Epidemiology and Management of Foliar Diseases in Asparagus (Asparagus Officinalis L.)

Epidemiology and Management of Foliar Diseases in Asparagus (Asparagus officinalis L.).

Jennifer Marie Foster

A Thesis Presented to The University of Guelph

In partial fulfilment of requirements for the degree of Doctor of Philosophy in Plant Agriculture

Guelph, Ontario, Canada

ABSTRACT

Epidemiology and Management of Foliar Diseases in Asparagus (Asparagus officinalis L.)

Jennifer Marie Foster Advisor: University of Guelph, 2018 Dr. Mary Ruth McDonald

Foliar diseases caused by Stemphylium vesicarium and Puccinia asparagi are an increasing problem in the major asparagus production regions of eastern and central Canada. Replicated and repeated controlled environment and field trials were conducted in Ontario to assess the disease reaction of host crops, compare fungicide efficacy and the Tom-Cast forecasting model, test fertilizer amendments and investigate the epidemiology of Pleospora herbarum (teleomorph of S. vesicarium) on asparagus fern. All of the lines of asparagus assessed were susceptible to infection by S. vesicarium, however, certain lines from the University of Guelph demonstrated quantitative resistance to rust (P. asparagi). Purple spot infection on spears was not consistently correlated with severity on ferns. Stemphylium vesicarium can cross-infect asparagus, but some host adaptation or specialization, possibly via host-specific toxins, appears likely. The pathogen was shown to colonize necrotic leaves of fall rye, which is often used as a cover crop in asparagus fields. Several fungicides demonstrated promising efficacy against rust, however, less control of

S. vesicarium was observed. The forecasting model Tom-Cast had limited success in both low and high fertility programs. The results show that forecasting models need to be validated locally, in asparagus cultivars relevant to production today. The development of pseudothecia on fern was correlated with fern yellowing and chlorophyll concentration in outdoor trials, however, no pseudothecia developed in controlled environment studies. Greater understanding of the environmental factors that contribute to pseudothecia maturity would enable growers to better time management strategies. The presented study has contributed to the understanding of the etiology and epidemiology of S. vesicarium in asparagus, and also validated and tested new tools for control of foliar disease in asparagus. An integrated disease management strategy that includes cultivar selection, timely application of fungicides and inoculum management may reduce crop losses and asparagus decline.

ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Mary Ruth McDonald, for her reassurance, support and guidance throughout my Ph.D. program. I always appreciated and enjoyed the time spent discussing my graduate project, plant pathology, and challenges with disease management and life.

Over the last 6 years, I have enhanced my depth of knowledge, and Dr. McDonald helped me gain more confidence in my abilities and understanding of plant pathology.

I also thank the other members of my advisory committee: Dr. Mary Hausbeck, Dr.

Katerina Jordan and Dr. David Wolyn for their patience and expertise in the study of plant pathology and plant breeding. Each member of my committee forced me to think differently about my assumptions and pushed my thesis further than I would have alone. I would especially like to thank Dr. Hausbeck for her warmth and invaluable timely advice.

I am grateful for the support of my colleagues. Brady Code was the first to inspire me and continues to be an indispensable ally. Also, I would like to thank Dr. Abhinandan Deora and

Ashley Dickson for their critical review of my ideas and their help in solving several problems associated with my graduate project. Finally, without the following summer students who spent days counting spots and spraying in sweat suits, I would have no data to analyze or interpret:

Stephen Boersma, Nykole Crevits, Katie Goldenhar, Marinda Gras, Kelvin Knip, Charlotte

Mackay, Scott Snyder, Anne-Miet Van Den Nieuwelaar, Janneke Van Den Nieuwelaar and Hans

Van Lith.

I am indebted to the support of Syngenta Canada Inc. and the Asparagus Farmers of Ontario that allowed this work to be completed. I would also like to thank my grower cooperators Rudy

Thiessen, Gary VanLeeuwen and Ken Wall who let me infect their fields year after year and often

were more interested in the research than even myself. Thank you again for not spraying through my trials. Also, thank you to Paul Banks for helping me set up and design my first experiments.

I would like to thank my parents, Steve and Cindy Foster, for their encouragement and understanding. You can finally stop asking me if I am done my thesis yet. I am also grateful for my dear friends, Matt Baker, Claire Cowan and Dr. Sofia Windstam for their unwavering support and mutual love of cheese, beer and Robyn. Nevertheless, she persisted.

TABLE OF CONTENTS

Abstract ...... ii Acknowledgements ...... iv Table of contents ...... vi List of Tables ...... x List of Figures ...... xiii CHAPTER ONE - LITERATURE REVIEW...... 1 1.1 Asparagus production in Ontario ...... 1 1.2 Asparagus decline ...... 3 1.3 Stemphylium vesicarium in asparagus ...... 4 1.4 Pathogenicity of Stemphylium vesicarium ...... 9 1.5 Puccinia asparagi in asparagus ...... 10 1.6 Management of Stemphylium vesicarium in asparagus ...... 13 1.6.1 Crop resistance ...... 13 1.6.2 Cultural control - sanitation ...... 15 1.6.3 Cultural control - improvement of crop environment ...... 17 1.6.4 Chemical control ...... 18 1.7 Management of asparagus rust ...... 23 1.7.1 Crop resistance ...... 23 1.7.2 Chemical control ...... 25 1.8 Conclusion ...... 27 CHAPTER TWO - PATHOGENICITY OF STEMPHYLIUM VESICARIUM AND SUSCEPTIBILITY OF ASPARAGUS (ASPARAGUS OFFICINALIS L.) ...... 29 Abstract ...... 29 2.1 Introduction ...... 30 2.2 Materials and methods ...... 32 2.2.1 Isolate collection and maintenance ...... 32 2.2.2 Inoculum preparation for pathogenicity assays ...... 34 2.2.3 Detached asparagus spear assays ...... 35 2.2.4 Pear fruit assay ...... 37 2.2.5 Rye pathogenicity assay ...... 38

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2.2.6 Asparagus field trials ...... 39 2.2.7 Data analysis ...... 40 2.3 Results ...... 41 2.3.1 Isolate phenotyping ...... 41 2.3.2 Detached asparagus spear assays ...... 41 2.3.3 Pear fruit assay ...... 43 2.3.4 Rye pathogenicity assay ...... 44 2.3.5 Asparagus field trials ...... 45 2.4 Discussion ...... 48 CHAPTER THREE - MANAGEMENT OF STEMPHYLIUM LEAF SPOT (STEMPHYLIUM VESICARIUM WALLR.) E.G. SIMMONS AND RUST (PUCCINIA ASPARAGI DC) OF ASPARAGUS (ASPARAGUS OFFICINALIS L.) WITH CULTIVAR SELECTION AND FUNGICIDES ...... 51 Abstract ...... 51 3.1 Introduction ...... 52 3.2 Materials and methods ...... 53 3.2.1 Asparagus cultivar susceptibility to SLS and rust ...... 53 3.2.2 Fungicide efficacy trials ...... 54 3.2.3 Data analysis ...... 55 3.3 Results ...... 56 3.3.1 Asparagus cultivar susceptibility to SLS and rust ...... 56 3.3.2 Fungicide efficacy trials ...... 57 3.4 Discussion ...... 61 CHAPTER FOUR - ASSESSING THE TOM-CAST FORECASTING MODEL FOR MANAGEMENT OF STEMPHYLIUM LEAF SPOT (STEMPHYLIUM VESICARIUM WALLR.) E.G. SIMMONS IN ASPARAGUS (ASPARAGUS OFFICINALIS L.) IN ONTARIO...... 63 Abstract ...... 63 4.1 Introduction ...... 64 4.2 Materials and methods ...... 67 4.2.1 Field trial design...... 67

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4.2.2 Fungicide applications ...... 68 4.2.3 Assessments and data analysis ...... 69 4.3 Results ...... 71 4.3.1 Fungicide applications and disease pressure ...... 71 4.3.2 Stemphylium leaf spot counts ...... 73 4.3.3 Disease progress ...... 75 4.3.4 Defoliation ...... 77 4.4 Discussion ...... 78 CHAPTER FIVE - THE EFFECT OF FERTILIZER, IRRIGATION AND FUNGICIDES ON FOLIAR DISEASE PROGRESS AND FERN HEALTH IN ASPARAGUS (ASPARAGUS OFFICINALIS L.) ...... 83 5.1 Introduction ...... 83 5.2 Materials and methods ...... 86 5.2.1 Site selection and description ...... 86 5.2.2 Field trial design and treatment application ...... 87 5.2.3 Assessments and data analysis ...... 88 5.3 Results ...... 90 5.3.1 Disease severity and progression ...... 90 5.3.2 Fern yellowing ...... 96 5.3.3 Defoliation ...... 100 5.3.4 Correlation analysis...... 103 5.4 Discussion ...... 105 CHAPTER SIX - DEVELOPMENT AND MANAGEMENT OF PLEOSPORA HERBARIUM IN DORMANT ASPARAGUS (ASPARAGUS OFFICINALIS L.) ...... 109 6.1 Introduction ...... 109 6.2 Materials and methods ...... 112 6.2.1 Pleospora herbarum maturity in fern ...... 112 6.2.2 Pleospora herbarum development in seedlings - outdoor experiment ...... 115 6.2.3 Pleospora herbarum development in seedlings - controlled environment ...... 117 6.2.4 Sanitation field trials ...... 118 6.2.5 Data analysis ...... 120

6.3 Results ...... 120 6.3.1 P. herbarum maturity in fern ...... 120 6.3.2 P. herbarum development in seedlings - outdoor experiment ...... 128 6.3.3 P. herbarium development in seedlings - controlled environment ...... 132 6.3.4 Sanitation field trial ...... 134 6.4 Discussion ...... 135 CHAPTER SEVEN - GENERAL DISCUSSION ...... 139 Literature Cited ...... 147 Appendix 1: Supplemental tables and figures for Chapter Two ...... 163 Appendix 2: Supplemental tables for Chapter Three ...... 236 Appendix 3: Supplemental tables for Chapter Four ...... 239 Appendix 4: Supplemental tables for Chapter Five ...... 249 Appendix 5: Supplemental tables for Chapter Six ...... 266

LIST OF TABLES

Table 1.1 Fungicides and fungicide mixtures registered in Canada for control of Stemphylium vesicarium in asparagus ...... 18

Table 1.2 Daily disease severity value (DSV) calculated from the hours of leaf wetness and average temperature during leaf wetness in the TOM-CAST model ...... 22

Table 1.3 Fungicides and fungicide mixtures registered in Canada for control of Puccinia asparagi in asparagus ...... 26

Table 2.1 Selected isolates of Stemphylium vesicarium, collected from asparagus and onion fields in Canada, assessed for pathogenicity on asparagus, pear and rye...... 34

Table 2.5 Purple spot incidence (%) and DSI (disease severity index) observed in six asparagus cultivars or lines at Simcoe Research Station, Simcoe, Ontario ...... 45

Table 2.6 Purple spot incidence (%) and DSI (disease severity index) observed in six asparagus cultivars or lines at Honeywood Research Facility, Plattsville, Ontario, n=5 ...... 46

Table 2.7 Stemphylium leaf spot DSI and sAUDPC (standardized area under the disease progress curve) observed in six asparagus cultivars or lines at two sites, n=5...... 47

Table 3.1 Fungicide treatment, application rate and product information applied every 14 days to asparagus fern infected with both Stemphylium vesicarium and Puccinia asparagi...... 55

Table 3.3 Field evaluation of foliar fungicides applied on a 14-day interval for rust control in a 3-year-old asparagus field, in Norfolk County, in 2011...... 59

Table 3.4 Field evaluation of foliar fungicides applied on a 14-day interval for rust and Stemphylium leaf spot control in a 4-year-old asparagus field, in Norfolk County, in 2011 ...... 60

Table 4.1 Number of fungicide applications timed according to either calendar-based application timing or the forecasting model TOM-CAST in asparagus...... 71

Table 4.2 Final Stemphylium leaf spot (Stemphylium vesicarium) counts on asparagus fern treated with foliar fungicides at four application timings...... 73

Table 5.1 Soil description at five field sites where fertilizer and foliar fungicide treatments were compared to control Stemphylium vesicarium in asparagus, 2015-16 ...... 86

Table 5.2 Location and planting description of asparagus field sites where fertilizer and fungicide treatments were compared for control of Stemphylium vesicarium, 2015-16 ...... 87

Table 5.3 Location, soil type, irrigation, number of applications and cumulative DSV recorded at trial sites ...... 91

Table 5.4 Interaction in disease severity index (DSI) between fertilizer and fungicide timing treatments at one site, irrigated with drip irrigation, 2016 ...... 94

Table 5.5 Standardized area under the disease progress curve (sAUDPC) of Stemphylium leaf spot calculated at 14 days after the last application on asparagus treated with chlorothalonil and azoxystrobin + difenoconazole according to the forecasting model TOM-CAST at either 15 or 30 DSV1...... 95

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Table 5.7 Spearman-rank correlation coefficient between disease response and agronomic treatments or characteristics ...... 104

Table 5.8 Pearson correlations coefficients between disease assessments and nutrient concentration measured at 42 days after cladophyll emergence from plots treated with 0 or 180 g N per ha...... 104

Table 6.2 Correlations (multivariate) between disease and dormancy parameters collected from field trials in 2015 and 2016, n=5...... 127

Table 6.4 Summary of significant fixed effects P ≤ 0.05 as determined from the restricted maximum likelihood (REML) covariance parameter estimates from data pooled between repetitions and asparagus cultivars Jersey Giant and Guelph Millennium sampled or assessed weekly in asparagus seedlings from mid-October to mid-December in 2016, n=20...... 129

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LIST OF FIGURES

Figure 1.1 Carbohydrate cycle in relation to production in established fields of asparagus in temperate climates...... 2

Figure 1.2 Purple spot lesions on asparagus spears (a), Stemphylium leaf spot lesions on asparagus fern cladophylls (b), and fern yellowing and defoliation caused by Stemphylium vesicarium (c)...... 4

Figure 1.3 Ascus and ascospores of Pleospora herbarum (modified from Simmons, 1985)...... 6

Figure 1.4 Conidia and conidiophores of Stemphylium vesicarium (modified from Simmons, 1969)...... 8

Figure 1.5 Rye cover crop stand in the spring seeded in an asparagus field the previous fall...... 9

Figure 1.6 Aecial (a), uredinial (b) and telial (c) pustules on asparagus fern caused by Puccinia asparagi...... 12

Figure 1.7 Slow-rusting asparagus lines surrounding the rust-susceptible ‘Tiessen’...... 25

Figure 4.1 Stemphylium leaf spot lesions counted per branch in the untreated control plots at six field trials. Error bars represent the standard error of the mean, n=4...... 72

Figure 5.1 Disease severity index observed in the untreated control in six field trials conducted in 2015 and 2016. Error bars are the standard error, n=16...... 91

Figure 5.2 Disease severity index observed in six field trials treated with fungicides according to the TOM-CAST forecasting model (*15DSV < 30 DSV and untreated, **15 and 30 DSV < untreated, ***15 DSV < 30 DSV < untreated, and ****15 DSV = 30 DSV and 30 DSV = untreated). Error bars are the standard error of the mean, n=16...... 93

Figure 5.3 Fern yellowing (%) observed in the untreated control in six field trials conducted in 2015 and 2016. Error bars are the standard error of the mean, n=16...... 97

Figure 5.4 Fern yellowing (%) observed in six field trials treated with fungicides according to the TOM-CAST forecasting model (* 15DSV < 30 DSV and untreated, ** 15 and 30 DSV < untreated, ***15 DSV < 30 DSV < untreated, **** 15 DSV = 30 DSV and 30 DSV = untreated, ***** 15 DSV = untreated and 30 DSV = untreated but 15 DSV ≠ 30 DSV). Error bars are the standard error of the mean, n=16...... 99

Figure 5.5 Cladophyll defoliation (%) observed in the untreated control in six field trials conducted in 2015 and 2016. Error bars are the standard error of the mean, n=16...... 100

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Figure 5.6 Cladophyll defoliation (%) observed in six field trials treated with fungicides according to the TOM-CAST forecasting model (* 15DSV < 30 DSV and untreated, ** 15 and 30 DSV < untreated, ***15 DSV < 30 DSV < untreated, **** 15 DSV = 30 DSV and 30 DSV = untreated, ***** 15 DSV = untreated and 30 DSV = untreated but 15 DSV ≠ 30 DSV). Error bars are the standard error of the mean, n=16...... 102

Figure 6.1 Air temperature (A), relative humidity (B), and rainfall (C) recorded at the Syngenta Canada Inc. research station in Oxford County, ON, in 2015 ...... 122

Figure 6.2 Air temperature (A), relative humidity (B), and rainfall (C) recorded at the Syngenta Canada Inc. research station in Oxford County, ON, in 2016 ...... 123

Figure 6.3 Stemphylium leaf spot and pseudothecia incidence (A) and severity (B), chlorophyll concentration (C), and cladophyll defoliation and fern yellowing (D) of asparagus fern in 2015, n=5. Error bars are the standard error of the mean...... 125

Figure 6.4 Stemphylium leaf spot and pseudothecia incidence (A) and severity (B), chlorophyll concentration (C), and cladophyll defoliation and fern yellowing (D) of asparagus fern in 2016, n=5. Error bars are the standard error of the mean ...... 126

Figure 6.5 Air temperature (A), relative humidity (B), and rainfall (C) recorded at the Syngenta Canada Inc. research station in Oxford County, ON, for the duration of outdoor seedling experiment in 2016 ...... 130

Figure 6.6 Chlorophyll concentration (A) and Stemphylium leaf spot lesions and pseudothecia (B), and proline (C) and sucrose (D) concentration in seedling ferns and asparagus crowns grown in pots outdoors from mid-October to mid-December in 2016, n=20. Error bars are standard error of the mean, and different letters denote Tukey’s HSD (P≤0.05) ...... 131

Figure 6.7 Chlorophyll concentration (A) and Stemphylium leaf spot lesions (B), and proline (C) and sucrose (D) concentration in seedling ferns and crowns of ‘Guelph Millennium’ asparagus at two temperatures, n=8. Error bars are standard error of the mean, and letters denote Tukey’s HSD (P≤0.05)...... 133

Figure 6.8 Incidence (A) and severity (B) of purple spot infection on asparagus spears harvested from plots where the fern was chopped either in the fall following fern senescence or spring following snow melt, n=12. Error bars are standard error of the mean. Letters denote Tukey’s HSD (P≤0.05) for the pair of bars. Spears were stored for 3 days and re-assessed for latent infection ...... 135

CHAPTER ONE

LITERATURE REVIEW

1.1 Asparagus production in Ontario

Asparagus (Asparagus officinalis L.) is a member of the Asparagaceae family, and the site of origin is not known, but is believed to have evolved either in Asia or the Mediterranean seacoast

(Bailey & Bailey, 1976). In North America, commercial production began in the late 1800s (Elmer et al., 1996), and today, the majority of both processing and fresh asparagus is produced in

California, Washington, Michigan and Ontario (NASS, 2016; Statistics Canada, 2016). In the

United States, the area planted decreased 11% from 10800 ha to 9600 ha from 2011 to 2016

(NASS, 2016). The production value of the market also declined in the United States from $92 million USD in 2011 to $75 million USD in 2016, largely due to the significant drop in production from California (NASS, 2016). In Canada, however, production has grown. From 2011 to 2016, the area of producing and non-producing fields grew by 27% from 1800 ha to nearly 2300 ha

(Statistics Canada, 2016). The farm gate value in Canada increased from $22 million CAD in 2011 to over $32 million CAD in 2016 (Statistics Canada, 2016). Most asparagus in Ontario is produced for fresh consumption, and specific guidelines are in place to market the high-value Canada No. 1 grade (CFIA, 2011). Any permanent defect which is the result of mechanical damage, insect feeding or disease would downgrade the spear from a Canada No. 1 to Canada No. 2.

