3 cm diameter were disinfested with 0.7% hypochlorite for 2 min and rinsed twice with deionized water.

Clear plastic boxes, measuring 31.5 x 25 x 10 cm, containing two 12-count egg cartons each were sprayed with 95% alcohol and placed in a fume hood under UV light for 20 min for sterilization. Disinfested fruit were placed in the egg cartons (Figure 3-2) and dried for 20 min in an air-flow hood. Before inoculation, the fruit were wounded with a sterile needle and then inoculated with a 10 µl conidial suspension at 105 conidia/ml.

Each isolate was inoculated on four fruit and four control fruit were treated with deionized water. To create a moist chamber (99 to 100% relative humidity), 70 ml of deionized water was added to the bottom of the boxes under the egg cartons. The boxes were kept at 23oC for 7 days and the disease incidence was evaluated visually.

Effect of Conidial Concentration on Resazurin Reduction

All eleven isolates were tested at four conidial concentrations 102, 103, 104, and

105 spores/ml adjusted using a hemacytometer. Two liquid media were used for this test, Complete Medium (CM) (Bennett and Lasure 1991) and HA medium (10g malt extract, 4g glucose, 4g yeast extract) (Mosbach et al. 2011) at pH 6.5 (adjusted with sodium hydroxide). A 4mM stock solution of resazurin was prepared by dissolving 0.2 g of resazurin salt (Sigma-Aldrich, St. Louis, MO) in 200 ml of deionized water and filter- sterilized using 0.22 µm pore filter (Corning Inc., Lowell, MA). A 40 µM solution was

prepared immediately before adding it to the plate. Two fungicides of the commercial formulations were tested: boscalid (Endura®, BASF, Research Triangle Park, NC) and pyraclostrobin (Cabrio® EG, BASF, Research Triangle Park, NC), at eight concentrations 0, 0.001, 0.01, 0.1, 0.5, 1, 5, and 10 µg/ml for boscalid and 0, 0.001,

0.01, 0.1, 0.5, 1, 10, and 50 µg/ml for pyraclostrobin. Salicylhydroxamic acid (SHAM)

(Acros Organics, New Jersey, USA) was added to all plates containing pyraclostrobin at

100 µg/ml, which was also the SHAM concentration per well. Three 96-well clear flat bottom microplates (Fisher Scientific, Nazareth, PA) were prepared for each concentration/medium/fungicide treatment with 80 µl of conidial suspension, 100 µl of medium amended with fungicide, and 20 µl of resazurin solution (40 µM), for a final volume of 200 µl per well. The plates were covered in aluminum foil and incubated in the dark at 25oC shaking at 300 rpm until evaluation. After 24h incubation, absorbance was read at 540 and 630 nm in a Multiskan® EX microplate reader (Thermo Scientific,

Vantaa, Finland) and resazurin reduction calculated. One row of wells containing only resazurin and medium was used as the control for each plate.

Evaluation of the Best Medium for the Reduction of Resazurin

Four liquid media were used for this test: Complete Medium (CM), Minimal

Medium (MM) (Bennett and Lasure 1991), HA medium at pH 5.5 (adjusted with hydrochloric acid) and HA at pH 6.5 (adjusted with sodium hydroxide). Eleven B. cinerea isolates were tested at 104 conidia/ml, and the same two fungicides, boscalid and pyraclostrobin (with addition of 100 µg/ml SHAM) were used. The fungicide and resazurin concentration, as well as the inoculation of the plates, incubation period, and measurements followed those above. Each medium/fungicide treatment (n=8) was performed in triplicate plates.

Time of Evaluation of the Microplates and its Effect on Resazurin Reduction

For testing the best time for evaluation of the plates, two media were used CM and HA at pH 6.5. All eleven isolates were tested at 104 spores/ml, and the plates were prepared with the fungicides at the concentrations described above. The 96-well microplates were incubated in the dark at 25oC shaking at 300 rpm, and evaluated every 2 h, from 16 to 32h after preparation, returning the plates to the shaker after each evaluation. Absorbance readings were taken at 540 and 630 nm in a Multiskan® EX microplate reader. Each medium/fungicide treatment was evaluated in triplicate plates, and the same replicate plate was either evaluated from 16 to 24h or from 24 to 32h.

Only five readings were taken in the same plate to avoid contamination.

Effect of SHAM and DMSO on the Respiration of Botrytis cinerea

To identify the effect of salicylhydroxamic acid (SHAM) on B. cinerea growth, the same eleven isolates were tested. Prior to addition to the amended medium, SHAM was diluted in dimethyl sulfoxide (DMSO) (0.1 g SHAM in 1 ml DMSO). Ten μl of SHAM was added to 10 ml of either CM or HA medium at pH 6.5, amended with pyraclostrobin (P) at concentrations of 0, 0.001, 0.01, 0.1, 0.5, 1, 10, or 50 µg/ml. The final concentration of SHAM in each well was 100 µg/ml. The effect of DMSO was also tested, to confirm absence of interference with the reduction of the dye, where 10 μl of the solvent was added to the amended medium mentioned above. The 96-well clear flat bottom microplates were prepared for each treatment with 80 µl of conidial suspension (104 conidia/ml), 100 µl of amended medium (P, P+SHAM, or P+DMSO), and 20 µl of resazurin solution (40 µM). One row containing only resazurin and the medium solution was used as a control for each plate, and every medium/fungicide treatment was

evaluated in triplicate plates. The plates were incubated as described above and evaluated after 24h incubation; absorbance also followed the previous description.

Resazurin Reduction Calculations

Resazurin, the oxidized form of the non-toxic dye is originally blue, after its reduction inside the living cells it becomes resorufin, fluorescent pink, or hydroresorufin, colorless. To calculate the percentage of reduction of the dye, equivalent to the fungal respiration rate, equation 3-1 provided by the manufacturer (Invitrogen 2009) was used, where the molar extinction coefficients can be found in Table 3-2:

% Reduction = (ƐOX)λ2 Aλ1 - (ƐOX) λ1 Aλ2 x 100 (3-1) (ƐRED)λ1 A’λ2 - (ƐRED)λ2 A’λ1

Where: ƐOX = molar extinction coefficient of alamarBlue oxidized form (BLUE) ƐRED = molar extinction coefficient of alamarBlue reduced form (RED) A = absorbance of test wells A’ = absorbance of negative control well (media + Resazurin) λ1 = 540nm λ2 = 630nm

The percent of reduction was calculated for each replicate plate and evaluated per experiment. A relative percent resazurin reduction for each treatment well was calculated by dividing values to the percent reduction of the positive control wells (B. cinerea conidial suspension + medium with resazurin) and multiplying by 100. The effective dose to provide 50% inhibition of the fungus (EC50) was obtained using this relative reduction on a sigmoidal analysis with four parameters on SigmaPlot 12.5 software (Systat Software, San Jose, CA, USA) and solving equation 3-2 to find X when

Y=50:

Y= Y0 + a (3-2) -([x-x ]/b) 1+e 0

Where: Y= Dose to inhibit germination by 50% (EC50) Y0= minimal % germination inhibition (in this case zero) a= range of % inhibition of germination X= log fungicide concentration X0= fungicide concentration at the maximum rate of change b= slope coefficient

The EC50 calculated from resazurin reduction was compared to the EC50 from a conidial germination inhibition assay with the same isolates and fungicide concentrations tested and correlated using SAS statistical software (Statistical Analysis

System, Cary, NC, USA).

Conidial Germination Test

To evaluate B. cinerea conidial germination inhibition, eleven isolates (Table 3-1) were cultivated on MYA for 7 days and a conidial suspension of 2.5x105 conidia/ml was prepared for each isolate. Each 10-cm diameter plate was divided into twelve 1.5 x 2 cm rectangles to guide droplets deposition. Two 7-µl drops of the spore suspension were placed diagonally in each square of the plate containing yeast bacto agar (YBA) or malt extract agar (MEA) amended with fungicides. YBA was amended with boscalid at 0,

0.001, 0.01, 0.1, 0.5, 1, 5, and 10 µg/ml; and MEA was amended with pyraclostrobin at

0, 0.001, 0.01, 0.1, 0.5, 1, 10, and 50 µg/ml. The effect of SHAM (100 µg/ml) and

DMSO was also evaluated when testing pyraclostrobin. Cultures were incubated at

24°C for 24h and conidial germination was evaluated microscopically. One hundred conidia were counted for each isolate and conidia were considered germinated if the germ tubes were equal to or longer than the conidial length. Percent inhibition was calculated relative to the control on non-amended YBA or MEA plate.

The EC50 was calculated for each isolate using Sigma Plot software and solving the Equation 3-2. Pearson’s correlation and analysis of variation of EC50 for the sensitive isolates from resazurin and conidial germination tests were calculated on SAS statistical software.

An estimate comparison of the resazurin-based assay (using either complete medium or HA medium), conidial germination, and mycelial growth tests was made for eleven isolates tested in one fungicide at eight different concentrations (Table 3-10).

The prices estimated are shown in US dollars and were based on the products used in this experiment. The space required was calculated measuring the volume of space occupied by the plates in the laboratory.

Results

Pathogenicity Tests

Ten of the 11 isolates produced gray mold symptoms in 100% of the strawberry fruit inoculated in the first experiment. Isolate 326 did not produce evident disease symptoms on the fruit inoculated in the first experiment. The experiment, then, was repeated using only isolates 25 (positive control) and 326. Isolate 326 produced symptoms on 100% of 4 fruit inoculated in the second experiment.

Effect of Conidial Concentration on Resazurin Reduction

Based on resazurin dye reduction within 24h, it was determined that the best conidial density of B. cinerea for the reduction of resazurin was 104 conidia/ml in both media and fungicides tested. On boscalid-amended medium (CM), isolates with lower conidia concentrations, 102 and 103 conidia/ml, did not reduce the resazurin dye, or just started to reduce it 24h after preparation of the plate (Figure 3-3). Conversely, 105 conidia/ml over-reduced the dye, forming hydroresorufin, a colorless compound and

precluding the calculation of EC50. In the plates without reduction or with over-reduction of the dye it was not possible to calculate EC50, since absorbance values are approximate the same. At 104 conidia/ml the resazurin reduction was visible and distinct for sensitive and resistant isolates (Figure 3-3) and was chosen for further tests. Similar results were observed on HA at pH 6.5 amended with boscalid or pyraclostrobin and

CM amended with pyraclostrobin (data not shown).

Evaluation of the Best Medium for the Reduction of Resazurin

HA medium at pH 5.5 was not a good medium for evaluation of resazurin reduction since the color change was not as distinct as with the other media and over- reduction was noted even though the fungicide and conidial concentration were the same for all media tested (Figure 3-4). Minimal medium (MM) showed lighter colored wells than complete medium or HA at pH 6.5, resulting in high readings of absorbance for MM. In the media where the change of color was not distinct (HA at pH 5.5) or the resazurin dye was too light (MM), it was not possible to calculate the EC50 for the isolates tested. The two best media for resazurin reduction were complete medium

(CM) and HA at pH 6.5, these showed the most difference between sensitive and resistant isolates. The EC50 values for the sensitive isolates on CM varied from 0.027 to

1.484 μg/ml on boscalid, and from 0.001 to 0.015 μg/ml on pyraclostrobin amended medium (Table 3-5). The same isolates on HA at pH 6.5 had EC50 values of 0.302 to

16.815 μg/ml on boscalid, and from 0.008 to 0.206 μg/ml on pyraclostrobin-amended medium. However, there was no significant difference (p=0.076) between the EC50 of sensitive isolates in CM compared to the EC50 values calculated in HA at pH 6.5 (Table

3-6). The EC50 values were highly correlated between those media amended with either

boscalid (r=0.75; Table 3-7) or pyraclostrobin (r=0.81; Table 3-8). Both CM and HA (pH

6.5) were used in further tests.

Time of Evaluation of the Microplates and its Effect on Resazurin Reduction

Five readings were made in each replicate plate every 2 hours, from 16 to 24h or from 24 to 32h, and a total of 9 readings were plotted and evaluated. Absorbance readings on HA medium at pH 6.5, as seen on Figure 3-5, were more stable overtime whereas those on complete medium (CM) had increased EC50 values of the sensitive isolates after 24h evaluation with both fungicides tested. Evaluations at 16 or 32h after preparation are not shown in the figure because the readings at 16h were low and not significantly different from those at 18h. Absorbance at 32h was high making the calculations of EC50 for the sensitive isolates not feasible (data not shown).

Effect of SHAM and DMSO on the Respiration of Botrytis cinerea

There was no effect of salicylhydroxamic acid (SHAM) and dimethyl sulfoxide

(DMSO) on the respiration of resistant isolates; however, when testing sensitive isolates in complete medium SHAM increased the range of EC50 values (Table 3-3), from 0.0318

(between 0.0004 and 0.0322 μg/ml) to 0.094 (between 0.0002 and 0.0942 μg/ml) when present. Similar results were observed on sensitive isolates on HA medium at pH 6.5

(Table 3-4). In Figure 3-6 the shift and increased EC50 value for the sensitive isolates can be observed with more wells remaining blue in the presence of SHAM.

Conidial Germination Test

There was no significant difference (p=0.076) between the EC50 values of the sensitive isolates when comparing the conidial germination test, and resazurin assay using complete medium (CM) or HA at pH 6.5 (Table 3-6). In conidial germination test, the sensitive isolates had the EC50 values from 0.029 to 0.799 μg/ml on boscalid-

amended medium and from 0.001 to 0.352 μg/ml on pyraclostrobin-amended medium

(Table 3-5).

Correlations between the EC50 values in conidial germination and the resazurin tests were all above 0.85 (Tables 3-7 and 3-8). Sensitive B. cinerea isolates on boscalid-amended medium had higher correlation of EC50 values between conidial germination and HA at 6.5 (r=0.94) than conidial germination and CM (r=0.89), whereas with pyraclostrobin the correlation was higher between conidial germination and CM

(r=0.99) than conidial germination and HA (r=0.85).

The SHAM effect on conidial germination was distinct (Table 3-9). On pyraclostrobin-amended medium, without SHAM or with dimethyl sulfoxide (DMSO) only, the sensitive isolates behave as resistant. In the presence of SHAM, sensitivity was observed with EC50 values from 0.001 to 0.352 μg/ml.