Traditionally, in the first year of production, asparagus seeds are planted into crown nursery fields and maintained free of disease and insects for the duration of the season (OMAFRA, 2017).

The following spring, the crowns are removed from the nursery and established into commercial fields. For the first 2 years after transplanting, the spears are not harvested in order to allow the

crowns to establish and build a reserve of carbohydrates. In the third year, spears are harvested for only a short period of time, which maximizes the amount of time in fern to contribute photosynthate to the developing crown (Fig. 1.1) (Elmer et al., 1996). Following the third year, spears are harvested for 6 to 8 weeks, depending on weather, stress to the crown and the market price (OMAFRA, 2017). Fields are expected to remain in production and profitable for > 15 years

(Elmer et al., 1996). The lengths of the harvest and fern development periods, total photosynthetic area (crop canopy) and fern health impact production and profitability of asparagus fields.

Figure 1.1 Carbohydrate cycle in relation to production in established fields of asparagus in temperate climates.

1.2 Asparagus decline

The longevity of an established asparagus field can be shortened by several abiotic and biotic factors. In the worst case, a reduction in yield can be observed as early as 5 years after establishment if fields are not properly managed, and this is referred to as asparagus decline (Elmer et al., 1996; Grogan & Kimble, 1959). Often symptoms of asparagus decline are not evident until a full harvest is taken from a field in year three or four (Elmer et al., 1996; Grogan & Kimble,

1959). Once a field has exhibited symptoms, very rarely can those fields be replanted (Elmer et al., 1996).

The primary cause of asparagus decline is biotic stress including several soil- and air-borne pathogens (Elmer et al., 1996). Soil-borne pathogens, such as Fusarium spp. (Grogan & Kimble,

1959) and Phytophthora spp. (Saude et al., 2008) affect the crown growth and development directly. When infection is severe, both Fusarium and Phytophthora root rot kill the crown resulting in large dead areas of the field (Groan & Kimble, 1959; Saude et al., 2008). On the contrary, foliar pathogens, such as Puccinia asparagi DC and Stemphylium vesicarium (Wallr.)

E.G. Simmons, affect the crown indirectly. Both asparagus rust (P. asparagi) and Stemphylium leaf spot (S. vesicarium) infect the fern and symptoms include premature fern yellowing and defoliation (Elmer et al., 1996). Premature defoliation reduces the annual photosynthetic potential of the fern and results in a net negative return of carbohydrate to the crown (Kahn et al., 1952). If left unmanaged, both soil-borne and foliar pathogens pose a threat to the longevity of the asparagus industry in Ontario.

1.3 Stemphylium vesicarium in asparagus

In 1946, a Stemphylium sp. was isolated from asparagus spears with purple lesions in

Toppenish, WA (Elmer et al., 1996). Subsequently, in the early- to mid-1980's, Stemphylium vesicarium (teleomorph Pleospora herbarum (Pers.) Rabenh.) was reported as the cause of purple spot on asparagus spears and Stemphylium leaf spot on asparagus fern in France, New Zealand,

Switzerland and the United States (Blanchard et al., 1984; Falloon, 1982; Falloon et al., 1984;

Gindrat et al., 1984; Johnson & Lunden, 1984; Lacy, 1982). Purple spot is now found in most production regions (Elmer et al., 1996). Although not formally reported, purple spot and

Stemphylium leaf spot have been recognized as diseases in Canada since the growers switched to no-tillage practices in the 1990s (K. Wall, personal communication). Symptoms of S. vesicarium include premature defoliation (Fig. 1.2c), but severe purple spot also reduces the quality and marketability of asparagus spears (Lacy, 1982). Stemphylium vesicarium therefore reduces yield both directly and indirectly in asparagus.

Figure 1.2 Purple spot lesions on asparagus spears (a), Stemphylium leaf spot lesions on asparagus fern cladophylls (b), and fern yellowing and defoliation caused by Stemphylium vesicarium (c).

The disease cycle and aetiology of S. vesicarium during the production season in asparagus is well documented. Stemphylium vesicarium is in the phylum Ascomycota, and similar to other sac fungi, the teleomorph P. herbarum reproduces sexually through the formation of asci in pseudothecia (Evans & Stephens, 1984; Falloon et al., 1984; Falloon et al., 1987; Miyabe, 1889).

The pseudothecia of P. herbarum overwinter on dry asparagus fern either in commercial fields or volunteer plants (Lacy, 1982), but little is understood regarding the conditions required for their development. In onion, the pseudothecia form in the winter when temperatures are between 5 and

15 ºC (Prados-Ligero et al., 2003; Llorente & Montesinos, 2004). The ascospores are ejected from the pseudothecia with turgor pressure and appear to be the primary source of inoculum that infect the asparagus spears in the spring (Hausbeck et al., 1999). Mature ascospores of P. herbarum are

32-35 mm x 13-15 µm with seven transverse and one longitudinal septa (Fig. 1.3 modified from

Simmons, 1985). Asci are bitunicate and approximately 160 x 25 µm (Simmons, 1985). Recently, the name Stemphylium vesicarium has been proposed over the sexual teleomorph, P. herbarum

(Woudenberg et al., 2017).

Figure 1.3 Ascus and ascospores of Pleospora herbarum (modified from Simmons, 1985).

Lesion development on spears is influenced by the environmental conditions. In Michigan, lesion counts peaked during periods of low temperature, low vapour pressure deficit (VPD) and high rainfall (Granke & Hausbeck, 2010). In addition, low VPD and high rainfall were also correlated with high concentrations of airborne ascospores (Granke & Hausbeck, 2010), and symptoms developed on the spears 48 hours after discharge was detected (Hausbeck et al., 1999).

The optimal temperature for S. vesicarium infection in either the fern or spears is unknown.

Symptoms of purple spot on asparagus spears are small, 1 to 2 mm in size, elliptical, somewhat

sunken purplish spots (Fig. 1.2a) (Lacy, 1982). Spears can be free of symptoms at harvest and through cold storage, however, often symptoms will appear once spears are removed from storage and placed on store shelves (E. Roddy, personal communication). The latency of S. vesicarium, however, is not well defined in asparagus.

Following harvest, asparagus spears elongate and branch to produce a tall, dense canopy, the fern, which is also susceptible to infection by the anamorph S. vesicarium. Symptoms appear as tan to brown lesions with dark purple margins (Fig. 1.2b) (Elmer et al., 1996) and are favoured by prolonged periods of leaf wetness, low VPD and rainfall (Falloon et al., 1987; Granke &

Hausbeck 2010). Stemphylium leaf spot in the fern is caused by ascospore infection early in the summer and conidia late in the summer (Hausbeck et al., 1999). Stemphylium vesicarium reproduces asexually with the rapid production of conidia (Evans & Stephens, 1984; Falloon et al., 1984; Falloon et al., 1987; Miyabe, 1889). The conidia of S. vesicarium are oval, olive-brown, with 1 to 5 transverse and 1 to 2 longitudinal septa (Fig. 1.4 modified from Simmons, 1969). The conidia of S. vesicarium are twice as long as they are wide which and distinguishable from those of S. botryosum (Simmons, 1969). Granke and Hausbeck (2010) found positive correlations between airborne concentration of conidia and the average temperature and number of hours of leaf wetness per day. Aerial conidia counts were negatively correlated with VPD (Granke &

Hausbeck, 2010). As expected, the number of leaf spots on asparagus ferns peaks in correlation with the highest concentration of ascospores and conidia (Hausbeck et al., 1999). A forecasting model may be used to appropriately time fungicide application, based on the association of infection and weather conditions.

Figure 1.4 Conidia and conidiophores of Stemphylium vesicarium (modified from Simmons, 1969).

Stemphylium vesicarium infects asparagus spears through open stomata or wounds

(Falloon et al., 1987: Johnson & Lunden, 1986; Lacy, 1982). Frequently, symptoms appear on the side of the spear which is facing into prevailing wind (Lacy, 1982). Since asparagus is grown on light soils, the wind easily picks up sand particles which wound the asparagus spear (Lacy, 1982).

In the tobacco sand region of Ontario, wind breaks were planted to prevent wind erosion, however, often the tree rows are not sufficient to eliminate the movement of sand at the soil surface. In addition to wind breaks, asparagus growers establish rye in the fall to reduce wind erosion the following spring during harvest (Fig. 1.5) (Brainard, 2012). In the spring, the rye cover crop is killed with herbicides, but it is unknown if S. vesiscarium can survive on the dead plant tissue since

Stemphylium spp. can be both pathogenic and saprophytic (Hudson, 1971; Kohl et al., 2013; Rossi et al., 2005).

Figure 1.5 Rye cover crop stand in the spring seeded in an asparagus field the previous fall.

1.4 Pathogenicity of Stemphylium vesicarium

Stemphylium vesicarium has a diverse host range including cultivated crops such as asparagus (Lacy, 1982), Allium spp. (Basallote et al., 1993; Miller et al., 1978; Rao & Pavgi 1973;

Rao & Pavgi 1975), parsley (Koike et al., 2013) and pear (Vilardell, 1988). Stemphylium botryosum has also been identified as pathogens in asparagus (Falloon et al., 1987; Leuprecht,

1990; Suzui, 1973). Despite the prevalence of S. vesicarium in asparagus, Stemphylium leaf blight of onion was not identified in Ontario until 2008 (Paibomesai et al., 2012). Also, brown spot of pear has not been identified in North America, yet it occurs widely in Europe (Llorente &

Montesinos, 2002).

In both asparagus and onion, small elliptical spots initially develop on the infected tissue, and under ideal conditions, they coalesce, which results in elongated lesions in asparagus and leaf dieback in onion (Lacy, 1982; Rao & Pavgi, 1975). Despite similar symptomology in onion and asparagus, it is unclear whether S. vesicarium cross-infects host crops (Falloon et al., 1987; Kohl et al., 2009; Shishkoff & Lorbeer, 1989; Tayviah, 2017). Falloon et al. (1987) inoculated non- wounded seedlings with isolates of Stemphylium spp. from asparagus and onion. Only those isolates collected from asparagus were able to produce symptoms on asparagus seedlings.

Conversely, Shishkoff and Lorbeer (1989) and Tayviah (2017) observed symptoms on onion leaves inoculated with isolates of S. vesicarium from both onion and asparagus. In pears, only a single isolate from onion produced symptoms on detached non-wounded pears leaves out of 52 isolates selected from onion and asparagus (Kohl et al., 2009). None of the 50 isolates from onion and asparagus were pathogenic to non-wounded pear fruit (Kohl et al., 2009). It is unknown whether S. vesicarium collected from infected onion and asparagus plants in Canada can cross- infect asparagus and pear.

Stemphylium spp. can colonize senesced plant tissue (Hudson, 1971). In pear orchards, researchers have isolated S. vesicarium from the dead and living leaves of weeds and grass species

(Kohl et al., 2013; Rossi et al. 2005; Rossi et al., 2008). Rossi et al. (2005) found the isolates did not lose their pathogenicity. Rye is considered a non-host, but S. vesicarium could potentially survive on the dead plant tissue as a saprophyte. This relationship has yet to be investigated.

1.5 Puccinia asparagi in asparagus

A second foliar pathogen that impacts early decline of asparagus is Puccinia asparagi DC, causing asparagus rust. The disease was first described in 1805 in France (Norton, 1913). The

pathogen was first reported in eastern North America in 1896; however it is likely P. asparagi was introduced from Europe before that time (Norton, 1913). Within a few years, rust was reported in most major asparagus production areas of North America, except the Pacific Northwest (Norton,

1913). Rust is now found in all areas where asparagus is produced in North America, Europe and the South Pacific (Elmer et al., 1996; Cheah et al., 2003).

Puccinia asparagi is an autoecious rust with four distinct spore stages; basidiospores, aeciospores, urediniospores, and teliospores (Elmer et al., 1996). In the spring, teliospores will germinate and produce basidiospores. The basidiospores will infect the unharvested asparagus plants and pycnia appear as oval, light green lesions 6 by 19 mm in size (Elmer et al., 1996). Aecia, which are light orange in colour, will develop on the same lesions shortly after infection (Fig. 1.6a)

(Elmer et al., 1996). The aecia produce aeciospores which are easily wind-blown and the primary source of inoculum in young fields (Elmer et al., 1996). Once an aeciospore infects an asparagus plant the fungus will start to produce uredinia (Fig. 1.6b) (Elmer et al., 1996). The uredinial stage can repeat itself every 10 to 14 days depending on moisture and humidity (Evans, 1942; Johnson,

1990). Urediniospores are also easily windblown and the dark red spores are usually observed in asparagus fields in early to late summer (Elmer et al., 1996). Basidiospores, aeciospores, and urediniospores all require moisture for infection to occur, and epidemics of rust usually occur from rain, prolonged dews, and/or sprinkler irrigation (Evans, 1942; Kahn et al., 1952). In late summer and early fall, the uredinia are replaced by telia which produce dark brown overwintering teliospores (Fig. 1.6c) (Elmer et al., 1996).

Figure 1.6 Aecial (a), uredinial (b) and telial (c) pustules on asparagus fern caused by Puccinia asparagi.

Since asparagus is usually harvested until early summer, pycnia and aecia are rarely observed in commercial asparagus fields. Aeciospore production is avoided in commercial fields since the spears are harvested until early summer (Elmer et al., 1996). However, in most regions where asparagus is produced, volunteer asparagus plants will grow in the fence rows, ditches, and abandoned fields. The volunteer plants are not harvested and can act as a host for the basidiospores and aeciospores in the spring (Elmer et al., 1996). Second year plantings are also a source of early season inoculum since they are only harvested for a few weeks in the spring (Elmer et al., 1996).

Rust will infect the stem, fern, and cladophyls, causing the asparagus to senesce prematurely (Kahn, 1952). As mentioned for S. vesicarium, the stress on the crown further intensifies asparagus decline and results in decreased yield in subsequent years (Kahn, 1952). The effect of asparagus decline is observed as a reduction in overall number of harvested spears the following year and low spear weight (Elmer et al., 1996). Yield reduction on rust-susceptible

‘Mary Washington’ was 19 and 50 percent after the first and second year of infection, respectively

(Elmer et al., 1996). Also, rust caused little or no yield reduction in partially resistant cultivars, such as Jersey Giant and UC-157 (Johnson & Lunden, 1992), and in Italy, ten commercial cultivars were screened for rust resistance and the commercial cultivars Marte and Grande had the least amount of rust (Fiume & Fiume, 2003). However, similar to S. vesicarum, the impact of rust on current commercial cultivars in North America is relatively unknown.

1.6 Management of Stemphylium vesicarium in asparagus

Disease control strategies such as crop resistance, cultural control and chemical control have been investigated and adopted widely in asparagus production. However, due to the change in cultivar selection and crop management in the last decade, the established control strategies may no longer be relevant. Cultural control methods, such as irrigation and nutrient management, have not been extensively studied. Also, it is unknown if the contribution of moisture stress, poor nutrition and S. vesicarium infection synergistically interact to deleteriously effect crop productivity and longevity in asparagus. Since asparagus is a perennial crop, likely an integrated strategy that includes crop resistance, cultural control and chemical control will be required to increase efficacy and prevent early decline.

1.6.1 Crop resistance

Breeding asparagus cultivars for resistance or tolerance would be the first means of S. vesicarium control in asparagus. Several cultivars were screened for their susceptibility to

Stemphylium leaf spot, and ‘Jersey Giant’, ‘Rutgers Beacon’ and ‘UC157’ were less susceptible to defoliation than ‘Aneto’, ‘Cito’ and ‘Desto’ (Broadhurst, 1996). None of the cultivars tested exhibited resistance to S. vesicarium in the field or growth room (Broadhurst, 1996). Growth habit,

branching and canopy structure were correlated with disease susceptibility (Broadhurst, 1996).

The cultivars with the greatest susceptibility were short with low branching and compact ferns

(Broadhurst, 1996). Broadhurst predicted that the compact fern growth provided an ideal microclimate that held suitable moisture for S. vesicarium infection (1996). For S. vesicarium specifically, prolonged periods of leaf wetness have contributed to disease severity in asparagus

(Falloon et al., 1987; Granke & Hausbeck, 2010; Hausbeck et al., 1999; Johnson & Lunden, 1986;

Menzies et al., 1991), pear (Llorente & Montesinos 2002; Montesinos et al., 1995) and garlic

(Prados-Ligero et al., 2003). In asparagus, the height of the first branch is highly correlated to the tightness of the spear tip (Hanna, 1935), and plant breeders are already selecting for this desirable phenotype. It is unknown, however, if selection of branch height has also inadvertently selected for foliar disease tolerance.

The current understanding of asparagus tolerance to Stemphylium leaf spot is outdated and not particularly relevant to Ontario asparagus production. Since 2000, the predominant asparagus cultivars have changed in Ontario, and most fields are now planted to ‘Guelph Millennium’, a cultivar with high yield and winter hardiness (CFIA, 2017). Growers, however, perceive that

‘Guelph Millennium’ is more susceptible to Stemphylium leaf spot than the older less popular cultivars (Hausbeck & Escobar-Ochoa, 2014). Little is understood about the actual susceptibility of ‘Guelph Millennium’ to S. vesicarium and whether or not growers need to modify their management of Stemphylium leaf spot in this cultivar specifically. Ideally, growers could utilize resistant cultivars to manage Stemphylium leaf spot and purple spot, but the susceptibility of asparagus cultivars suitable for production in Canada has not been evaluated.

1.6.2 Cultural control - sanitation

Most asparagus in North America is grown using no-tillage practices to reduce wind erosion and the subsequent sand blasting to asparagus spears (Lacy, 1982). The infected fern stays standing in the field throughout the winter and is mowed the following spring prior to harvest. As a result, the pseudothecia of P. herbarum overwinter in the fields on dormant fern (Evans &

Stephens, 1984; Lacy, 1982). Regardless of the number of fungicide applications made to the fern in the previous season and the level of control observed, the inoculum does not appear to be reduced in the following spring (K. Wall, personal communication). Therefore, to improve purple spot control, methods aimed to reduce the source of primary inoculum are needed. Burial of asparagus residue reduced purple spot infection on the spears the following season (Johnson,

1990). As well, Falloon et al. (1984) noted less disease on spears from fields where the residue was removed. However, the obvious method to reduce inoculum, burial of the infected residue, is not a practical solution in no-till production.

In other perennial cropping systems, such as tree fruit, leaf litter from the previous season is associated with primary inoculum in the spring. In apple scab, the shredding of leaves in the fall and spring reduced the risk of scab by 80 to 90%, provided that all was shredded (Sutton et al.,

2000). Similarly, leaf shredding or leaf removal in pear orchards reduced the release of ascospores of Pleospora allii (Llorente et al., 2006). Sanitation provided similar control of brown spot as compared to a standard fungicide program, yet, the addition of sanitation to a fungicide program did not increase control compared to a standard fungicide program (Llorente et al., 2010).

Shredding dormant fern in the fall, rather than in the spring, and applying foliar amendments may reduce the primary inoculum in asparagus. The level of P. asparagi inoculum was reduced when infected plants were cut and removed from the field in late fall (Elmer et al., 1996; Fantino et al.,

1990). However, asparagus is often grown on sandy soil and the standing fern prevents wind erosion and catches snow through the winter.

Since the burial of fern is impractical in current no-tillage asparagus production, increasing the speed of decomposition of fern in the spring could help reduce infection at harvest. Other sanitation methods, such as the application of urea, have been tested in pear to control S. vesicarium (Llorente et al., 2010). Urea can be used to increase the speed of leaf decomposition

(Burchill, 1968), eliminating the need to physically remove the leaves or ferns from the field. Also, applications of urea after harvest but before leaf-fall restricted perithecial production by V. inaequalis (Burchill, 1968). Urea applied in November or April reduced the number of aerial ascospores of V. inaequalis by 50 and 66%, respectively (Sutton et al., 2000). In the asparagus production cycle, urea can be applied in either the late fall or early spring. Urea applications have proven to be beneficial in increasing the breakdown of residue in subtropical asparagus (Fantino et al., 1990), however, it is not known if urea has a direct impact on S. vesicarium inoculum in temperate climates.

Several studies at the University of Guelph have investigated dormancy of asparagus in

Ontario with respect to winter-hardiness and crown survival (Landry & Wolyn, 2011; Landry &

Wolyn, 2012; Panjtandoust & Wolyn, 2016). Early dormancy increases crown survival (Landry &

Wolyn, 2011), but as the fern goes dormant in the fall, the severity of Stemphylium leaf spot and the presence of pseudothecia appear to increase. Ideally, the fern could be mulched in the fall prior to the development of pseudothecia, however, removing the fern prior to dormancy has negative impact on crown health the following spring (Kelly & Bai, 1999). If the production of pseudothecia is related to dormancy, then growers could apply timely applications of amendments to reduce spring inoculum.

1.6.3 Cultural control – improvement of crop environment

Nutrient deficiencies can result in stress, and plants under abiotic stress have increased susceptibility to disease. Nitrogen, specifically, is the most extensively studied nutrient in plant disease research, and has been attributed to both increasing and decreasing the disease severity in several crops (Tompkins et al., 1992; Jensen & Munk, 1997) and necrotrophs (Howard et al., 1994;

Leitch & Jenkins, 1995; Long et al., 2000; Simon et al., 2003; Talukder et al., 2005). When nitrogen was applied in excess, the severity of foliar disease increased due to the enhanced production of lush susceptible host tissue (Long et al., 2000; Tompkins et al., 1992). Also, the denser canopy created an environment that promoted leaf wetness (Leitch & Jenkins, 1995).

Interestingly, in cereals, when nitrogen was applied later in the season, the disease severity was higher than that in the control (Neumann et al., 2004). Asparagus growers are advised to apply nitrogen fertilizer in a single dose following harvest to promote rapid plant growth to build a healthy crown. Inadvertently, the dense canopy is also creating an environment conducive to foliar disease. It is understood, that a balanced fertility program decreases disease by enabling the plant to grow out of the disease, increasing plant tolerance, or inhibiting pathogen activity (Huber &

Watson, 1974). Consequently, growers have been experimenting with the use of foliar fertilizer throughout the season, with varying rumoured success. For soil-borne disease in asparagus, the role of nitrogen fertilizer on disease progress is well studied (Elmer, 1989). However, since S. vesicarium is a foliar pathogen in asparagus, it is unknown if the application of nitrogen would increase or decrease Stemphylium leaf spot and premature defoliation.

rots, such as Phytophthora and Fusarium (Elmer et al., 1996). However, during periods of drought, asparagus grown on light soil types are most greatly affected, and the fern defoliates prematurely and senesces prior to dormancy (Panka & Rolbiecki, 2008). The potential impact of S. vesicarium infection on asparagus, in addition to moisture stress, is unknown. Also, asparagus growers in

Ontario have recently adopted the use of irrigation to prevent moisture stress during periods of drought. In dense foliar canopies, however, overhead irrigation is often attributed to a rise in foliar disease (Rotem & Palti, 1969). The impact of irrigation, either drip or overhead, on S. vesicarium infection of asparagus, is unknown.

1.6.4 Chemical control

Currently, only three fungicides are registered for control of Stemphylium leaf spot in asparagus in Canada (Table 1.1). Among the registered fungicides, azoxystrobin and trifloxystrobin belong to the Quinone-outside inhibitors (QoI) and chlorothalonil belongs to the multi-site fungicides. Each fungicide class has a unique mode of action, and often within the classes the different chemistries will differ in intrinsic activity against plant pathogenic fungi.

Table 1.1 Fungicides registered in Canada for control of Stemphylium vesicarium in asparagus. Active ingredient FRAC1 Resistance Target site Group name (Tradename) code risk azoxystrobin ...... complex III: cytochrome QoI2 11 high bc1 at Qo site chlorothalonil ...... multi-site chloronitriles M05 low trifloxystrobin ...... complex III: cytochrome QoI 11 high bc1 at Qo site 1Fungicide Resistance Action Committee 2Quinone outside inhibitor

The Quinone outside Inhibitor (QoI) fungicides, also referred to as strobilurins, affect cell respiration in fungal plant pathogens (FRAC, 2017). In order to spread and survive, fungi use glucose from the host plant for energy production. QoI fungicides interrupt this reaction but target the QoI target site. QoI fungicides are protectants with translaminar and/or systemic movement in the plant (Gullino et al., 2000). Most QoI fungicides are mobile in the xylem, however, the different active ingredients will vary in their distribution within the treated plant. Azoxystrobin, for example, spreads toward the leaf tip, but evenly distributes throughout the treated leaf (Bartlett et al., 2011). The group of QoI fungicides have a relatively broad spectrum of activity, however, the spectrum will vary by chemical group and active ingredient (Gullino et al., 2000). In general, though, the QoI have activity on not just Basidiomycete and Ascomycete fungi, but also control certain water molds like downy mildew (Bartlett et al., 2011). The resistance risk of QoI fungicides is high, due to the single-site mode of action (Brent & Holloman, 2007). The Fungicide Resistance

Action Committee (FRAC) recommend QoI fungicides are alternated and/or mixed with fungicides from a different cross-resistant group. Specifically, in certain crops and pathosystems, the number of applications within a season or crop cycle should also be limited in order to delay the development of resistance to QoI fungicides (FRAC, 2017).

As the name implies, fungicides with multi-site modes of action have more than one target site in the pathogen. Typically, multi-site chemistries are older contact fungicides, such as chlorothalonil and mancozeb, which control a broad spectrum of plant pathogens including fungi, oomycetes and bacteria (Gisi & Sierotzki, 2008; McGrath, 2001). For example, chlorothalonil has a multi-site action since it reduces fungal intracellular glutathione levels, which effects a number of biochemical pathways that utilize glutathione (Gisi & Sierotzki, 2008). Most multi-site fungicides are protectants and should be applied prior to infection. However, certain multi-site

fungicides, such as copper, also have curative activity against specific plant pathogens. Even though chemistries with the FRAC code M are old, multi-site fungicides play an important role in resistance management (Brent & Holloman, 2007). Due to their low resistance risk, multi-site fungicides are often mixed with single-site fungicides to help prevent or delay the development of resistance (Brent & Holloman, 2007).

Recently, several carboxamide fungicides have been registered for use in arable and vegetable crops in Canada, but this class of chemistry has not been assessed for Stemphylium leaf spot control in asparagus. The carboxamide fungicides are a group of single-site fungicides that affect cell respiration in fungal plant pathogens (Avenot & Michalides, 2010). In order to spread and survive, fungi use glucose from the host plant for energy production. Carboxamides act similar to the QoI, but they inhibit succinate dehydrogenase (Complex II) in the mitochondrial electron transport chain (Avenot & Michalides, 2010). Without energy, the cells are unable to function, which starves the pathogen and prevents the spread and growth of the fungi in the host.

Carboxamide fungicides are protectants, and depending on the chemistry, the movement in the plant can be translaminar and/or systemic. The first carboxamides had a limited spectrum, however, the subsequent generation of carboxamides are considered broad spectrum. Due to their single-site mode of action, the resistance risk of carboxamide fungicides are considered medium to high (FRAC, 2017). In certain crops, like cereals, carboxamide fungicides are recommended in mixtures with another fungicide with a different mode of action. Also, the number of applications within a crop is restricted to delay the development of resistance to carboxamide fungicides

(FRAC, 2017).

In Ontario, fungicide spray programs in asparagus are initiated once the cladophylls (syn. phylloclads, cladodes) have fully emerged on the branches, and fungicides are re-applied on a

14-day interval. Regardless of the number of foliar applications made, growers are not achieving at best adequate control of Stemphylium leaf spot. In other regions, forecasting models have been tested in the field to predict infection periods and time fungicide sprays accordingly to control

Stemphylium leaf spot in asparagus (Eichhorn et al., 2009; Hausbeck et al., 2008; Meyer et al.,

2000). Most recently, growers in Michigan have adopted the forecasting model TOM-CAST for use in asparagus to control Stemphylium leaf spot (Meyer et al., 2000). TOM-CAST is a simplified version of the FAST model, and both models were developed for Alternaria control in tomatoes.

Specifically, the TOM-CAST program is used to calculate a disease severity value (DSV) for a 24 h period based on the hours of leaf wetness and the average temperature during the period of leaf wetness (Table 1.2). Once the field has accumulated a pre-determined DSV, a fungicide is applied.

In asparagus, TOM-CAST was tested for suitability to improve management of Stemphylium leaf spot because the concentration of airborne Alternaria sp. and P. herbarum conidia and ascospores were found to be correlated (Granke & Hausbeck, 2012). In Michigan, the fungicides chlorothalonil and mancozeb were applied on a calendar-based re-application interval or once

TOM-CAST had accumulated 15 DSV (Meyer et al., 2000). The TOM-CAST program reduced fungicide sprays by 60% compared to an application every 7 days without compromising fern health (Meyer et al., 2000). In Ontario, if growers time fungicide applications according to a forecasting model, there should be decreased symptoms of Stemphylium leaf spot.

Table 1.2 Daily disease severity value (DSV) calculated from the hours of leaf wetness and average temperature during leaf wetness in the TOM-CAST model. Average temperature (ºC) during leaf Hours of leaf wetness per day wetness hours 13 to 17 ...... 0 to 6 7 to 15 15 to 20 21 + 18 to 20 ...... 0 to 3 4 to 8 9 to 15 16 to 22 23 + 21 to 25 ...... 0 to 2 3 to 5 6 to 12 13 to 20 21 + 26 to 29 ...... 0 to 3 4 to 8 9 to 15 16 to 22 23 + Daily DSV 0 1 2 3 4

Contact and systemic fungicides have not been directly compared in a TOM-CAST spray program in asparagus. However, it is understood that fungicides differ in their efficacy and suitability within a forecasting model (Meyer et al., 2000). In Germany, TOM-CAST was evaluated at 15, 20, 25, 30 and 35 DSV (Eichhorn et al., 2009). Fungicides applied at the lowest

DSV provided increased control of Stemphylium leaf spot, and a 20 DSV model was recommended when using a protectant fungicide (Eichhorn et al., 2009). A curative fungicide was required to maintain control if a 20 DSV was surpassed due to unfavourable application conditions

(Eichhorn et al., 2009). In carrots, copper (contact, curative and protectant fungicide), chlorothalonil (contact and protectant fungicide) and azoxystrobin (systemic, curative and protectant fungicide) were compared when applied according to TOM-CAST at 10, 15 and 20

DSV for control of foliar fungal blights (Dorman et al., 2009). All of the fungicides had similar disease incidence and severity to the equivalent calendar treatment when TOM-CAST was applied at 10 and 15 DSV (Dorman et al., 2009). However, the fungicides differed once they were applied at a 20 DSV threshold, and only azoxystrobin provided adequate control (Dorman et al., 2009). In

Canada, only the contact fungicide chlorothalonil and the QoI fungicides azoxystrobin and trifloxystrobin are registered for control of S. vesicarium in asparagus. The QoI fungicides have

not been assessed in asparagus for their effectiveness when used within the forecasting model

TOM-CAST in North America.

Certain fungicides are best suited for use in a forecasting model, yet, little information is available to determine which are most effective in asparagus. If a fungicide had enhanced efficacy or even curative activity, perhaps an increased DSV could be used to prompt an application, reducing the number of applications within a season. The predominant cultivar in Ontario is

‘Guelph Millennium’, whereas TOM-CAST was validated in asparagus before this cultivar was commercially available. Research on the effectiveness of TOM-CAST in ‘Guelph Millennium’ is warranted. Also, little is understood regarding the usefulness of systemic fungicides applied within a forecasting model, especially in asparagus.

1.7 Management of asparagus rust

Resistance of asparagus to rust has been studied extensively, however, the resistant cultivars frequently lack desirable horticultural characteristics or are not well adapted for the environment in Ontario. Also, several chemical control options are available for use in the United

States, but there is a lack of products registered in Canada. Currently, growers rely on protectant foliar fungicides and cultivar selection to control rust in asparagus in Canada. However, it is unclear which new cultivars have decreased susceptibility to foliar disease and if any unregistered fungicides might provide adequate control.

1.7.1 Crop resistance

Since the 1990s, several asparagus breeding programs have tried to identify and develop rust resistant cultivars (Blanchette et al., 1982; Hepler et al., 1957; Norton, 1913). In 1913, Norton

noticed that all of the cultivars screened were infected by P. asparagi, but some lines appeared to be less susceptible than others. Rust resistance in asparagus is quantitative which results in differences in susceptibility to infection among cultivars and crosses between resistant and susceptible parents (Hepler et al., 1957; Johnson, 1986; Johnson, 1989; Johnson & Peaden, 1993;

Norton, 1913). The quantitative resistance in asparagus is referred to as slow-rusting (Fig. 1.7).