Both mycelial growth and conidial germination tests are more expensive and require more time and space than the resazurin-based assay (Table 3-10). To test eleven isolates in one fungicide at eight different concentrations, the number of plates necessary for the mycelial growth assay (n=264) is eleven times higher than the required for conidial germination (n=24), and 88 times higher than the resazurin-based assay (n=3). In the mycelial growth assay, 5280 ml of media are used, while the conidial germination uses 960 ml and the resazurin assay 28.8 ml. The price and time of the resazurin-based assay using HA media are the lowest observed, US$37.45 and 26h

27min. Also, the space required for the resazurin assay (703 cm3) is smaller than the required by the mycelial growth (13248 cm3) or the conidial germination (3800 cm3) tests.

Discussion

Numerous advantages can be seen for the resazurin reduction assay over the other fungicide sensitivity tests. It is faster, more accurate, cheaper, and requires less space. To evaluate 11 isolates, one fungicide, and 8 fungicide concentrations in triplicate: 264 petri dishes would be needed for the mycelial growth test, 24 petri dishes for conidial germination assay (11 isolates per plate) or 3 microtiter plates for the resazurin reduction assay. Tremblay et al. (2003) used 100 times less medium in the resazurin test, whereas this study estimated 180 times less medium used when compared with mycelial growth and 33 times less when compared with conidial germination. The total price calculated for the mycelial growth assay is approximately

US$119.25 and for the resazurin assay US$37.45; Cox et al. (2009) estimated US$4 per isolate on resazurin test versus US$15 on mycelial growth. The time of evaluation of one isolate is 45 min to 1h in conidial-germination assay (Vega et al. 2012) as opposed to 15 min for the three 12-isolate plates. Thus, the total time necessary for the resazurin assay is at least 4h lower than the other methodologies. This study then confirms that the resazurin assay is less expensive, faster and requires less laboratory space than the usual methodologies.

To adapt a resazurin based assay for testing the sensitivity of B. cinerea to respiration-inhibitor fungicides several components needs to be considered. The factors that interfere with the reduction of resazurin were evaluated in this experiment. The conidial concentration, medium, time of evaluation of the plates, and the effect of SHAM were tested to optimize a resazurin reduction assay for B. cinerea isolates from nursery transplants.

The best B. cinerea density for reduction of resazurin, 104 conidia/ml, was lower than with Alternaria alternata (Vega et al. 2012), Monilinia fructicola (Cox et al. 2009), and Verticillium dahliae (Rampersad 2011), and also lower than a fluorometric-based test with B. cinerea that used 105 conidia/ml (Pelloux-Prayer et al. 1998). With an automated quantitative assay that tested B. squamosa sensitivity to several fungicides,

Tremblay et al. (2003) concluded that the pathogen has to sporulate well to optimize and use the microtiter assay. In higher conidial concentrations, 105 conidia/ml, it was observed an over-reduction of the dye that can be explained by the rapid growth of the fungus; however, with lower concentrations, 102 and 103 conidia/ml, the number of conidia per well was not enough to reproduce and show results in 24h. Perhaps, the plates with lower concentrations could be incubated for longer times, but the objective of this experiment was to obtain quick results.

Medium is another factor to be considered when optimizing the assay. HA is a specific medium used for sporulation of Botrytis and should be adjusted to pH 5.5 prior to autoclaving to avoid growth of bacteria. The fact that HA medium at pH 5.5 was not a good medium for the reduction of resazurin is because the dye is sensitive to alteration of pH in the environment (Bueno et al. 2002; Promega 2013). However, HA at pH 6.5 was as good as Complete Medium (CM) for fungal growth. Consequently, if one medium is to be chosen over another, HA (pH 6.5) would be selected because it is 1h

30 min faster to prepare than CM, and is US$0.07 more economic than CM. The light color of Minimal Media can be explained by its lack of nutrients making the absorbance readings inaccurate.

Some work argues that readings over time should not be made on the same plate due to possible contamination when opening the plate (Rampersad 2011). No contamination was found in the plates evaluated even after five evaluations per plate; this could be verified because the control wells remained blue throughout the evaluation. The decision on evaluating the plates over time was to determine the log growth stage of the fungus and the highest resazurin reduction, which was after 24h.

Evaluations at 24h after preparation was chosen as the best time for evaluation due to the more steady EC50 values observed for the sensitive isolates.

Salicylhydroxamic acid (SHAM) is added in vitro to medium amended with QoI fungicides (eg. pyraclostrobin, azoxystrobin, picoxystrobin, trifloxystrobin) to inhibit the alternative fungal respiration. The SHAM effect on the resazurin assay was performed because both compounds have activity on mitochondria (Promega 2013). In contrast to

Alternaria spp. respiration (Vega et al. 2012), SHAM interfered with B. cinerea respiration as seen in conidial germination; however, in resazurin reduction, it was visual but not consistent with the EC50 calculations. This may be possible to correct by the use of a different wavelength filter for absorbance readings or with the use of technical grade pyraclostrobin. In the absence of SHAM in pyraclostrobin-amended medium, B. cinerea can alternatively respire and grow, consequently increasing the reduction of resazurin dye and the EC50 value calculated for sensitive isolates. DMSO, the solvent used to dilute SHAM, had no effect on the respiration of the fungus.

The conidial germination inhibition assay evaluates the growth of the fungus after the contact with the fungicide, the resazurin reduction analyses the metabolic activity of the fungi while germinating and incorporating the fungicide (Vega et al. 2012). There is

a strong correlation between the EC50 for both tests and no significant difference between them, demonstrating that the resazurin assay can be used to evaluate fungicide sensitivity of B. cinerea. Moreover, examining the actual EC50 values, the inhibition of fungal growth in the resazurin test should be higher, due to its greater sensitivity, as reported by Rampersad (2011); however, it was not in agreement with my results, which may change with the use of different wavelength to measure absorbance.

Technical grade fungicides are the pure active ingredient known as unformulated fungicide. Commercial formulations though are a mixture of the active ingredient and other substances, like adjuvants that aid the action and stability of the active ingredient when sprayed in the field. Both fungicides used in this project were commercial formulations; however, the effect of the adjuvants on the reduction of resazurin was not evaluated, which could potentially give fluctuating results from test to test, i.e. the EC50 values of the isolate 24. The fungicides studied in this project have its mode of action on fungal respiration, since resazurin is also reduced in the mitochondria (Promega 2013); boscalid is a succinate dehydrogenase inhibitor fungicide, whereas pyraclostrobin is a quinone outside inhibitor, both inhibiting the respiration of the fungus at different points

(FRAC 2015). With the results from this study and even with the variable EC50 results, the resazurin-based assay was considered a viable test to replace a plate assay.

According to the manufacturer (Invitrogen 2009), the resazurin dye has its peak absorbance at 570nm and 600 nm, while its peak fluorescence is at 530-560nm excitation and 590nm emission. The only phase that the dye is fluorescent is in its reduced form, resorufin. When making the calculations to determine percentage of reduction of the dye, using different wavelength could lead to different reduction

calculations. The wavelengths 540 and 630nm used in the absorbance readings were the same as those used in previous work with Verticillium dahliae (Rampersad 2011), but different from the wavelengths 570 and 600nm used by Vega et al. (2012) and Cox et al. (2009), which are the recommended by the manufacturer. In a future work, the same wavelengths used by Vega et al. (2012) and Cox et al. (2009) are going to be tested to evaluate possible differences in results.