Transgressive segregation in the population was not observed in populations from highly resistant parents, but it was observed in some populations from moderately resistant parents (Hepler et al.,

1957; Johnson & Peaden, 1993). Johnson and Peade (1993), estimated heritability was 55%. ‘Mary

Washington’ was at first considered a rust-resistant cultivar prior to 1919 (Norton, 1913), and the male parents used to develop rust-resistant cultivars ‘Jersey Centennial’ and ‘Jersey Giant’ from

Rutgers University were selected from a 15-year-old field of ‘Mary Washington’ in 1960 (Ellison

& Kinelski, 1985; Ellison et al., 1981). Severe outbreaks of rust were observed in ‘Mary

Washington’ in Illinois in the 1940s and 1950s, and it is not known if a different strain of P. asparagi overcame the partial resistance or if seed had been unintentionally selected for susceptibility from open-pollinated fields (Hepler et al., 1957). However, considerable heterogeneity in rust resistance was found within both open-pollinated and clonal asparagus cultivars (Johnson, 1989). Age-related resistance has also been observed in asparagus, and regardless of inherent resistance, as stems aged, resistance to rust increased (Johnson, 1986).

Figure 1.7 Slow-rusting asparagus lines surrounding the rust-susceptible ‘Tiessen’.

1.7.2 Chemical control

Currently, only five fungicides are registered for control of rust in asparagus in Canada

(Table 1.3). Among the registered fungicides, myclobutanil, propiconazole and tebuconazole belong to the DeMethylation Inhibitors (DMI), trifloxystrobin belongs to the QoI, and chlorothalonil and metiram belong to the multi-site fungicides. Similar to Stemphylium leaf spot, the fungicide classes differ in their mode of action, but the chemistries within the classes will differ in intrinsic activity against rusts.

Table 1.3 Fungicides registered in Canada for control of Puccinia asparagi in asparagus. FRAC1 Resistance Active ingredient Group name code risk chlorothalonil ...... chloronitriles M05 low metiram ...... dithio-carbamates M03 low myclobutanil ...... DMI2 3 medium propiconazole ...... DMI 3 medium tebuconazole ...... DMI 3 medium trifloxystrobin ...... QoI3 11 high 1Fungicide Resistance Action Committee 2De-Methyl Inhibitor 3Quinone outside Inhibitor

The site-specific DMI fungicides inhibit the C14 demethylation step within fungal sterol biosynthesis (FRAC, 2017). DMI fungicides are protectant and curative, and are generally more mobile in plant tissues than other fungicides (Gullino et al., 2000). Difenoconazole, for example, is translaminar and propiconazole is xylem mobile (Tsuda et al., 2004). DMI fungicides are relatively broad spectrum, but known for their activity against leaf spots and rust (Gullino et al.,

2000). Due to their single-site mode of action, the resistance risk of DMI fungicides are considered medium (FRAC, 2017). Resistance to DMI fungicides is well characterized, and is referred to as shifting-type resistance (Brent & Holloman, 2007). The population sensitivity shifts over time, but with increased application rates or reduced selection pressure, a partial recovery is observed. The application of DMI fungicides is recommended in mixtures with fungicides from a different mode of action (Brent & Holloman, 2007). In crops with multiple applications in a season, DMI fungicides are recommended in alternation or block programs in order to delay the development of resistance (FRAC, 2017).

Traditionally, fungicides have been applied to rust-susceptible cultivars to manage disease progression in a season, but sometimes fungicide applications are also required on rust-resistant cultivars when inoculum levels are high and the weather is conducive for disease development

(Elmer et al., 1996). When rust was first reported in North America, few effective fungicides were available, and growers relied on protectant fungicides (Evans, 1942; Hall & Sirrine, 1900).

Although protectant fungicides are still useful components of a rust management program, systemic fungicides have been evaluated recently for efficacy against rust in asparagus. Generally, among the fungicide classes, the DMI and QoI fungicides have provided the greatest control of rust in asparagus (Cheah & Horlock, 2007; Davis, 2002; Foster & Hausbeck, 2008; Hausbeck et al., 2000; Hausbeck & Cortright, 2004; Mullen & Viss, 1996). Also, fungicide programs applied on a 14-day interval had less disease than fungicides applied on a 21-day interval (Hausbeck &

Cortright, 2002). Of the systemic fungicides that have been evaluated, only myclobutanil, propiconazole, tebuconazole and trifloxystrobin are registered for control of asparagus rust in

Canada.

1.8 Conclusion

Foliar disease impacts the yield and quality of asparagus spears, and affect the longevity of asparagus fields in Ontario. Both S. vesicarium and P. asparagi are difficult to control, and current management tools are not providing adequate protection. Currently, growers rely on the application of foliar fungicides to the fern, but clearly multiple management tools need to be integrated in order to reduce the long term effects of foliar disease in asparagus. The impact of sanitation, fertilizer, irrigation and crop resistance on Stemphylium leaf spot and purple spot has not been studied extensively. Also, cultivars with rust tolerance are outdated and no longer relevant. Since the first sale of ‘Guelph Millennium’, the University of Guelph asparagus breeding program has continued to develop additional lines and cultivars suitable for production in Ontario.

However, the tolerance of these cultivars to both S. vesicarium and P. asparagi is unknown.

Furthermore, asparagus growers in the United States utilize a forecasting model to best time the application of foliar fungicides to control Stemphylium leaf spot. This model, though, was validated on asparagus cultivars that are no longer produced in Ontario. An effective integrated management program must be developed that increases disease control while maintaining the economic viability of asparagus production in Ontario.

The overall objective of this research is to investigate the development of both primary and secondary inoculum of S. vesicarium with an emphasis on improving the management of foliar pathogens in asparagus. The specific objectives are:

1) determine the pathogenicity of S. vesicarium in host and non-host crops, and test new lines for resistance to rust

2) compare management tools for control of foliar disease, including fungicides, the TOM-CAST forecasting model, fertilizer and irrigation

3) investigate the occurrence of P. herbarum on senescing fern and compare sanitation methods to reduce primary inoculum

These studies will contribute to the management of foliar diseases of asparagus in the Great Lakes region and maintain the high value of the asparagus market to Ontario agriculture.

CHAPTER TWO

PATHOGENICITY OF STEMPHYLIUM VESICARIUM AND SUSCEPTIBILITY OF

ASPARAGUS (ASPARAGUS OFFICINALIS L.)

Abstract

Diseases caused by Stemphylium vesicarium are an increasing problem in the major asparagus and onion production regions of eastern and central Canada. Replicated and repeated controlled environment and field trials were conducted in Ontario to assess the disease reaction of asparagus cultivars and pears to determine if isolates from onion and asparagus can cross infect other hosts and to assess the ability of the pathogen to survive in non-host species. All of the lines of asparagus assessed were susceptible to infection by S. vesicarium. There were small differences in susceptibility among cultivars and in aggressiveness among isolates. These differences were not strongly associated with host crop. In asparagus, infection on spears was not consistently correlated with severity on ferns. All isolates from asparagus and onion produced typical symptoms on wounded, but not unwounded, pear fruit. Finally, the pathogen was shown to colonize and sporulate on necrotic leaves of fall rye, which is often used as a cover crop in asparagus fields.

Stemphylium vesicarium can cross-infect asparagus, but some host adaptation or specialization, possibly via host-specific toxins, appears likely.

2.1 Introduction

Stemphylium vesicarium (Wallr.) E.G. Simmons (teleomorph: Pleospora herbarum, syn.

P. alli) is the most important foliar pathogen of asparagus in Ontario. Symptoms begin as small elliptical lesions on the infected tissue. The spots coalesce, resulting in elongated lesions (Lacy,

1982). The pathogen was identified for the first time in Ontario in the 1990’s, although it had been present in Michigan since the 1980’s (Lacy, 1982). Stemphylium vesicarium is also a pathogen of onions, but was not reported in Ontario until 2008 (Paibomesai et al., 2012). The long time interval between reports on asparagus and onion is not surprising because production of these crops is geographically separated- asparagus in southwestern Ontario and onion in the Holland Marsh.

Stemphylium vesicarium has a diverse host range including cultivated crops such as asparagus (Lacy, 1982), Allium spp. (Basallote et al., 1993; Miller et al., 1978; Rao & Pavgi, 1973;

Rao & Pavgi 1975), parsley (Koike et al., 2013) and pear (Vilardell, 1988). The disease is known as purple spot on asparagus spears, Stemphylium leaf spot on asparagus fern, Stemphylium leaf blight on onion and brown spot on pear. Other Stemphylium spp. have also been identified as pathogens in asparagus (Falloon et al., 1987; Leuprecht, 1990; Suzui, 1973) and onion (Zheng et al., 2009). Brown spot of pear has not been identified in North America, but is widely distributed in Europe (Llorente & Montesinos, 2002).

Despite similar symptomology on onion and asparagus, it is unclear whether S. vesicarium cross-infects between these host crops. In one report, seedlings of asparagus were inoculated with isolates of Stemphylium spp. from asparagus and onion, but only isolates from asparagus produced symptoms on asparagus (Falloon et al., 1987). In another report, symptoms developed on onion leaves inoculated with isolates of S. vesicarium from both onion and asparagus (Shishkoff &

Lorbeer, 1989). In pear, only 1 of 52 isolates from onion or asparagus produced symptoms on

detached pears leaves, and no isolate produced symptoms on pear fruit (Kohl et al., 2008). In addition, S. vesicarium was recently identified as a pathogen of parsley, and the isolates collected from parsley caused symptoms on other Apiaceae crops and artificially wounded pears, but not

Allium spp. and unwounded pears (Koike et al., 2013).

In asparagus, S. vesicarium infects the emerging spears in spring, reducing the quality and resulting in premature defoliation, which can greatly reduce subsequent harvests (Hausbeck et al.,

1999). The primary inoculum is air-borne ascospores from pseudothecia produced on crop residue from the previous year (Evans & Stephens, 1984; Falloon, 1982; Falloon et al., 1987; Hausbeck et al., 1999). The etiology of S. vesicarium in asparagus is considered well understood.

None of the asparagus cultivars commonly grown in Ontario are known to be resistant to

S. vesicarium (K. Wall, personal communication). Several cultivars were screened for susceptibility to Stemphylium leaf spot, and ‘Aneto’, ‘Cito’ and ‘Desto’ were more susceptible to defoliation than ‘Jersey Giant’, ‘Rutgers Beacon’ and ‘UC157’ (Broadhurst, 1996). The least susceptible cultivars had tall growth habit, high branching, and relatively open canopies whereas the most susceptible cultivars were short with more compact ferns and reduced branching

(Broadhurst, 1996). Since 2000, asparagus growers in Ontario have switched from producing the

‘Jersey’ cultivars to ‘Guelph Millennium’ a hybrid with high yield and superior winter hardiness

(Landry & Wolyn, 2011). However, ‘Guelph Millennium’ appears to be more susceptible than the other common lines. Broadhurst (1996) predicted that short, compact fern growth provides an ideal microclimate that holds suitable moisture for S. vesicarium infection. Interestingly, the height to the first branch in ‘Guelph Millennium’ is shorter than that in ‘Jersey Giant’ (CFIA, 2017). In asparagus, the height of the first branch is highly correlated to the tightness of the spear tip (Hanna,

1935), and plant breeders are already selecting for this desirable phenotype. It is unknown, however, if selection of branch height has also selected for foliar disease tolerance.

Generally, Stemphylium spp. are weak pathogens that colonize senesced plant tissue (Black et al., 2012; Ellis, 1971; Hudson, 1971). Wounding, however, even by erosion of the wax layer by wind-blown soil, can increase disease incidence in asparagus (Johnson & Lunden, 1986; Lacy,

1982). In pear orchards, S. vesicarium has been isolated from the dead leaves of weeds and grass species (Kohl et al., 2013; Rossi et al., 2005; Rossi et al., 2008), and isolates of S. vesicarium collected from seven orchard grass species were pathogenic on pear leaves (Rossi et al., 2005).

Asparagus growers plant rye (Secale cereale) in the fall and kill it with herbicide in spring to provide a dense plant cover to minimize wind erosion during harvest (Brainard, 2012). Even though rye is considered a non-host, S. vesicarium could survive on the dead plant tissue as a saprophyte.

Little is known about the prevalence of S. vesicarium in Ontario, despite its impact on asparagus and onion production and the lack of effective control measures. The objectives of this research were to assess the pathogenicity and host specificity of S. vesicarium isolates on asparagus, pear and rye, and to assess the disease reaction of common asparagus cultivars to S. vesicarium.

2.2 Materials and methods

2.2.1 Isolate collection and maintenance

From 2012 to 2016, 67 fields across five counties in Ontario (ON) and two counties in

Nova Scotia (NS) were scouted for symptoms caused by S. vesicarium in asparagus and onion (Fig and Table in Appendix). Symptomatic onion leaves and asparagus ferns were collected, surface

sterilized and the pathogen isolated onto potato dextrose agar (PDA). The resulting fungal colonies were purified using hyphal tip culture and single-spore isolation. In addition, P.D. Hildebrand contributed five isolates of S. vesicarium collected from commercial onion fields in Kings County,

NS. The isolates were identified based on morphological characteristics, where conidia are olive- brown, oval to ovoid, and are borne on conidiophores that are pale to brown with dark edges and bands (Table A1.32 and Tayviah, 2017). The conidia have 1-5 transverse septa and are constricted at 1-3 transverse septa (Simmons, 1969). These identifications were confirmed using PCR targeting the gpd gene (Tayviah, 2017). The isolates were maintained in long-term storage on half strength PDA and water agar slants at 5 ± 1°C. The identifier for each isolate indicated the province where it was collected (O-Ontario and N-Nova Scotia) and the host plant (A-asparagus or O-onion)

(Table 2.1). In preparation for each experiment, the selected isolates were transferred onto PDA from long-term storage and incubated for 7 days at 20 ± 2°C.

Table 2.1 Selected isolates of Stemphylium vesicarium, collected from asparagus and onion fields in Canada, assessed for pathogenicity on asparagus, pear and rye. Year Code Collection location Crop Source collected OA20 2012 Norfolk County, ON asparagus J.M. Foster OA46 2013 Norfolk County, ON asparagus J.M. Foster OA48 2014 Essex County, ON asparagus J.M. Foster OA56 2014 Norfolk County, ON asparagus J.M. Foster NA51 2014 Kings County, NS asparagus J.M. Foster NA52 2014 Kings County, NS asparagus J.M. Foster OO27 2013 Simcoe County, ON bulb onion J.M. Foster OO31 2013 Simcoe County, ON bulb onion J.M. Foster OO55 2014 York Regional Municipality, ON bulb onion C.S. Tayviah NO35 2013 Kings County, NS bulb onion P.D. Hildebrand NO36 2013 Kings County, NS bulb onion P.D. Hildebrand

2.2.2 Inoculum preparation for pathogenicity assays

Eleven isolates were selected from long-term storage for use in the pathogenicity assays

(Table 2.1). Isolates OA46, OA48, OA56, NA51, NA52, OO27 and NO36 were evaluated for pathogenicity on asparagus spears. Isolates OA20, OA48, OA56, OO31, NO35 and NO36 were evaluated for pathogenicity to pear fruit, and OA46, OA48 and OO55 were evaluated for pathogenicity to rye grass.

Agar plugs were transferred from stock plates onto unclarified V8 agar (UCV8, 16 g agar,

30 mM CaCO3, 160 ml unfiltered V8 juice, and 840 ml distilled water) and incubated for 7 days under constant fluorescent light at 20 ± 2°C. Ten drops of sterile water were placed around the outer edge of the culture, and a sterile inoculation rod was used to gently mat down the mycelia.

Plates were then placed under UV light at 20 ± 2°C for 12 hours, followed by darkness for another

12 hours to induce the production of conidia. Conidial inoculum was prepared by flooding actively sporulating cultures with sterile distilled water and dislodging the conidia with a sterile inoculation rod or microscope slide. The suspensions were filtered through sterile cheesecloth to remove

mycelia fragments. A drop of the surfactant Tween 20 (J.T Baker Inc., Philipsburg, NJ, USA) was added to every 10 ml of conidial suspension for improved spore wetting. The concentration of the conidial suspension was estimated using a Neubauer hemacytometer and adjusted accordingly.

Assays were designed to determine the pathogenicity of a subset of the collected isolates to the known hosts in Canada: onion and asparagus. An assay with pear fruit was also conducted to confirm the pathogenicity on a host described outside of Canada.

2.2.3 Detached asparagus spear assays

Two asparagus assays were conducted to screen commercial cultivars and lines to determine if isolates of S. vesicarium collected from onion and asparagus were pathogenic to wounded asparagus spears. Commercial standards, newly released cultivars and experimental breeding lines were included in the assays. Both of the assay included the commercial standard

'Guelph Millennium' and two experimental lines UG010 and ‘Guelph Equinox’ (formerly coded

UG020). All experimental lines were from the University of Guelph asparagus breeding program.

The first assay also included lines ‘Guelph Eclipse’ (formerly coded UG005) and ‘Asparabest’

(formerly coded UG009), while the second assay included the commercial standard 'Jersey Giant', an old standard 'Tiessen', and the line UG023.

Asparagus spears for the first and second assays were harvested from the asparagus breeding program trials at the University of Guelph Simcoe Research Station (first assay) and the two field cultivar trials described below (second assay). Spears were stored for 2 weeks at 2 ± 1°C and 95% RH in open plastic bins. A day prior to inoculation, spears were returned to room temperature, surface-disinfested with 10% bleach (6.15% NaClO) solution for 10 min, and rinsed with sterile distilled water.

A subset of the collected isolates were inoculated onto each asparagus cultivar or line. The isolates were chosen to account for both the sampling geography and host crop. There were five isolates selected from asparagus: OA46, OA48, OA56, NA51, NA52 and three selected from onion: OO27, OO55, and NO36. All of the spears were wounded prior to inoculation, except the non-wounded, non-inoculated control. Sterile water was used as the negative control on wounded and non-wounded spears. Spears were placed onto plastic racks in sterile humidity chambers, and sterile water was poured into the bottom of the chamber to cover the surface. Each spear was wounded at five locations on the spear to a depth of 1 to 2 mm using a sterile needle. The first wound was located 2.5 cm from the spear tip and wounds were spaced every 2.5 cm. A 10-µl drop of conidial suspension (1 x 105 conidia/ml) was pipetted onto each wound site. Chambers were sealed with plastic lids and maintained at room temperature (20 ± 2°C) and constant fluorescent light. Treatments were arranged in a randomized complete block design, and each block was contained within an individual humidity chamber. One experimental unit consisted of one spear and each isolate and cultivar combination was replicated four times. Both assays were repeated with spears collected from a second harvest.

Lesion development on the spears was evaluated on a 1 to 6 scale (1 = no lesion, 2 = 1 mm lesion length, 3 = 2 mm lesion length, 4 = 3 mm lesion length, 5 = 3.1 to 7 mm lesion length and

6 =7.1 to 10 mm lesion length). The area under the disease progress curve (AUDPC) was calculated by inserting the disease score into the equation by Shaner and Finney (1977) to describe the cumulative disease severity throughout the assessment period. Upon conclusion of the experiment, 10% of the symptomatic spears were collected to isolate and verify the pathogen as described above.

2.2.4 Pear fruit assay

An assay was conducted to test the pathogenicity of S. vesicarium on the pear cultivar

‘Hargold’. The cultivar was selected since it was easy to obtain untreated pear fruit. The fruit were harvested August 11, 2015 from a commercial orchard located near Grimsby, Ontario, and stored in a cooler at 2 ± 1°C and 95 % RH for 2 weeks. One day prior to inoculation, fruit were returned to room temperature, surface-disinfested with 10% bleach (6.15% NaClO) solution for 10 min, and rinsed with sterile distilled water.

It was difficult to produce consistent spore suspensions from all isolates of S. vesicarium, which influenced the isolates and conidial concentrations that were used in each repetition. The lowest concentration produced was 2.25 x 105 conidia/ml. Isolates that produced more conidia, were assessed at the initial suspension concentration and then the concentration was adjusted to the standard 2.25 x 105 conidia/ml concentration. All isolates, and the specified concentration, were replicated five times, and an experimental unit consisted of one pear fruit. Pears were either wounded to a depth of 1 to 2 mm or left non-wounded. A 10-µl drop of the conidial suspension was pipetted onto either the surface or the pear or at the wound site. A non-inoculated and water- only control was included in both the wounded and non-wounded pear fruit. Fruits were placed onto plastic racks in sterile humidity chambers, and sterile water was poured into the bottom of the chamber to cover the surface. Chambers were sealed with plastic lids and maintained at room temperature (20 ± 2°C). Treatments were arranged in a randomized complete block design, and each block was contained within an individual humidity chamber.

Brown spot severity was evaluated by measuring the lesion diameter every day for 12 days.

The AUDPC was estimated as described above. Upon conclusion of the experiment, 10% of the

symptomatic fruit were collected to isolate and verify the causal pathogen. The assay was conducted three times concurrently in different lab spaces.

2.2.5 Rye pathogenicity assay

An assay was conducted to test the pathogenicity of S. vesicarium on fall rye. The study was arranged in a randomized complete block design with four replicates (pots). Five rye seeds were planted into each tall, narrow plastic pot (19-cm-tall SC7 Ray Leach Cone-tainers, Stuewe and Sons, Inc. Corvallis, OR) filled with soil-less medium (Promix PGX, Premier Tech

Horticulture, Rivière-du-Loup, QC). Seedlings were maintained in a growth chamber at 22 / 18 °C day/night cycle with 16-h photoperiod (Standard F54T5/80/HO/PS, 325 µm/m2/second). One month after seeding, plants were inoculated with conidial suspensions of isolates OA46, OA48 and OO55 (1 x 105 conidia ml-1), plus sterile-water and a no water controls. Each isolate and control were replicated four times and arranged in a randomized complete block design.

Immediately after inoculation, plants were bagged to maintain humidity and returned to the growth chamber. At 14 days after inoculation, the plants were visually examined for symptoms of disease.

Each seedling was divided into green and brown tissue and these were cut into 2-cm-long pieces.

The leaf pieces were surface sterilized in 10% bleach for 10 min, rinsed with sterile water, and placed in a flow hood to dry. Five pieces each of green and brown tissue per pot were placed onto

PDA medium, incubated under constant fluorescent light at 20°C ± 2 °C for 7 d, and examined for cultures of S. vesicarium based on culture morphology and pigmentation. The experiment was repeated over time.

2.2.6 Asparagus field trials

Field trials were established in 2011 at the University of Guelph Simcoe Research Station in Norfolk County, ON, on a silt loam soil (23% sand, 73% silt, 4% clay, pH 7.4 and 1.7% OM) and Syngenta Canada Inc. Honeywood Research Farm, Plattsville, ON, on a silt loam soil (34% sand, 58% silt, 8% clay, pH 6.5 and 2.5% OM). In mid-March, seeds were planted in 288-cell plug trays and the plugs were transplanted 4 weeks later into 50-cell trays filled with Sunshine mix # 5

(Sun Gro Horticulture, Agawam, MA). Seedlings of 'Guelph Millennium', 'Jersey Giant' and

'Tiessen' and lines UG010, ‘Guelph Equinox’ and UG023 were then grown in a greenhouse for 10 weeks, placed outside for 7 days to acclimate and transplanted by hand at each site. Each plot consisted of one 6-m-long row, with 1.5 m between rows, plants spaced 0.3 m apart within a row, and 2 m between blocks. Plots, each containing 19 plants, were arranged in a randomized complete block design with five replicates.

In 2011-2014, the trials were fertilized with 50 kg N ha-1 in June, July and August. In 2015 and 2016, the trials were fertilized with 100 kg N ha-1 and 100 kg K ha-1 immediately following the final harvest. Weeds and insects were managed according to standard commercial practice and integrated pest management recommendations (OMAFRA, 2015).

From 2014 to 2016, spears were harvested three times weekly for 6 weeks. Spears harvested when no rain event had occurred were collected and held at 4 °C to be used in other studies (described previously). One day following most rain events, 10 spears per plot were collected and assessed for purple spot incidence and severity. Harvested spears were 25 ± 5 cm in length. Spears were assessed four (2014), eight (2015) and 13 (2016) times per year at the

Plattsville site and three (2014), four (2015) and 12 (2016) times at the Simcoe site. Each spear was rated on a scale of 0 to 4, where 0 = no lesions, 1 = 1 to 20 lesions, 2 = 21 to 50 lesions, 3 =

51 to 90 lesions and 4 > 90 lesions (Falloon et al. 1987). A disease severity index (DSI) was calculated according to the following formula: DSI = [∑[(class no.)(no. of spears in each class)]]/[(total no. of spears per sample)(no. classes - 1)] x 100 (Kobriger & Hagedorn, 1983).

After harvest was complete in 2015 and 2016, ferns were assessed weekly for the presence of Stemphylium leaf spot on the branches and cladophylls by rating 10 randomly selected branches per plot on a scale of 0 to 5, where 0 = no lesions, 1 = 1 to 20 lesions, 2 = 21 to 50 lesions, 3 = 51 to 90 lesions, 4 = 90 to 200 lesions, and 5 > 200 lesions (modified from Falloon et al. 1987). A

DSI and AUDPC were calculated as described (Kobriger & Hagedorn, 1983; Shaner & Finney

1977). Twelve weeks after cladophyll emergence, AUDPC was calculated.

2.2.7 Data analysis

Statistical analyses were conducted using SAS v.9.4 (SAS Institute Inc., Cary, NC). Mixed model analysis of variance was used to assess the disease data (PROC GLIMMIX). No outliers were identified in these data sets based on Lund’s test. The normality of each data set was assessed using PROC UNIVARIATE. The restricted maximum likelihood (REML) covariance parameter estimates were used to analyze parameters. In the detached spear assays, variance was portioned into random (block and repetition) and fixed (isolate, cultivar and cultivar × isolate) effects. In the pear fruit assay, variance was portioned into random (block) and fixed (isolate) effects within each repetition. The non-inoculated controls were removed from the ANOVA. Variance in the field trial data was partitioned into random effects (block) and fixed effects (location, year, location × year, cultivar, location × cultivar, year × cultivar and location × year × cultivar). Means were separated using Tukey’s HSD (P ≤ 0.05).

2.3 Results

2.3.1 Isolate phenotyping

Symptoms of S. vesicarium were confirmed on onions and asparagus, and isolates were obtained from plants showing distinct symptoms. Specifically, isolates were obtained from the 20 commercial onion fields previously surveyed in Ontario (Paibomesai et al., 2012), four onion fields in Ontario in 2014 and the five commercial onion fields surveyed in Nova Scotia. Isolates were also obtained from 16 commercial asparagus fields in Ontario and two commercial asparagus fields in Nova Scotia that exhibited symptoms of purple spot. The conidia were oblong in shape with size averaging 21 x 45 µm and with 5 transverse septa and 1 complete longitudinal septa.

2.3.2 Detached asparagus spear assays

In both assays, each of the asparagus cultivars and lines were susceptible to S. vesicarium, and no symptoms were observed on the wounded and non-wounded control spears (data not shown). Lesions developed at the wound site and grew lengthwise along the spear. All isolates were pathogenic on asparagus spears, regardless of the host or location from which the isolate was collected (Table 2.2).

No interaction between isolate and cultivar was observed for any of the three parameters in the first assay (Table A1.1, Table A1.2 and Table A1.3). The effects of isolate and cultivar were not significant for disease incidence in the first assay. For DSI and AUDPC, though, the effect of isolate was significant for both parameters. Also, the fixed effect of cultivar was significant for

DSI. The fixed effects of isolate, cultivar and cultivar × isolate were insignificant for all parameters in the second assay (Table A1.4, Table A1.5 and Table A1.6). The means for incidence, DSI and

AUDPC are presented for cultivar and isolate separately for both assays (Table 2.2 and Table 2.3).

In the first assay, DSI was lower on NO36 than for most of the other isolates (Table 2.2). Among the cultivars, ‘Guelph Eclipse’ had a lower final DSI than UG010 and ‘Asparabest’ (Table 2.3).

Table 2.2 Differences in final incidence, final disease severity index (DSI) and area under the disease progress curve (AUDPC) on artificially wounded spears of asparagus inoculated with isolates of Stemphylium vesicarium from asparagus and onion. Isolate Incidence (%) DSI (0-100)1 AUDPC1 NA52 100 80 a 170 a OA46 100 78 a 176 a OA56 100 74 a 159 a NA51 100 74 a 154 ab OA48 100 73 ab 149 ab OO55 99 63 ab 127 ab NO36 96 47 b 100 b Standard error 1.8 4.4 24.4 P-value 0.5513 0.0210 0.0166 Second Assay OA46 88 54 96 OO27 86 27 36 Standard error 4.4 7.2 16.9 P-value 0.6672 0.3020 0.2440 1Means in a column followed by the same letter do not differ using Tukey’s multiple range test at P ≤ 0.05, n=8.

Table 2.3 Differences in disease severity index (DSI) and area under the disease progress curve (AUDPC) of asparagus spears of several cultivars and lines wounded and inoculated with isolates of Stemphylium vesicarium. Cultivar or line Incidence (%) DSI (0-100) AUDPC First Assay Guelph Eclipse 97 60 a1 125 Guelph Equinox 100 69 ab 142 Guelph Millennium 99 70 ab 148 UG010 100 75 b 167 Asparabest 99 76 b 158 Standard error 1.0 2.0 24.1 P-value 0.1480 0.0211 0.1754 Second Assay Guelph Equinox 86 35 58 Guelph Millennium 86 39 62 Tiessen 90 38 65 UG023 79 41 67 Jersey Giant 93 42 70 UG010 89 45 76 Standard error 6.9 5.1 5.1 P-value 0.6672 0.3020 0.2440 1Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=8.

2.3.3 Pear fruit assay

Symptoms were observed as early as 2 days after inoculation. No symptoms developed on non-wounded pear fruit (data not shown), but all isolates were able to cause symptoms on wounded pear fruit (data not shown). In the first assay, the fixed effect of isolate was significant for disease incidence, but not lesion size or sAUDPC (Table A1.7, Table A1.8 and Table A1.9). In the second assay, the fixed effect of isolate was significant for disease incidence, lesion size and sAUDPC

(Table A1.10, Table A1.11 and Table A1.12). In the third assay, though, the fixed effect of isolate was not significant for any parameter (Table A1.13 and Table A1.14). The means for incidence,

DSI and sAUDPC are presented for cultivar and isolate separately for the three assays since each assay included a different combination of isolates (Table 2.5).

Generally, high concentrations of conidia of S. vesicarium produced larger lesions than low concentrations of S. vesicarium (data not shown). Specifically, isolate OO31 did not produce lesions at 2.25 x 105 conidia mL-1 (data not shown) but did produce disease symptoms at 4.5 x 105 conidia mL-1 (Table 2.4). In the second repetition, when the concentration of conidia was diluted to 2.25 x 105 conidia mL-1, NO35 produced larger lesions than OA48, OA56, NO36 and OA20.

Table 2.4 Incidence, lesion diameter (mm) and sAUDPC (standardized area under the disease progress curve) recorded and calculated 12 days after inoculation with a conidial suspension (2.25 x 105 conidia mL-1) on wounded ‘Hargold’ pear fruit. Incidence (%)1 Lesion diameter (mm)1 sAUDPC1 Isolate Repetition Repetition Repetition 1 2 3 1 2 3 1 2 3 NO35 100 a 20 a 2.8 a OA48 80 ab 100 4 b 8 1.0 b 1.5 OA56 60 ab 2 b 0.8 b NO36 80 a 40 ab 100 8 2 b 18 1.3 0.6 b 2.3 OA20 100 a 20 b 100 6 0 b 7 1.4 0.4 b 1.3 OO31 0 b 0 0.0 Standard error 16.0 20.0 0.0 3.0 1.3 2.9 0.42 0.20 0.24 P-value 0.0007 0.0195 NA 0.1467 <0.0001 0.0931 0.1438 <0.0001 0.1211 1Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=5.

2.3.4 Rye pathogenicity assay

Symptoms were not observed on rye plants inoculated with S. vesicarium, but cultures were obtained from the necrotic, brown tissue. Stemphylium vesicarium was isolated from 63 (SE 18.3),

13 (SE 12.5) and 75 (SE 16.4) % of the rye inoculated with isolates OA46, OA48 and OO55, respectively. The control plants did not develop fungal growth or growth from contaminants.

Stemphylium vesicarium was not recovered from green leaf tissue.

2.3.5 Asparagus field trials

For disease incidence and DSI on the spears, the fixed effects of year, location, year × location, cultivar and location × cultivar were significant (Table A1.15 and Table A1.16), and locations were then analyzed separately. At the Simcoe location, the fixed effect of year × cultivar was insignificant for incidence but significant for DSI (Table A1.17 and Table A1.18). The means for incidence are combined among years and then separated among years for DSI (Table A1.19,

Table A1.20, Table A1.21 and Table 2.5). The fixed effect of cultivar was significant for the combined incidence and the DSI in all three years. At the Plattsville location, the fixed effect of year × cultivar was insignificant for both incidence and DSI on the spears (Table A1.22 and Table

A1.23). The means for both incidence and DSI were combined among years, and the fixed effect of cultivar was insignificant (Table 2.6).

Table 2.5 Purple spot incidence (%) and DSI (disease severity index) observed in six asparagus cultivars or lines at Simcoe Research Station, Simcoe, Ontario. Incidence DSI2 Cultivar or Line (%)1 2014 2015 2016 Guelph Millennium 37 a3 6.4 a 15.2 ab 14.2 a Guelph Equinox 38 a 7.8 a 11.2 a 17.8 ab Tiessen 37 a 6.6 a 15.8 ab 16.0 ab UG010 44 a 8.6 ab 14.2 ab 18.8 b UG023 56 b 19.0 bc 21.6 b 25.4 c Jersey Giant 62 b 22.6 c 18.4 ab 27.4 c Standard error 3.3 2.38 1.91 1.34 P-value <0.0001 0.0002 0.0155 <0.0001 1Data from 2014 to 2016 were combined as no interaction was observed between cultivar and year (P=0.0545). 2Data from 2014 to 2016 were not combined as an interaction was observed between cultivar and year (P=0.0337). 3Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=5.

Table 2.6 Purple spot incidence (%) and DSI (disease severity index) observed in six asparagus cultivars or lines at Honeywood Research Facility, Plattsville, Ontario, n=5. Cultivar or Line Incidence (%)1 DSI2 UG010 66 25.5 UG023 69 28.1 Jersey Giant 72 29.5 Guelph Millennium 74 30.9 Guelph Equinox 71 31.6 Tiessen 73 32.1 Standard error 2.5 1.71 P-value 0.2077 0.1220 1Data from 2014 to 2016 were combined as no interaction was observed between cultivar and year (P=0.9742). 2Data from 2014 to 2016 were combined as no interaction was observed between cultivar and year (P=0.9997).

For disease incidence in the fern, all fixed effects were significant (Table A1.24), but for

DSI the fixed effects of year × location × cultivar, location × cultivar, year × cultivar and year were insignificant (Table A1.26). The sAUDPC were then assessed by location because there was an interaction between location and cultivar (Table A1.27). At the Plattsville location, neither the fixed effects of year × cultivar or cultivar were significant. At Simcoe, though, the fixed effect of cultivar was significant (Table A1.28). The means for DSI were combined between locations, but separated between locations for sAUDPC (Table 2.7).