Besides all the advantages of the resazurin reduction test, some disadvantages can be noted such as the dependence on spores and the use of a nutrient-rich liquid medium that is very sensitive to contaminants; thus, only pure cultures can be used rather than field samples (Cox et al. 2009). Another point observed from my results is the viability of the spores. Old cultures with less viable spores would give incorrect results for the isolate tested, showing that the best conditions are the use of recently sporulated colonies of B. cinerea instead of a 2-week old colony.

The resazurin reduction assay is a good alternative for screening sensitive and resistant isolates of B. cinerea, especially when evaluating a large number of isolates. If used with this purpose, it provides a quick screening for sensitivity to respiration- inhibitor fungicides on the same microplate, at least 4h faster than the conidial germination assay. However, for determination of EC50 of the isolates the test has to be more precise and fine adjustments have to be made depending on the fungus tested.

From this study, different wavelengths and the use of technical grade fungicides are still going to be tested. Future work can be done testing other respiration-inhibitors (e.g. fluopyram, and penthiopyrad) that are used on strawberries for B. cinerea management.

It was confirmed that the resazurin-based assay is a useful assay to evaluate fungicide

resistance of B. cinerea to respiration-inhibitor fungicides and that this assay is faster, more affordable, and requires less space than either conidial germination or mycelial growth assays.

Figure 3-1. Pink fruit harvested from the field (left), selected by size, and with the calix removed (right). February 9, 2015. Courtesy of Michelle Souza Oliveira.

Figure 3-2. Strawberry fruit in egg cartons ready to be inoculated. Each fruit was inoculated with one isolate and bottom right fruit was the control, treated with water. February 9, 2015. Courtesy of Michelle Souza Oliveira.

Figure 3-3. Resazurin reduction by four conidial concentrations of Botrytis cinerea, from 102 to 105 conidia/ml, on boscalid-amended medium. Blue color indicates no reduction, pink indicates reduction, and bleach color indicates over-reduction of the dye. Sensitive isolates are 22, 24, 25, 26, 31, and 326. Resistant isolates are 13, 71, 151, 203, and 307. March 6, 2015. Courtesy of Michelle Souza Oliveira.

Figure 3-4. Resazurin reduction of Botrytis cinerea isolates (104 conidia/ml) on four liquid media, Complete Medium (CM), Minimal Medium (MM), HA at pH 5.5 and 6.5, amended with boscalid. The blue color indicates no reduction, pink indicates reduction, and the bleach color indicates over-reduction of the dye. Sensitive isolates are 22, 24, 25, 26, 31, and 326. Resistant isolates are 13, 71, 151, 203, and 307. June 13, 2015. Courtesy of Michelle Souza Oliveira.

Figure 3-5. EC50 (μg/ml) of sensitive Botrytis cinerea isolates in a resazurin assay with pyraclostrobin- and boscalid-amended media evaluated from 18 to 30h after preparation of the plates.

Figure 3-6. The effect of salicylhydroxamic acid (SHAM) on pyraclostrobin sensitivity in a resazurin-reduction assay. The blue color indicates no reduction and pink indicates reduction of the dye. Sensitive isolates are 22, 24, 25, 26, and 31. Resistant isolates are 13, 71, 151, 203, 307, and 326. February 6, 2015. Courtesy of Michelle Souza Oliveira.

Table 3-1. Botrytis cinerea isolates from strawberry nursery transplants and their sensitivity to boscalid and pyraclostrobin. Sensitivitya Isolate Region Nursery Boscalid Pyraclostrobin 13 California Cr R R 22 Nova Scotia HB S S 24 Nova Scotia HB S S 25 Nova Scotia HB S S 26 Nova Scotia HB S S 31 Nova Scotia HB S S 71 North Carolina NC R R 151 Nova Scotia CK R R 203 Ontario EG R R 307 North Carolina B R R 326 Nova Scotia G S R a Sensitivity previously tested by inhibition of mycelial growth and conidial germination on an amended agar assay (see Chapter 2). R=resistant, S=sensitive.

Table 3-2. Molar extinction coefficient for Resazurin (Invitrogen 2009).

Wavelength (λ) ƐRED ƐOX 540 nm 104,395 47,619 630 nm 5,494 34,798

Table 3-3. The EC50 (μg/ml) values of Botrytis cinerea isolates in a resazurin reduction assay and the effect of salicylhydroxamic acid (SHAM) and dimethyl sulfoxide (DMSO) on pyraclostrobin-amended medium (complete medium). EC value (μg/ml) Isolatea 50 Pyraclostrobin Pyraclostrobin+ SHAMb Pyraclostrobin+DMSO 13 >50 >50 >50 22 0.0025 0.0942 0.0004 24 0.0020 0.0070 0.0001 25 0.0004 0.0351 0.0044 26 0.0322 0.0002 0.5 x10-5 31 0.0064 0.0143 2.9 x10-5 71 >50 >50 >50 151 >50 >50 >50 203 >50 >50 >50 307 >50 >50 >50 326 >50 >50 >50 Note: EC50 is the effective fungicide dose that reduces fungal growth by 50%. Fungicide used was Cabrio® (pyraclostrobin). a Sensitive isolates are 22, 24, 25, 26, and 31. Resistant isolates are 13, 71, 151, 203, 307, and 326 (Table 3-1). b SHAM was used at 100 μg/ml

Table 3-4. The EC50 values (μg/ml) of sensitive Botrytis cinerea isolates in a resazurin- reduction assay and the effect of salicylhydroxamic acid (SHAM) and dimethyl sulfoxide (DMSO) on a pyraclostrobin-amended medium (HA at pH 6.5). EC (μg/ml) Isolate 50 Pyraclostrobin Pyraclostrobin+SHAMa Pyraclostrobin+DMSO 22 0.1164 0.0800 0.2003 24 4.0421 0.0076 ...b 25 0.0459 0.2064 0.0564 26 0.0006 0.1144 ... 31 0.0912 0.0168 7.3283 Note: EC50 is the effective fungicide dose that reduces fungal growth by 50%. Fungicide used was Cabrio® (pyraclostrobin). a SHAM was used at 100 μg/ml b Two of the EC50 values could not be calculated due to over-reduction of the dye

Table 3-5. The EC50 values (μg/ml) of Botrytis cinerea isolates on boscalid and pyraclostrobin- amended medium comparing a resazurin reduction assay and the conidial germination test.

EC50 values (μg/ml) Boscalid Pyraclostrobin Isolatea Conidial Resazurin Resazurin Conidial Resazurin Resazurin germination (CM) (HA 6.5) germination (CM) (HA 6.5) 13 >10 >10 >10 >50 >50 >50 22 0.029 0.103 0.302 0.077 0.004 0.080 24 0.799 1.484 >10 0.002 0.003 0.008 25 0.027 0.027 0.548 0.352 0.015 0.206 26 0.194 0.102 8.882 0.001 0.001 0.114 31 0.123 0.175 0.623 0.011 0.001 0.017 71 >10 >10 >10 >50 >50 >50 151 >10 >10 >10 >50 >50 >50 203 >10 >10 >10 >50 >50 >50 307 >10 >10 >10 >50 >50 >50 326 0.163 0.839 2.549 >50 >50 >50 Note: CM=Complete Medium, HA 6.5= HA medium at pH 6.5. EC50 is the effective fungicide dose that reduces fungal growth by 50%. Fungicides used were Endura® (boscalid) and Cabrio® (pyraclostrobin). a Sensitive isolates are 22, 24, 25, 26, and 31. Resistant isolates are 13, 71, 151, 203, 307, and 326 (Table 3-1).