Table 2.7 Stemphylium leaf spot DSI and sAUDPC (standardized area under the disease progress curve) observed in six asparagus cultivars or lines at two sites. sAUDPC2 Cultivar or Line DSI1 Simcoe Plattsville Jersey Giant ...... 19 a3 5.4 a 3.0 Tiessen ...... 18 a 7.1 ab 2.6 Guelph Millennium ...... 25 a 7.8 b 2.9 UG010 ...... 19 a 7.9 b 2.6 ‘Guelph Equinox’ ...... 23 a 8.3 b 3.0 UG023 ...... 25 a 8.7 b 3.3 Standard error 1.9 0.52 0.48 P-value 0.0166 0.0009 0.9287 1Data were combined between sites, years and cultivars since no interaction was observed among sites, years and cultivars (P=0.9792) or sites and cultivars (P=0.0521). 2Data were separated between sites as an interaction between site and cultivar was observed (P=0.0239). 3Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=5.

All six cultivars developed characteristic symptoms on spears, branches and cladophylls, but there was no clear pattern of disease response among cultivars over sites and years. At Simcoe,

‘Guelph Millennium’, ‘Guelph Equinox’, ‘Tiessen’ and UG010 had lower purple spot incidence on the spears than UG023 and ‘Jersey Giant’ (Table 2.7). In all three years, ‘Guelph Equinox’ had less DSI than UG023, but no other trends were observed among years. In the fern, differences were not found in the means separation test for DSI combine between sites. At Simcoe, the ‘Jersey

Giant’ had a lower sAUDPC than ‘Guelph Millennium’ and all of the UG lines. Severity (DSI) on spears and ferns were negatively correlated (r = -0.44, P = 0.0004) at Simcoe and positively correlated (r = 0.44, P = 0.0002) at Plattsville.

Asparagus rust developed on ferns at the Simcoe site but not at Plattsville, and all six cultivars or lines were susceptible (data not shown). The DSI of rust and Stemphylium leaf spot were negatively correlated (r = -0.82, P < 0.0001).

2.4 Discussion

In the current study, all cultivars and lines of asparagus assessed were susceptible to S. vesicarium in both controlled environment and field trials. Small differences in susceptibility were identified, but no cultivars were resistant.

Toxins produced by S. vesicarium are known to affect the disease reaction of onion cultivars, and differences in aggressiveness among isolates has been attributed to the reaction of cultivars to host-specific toxins in pear (Montesinos et al., 1995; Pattori et al., 2006; Singh et al.,

1999). Differences in reaction to initial infection and subsequent disease development in asparagus cultivars may relate to differential response to host-specific toxins. Investigation into the host- specific toxins produced by S. vesicarium would assist plant breeders in cultivar selection for resistance in asparagus.

Isolates of S. vesicarium were collected from infected asparagus spears and fern. Overall, isolates differed in infection success on asparagus spears. Although not always significant, isolates from onion were generally less aggressive on asparagus spears than isolates from asparagus.

Stemphylium vesicarium was identified only by conidial morphology and sequencing of the gpd gene. The genus and species of the isolates in the presented study should be confirmed using typical techniques as prescribed by Inderbitzin et al. (2009): sequencing of rDNA-ITS, EF-

1α and vmaA-vpsA.

In the current study, wounding was required to initiate infection by S. vesicarium and symptom development on both asparagus spears and pear fruit. The pathogen is known to infect asparagus spears through wounds caused by sand-blasting and open stomata (Fallon et al., 1984;

Falloon et al., 1987; Johnson & Lunden, 1986; Lacy, 1982; Sutherland et al., 1989). Isolates collected from pear, however, were able to infect both pear fruit and leaves without wounding

(Kohl et al., 2009; Llorente et al. 2006). In a study where pear fruit and leaves were not wounded prior to inoculation, only 1 of 52 isolates from onion and asparagus were pathogenic on pear leaves and none were pathogenic on pear fruit (Kohl et al., 2009). Taken together, this indicates that isolates adapted to other hosts were only able to cause symptoms on wounded pears, whereas isolates adapted to pear do not require wounding to produce symptoms.

The reaction of the asparagus lines and cultivars to purple spot differed among the controlled environment studies and both field sites. Similar to previous studies, in the detached spear assay, infection occurred only at the site of wounding (Falloon et al., 1987; Johnson &

Lunden, 1986; Lacy, 1982). In attached spear studies, like the field trials, the spears can be infected through either natural openings or wounding (Falloon et al., 1987). If a mechanism of resistance was physical, such as stomatal opening and closing, wounding of the spears would artificially circumvent the mechanism. Further studies could investigate the reaction of asparagus in attached spear assays to establish which cultivars are resistant in the absence of artificial wounding. In addition, the asparagus rust outbreak at Simcoe influenced the results of the Stemphylium leaf spot severity at this site. These results demonstrate the difficulty of screening cultivars for resistance when multiple pathogens are present, and also demonstrate the importance of conducting trials across multiple sites for plant breeding research.

In the current study, S. vesicarium was shown to colonize and sporulate on necrotic leaves of rye, which is normally considered to be a non-host. Inoculation with S. vesicarium did not produce visible symptoms on fall rye, but the pathogen was later re-isolated from dead, surface- sterilized rye leaves. This indicated that S. vesicarium was able to survive as a saprophyte on dead fall rye tissue, which may provide a reservoir of overwintering inoculum for the pathogen in asparagus. This observation supports the results of studies conducted in pear orchards, where

isolates from non-host tissue were able to re-infect pear fruit (Kohl et al., 2009; Kohl et al. 2013;

Rossi et al., 2005). The potential for S. vesicarium to survive on living or senesced weed species and cover crops should be assessed as to further understand the etiology in asparagus.

Future research is required in asparagus to investigate the host-specific toxins produced, mechanisms of resistance, and the importance of non-host crops on the etiology of S. vesicarium.

CHAPTER THREE

MANAGEMENT OF STEMPHYLIUM LEAF SPOT (STEMPHYLIUM VESICARIUM

WALLR.) E SIMMONS AND RUST (PUCCINIA ASPARAGI DC) OF ASPARAGUS

(ASPARAGUS OFFICINALIS) WITH CULTIVAR SELECTION AND FUNGICIDES

Abstract

Stemphylium leaf spot (SLS) and rust are foliar diseases of asparagus fern. If left unmanaged, both SLS and rust can contribute to early decline of an asparagus field and a reduction in yield in subsequent seasons. Currently, growers rely on protectant fungicides and cultivar selection to control foliar disease in asparagus in Canada. However, it is unclear which new cultivars are susceptible to foliar disease and if any new fungicides might provide adequate control.

The objectives of this research were to: (1) assess cultivars and lines for sensitivity to foliar disease, and (2) evaluate fungicides for control of foliar disease in asparagus. A cultivar trial was established in 2011 to compare six cultivars and lines. Also in 2011, thirteen fungicides and combinations of fungicides were assessed for control of foliar disease in ‘Guelph Millennium’. In both studies, the fern was assessed for symptoms of SLS and rust. ‘Jersey Giant’ and ‘Guelph

Equinox’ and lines UG010 and UG023 had lower rust severity (as measured as standardized area under the disease progress curve, sAUDPC) than ‘Guelph Millennium’ and ‘Tiessen’. ‘Jersey

Giant’ also had lower SLS disease severity index (DSI) and sAUDPC than ‘Guelph Millennium’ and the lines ‘Guelph Equinox’ and UG023. There was a negative correlation between SLS and rust for both sAUDPC and DSI assessments. In the fungicide screening trials, differences were observed in rust lesion counts and sAUDPC. In a 3-year-old field, azoxystrobin/cyproconazole, azoxystrobin/difenoconazole, benzovindiflupyr, cyproconazole, tebuconazole, and the tank- mixture of mineral oil + pigment + propiconazole had lower sAUDPC than the untreated control.

In the 4-year-old field, only azoxystrobin/cyproconazole, azoxystrobin/difenoconazole, and cyproconazole had lower sAUDPC than the untreated control. Further research is warranted to investigate rust management programs with fungicides on slow-rusting asparagus cultivars.

3.1 Introduction

Foliar diseases cause premature defoliation, early senescence and a reduction in marketable yield in asparagus (Elmer et al., 1996; Hausbeck et al., 2008). In Ontario, both Stemphylium leaf spot (SLS) and rust are foliar diseases that occur annually in most asparagus fields. Currently, growers rely on cultivar selection and the application of foliar fungicides to control both SLS and rust (Ellison & Kinelski, 1985; Elmer et al., 1996; Hausbeck et al., 2008). The University of

Guelph asparagus breeding program released ‘Guelph Millennium’ over 10 years ago. Since then, the majority of fields in Ontario have shifted to producing ‘Guelph Millennium’, and the asparagus breeding program continues to develop new lines. These cultivars are high yielding and exhibit superior winter hardiness, yet little is understood regarding the susceptibility of ‘Guelph

Millennium’ or the lines to SLS or rust.

Extensive studies conducted in Michigan, have identified several fungicides that control

SLS or rust (Foster & Hausbeck, 2009; Hausbeck & Cortright, 2004; Hausbeck et al., 2008), yet, few fungicides are registered for control of these diseases in asparagus. In Ontario, only azoxystrobin (Fungicide Resistance Action Committee, FRAC, group 11), chlorothalonil (FRAC group M5) and trifloxystrobin (FRAC group 11) are registered for control of SLS. Chlorothalonil and trifloxystrobin are also registered for control of rust, as are metiram (FRAC group M3), myclobutanil (FRAC group 3), propiconazole (FRAC group 3), and tebuconazole (FRAC group

3). Currently, both chlorothalonil and metiram are under re-evaluation at the Pest Management

Regulatory Agency (PMRA) in Canada. Also, site-specific fungicides, such as group 3 and group

11 fungicides, have demonstrated greater potency against both SLS and rust pathogens than traditional multi-site fungicides (Hausbeck et al. 2008). The FRAC guidelines recommend the use of fungicide mixtures to delay or prevent the development of resistance to site-specific fungicides

(Brent & Hollomon, 2007). No fungicide mixtures are registered for control of SLS or rust, and neither pre-formulated mixtures nor tank-mixtures have been tested for control of SLS or rust in

Ontario. FRAC also recommends the alternation of different modes of action to prevent resistance

(Brent & Hollomon, 2007), yet only two and four fungicide groups are registered for control of

SLS and rust, respectively.

The purpose of this study was to determine the susceptibility of six asparagus cultivars and lines, and to screen unregistered fungicides and fungicide combinations for control of SLS and rust.

3.2 Materials and methods

3.2.1 Asparagus cultivar susceptibility to SLS and rust

Field trials were established in 2011 at the University of Guelph Simcoe Research Station in Norfolk County, Ontario, Canada as described in CHAPTER TWO. The fern was assessed weekly following harvest for the presence of SLS and rust on the branches. The disease severity indices (DSI) for SLS and rust were determined by rating ten branches per plot on a scale of 0 to

5, 0 = no lesions, 1 = 1 to 20 lesions, 2 = 21 to 50 lesions, 3 = 51 to 90 lesions, 4 = 90 to 200 lesions, and 5 > 200 lesions (modified from Falloon et al., 1987) and calculated with the following formula: DSI = [∑[(class no.)(no. of branches in each class)]]/[(total no. of branches per sample)(no. classes - 1)] x 100 (Kobriger & Hagedorn, 1983). The AUDPC was calculated according to the methods described by Shaner & Finney (1977) and standardized by the methods

described by Simko & Piepho (2012). The fern was also assessed weekly for yellowing (% incidence) and defoliation (% cladophyll drop).

3.2.2 Fungicide efficacy trials

Two trials were conducted in commercial asparagus fields in Norfolk County, Ontario,

Canada on sandy soil. The first field site, referred to as 4-year-old field, was established in 2009 and the second field site, referred to as 3-year-old field, was established in 2008. Both fields were established with ‘Guelph Millennium’ asparagus crowns spaced approximately 0.2 m in the row and 1.2 m between rows. The fields were maintained according to commercial practice

(OMAFRA, 2015).

Each treatment plot was a single 6-m-long row separated by an untreated buffer row to prevent spray drift among treatments. Thirteen fungicides and fungicide combinations were compared to an untreated control (Table 3.1). Fungicides were selected to represent a range of fungicide groups and known activity against other rust species. Treatments were applied to four replications, arranged in a randomized complete block design. Untreated plots were established for comparison. Blocks were separated by a 2-m-long space of untreated fern to prevent spray drift among replications. Fungicide treatments were applied with a CO2-propelled backpack sprayer

(Bellspray Inc., Opelousas, Louisiana, U.S.A.) and a 3-nozzle boom equipped with 50 mesh screens and 11005XR nozzles spaced 50 cm apart, calibrated to deliver 400 l/ha. Treatments were initiated once the cladophylls had fully emerged and subsequent applications were made every 14 days. Six applications were made in total. Several types of disease severity assessments were made.

The rust and SLS lesions were counted twice in the treatment window on five randomly selected whole ferns per plot. At the end of September, rust severity was assessed using the following scale:

1=no foliar infection, 2=trace to 10% foliar infection, 3=10-20% foliar infection, 4=20-30% foliar infection, 5=30-40% foliar infection, 6=40-50% foliar infection, 7=50-65% foliar infection, 8=65-

80% foliar infection, 9=80-90% foliar infection, and 10=90-100% foliar infection. The defoliation

(%) was also visually estimated at this time. The AUDPC was calculated according to the methods described by Shaner and Finney (1977) and standardized by the methods described by Simko and

Piepho (2012).

Table 3.1 Fungicide treatment, application rate and product information applied every 14 days to asparagus fern infected with both Stemphylium vesicarium and Puccinia asparagi. Treatment g ai/ha (L/ha) Product and formulation untreated control - - azoxystrobin/cyproconazole 100/40 Quadris Xtra 280SC1 azoxystrobin/difenoconazole 250/125 Quadris Top 325SC1 benzovindiflupyr 75 Aprovia 100EC1 chlorothalonil 1700 Bravo 500SC1 cyproconazole 40 Alto 100SL1 fluazinam 500 Allegro 500F2 metiram 2600 Polyram 80DF3 mineral oil + proprietary pigment (18.7 + 1.2) Civitas + Harmonizer4 mineral oil + proprietary pigment + (18.7 + 1.2) + 62.5 Civitas + Harmonizer + Tilt propiconazole propiconazole 62.5 Tilt 250EC1 pyraclostrobin 112 Cabrio 20WG3 pyraclostrobin/boscalid 111/219 Pristine 38WG3 tebuconazole 125 Folicur 432F5 1Syngenta Canada Inc., Guelph, ON 2ISK Biosciences Corp., Kearney, MO 3BASF Canada Inc., Mississauga, ON 4Suncor Energy Inc., Mississauga, ON 5Bayer Cropscience Canada, Calgary, AB

3.2.3 Data analysis

Statistical analyses were conducted using SAS v.9.4 (SAS Institute Inc., Cary, NC). Data from disease assessments were analyzed with an analysis of variance (ANOVA) in PROC MIXED.

The restricted maximum likelihood (REML) covariance parameter estimates were used to analyze parameters. The Shapiro-Wilk test was used to test the normality of residuals and distribution of error was determined by residual plots. No outliers were identified in these data sets based on

Lund’s test. The normality of each data set was assessed using PROC UNIVARIATE. Data were transformed with either a square-root or log + 1 transformation if required. Variance in both the fungicide screen and cultivar screen was partitioned into random (block) effects and fixed (year, year × cultivar, cultivar and fungicide) effects. Means were separated using a Tukey’s HSD

(P=0.05). Pearson’s correlations were estimated using the PROC CORR procedure in SAS.

3.3 Results

3.3.1 Asparagus cultivar susceptibility to SLS and rust

The fixed effect of year × cultivar for all parameters was not significant for either disease

(Table A2.1, Table A2.2, Table A2.3, Table A2.4, Table A2.5 and Table A2.6). Data were pooled over years for both diseases and all parameters. For all estimates, the fixed effect of cultivar was significant, and means are presented (Table 3.2). ‘Jersey Giant’ and ‘Tiessen’ had a lower final

SLS DSI than ‘Guelph Millennium’. Also, ‘Jersey Giant’ had a lower SLS sAUDPC than UG010,

‘Guelph Equinox’, UG023 and ‘Guelph Millennium’. The final SLS DSI and sAUDPC were positively correlated (r= 0.80, P<0.0001). The line ‘Guelph Equinox’ had the lowest final rust DSI, and had a lower rust DSI than ‘Tiessen’. All three UG lines and ‘Jersey Giant’ had a lower rust sAUDPC than ‘Guelph Millennium’ and ‘Tiessen’. The final rust DSI and sAUDPC were positively correlated (r= 0.87, P<0.0001). These results indicate the UG lines have a similar slow- rusting phenotype as ‘Jersey Giant’. The final DSI and sAUDPC were negatively correlated between SLS and rust (r= -0.82, P<0.0001 and r= -0.64, P<0.0001, respectively).