Table 3-6. Analysis of variation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in a conidial germination test and a resazurin reduction assay with boscalid- and pyraclostrobin-amended medium. Source of variation Degrees of freedom SS MS F Value P value Testa 2 47.145 23.573 2.840 0.076 Fungicideb 1 27.025 27.025 3.260 0.082 Test*Fungicide 2 38.211 19.105 2.300 0.119 aThe tests evaluated were: conidial germination, resazurin reduction on complete medium, and resazurin reduction on HA at pH 6.5. bFungicides used were Endura® (boscalid) and Cabrio® (pyraclostrobin).

Table 3-7. Correlation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in a conidial germination test and resazurin reduction assay with boscalid-amended medium. Assay Conidial germination Resazurin (CM) Resazurin (HA) Assay rc P r P r P Conidial germination ...... 0.89 0.017 0.94 0.006 Resazurin (CMa) 0.89 0.017 ...... 0.75 0.086 Resazurin(HAb) 0.94 0.006 0.75 0.086 ... … a CM = Complete Medium b HA = HA medium at pH 6.5 c Pearson’s correlation coefficients

Table 3-8. Correlation of the EC50 values (μg/ml) of Botrytis cinerea sensitive isolates in conidial germination test and resazurin-reduction assay with pyraclostrobin-amended medium. Assay Conidial germination Resazurin (CM) Resazurin (HA) Assay rc P r P r P Conidial germination ...... 0.99 0.002 0.85 0.068 Resazurin (CMa) 0.99 0.002 ...... 0.81 0.098 Resazurin (HAb) 0.85 0.068 0.81 0.098 ... … a CM = Complete Medium b HA = HA medium at pH 6.5 c Pearson’s correlation coefficients

Table 3-9. Salicylhydroxamic acid (SHAM) and dimethyl sulfoxide (DMSO) effect on the EC50 values (μg/ml) of Botrytis cinerea isolates in conidial germination assay with pyraclostrobin-amended medium. EC values (μg/ml) Isolatea 50 Pyraclostrobin Pyraclostrobin+SHAMb Pyraclostrobin+DMSO 13 >50 >50 >50 22 >50 0.077 >50 24 >50 0.002 >50 25 >50 0.352 >50 26 >50 0.001 >50 31 >50 0.011 >50 71 >50 >50 >50 151 >50 >50 >50 203 >50 >50 >50 307 >50 >50 >50 326 >50 >50 >50 a Sensitive isolates are 22, 24, 25, 26, and 31. Resistant isolates are 13, 71, 151, 203, 307, and 326. b The concentration of SHAM was 100 μg/ml

Table 3-10. Price, time and space required in three different methodologies used to evaluate sensitivity of eleven Botrytis cinerea isolates to one fungicide in eight fungicide concentrations. Mycelial growth Conidia germination Resazurin (CM) Resazurin (HA) Number of plates 264 (60 mm) 24 (100 mm) 3 (96-well) 3 (96-well) Amount of media 5280 ml 960 ml 28.8 ml 28.8 ml P rice (US dollars) M edia $26.75 $2.22 - $5.70b $0.09 $0.02 Plates $40.00 $2.60 $16.04 $16.04 Resazurin dye - - $0.02 $0.02 Pipette tips - $1.90 $5.70 $5.70 Labora $52.50 $55.83 $25.67 $15.67 Total $119.25 $62.53 - $66.03 $47.52 $37.45 Time Preparation of the media 15 min 15 min 1h + 15minc 15 min Autoclave 30 min 30 min 30 min + 30 mind 30 min Cooling time 30 min + 30 mine 30 min + 30 mine 30 min 30 min Preparation spore suspension - 1h 1h 1h Preparation of the plates 1h 20 min 10 min 10 min Evaluation time 48h 24h 24h 24h Evaluation period 4h 4h 9 min 9 min Total 54h 45 min 31h 5 min 28h 4 min 26h 34 min Space required Volume (cm3) 13248 3800 702 702 a The labor price is based on a rate of US$10/hour. bThe price range depends on the media used: US$2.22 for malt extract agar or US$5.70 for yeast bacto agar. cPreparation of stock solutions before making the media takes 1h and preparation of the media 15 min. dStock solution need to be autoclaved for 30 min before added to the media, which is also autoclaved for 30 min. eThe cooling time is separated in 30 min before addition of fungicide and 30 min to solidify.

CHAPTER 4 CONCLUSIONS

Botrytis cinerea, causal agent of gray mold on strawberry fields, was detected at high frequency on transplants from all the nurseries sampled, and some isolates were determined to be resistant to multiple fungicides. Fungicide resistant B. cinerea isolates most likely resulted from non-target sprays used to control multiple pathogens in the nursery. Because most of the fungicides used in Florida are also used in nurseries, many B. cinerea isolates from quiescent infections were resistant, increasing the risk of resistance in the commercial strawberry fields of Florida.

Fluopyram (registration pending) and fludioxonil were the only fungicides for which resistance was not detected and thus they should not be used in nurseries to avoid selection of resistant populations. Fludioxonil is the most effective fungicide, but should be used judiciously to control B. cinerea, when conditions are favorable for gray mold development and limited to two times per season to avoid selection of resistant populations. Boscalid and pyraclostrobin (Pristine) as well as pyrimethanil (Scala) had high frequencies of resistant isolates and should no longer be recommended to control

B. cinerea in strawberry in Florida.

These findings warrant improvement of existing management practices for gray mold, with early detection and control of the disease to avoid further economic losses, and a cautious use of high risk fungicides in nurseries. They also reinforce the need for an integrated approach between strawberry nurseries and production fields for the management of gray mold.

The optimization of the resazurin-based assay suggests that this rapid, cost- effective and reliable technique can be used on a large scale to evaluate the sensitivity

of B. cinerea to respiration-inhibitor fungicides. The optimal conditions for evaluation of

B. cinerea reduction of resazurin were HA medium at pH 6.5, spore concentration of 104 conidia/ml, and evaluation of the microplates at 24h after preparation. Moreover, salicylhydroxamic acid (SHAM) is required when evaluating pyraclostrobin effect on B. cinerea respiration and conidial germination. The resazurin-reduction assay can be used to determine the EC50 of fungal isolates and is another possible way to screen for fungicide sensitivity in a large fungal population.

LIST OF REFERENCES

Agricultural Statistics, U. 2014. Vegetables, 2013 Summary;2014 ASI 1621-25;ISSN 0884-6413.

Agrios, G. N. 2005. Plant Pathology. 5th ed. ed. Elsevier Academic Press, Amsterdam ; Boston.

Amiri, A., and Peres, N. A. 2014. Diversity in the erg27 gene of Botrytis cinerea field isolates from strawberry defines different levels of resistance to the hydroxyanilide fenhexamid. Plant Disease 98:1131-1137.

Amiri, A., Heath, S. M., and Peres, N. A. 2013. Phenotypic characterization of multifungicide resistance in Botrytis cinerea isolates from strawberry fields in Florida. Plant Disease 97:393-401.

Amiri, A., Heath, S. M., and Peres, N. A. 2014. Resistance to fluopyram, fluxapyroxad, and penthiopyrad in Botrytis cinerea from strawberry. Plant Disease 98:532-539.

Angelini, R. M. D., Habib, W., Rotolo, C., Pollastro, S., and Faretra, F. 2010. Selection, characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid. European Journal of Plant Pathology 128:185-199.

Bardas, G. A., Veloukas, T., Koutita, O., and Karaoglanidis, G. S. 2010. Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs, QoIs and fungicides of other chemical groups. Pest Management Science 66:967-973.

Baroffio, C. A., Siegfried, W., and Hilber, U. W. 2003. Long-term monitoring for resistance of Botryotinia fuckeliana to anilinopyrimidine, phenylpyrrole, and hydroxyanilide fungicides in Switzerland. Plant Disease 87:662-666.

Bartlett, D. W., Clough, J. M., Godfrey, C. R. A., Godwin, J. R., Hall, A. A., Heaney, S. P., and Maund, S. J. 2001. Understanding the strobilurin fungicides. Pesticide Outlook 12:143-148.

Bennett, J. W., and Lasure, L. L. 1991. More gene manipulations in fungi. Academic Press, Inc.: San Diego, California, USA; London, England, Uk. Illus.

Brown, M. 2003. A Florida strawberry production and marketing enterprise. Pages 31- 42 in: The Strawberry: a Book for Growers, others. N. F. Childers, ed. N.F. Childers Publications, Gainesville-FL.

Bueno, C., Villegas, M. L., Bertolotti, S. G., Previtali, C. M., Neumann, M. G., and Encinas, M. V. 2002. The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reaction. Photochemistry and Photobiology 76:385-390.

Bulger, M. A., Ellis, M. A., and Madden, L. V. 1987. Influence of temperature and wetness duration on infection of strawberry flowers by Botrytis cinerea and disease incidence of fruit originating from infected flowers. Phytopathology 77:1225-1230.

Chatzidimopoulos, M., Ganopoulos, I., Madesis, P., Vellios, E., Tsaftaris, A., and Pappas, A. C. 2014. High-resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid. Plant Pathology 63:1336-1343.

Ciliberti, N., Fermaud, M., Languasco, L., and Rossi, V. 2015. Influence of fungal strain, temperature, and wetness duration on infection of grapevine inflorescences and young berry clusters by Botrytis cinerea. Phytopathology 105:325-333.

Cordova, L., Zuniga, A., Mertely, J., and Peres, N. 2014. Evaluation of products for the control of Botrytis fruit rot in annual strawberry, 2013-14. Plant Disease Management Report.

Cox, K. D., Quello, K., Deford, R. J., and Beckerman, J. L. 2009. A rapid method to quantify fungicide sensitivity in the brown rot pathogen Monilinia fructicola. Plant Disease 93:328-331.

Darnell, R. L. 2003. Strawberry growth and development. Pages 3-9 in: The Strawberry: a Book for Growers, others. N. F. Childers, ed. N.F. Childers Publications, Gainesville-FL.

Davis, R. M. 1997. Compendium of Lettuce Diseases. The American Phytopathological Society, St. Paul, MN.

Deising, H. B., Reimann, S., and Pascholati, S. F. 2008. Mechanisms and significance of fungicide resistance. Brazilian Journal of Microbiology 39:286-295.

Elad, Y., Williamson, B., Tudzynski, P., and Delen, N. 2007. Botrytis spp. and diseases they cause in agricultural systems. Pages 1-8 in: Botrytis: biology, pathology and control. Springer, Dordrecht ; Boston.

Ellis, M. A., and Grove, G. G. 1982. Fruit rots cause losses in Ohio strawberries. Ohio Report on Research and Development 67:3-4.

Esterio, M., Ramos, C., Walker, A. S., Fillinger, S., Leroux, P., and Auger, J. 2011. Phenotypic and genetic characterization of Chilean isolates of Botrytis cinerea with different levels of sensitivity to fenhexamid. Phytopathologia Mediterranea 50:414-420.

FAOSTAT. 2015. Strawberry production quantities by country. Food and Agriculture Organization of the United Nations - Statistic Division, http://faostat3.fao.org/.

Fernandez-Ortuno, D., Chen, F. P., and Schnabel, G. 2012. Resistance to pyraclostrobin and boscalid in Botrytis cinerea isolates from strawberry fields in the Carolinas. Plant Disease 96:1198-1203.

Fernandez-Ortuno, D., Chen, F. P., and Schnabel, G. 2013. Resistance to cyprodinil and lack of fludioxonil resistance in Botrytis cinerea isolates from strawberry in North and South Carolina. Plant Disease 97:81-85.

Fernandez-Ortuno, D., Grabke, A., Bryson, P. K., Amiri, A., Peres, N. A., and Schnabel, G. 2014. Fungicide resistance profiles in Botrytis cinerea from strawberry fields of seven southern US states. Plant Disease 98:825-833.

Fillinger, S., Leroux, P., Auclair, C., Barreau, C., Al Hajj, C., and Debieu, D. 2008. Genetic analysis of fenhexamid-resistant field isolates of the phytopathogenic fungus Botrytis cinerea. Antimicrobial Agents and Chemotherapy 52:3933-3940.

Forcelini, B. B. 2013. Effect of inoculum concentration, temperature and wetness duration on anthracnose fruit rot development on strawberry cultivars with different levels of susceptibility. University of Florida, Gainesville-FL.

Fraaije, B. A., Bayon, C., Atkins, S., Cools, H. J., Lucas, J. A., and Fraaije, M. W. 2012. Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat. Molecular Plant Pathology 13:263-275.

FRAC. 2015. Fungicide Resistance Action Committee (FRAC) Code List 2015: Fungicides sorted by mode of action (including FRAC Code numbering). CropLife International, Brussels, Belgium.

Gilreath, J. P., Santos, B. M., Noling, J. W., Locascio, S. J., Dickson, D. W., Rosskopf, E. N., and Olson, S. M. 2006. Performance of containerized and bare-root transplants with soil fumigants for Florida strawberry production. Horttechnology 16:461-465.

Grabke, A., and Stammler, G. 2015. A Botrytis cinerea population from a single strawberry field in Germany has a complex fungicide resistance pattern. Plant Disease 99:1078-1086.

Grabke, A., Fernandez-Ortuno, D., and Schnabel, G. 2013. Fenhexamid resistance in Botrytis cinerea from strawberry fields in the Carolinas is associated with four target gene mutations. Plant Disease 97:271-276.

Gronover, C. S., Kasulke, D., Tudzynski, P., and Tudzynski, B. 2001. The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea. Molecular Plant-Microbe Interactions 14:1293-1302.

Grunwald, N. J., Garbelotto, M., Goss, E. M., Heungens, K., and Prospero, S. 2012. Emergence of the sudden oak death pathogen Phytophthora ramorum. Trends in Microbiology 20:131-138.

Hochmuth, G., Cantliffe, D., Chandler, C., Stanley, C., Bish, E., Waldo, E., Legard, D., and Duval, J. 2006. Containerized strawberry transplants reduce establishment- period water use and enhance early growth and flowering compared with bare- root plants. HortTechnology 16:46-54.

Horst, R. K. 1983. Compendium of Rose Diseases. American Phytopathological Society in cooperation with Dept. of Plant Pathology, Cornell University, St. Paul, Minn., USA.

Invitrogen. 2009. alamarBlue Assay. Thermo Fisher Scientific, http://www.thermofisher.com/.

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 Management Science 67:474-482.