Table 3.2 Final disease severity index (DSI), standardized area under the disease progress curve (sAUDPC), final fern yellowing and final defoliation observed in asparagus infected with Stemphylium leaf spot and rust. Stemphylium Rust Yellowing Defoliation Cultivar or line DSI sAUDPC2 DSI sAUDPC2 (%) (%) UG023 30 bc3 8.7 b 23 ab 7.2 a 54 a 39 a Guelph Equinox 30 bc 8.3 b 20 a 7.9 a 63 ab 55 ab Jersey Giant 18 a 5.4 a 23 ab 7.7 a 56 a 60 ab UG010 24 abc 7.9 b 24 ab 6.4 a 68 ab 61 ab Guelph Millennium 32 c 7.8 b 25 ab 11.9 b 80 ab 76 b Tiessen 20 ab 7.1 ab 34 b 14.2 b 83 b 79 b Standard error 2.6 0.51 3.1 1.0 6.4 6.2 P-value 0.0018 0.0009 0.0227 <0.0001 0.0010 0.0096 1Disease assessments were made weekly following full cladophyll emergence. Final assessments were made Oct 13, 2015 and October 5, 2016. Data were combined between the two years since no interaction was observed between cultivar and year. 2Standardized area under the disease progress curve calculated from Stemphylium or rust DSI collected over time. 3Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=5.

The asparagus cultivars and lines also differed in final fern yellowing and defoliation

(Table 3.2). The line UG023 and ‘Jersey Giant’ had less fern yellowing than ‘Tiessen’. Fern yellowing and final SLS DSI were positively correlated (r= 0.45, P=0.0003), however, fern yellowing is also an indicator of dormancy. The line UG023 had the least defoliation and had less defoliation than ‘Guelph Millennium’ and ‘Tiessen’. Defoliation and final rust DSI were positively correlated (r= 0.34, P=0.0077). Also, fern yellowing and defoliation were positively correlated (r=

0.65, P<0.0001).

3.3.2 Fungicide efficacy trials

In the 3-year-old field, the fixed effect of fungicide treatment was significant for all parameters (Table A2.7, Table A2.8 and Table A2.9.). Azoxystrobin/cyproconazole, azoxystrobin/difenoconazole, benzovindiflupyr, cyproconazole, pyraclostrobin, tebuconazole, and

the tank-mixture of mineral oil + proprietary pigment + propiconazole had less rust than the untreated control in June (Table 3.3). However, by July, all fungicides had less rust than the untreated control except chlorothalonil, fluazinam, pyraclostrobin, pyraclostrobin/boscalid, and the tank-mixture of mineral oil + proprietary pigment. Only azoxystrobin/cyproconazole provided greater than 80 % control, which is the threshold established by the Pest Management Regulatory

Agency (PMRA) in Canada. All treatments except pyraclostrobin/boscalid had a lower rust severity than the untreated control by September. Also, all treatments that contained a FRAC group

3 fungicide had a lower sAUDPC than the untreated control. Benzovindiflupyr and metiram were the only other non-group 3 fungicides with a lower AUDPC than the untreated control. The final rust counts were positively correlated with the sAUDPC (r= 0.77, P<0.0001). The rust severity was moderately correlated with the final rust counts (r= 0.50, P<0.0001) and rust sAUDPC (r=

0.48, P=0.0002).

Table 3.3 Field evaluation of foliar fungicides applied on a 14-day interval for rust control in a 3-year-old asparagus field, in Norfolk County, in 2011. Rust Rust lesions/plant Treatment and g a.i./ha (L/ha) severity sAUDPC1 27 Jun 25 Jul 23 Sep untreated control 210 d2 250 c 10 161 f azoxystrobin/difenoconazole 325 50 ab 75 abc 3 46 a azoxystrobin/cyproconazole 140 63 abc 50 a 3 51 ab cyproconazole 40 72 abcd 52 ab 6 56 abc benzovindiflupyr 75 50 a 83 abc 3 59 abc tebuconazole 125 83 abc 99 abc 5 70 abcd mineral oil (18.7) + pigment (1.2) + propiconazole 125 98 abcd 105 abc 6 78 abcde propiconazole 125 141 cd 156 abc 7 113 abcdef metiram 2600 156 bcd 141 abc 6 114 bcdef mineral oil (18.7) + pigment (1.2) 182 cd 204 c 5 129 cdef chlorothalonil 1700 137 bcd 278 c 8 134 def fluazinam 500 181 cd 172 bc 8 135 def pyraclostrobin 112 95 bcd 376 c 7 146 ef pyraclostrobin/boscalid 330 283 d 300 c 8 195 f Standard error 39.5 32.0 18.0 P-value 0.0016 <0.0001 <0.0001 1Standardized area under the disease progress curve calculated from rust counts. 2Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=4.

In the 4-year-old field, the fixed effect of fungicide treatment was significant for all parameters (Table A2.10, Table A2.11, Table A2.12, Table A2.13 and Table A2.14). Rust was not observed in the untreated control until August (data not shown), and was observed later in the 4- year-old field than the 3-year-old field. By September, all treatments had less rust than the untreated control except pyraclostrobin/boscalid, pyraclostrobin and propiconazole (Table 3.4).

No differences were observed among the treatments in sAUDPC. The final rust counts were positively correlated with both the sAUDPC (r= 0.89, P<0.0001) and rust severity (r= 0.58,

P<0.0001). No treatments had less SLS than the untreated control. SLS lesion counts and SLS

sAUDPC were positively correlated (r= 0.88, P<0.0001), as expected. Rust and SLS counts were not correlated. The combination of mineral oil, proprietary pigment and propiconazole, pyraclostrobin/boscalid, benzovindiflupyr, azoxystrobin/difenoconazole, azoxystrobin/cyproconazole, fluazinam, cyproconazole and metiram had less defoliation than the untreated control. Also, all treatments provide > 80 % control of defoliation except chlorothalonil, cyproconazole, propiconazole, and the tank-mixture of mineral oil + pigment. Both final rust counts (r= 0.41, P=0.0020) and SLS counts (r= 0.31, P=0.0234) were weakly correlated with defoliation.

Table 3.4 Field evaluation of foliar fungicides applied on a 14-day interval for rust and Stemphylium leaf spot control in a 4-year-old asparagus field, in Norfolk County, in 2011. Rust Rust Rust Purple Defol- severity Treatment and g a.i./ha (L/ha) lesions/plant sAUDPC spots/fern iation (0 to 10) 1 23 Aug 20 Sep 28 Sep 20 Sep (%) untreated control 33 abc2 90 e 7 25 c 84 abcd 83 b mineral oil (18.7) + pigment (1.2) + propiconazole 125 8 a 13 abc 2 4 abc 85 bcd 6 a pyraclostrobin/boscalid 330 12 abc 57 de 3 13 bc 74 abc 6 a benzovindiflupyr 75 9 ab 4 ab 1 3 abc 40 a 9 a azoxystrobin/difenoconazole 325 2 a 5 ab 1 1 ab 43 ab 11 a azoxystrobin/cyproconazole 140 8 a 5 a 1 3 ab 74 bcd 14 a fluazinam 500 7 a 18 bcd 2 5 abc 114 bcd 14 a cyproconazole 40 4 a 1 a 2 1 a 137 bcd 21 a metiram 2600 7 a 13 abcd 2 4 abc 147 cd 23 a tebuconazole 125 4 a 8 ab 1 2 abc 136 bcd 27 ab pyraclostrobin 112 50 c 46 cde 5 21 bc 131 bcd 30 ab chlorothalonil 1700 9 ab 44 bcd 4 9 abc 175 d 39 ab mineral oil (18.7) + pigment (1.2) 44 bc 50 bcd 4 20 bc 101 bcd 43 ab propiconazole 125 14 abc 62 cde 5 14 bc 156 bcd 54 ab Standard error 9.0 18.1 4.1 27.7 10.8 P-value 0.0034 0.0206 0.0007 0.0241 0.0017 1Standardized area under the disease progress curve calculated from rust counts. 2Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=4.

3.4 Discussion

Foliar disease in asparagus is affected by the age of a field and the environment (Elmer et al., 1996). Both rust and SLS are endemic in Ontario asparagus fields. Foliar disease causes premature defoliation and senescence, and both reduce the photosynthetic potential of the crop which ultimately affects the amount of carbohydrates moving to the crown prior to dormancy

(Meyer et al., 2000). Due to the increase in disease severity observed in Ontario, and a lack of registered fungicides, an approach that includes the application of multiple fungicide groups and host resistance is required to achieve satisfactory disease management.

The lines UG010, ‘Guelph Equinox’ and UG023 were identified as slow-rusting phenotypes, similar to ‘Jersey Giant’. UG023 also had the greatest retention of cladophylls.

Interestingly, rust and SLS were negatively correlated. Due to the inverse relationship, it is difficult to draw conclusions about resistance or susceptibility from the SLS data. However, the lines with similar sAUDPC to the standard slow-rusting cultivar, ‘Jersey Giant’, are promising additions to a rust management program.

In the fungicide trials, rust occurred earlier in the 3-year-old field than the 4-year-old field.

In the former, the spears were harvested for just 2 weeks, whereas a full harvest was made in the latter. Since the fern emerged earlier in the 3-year-old field, the primary inoculum was able to establish earlier in the season. In turn, the polycyclic part of the epidemic was also initiated earlier, which resulted in earlier and greater disease pressure in the 3-year-old field than the 4-year-old field. None of the fungicides tested provided > 70 % control of rust pustules in the field with the greatest severity. In the field with the lowest rust severity, several fungicides provided > 70 % control, including the registered fungicides metiram and tebuconazole. In periods of high disease pressure, or in young asparagus fields, a 7- to 10-day application interval may be required to obtain

adequate disease control (Hausbeck et al., 2008). However, only half of the registered fungicides recommend an interval shorter than 14 days: metiram, myclobutanil and trifloxystrobin.

Several fungicide mixtures and different modes of action were identified with activity against SLS and rust. The pre-mixtures azoxystrobin/cyproconazole and azoxystrobin/difenoconazole provided good control of rust in both field trials. In the 4-year-old field, these fungicide mixtures also had lower SLS than the registered standard chlorothalonil. In the 3-year-old field, the tank-mixture of mineral oil + pigment + propiconazole had lower rust counts in June than the individual components of the mixture.

SLS was present at both fungicide field sites, but due to the high rust severity, it was difficult to discern any differences. In order to conduct trials for SLS, and to obtain useful conclusions, fungicides which controlled rust but had limited activity against SLS, such as cyproconazole or tebuconazole, could be applied to manage rust. Likewise, slow-rusting lines that were identified as susceptible to SLS, such as ‘Guelph Equinox’ and UG023, could also be used in SLS fungicide screening experiments.

CHAPTER FOUR

ASSESSING THE TOM-CAST FORECASTING MODEL FOR MANAGEMENT OF

STEMPHYLIUM LEAF SPOT (STEMPHYLIUM VESICARIUM WALLR.) E.G.

SIMMONS IN ASPARAGUS (ASPARAGUS OFFICINALIS L.) IN ONTARIO

Abstract

In the last 5 years, asparagus acreage in Canada has increased by over 25 %. Stemphylium leaf spot, caused by Stemphylium vesicarium, has emerged as the predominant foliar pathogen of asparagus. Typically, contact fungicides are applied every 14 days, however, regardless of the number of applications, growers are not achieving adequate control of the disease. The TOM-

CAST forecasting model is used widely in Michigan asparagus fields, but has never been assessed for suitability in Ontario or in the popular cultivar, Guelph Millennium. Six field trials were conducted in 2012 and 2013 to evaluate the TOM-CAST forecasting model in two asparagus cultivars. The fungicides chlorothalonil or azoxystrobin/difenoconazole were applied according to the forecasting model or on a 14-day interval. The effectiveness of the forecasting model differed between sites and cultivars. Even though TOM-CAST is used in all cultivars in Michigan, TOM-

CAST was not effective on ‘Guelph Millennium’. In ‘Jersey Giant’, however, TOM-CAST with a

20 DSV spray threshold improved control of Stemphylium leaf spot without increasing the number of sprays, compared to a 14-day treatment. The results in ‘Guelph Millennium’ differed between sites. At one site, TOM-CAST maintained similar levels of Stemphylium leaf spot, but increased the number of applications, compared to a 14 day application interval. Of more concern, none of the fungicide treatments differed greatly from the untreated control at the other site. Our results show that forecasting models need to be validated locally, in asparagus cultivars relevant to production today.

4.1 Introduction

Stemphylium leaf spot (SLS) on asparagus fern and purple spot on asparagus spears are diseases caused by the fungus Stemphylium vesicarium (Wallr.) E.G. Simmons and its teleomorph

Pleospora herbarum, respectively (Simmons, 1969). Stemphylium vesicarium and other

Stemphylium spp. have been reported as pathogens of asparagus in all major production regions of the world (Falloon et al., 1987; Gonzalez, 2012; Graf et al., 2016; Johnson & Lunden, 1984; Lacy,

1982).

Asparagus is a perennial crop. The marketable spear emerges in the spring and is harvested for a period of six to eight weeks. After that time, the spears are allowed to grow into ferns, with modified stems, called cladophylls. The fern dies in the late fall, and the residue is either left to stand in the field, or mowed. Stemphylium vesicarium infects the stems, ferns and cladophylls of asparagus, and pseudothecia develop on the dead fern over the winter. Standard practice is to mulch the fern in the spring and leave it in the field, which contributes to the primary inoculum for purple spot on the emerging spears (Evans & Stephens, 1984). Similar to Michigan, growers in Ontario have adopted no-till production, and the inoculum accumulates year over year

(Hausbeck et al., 1999; Lacy, 1984). Even though ascospores produced in the pseudothecia are known to be the source of primary inoculum, reducing or eliminating the overwintering pseudothecia is difficult. Growers are only able to manage the secondary infection which occurs in the fern following harvest and when the infection by S. vesicarium is severe, the fern defoliate which reduces the photosynthetic area and the amount of carbohydrate returning to the crown

(Eichhorn et al., 2009; Elmer et al., 1996; Meyer et al., 2000).

The production of asparagus in Ontario has increased in recent years (Statistics Canada,

2016), and growers have relied on cultural management and the application of foliar fungicides to

the fern to control S. vesicarium. In Ontario, fungicide spray programs usually start once the cladophylls have emerged, and applications are re-applied on a 14-day interval. Regardless of the number of foliar fungicides applied, growers are not achieving adequate control of the disease.

Forecasting models have been tested in the United States and Germany to predict infection periods and time fungicide sprays accordingly, to control SLS in asparagus (Eichhorn et al., 2009;

Hausbeck et al., 2008; Meyer et al., 2000). Most recently, growers in Michigan have adopted the forecasting model TOM-CAST for control of SLS in asparagus based on the work conducted by

Meyer et al. (2000). The program TOM-CAST provides a disease severity value (DSV) between

0 and 4 for each 24 h period, determined by the hours of leaf wetness and the average temperature during the period of leaf wetness. Once the cumulative DSVs for a field reach a pre-determined value, a fungicide is applied. The fungicides chlorothalonil and mancozeb were applied on a calendar-based timing or once TOM-CAST had accumulated 15 DSV (Meyer et al., 2000).

Following the TOM-CAST program reduced fungicide sprays by 60% compared to a fungicide application every 7 days, without compromising fern health (Meyer et al., 2000). If growers time fungicide applications according to a forecasting model, there should be decreased SLS and a reduction of premature defoliation of the fern.

Contact and systemic fungicides have not been directly compared in a TOM-CAST spray program in asparagus. It is understood that fungicides differ in their efficacy and suitability within a forecasting model (Meyer et al., 2000). In Germany, TOM-CAST was evaluated at 15, 20, 25,

30, and 35 DSV decision points (Eichhorn et al., 2009). Fungicides applied with TOM-CAST at lower DSV decision points provided improved control of SLS, and a 20 DSV model was recommended when using a protectant fungicide (Eichhorn et al., 2009). If a 20 DSV was surpassed due to unfavourable application conditions, it was found that a curative fungicide was

required to maintain control (Eichhorn et al., 2009). In carrots, copper (contact, curative and preventive fungicide), chlorothalonil (contact, preventive fungicide) and azoxystrobin (systemic, curative and preventive fungicide) were applied according to TOM-CAST at 10, 15 and 20 DSV and compared for control of foliar blight (Dorman et al., 2009). All of the fungicides provided similar control when TOM-CAST was applied at 10 and 15 DSV (Dorman et al., 2009). However, the fungicides differed once they were applied at a 20 DSV threshold, and only azoxystrobin provided adequate control (Dorman et al., 2009).

Certain fungicides are best suited for use in a forecasting model, yet little information is available to determine which are most effective in asparagus. In Canada, only the contact fungicide chlorothalonil and the Quinone outside inhibitor (QoI) fungicides azoxystrobin and trifloxystrobin are registered for control of S. vesicarium in asparagus. However, when unregistered fungicides were screened, the combination of QoI and a different mode of action had less SLS and defoliation than the individual components (Foster & McDonald, 2015; Hausbeck & Escobar-Ochoa, 2014).

Also, Foster and McDonald (2015) and Hausbeck and Escobar-Ochoa (2014) found the combination of azoxystrobin/difenoconazole had less SLS than the registered standard chlorothalonil. If a fungicide had increased efficacy or even curative activity, perhaps an increased

DSV could be used to prompt an application, reducing the number of applications within a season.

This strategy, though, increases the risk for the development of fungicide resistance (FRAC, 2016).

The asparagus cultivars grown in Ontario have changed since 2000. The predominant cultivar is now ‘Guelph Millennium’, a cultivar with high yield and winter hardiness (CFIA, 2016).

However, ‘Guelph Millennium’ is more susceptible to SLS than the traditional cultivars established in Ontario (Foster & McDonald 2017; Hausbeck and Escobar-Ochoa 2014). Even

though ‘Guelph Millennium’ is more susceptible to SLS, little is understood about whether or not growers need to modify their management of SLS in this cultivar specifically.