Jarvis, W. R. 1962. Infection of strawberry and raspberry fruits by Botrytis cinerea Fr. Annals of Applied Biology 50:569-&.

Kim, Y. K., and Xiao, C. L. 2010. Resistance to pyraclostrobin and boscalid in populations of Botrytis cinerea from stored apples in Washington state. Plant Disease 94:604-612.

Kirk, P. 2015. Index Fungorum. http://www.indexfungorum.org/Names/NamesRecord.asp?RecordID=217312.

Konstantinou, S., Sarmis, G., and Karaoglanidis, G. S. 2014. Population structure and fungicide resistance profile of Botrytis spp. causing damping-off disease in stone and pome fruit rootstock seedlings. Phytopathology 104:62-63.

Laleve, A., Fillinger, S., and Walker, A. S. 2014. Fitness measurement reveals contrasting costs in homologous recombinant mutants of Botrytis cinerea resistant to succinate dehydrogenase inhibitors. Fungal Genetics and Biology 67:24-36.

Legard, D. E., Xiao, C. L., Mertely, J. C., and Chandler, C. K. 2001. Management of Botrytis fruit rot in annual winter strawberry using captan, thiram, and iprodione. Plant Disease 85:31-39.

Legard, D. E., Ellis, M., Chandler, C. K., and Price, J. F. 2003. Integrated management of strawberry diseases in winter fruit production areas. Pages 111-124 in: The Strawberry: a Book for Growers, others. N. F. Childers, ed. N.F. Childers Publications, Gainesville-FL.

Legard, D. E., MacKenzie, S. J., Mertely, J. C., Chandler, C. K., and Peres, N. A. 2005. Development of a reduced use fungicide program for control of Botrytis fruit rot on annual winter strawberry. Plant Disease 89:1353-1358.

Leroux, P., Gredt, M., Leroch, M., and Walker, A. S. 2010. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Applied and Environmental Microbiology 76:6615-6630.

Leroux, P., Fritz, R., Debieu, D., Albertini, C., Lanen, C., Bach, J., Gredt, M., and Chapeland, F. 2002. Mechanisms of resistance to fungicides in field strains of Botrytis cinerea. Pest Management Science 58:876-888.

Li, X., Fernandez-Ortuno, D., Grabke, A., and Schnabel, G. 2014a. Resistance to fludioxonil in Botrytis cinerea isolates from blackberry and strawberry. Phytopathology 104:724-732.

Li, X., Fernandez-Ortuno, D., Chen, S., Grabke, A., Luo, C.-X., Bridges, W. C., and Schnabel, G. 2014b. Location-specific fungicide resistance profiles and evidence for stepwise accumulation of resistance in Botrytis cinerea. Plant Disease 98:1066-1074.

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd ed. ed. APS Press, St. Paul, Minn., USA.

MacKenzie, S. J., and Peres, N. A. 2012. Use of leaf wetness and temperature to time fungicide applications to control Botrytis fruit rot of strawberry in Florida. Plant Disease 96:529-536.

Martin, R. R., James, D., and Levesque, C. A. 2000. Impacts of molecular diagnostic technologies on plant disease management. Pages 207-239 in: Annual Review of Phytopathology, vol. 38. Annual Reviews {a} , 4139 El Camino Way, Palo Alto, CA, 94303-0139, USA.

Mercier, J., Kong, M., and Cook, F. 2010. Fungicide resistance among Botrytis cinerea isolates from California strawberry fields. Plant Health Progress:PHP-2010-0806- 2001-RS.

Mertely, J. C., and Peres, N. A. 2009. Botrytis fruit rot of gray mold of strawberry. in: EDIS Plant Pathology Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Gainesville-FL.

Mertely, J. C., MacKenzie, S. J., and Legard, D. E. 2002. Timing of fungicide applications for Botrytis cinerea based on development stage of strawberry flowers and fruit. Plant Disease 86:1019-1024.

Mosbach, A., Leroch, M., Mendgen, K. W., and Hahn, M. 2011. Lack of evidence for a role of hydrophobins in conferring surface hydrophobicity to conidia and hyphae of Botrytis cinerea. BMC Microbiology 11:(13 January 2011).

Moslonka-Lefebvre, M., Finley, A., Dorigatti, I., Dehnen-Schmutz, K., Harwood, T., Jeger, M. J., Xu, X. M., Holdenrieder, O., and Pautasso, M. 2011. Networks in plant epidemiology: from genes to landscapes, countries, and continents. Phytopathology 101:392-403.

Mwangi, M., and Nakato, V. 2009. Key factors responsible for the Xanthomonas wilt epidemic on banana in East and Central Africa. in: Acta Horticulturae. D. Jones and I. van der Bergh, eds. International Society for Horticultural Science (ISHS), Leuven, Belgium.

Myresiotis, C. K., Karaoglanidis, G. S., and Tzavella-Monari, K. 2007. Resistance of Botrytis cinerea isolates from vegetable crops to anilinopyrimidine, phenylpyrrole, hydroxyanilide, benzimidazole, and dicarboximide fungicides. Plant Disease 91:407-413.

Myresiotis, C. K., Bardas, G. A., and Karaoglanidis, G. S. 2008. Baseline sensitivity of Botrytis cinerea to pyraclostrobin and boscalid and control of anilinopyrimidine- and benzimidazole-resistant strains by these fungicides. Plant Disease 92:1427- 1431.

O'Dell, C. 2003. Ten years experience with plasticulture strawberries for colder areas. N.F. Childers Publications, Gainesville, FL.

Oliveira, M. S. 2014. Botrytis cinerea. http://wiki.bugwood.org/Botrytis_cinerea, Bugwood Wiki.

Oliver, R. P. a., and Hewitt, H. G. a. 2014. Fungicides in crop protection. 2nd edition ed. CABI, Boston, MA; Wallingford, Oxfordshire.

Park, S., Lee, D., Chung, H., and Koh, Y. 1995. Gray mold neck rot of onion caused by Botrytis allii in Korea. Korean Journal of Plant Pathology 11:348-352.

Pasche, J. S., Wharam, C. M., and Gudmestad, N. C. 2004. Shift in sensitivity of Alternaria solani in response to Q(o)I fungicides. Plant Disease 88:181-187.

Patel, J. S., Gudmestad, N. C., Meinhardt, S., and Adhikari, T. B. 2012. Pyraclostrobin sensitivity of baseline and fungicide exposed isolates of Pyrenophora triticirepentis. Crop Protection 34:37-41.

Pavan, W., Fraisse, C. W., and Peres, N. A. 2011. Development of a web-based disease forecasting system for strawberries. Computers and Electronics in Agriculture 75:169-175.

Pavan, W., Fraisse, C. W., and Peres, N. A. 2012. The strawberry advisory system: a web-based decision support tool for timing fungicide applications in strawberry. University of Florida Institute of Food and Agricultural Sciences (UF/IFAS) Extension, Gainesville.

Pearson, R. C., and Goheen, A. C. 1988. Compendium of Grape Diseases. APS Press, St. Paul, Minn., USA.

Pelloux-Prayer, A. L., Priem, B., and Joseleau, J. P. 1998. Kinetic evaluation of conidial germination of Botrytis cinerea by a spectrofluorometric method. Mycological Research 102:320-322.

Peres, N. A. 2015a. Two new fungicides might be available for strawberry this season. Page 13 in: Berry Vegetable Times IFAS Extension.

Peres, N. A. 2015b. Fungicides recommended for control of Anthracnose and Botrytis Fruit Rots in Florida. AgroClimate, http://agroclimate.org/tools/Strawberry- Advisory-System/.

Pettit, R. K., Weber, C. A., Kean, M. J., Hoffmann, H., Pettit, G. R., Tan, R., Franks, K. S., and Horton, M. L. 2005. Microplate alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrobial Agents and Chemotherapy 49:2612-2617.

Pimentel, D. 2008. Environmental and economic cost of the application of pesticides primarily in the United States. Pages 161-182 in: Food, Energy, and Society. D. Pimentel and M. H. Pimentel, eds. CRC Taylor and Francis Group, LLC, Boca Raton-FL.

Pritt, M. P. 2003. Perennial strawberry production and marketing. Pages 140-166 in: The Strawberry : a Book for Growers, others. N. F. Childers, ed. N.F. Childers Publications, Gainesville-FL.

Promega. 2013. CellTiter-Blue® Cell Viability Assay. in: Technical Bulletin Promega Corporation, Madison, WI.

Rahman, M., Ojiambo, P., and Louws, F. 2015. Initial inoculum and spatial dispersal of Colletotrichum gloeosporioides, the causal agent of strawberry anthracnose crown rot. Plant Disease 99:80-86.

Rampersad, S. N. 2011. A rapid colorimetric microtiter bioassay to evaluate fungicide sensitivity among Verticillium dahliae isolates. Plant Disease 95:248-255.

Russell, P. E. 2002. Sensitivity baselines in fungicide resistance research and management. Crop Life International, Belgium.

Samuel, S., Papayiannis, L. C., Leroch, M., Veloukas, T., Hahn, M., and Karaoglanidis, G. S. 2011. Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts. Pest Management Science 67:1029- 1036.

Santos, B. M., Peres, N. A., Price, J. F., Chandler, C. K., Whitaker, V. M., Stall, W. M., Olson, S. M., Smith, S. A., and Simonne, E. H. 2012. Strawberry production in Florida. Pages 271-282 in: Vegetable Production Handbook for Florida. S. M. Olson and B. Santos, eds. University of Florida - IFAS Extension, Gainesville-FL.

Shim, C. K., Kim, M. J., Kim, Y. K., and Jee, H. J. 2014. Evaluation of lettuce germplasm resistance to gray mold disease for organic cultivations. Plant Pathology Journal 30:90-95.

Sierotzki, H., Frey, R., Wullschleger, J., Palermo, S., Karlin, S., Godwin, J., and Gisi, U. 2007. Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance. Pest Management Science 63:225-233.

Soufi, Z., and Komor, E. 2014. Latent infection of asymptomatic Hawaiian sugarcane cultivars with 16SrI and 16SrXI phytoplasmas. Journal of General Plant Pathology 80:255-263.

Strand, L. L. 2008. Integrated pest management for strawberries. 2nd edition ed. University of California, Division of Agriculture and Natural Resources, Oakland- CA.

Sumner, D. R., Hanlin, R. T., and Gay, J. D. 1994. A bulb rot of Vidalia sweet onion caused by Botrytis tulipae in Georgia. Plant Disease 78:1218-1218.

Tremblay, D. M., Talbot, B. G., and Carisse, O. 2003. Sensitivity of Botrytis squamosa to different classes of fungicides. Plant Disease 87:573-578.

U.S.Government. 2008. U.S. Code - Plant pest and disease management and disaster prevention. in: 7, L. I. Institute, ed. Cornell University of Law School, Ithaca-NY.

U.S.Government. 2015. Eletronic Code of Federal Regulations. in: 7, N. A. a. R. A. s. O. o. t. F. R. (OFR), ed. Government Publishing Office, Washington.

USDA. 2013. Plants for Planting Manual. Page 698 USDA - Animal and Plant Health Inspection Service, http://www.aphis.usda.gov/import_export/plants/manuals/ports/downloads/plants _for_planting.pdf.

Vega, B., Liberti, D., Harmon, P. F., and Dewdney, M. M. 2012. A rapid resazurin-based microtiter assay to evaluate Qol sensitivity for Alternaria alternata isolates and their molecular characterization. Plant Disease 96:1262-1270.

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

Veloukas, T., Markoglou, A. N., and Karaoglanidis, G. S. 2013. Differential effect of sdhB gene mutations on the sensitivity to SDHI fungicides in Botrytis cinerea. Plant Disease 97:118-122.

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

Veloukas, T., Kalogeropoulou, P., Markoglou, A. N., and Karaoglanidis, G. S. 2014. Fitness and competitive ability of Botrytis cinerea field isolates with dual resistance to SDHI and QoI fungicides, associated with several sdhB and the cytb G143A mutations. Phytopathology 104:347-356.

Weber, R. W. S. 2011. Resistance of Botrytis cinerea to multiple fungicides in northern German small-fruit production. Plant Disease 95:1263-1269.

Wedge, D. E., Smith, B. J., Quebedeauxc, J. P., and Constantinc, R. J. 2007. Fungicide management strategies for control of strawberry fruit rot diseases in Louisiana and Mississippi. Crop Protection 26:1449-1458.

Whitaker, V. M., Boyd, N. S., Peres, N. A., and Smith, H. A. 2015. Strawberry Production. Pages 189-199 in: Vegetable Production Handbook of Florida. G. E. Vallad, J. H. Freeman and P. J. Dittmar, eds. University of Florida - IFAS Extension, Gainesville-FL.

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.

Yin, Y. N., Kim, Y. K., and Xiao, C. L. 2012. Molecular characterization of pyraclostrobin resistance and structural diversity of the cytochrome b gene in Botrytis cinerea from apple. Phytopathology 102:315-322.

Yukita, K. 2005. Field survey and infection time of blossom end rot of apple caused by Botrytis cinerea in Aomori Prefecture. Annual Report of the Society of Plant Protection of North Japan:88-92.

BIOGRAPHICAL SKETCH

Michelle Souza Oliveira was born and raised in Uberlandia-MG, Brazil. She enrolled at the University of Sao Paulo in 2007 where she would get her bachelor’s degree in agricultural engineering. In 2008 she started her second major in agricultural education. During her undergraduate studies she also developed research in plant breeding, animal biotechnology, and bioinformatics at the Center of Nuclear Energy on

Agriculture (CENA-USP) and at the University. Her final project was funded by FAPESP

(Sao Paulo Research Foundation) and was focused on horticultural fruit crops with the development of herbaceous and semi-ligneous cuttings of rootstocks of grapevine for scion stock production. In fall 2011, she started an internship at the Gulf Coast

Research and Education Center (GCREC) at University of Florida and developed research on the potential spread of Phytophthora from tailwater ponds and irrigation sources in commercial strawberry fields, which was published in the Proceedings of the

Florida State Horticultural Society. In 2012 she finished both her bachelor’s and in May

2013 she was admitted for the Master of Science in plant pathology at the University of

Florida. She focused her project on the study of the initial inoculum and fungicide sensitivity of Botrytis cinerea isolates that was partially funded by the Florida Strawberry

Growers Association (FSGA). During her master’s degree she did an Internship at the

Plant Diagnostic Center in Gainesville (PDC-GNV), presented her work at four meetings, received third place in a poster competition, and was granted three travel awards.

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