Since the predominant cultivar in Ontario is ‘Guelph Millennium’, research on the effectiveness of fungicides in this cultivar is warranted. Furthermore, the forecasting model TOM-

CAST was validated in no-till asparagus in Michigan, however, its effectiveness for ‘Guelph

Millennium’ has not been confirmed. Also, little is understood regarding the usefulness of systemic fungicides applied within a forecasting model, especially in asparagus. The objective of this research was to determine the effectiveness of systemic fungicides applied according to the TOM-

CAST forecasting model for control of SLS in the asparagus cultivar Guelph Millennium.

4.2. Materials and methods

4.2.1 Field trial design

Trials were conducted in two commercial asparagus fields (sites) located in Elgin County,

Ontario. Site 1 had sandy soil. One-year-old ‘Jersey Giant’ crowns were planted in 2007 and

‘Guelph Millennium’ 8-week-old seedlings were planted in 2008. Transplants and crowns were spaced 20 cm apart within the rows and 1.2 m between rows. The first full seasons of harvest at

Site 1 were 2010 and 2012 in the ‘Jersey Giant’ and ‘Guelph Millennium’ fields, respectively. Site

2 had sandy loam soil. At this site, ‘Guelph Millennium’ seeds were planted in 2004. The resulting asparagus plants were thinned to 20 cm spacing within the row and 1.2 m between rows. The first full season of harvest at Site 2 was in 2009.

In 2012, field trials were conducted in both cultivars located at Site 1. In 2013, the trials at

Site 1 were repeated, but placed in different sections of the field. Also, in 2013, two field trials were conducted at Site 2, in different sections of the field. The six field trials were designated by

their site (Site 1 or Site 2), the cultivar (‘Guelph Millennium’ or ‘Jersey Giant’), and at Site 2, by the location in the field (north or south). Field trials were initiated once the cladophylls were emerging (Meyer et al. 2000). Each experimental unit was one 6-m-long row and treatments were arranged in a randomized complete block design. Treatments were combinations of fungicide and application timing, as well as an untreated control, for a total of nine treatments. Each treatment was replicated four times and separated by an unsprayed buffer row. Blocks were separated by 2 m of unsprayed fern. The fungicide applications were made with a compressed CO2-propelled backpack sprayer and three-nozzle boom equipped with flat-fan nozzles (TeeJet XR11005) operated at 241 kpa and calibrated to deliver 400 L/ha.

4.2.2 Fungicide applications

Chlorothalonil (Bravo 500SC, Syngenta Canada Inc., Guelph, ON) and azoxystrobin/difenoconazole (Quadris Top 325SC, Syngenta Canada Inc., Guelph, ON) treatments were applied on a 14-day interval or according to the TOM-CAST forecasting model. Hourly temperature and leaf wetness were measured from sensors (HOBO USB Micro Station Data

Logger-H21-USB, Onset Computer Corporation, Bourne, MA) placed at mid-fern height within the canopy to calculate DSV. Calendar-based sprays were initiated following the final harvest when the plants had produced secondary branching and the cladophylls were beginning to emerge.

The first TOM-CAST sprays were applied with the accumulation of 15, 20 or 30 DSV following cladophyll emergence (Meyer et al., 2000). Subsequent sprays were applied after the accumulation of 15, 20, or 30 DSV since the last fungicide application in the respective treatment. At Site 1 in

2012, the final applications of 14-day, 15 DSV, 20 DSV, and 30 DSV treatments were made 56,

62, 56 and 56 days after cladophyll emergence, respectively. In 2013, the final applications of 14-

day, 15 DSV, 20 DSV and 30 DSV treatments were made 42, 50, 46 and 50 days after cladophyll emergence, respectively. At Site 2 in 2013, the final applications of 14-day, 15 DSV, 20 DSV and

30 DSV treatments were made 42, 50, 50 and 50 days after cladophyll emergence.

4.2.3 Assessments and data analysis

Twenty branches per plot were randomly selected and removed from individual fern. The number of SLS lesions on each branch was counted every 14 days following the emergence of cladophylls. In 2012 at Site 1, 2013 at Site 1 and 2013 at Site 2, the final lesion counts were conducted 84, 42 and 70 days after cladophyll emergence, respectively. The area under the disease progress curve (AUDPC) was calculated from the SLS lesion counts using the following equation:

AUDPC = Σ [((Yi + Yi-1) (Xi – Xi-1))/2] where Yi is number of SLS lesions at day Xi and Yi-1 is number of SLS lesions at day Xi-1 (Shaner & Finney, 1977). The AUDPC was standardized

(sAUDPC) by dividing the AUDPC by the number of days the treatments were assessed (Simko

& Piepho, 2012). In 2013, defoliation (% of cladophylls dropped from branch) occurred and was visually estimated 84 days after cladophyll emergence at both sites. Defoliation did not occur in

2012 during the assessment period.

The six field trials were first analyzed as randomized complete block designs, and fungicide treatments and timings were compared to the untreated control at both locations for the parameters SLS counts, sAUDPC and defoliation. Parameters were subjected to analysis of variance (ANOVA) using SAS PROC GLIMMIX (v. 9.4, SAS Institute Inc., Cary, NC). The restricted maximum likelihood (REML) covariance parameter estimates were used to analyze parameters. Variance for SLS counts and sAUDPC was partitioned into random (block(location), block × location × treatment) and fixed effects (treatment, location and location × treatment ), and

variance for defoliation was portioned into random (block(location)) and fixed effects (location, site, location × site, treatment, treatment × location, treatment × site, treatment × location × site).

The six field trials were then analyzed as a factorial design with two factors: fungicide and application timing. The parameter SLS counts was subjected to factorial ANOVA. Variance was portioned into random effects (block(location) and location × fungicide × application timing) and fixed effects (fungicide, application timing and fungicide × application timing). The untreated control was removed from the ANOVA to make a balanced factorial.

The field trials at Site 1 were also analyzed as a split plot factorial design with three factors: cultivar, fungicide and application timing. Cultivar was treated as whole plot factors and fungicide and application were treated as subplot factors arranged in a randomized complete block design.

The parameters SLS counts and sAUDPC were subjected to ANOVA. Variance was portioned into random effects (block(year × cultivar), block × cultivar, block × fungicide, and block × application timing) and fixed effects (cultivar, fungicide, application timing, cultivar × fungicide, cultivar × application timing, fungicide × application timing, and cultivar × fungicide × application timing). The untreated control was removed from the ANOVA to make a balanced factorial.

Tukey’s HSD (HSD) was used for separation of means when treatments were found to be statistically significant in ANOVA analysis (P≤0.05). No outliers were identified in these data sets based on Lund’s test. The assumptions of normality for each data set were assessed using the

Shapiro-Wilk statistic. Homogeneity and the distribution of error were tested using Levene’s test and by visual assessment of the residual plots, respectively. The final lesion count, sAUDPC and defoliation data were either square-root or log + 1 transformed to improve the fit to a normal distribution. Non-transformed means and standard errors are presented.

4.3 Results

4.3.1 Fungicide applications and disease pressure

The disease forecasting models triggered different numbers of sprays for most site-years.

Applications made on a 14-day schedule resulted in 5, 4 and 4 applications at Site 1 in 2012 and

2013, and Site 2 in 2013, respectively (Table 4.1). At both sites, the 15 DSV TOM-CAST model triggered more applications, 20 DSV TOM-CAST triggered a similar number of applications, and

30 DSV TOM-CAST triggered fewer applications than the 14-day application.

Table 4.1 Number of fungicide applications timed according to either calendar-based application timing or the forecasting model TOM-CAST in asparagus. Number of sprays Treatment schedule Site 1 Site 2 2012 2013 2013 untreated 0 0 0 14-day interval 5 4 4 15 DSV TOM-CAST 6 5 7 20 DSV TOM-CAST 5 4 6 30 DSV TOM-CAST 3 2 4

SLS was present on the portion of the stem below the first branch prior to the first application at Site 1 in both years but 14 days after cladophyll emergence at Site 2. Disease was first detected on the branches in 2012 in both cultivars by 14 days after cladophyll emergence (Fig.

4.1). In 2013, however, SLS was first detected on the branches by 14 and 28 days after cladophyll emergence in ‘Guelph Millennium’ and ‘Jersey Giant,’ respectively. At Site 2, trace amounts of

SLS were detected on the branches by 14 days after cladophyll emergence.

Figure 4.1 Stemphylium leaf spot lesions counted per branch in the untreated control plots at six field trials. Error bars represent the standard error of the mean, n=4.

Overall disease pressure was lower in 2012 than in 2013. Lesion counts were higher in

2013 than in 2012 at Site 1, and the duration of assessments was therefore shortened since the

SLS counts in the untreated control dropped following premature defoliation (Fig. 4.1). The highest cumulative DSV were recorded at Site 2 in 2013, however, the highest SLS lesion counts were observed at Site 1 in 2013 (Table 4.2).

4.3.2 Stemphylium leaf spot counts

From the ANOVA, the fixed effects location, treatment, and location × treatment for SLS counts were significant (Table A3.1). Locations were analyzed separately, and the fixed effect of treatment was significant at four of six field sites (Table A3.2, Table A3.3, Table A3.4, Table

A3.5, Table A3.6 and Table A3.7). In half of the field trials, certain treatments had less disease than the untreated control, however, none of the treatment combinations were consistent across locations (Table 4.2). Rarely were SLS counts lower in the fungicide treated plots than the untreated control.

Table 4.2 Final Stemphylium leaf spot (Stemphylium vesicarium) counts on asparagus fern treated with foliar fungicides at four application timings. Final disease severity (lesions/branch)1 Fungicide Site 1 Site 2 treatment and ‘Jersey Giant’ ‘Guelph Millennium’ ‘Guelph Millennium’ timing 20122 20133 20122 20133 2013 2013 (south)3 (north)2 untreated 12 94 b4 41 b 88 a 48 b 66 chlorothalonil 14-day 11 35 ab 15 ab 132 ab 29 a 50 15 DSV 6 43 ab 16 ab 109 ab 31 a 46 20 DSV 4 22 a 19 ab 147 ab 31 ab 39 30 DSV 11 74 ab 29 ab 262 ab 28 a 41 azoxystrobin/ difenoconazole 14-day 9 32 ab 10 a 48 a 30 a 56 15 DSV 4 44 ab 8 a 65 a 33 ab 50 20 DSV 8 28 ab 14 a 124 ab 31 ab 54 30 DSV 8 55 ab 10 a 314 b 35 ab 55 Standard error 2.4 13.9 5.6 42.0 3.5 6.7 P-value 0.0902 0.0341 0.0047 0.0082 0.0232 0.2567 1Final disease assessment was made 84 days after cladophyll emergence (2012, Site 1), 42 days after cladophyll emergence (2013, Site 1), and 70 days after cladophyll emergence (2013, Site 2). 2Data were square-root transformed to satisfy normality assumptions. Presented means and standard error are non-transformed. 3Data were log transformed to satisfy normality assumptions. Presented means and pooled standard error are non-transformed. 4Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=4.

In the factorial ANOVA, and interaction was not observed between location, fungicide and application timing, but there was an interaction between location and timing (Table A3.8). The six field trials were then analyzed separately. In two of the trials, fungicide, application timing and fungicide × application timing were not significant (Table A3.9 and Table A3.14). In 2012, in the trial conducted in ‘Guelph Millennium’, the fixed effect of fungicide was significant (Table

A3.11). The fungicide azoxystrobin/difenoconazole had lower SLS counts than chlorothalonil

(P=0.0103). In 2013, at Site 1, the fixed effect of application timing was significant in both the

‘Jersey Giant’ and ‘Guelph Millennium’ field (Table A3.10 and Table A3.12). In both fields, the

20 DSV application timing had lower SLS counts than the 30 DSV application (P=0.0484 and

P=0.0039, respectively). At Site 2, the fixed effect of fungicide was significant in only the north field (Table A3.13). The fungicide chlorothalonil had lower SLS counts than azoxystrobin/difenoconazole (P=0.0225).

From the split-block factorial ANOVA, the fixed effect of year × cultivar × fungicide × application timing was significant (Table A3.15) and years were then analyzed separately. In 2012, all fixed effects were not significant (Table A3.16). In 2013, the fixed effects of cultivar, application timing and cultivar × application timing were significant (Table A3.17). At the two highest DSV, final disease severity was greater in ‘Guelph Millennium’ than in ‘Jersey Giant’

(Table 4.3). ‘Guelph Millennium’ was consistently more susceptible to SLS than ‘Jersey Giant.’

Table 4.3 Factorial analysis of the final disease severity between cultivar and application timing sprayed and assessed in asparagus fern for control of Stemphylium leaf spot (Stemphylium vesicarium), 2013. Final disease severity (lesions/branch)1,2 Factors and interaction ‘Guelph ‘Jersey Millennium’ Giant’ Untreated control 66 94 Application timing 14-day interval 98 ab 34 ab 15 DSV 94 ab 44 ab 20 DSV 128 b 25 a 30 DSV 285 c 64 ab Standard error 21.8 P-value 0.0019 1Final disease assessment was made 42 days after cladophyll emergence. 2Means followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=8. The untreated control is presented as a reference but was excluded from the ANOVA.

4.3.3 Disease progress

All three fixed effects, location, treatment and location × treatment were significant in the

ANOVA (Table A3.18), and the field trials were then analyzed separately. In three of the trials, the fixed effect of treatment was significant in the ANOVA (Table A3.20, Table A3.21 and Table

A3.22). Means for all locations are summarized in Table 4.4. At Site 1, in 2013 ‘Jersey Giant’, the

20 DSV application timing had a lower sAUDPC than the untreated control regardless of fungicide

(Table 4.4). In ‘Guelph Millennium’, the results in 2012 differed from those of 2013. In 2012, all application timing and fungicide combinations had a lower sAUDPC than the untreated control.

In 2013, all application timing and fungicide combinations were similar to the untreated control.

Table 4.4 Standardized area under the disease progress curve calculated from Stemphylium leaf spot (Stemphylium vesicarium) lesion counts on asparagus fern treated with foliar fungicides at four application intervals. Site 1 Site 2 Fungicide ‘Jersey Giant’ ‘Guelph Millennium’ ‘Guelph Millennium’ treatment and 2013 2013 timing 20121 20131 20121 20132 (south)1 (north) Untreated control 9 29 b3 23 b 129 ab 24 26 chlorothalonil 14-day 7 11 ab 9 a 66 a 15 20 15 DSV 7 17 ab 7 a 51 a 16 20 20 DSV 5 7 a 9 a 69 ab 13 19 30 DSV 10 26 ab 11 a 125 ab 19 17 azoxystrobin/ difenoconazole 14-day 10 12 ab 8 a 26 a 16 21 15 DSV 6 12 ab 8 a 76 ab 16 22 20 DSV 10 8 a 8 a 94 ab 19 22 30 DSV 7 16 ab 7 a 171 b 20 22 Standard error 1.6 3.8 2.0 20.7 3.0 3.1 P-value 0.3126 0.0117 0.0002 0.0044 0.3652 0.7994 1Data were log transformed to satisfy normality assumptions. Presented means and pooled standard error are non-transformed. 2Data were square-root transformed to satisfy normality assumptions. Presented means and standard error are non-transformed. 3Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=4.

From the split-block factorial ANOVA, the fixed effect of year × cultivar × application timing was significant (Table A3.25), and years were then analyzed separately. Similar to the results for SLS counts, in 2012, all fixed effects were not significant (Table A3.26), but again in

2013, the fixed effects of cultivar, application timing and cultivar × application timing were significant (Table A3.27). In the three DSV application timing treatments, final disease severity was greater in ‘Guelph Millennium’ than in ‘Jersey Giant’ (Table 4.5). In the application timing of 14-day interval, no differences were observed between the cultivars.

Table 4.5 Standardized area under the disease progress curve among fungicide applications timed according to a calendar-based schedule or the forecasting model TOM-CAST at different DSV for control of Stemphylium leaf spot (Stemphylium vesicarium) in 2013. Fungicide timing ‘Jersey Giant’ ‘Guelph Millennium’ Untreated control 29 129 14-day interval 12 a1 48 abc 15 DSV 15 a 63 bc 20 DSV 8 a 80 c 30 DSV 22 ab 148 d Standard error 10.2 P-value 0.0002 1Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=8. The untreated control is presented as a reference but was excluded for the means separation test.

4.3.4 Defoliation

The fixed effects of site, treatment and site × treatment were significant in the ANOVA

(Table A3.28), and the field sites were then analyzed separately. At both sites, the fixed effect of treatment was significant in the ANOVA (Table A3.29 and Table A3.30). Means for both sites are summarized in Table 4.6. At Site 1, only azoxystrobin/difenoconazole applied on a 14-day interval or at 15 and 20 DSV had less defoliation than the untreated control (Table 4.6). At Site 2, all treatments had less defoliation than the untreated control, and most of the DSV application timing treatments had less defoliation than the standard treatment of chlorothalonil applied on a 14-day interval.

Table 4.6 Defoliation of asparagus cladophylls, estimated 84 days after cladophyll emergence, in four fields treated with foliar fungicides timed according to the forecasting model TOM-CAST in 2013. Fungicide timing Site 11 Site 21 Untreated control 99 b2 91 d chlorothalonil 14-day 100 b 56 cd 15 DSV 99 b 4 a 20 DSV 100 b 17 ab 30 DSV 100 b 19 ab azoxystrobin/ difenoconazole 14-day 53 a 39 bc 15 DSV 55 a 21 ab 20 DSV 45 a 19 ab 30 DSV 88 b 33 bc Standard error 5.4 6.3 P-value <0.0001 <0.0001 1Data were square-root transformed to satisfy normality assumptions. Presented means and standard error are non-transformed. 2Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=8.

4.4 Discussion

Failure of a management program to control foliar disease in asparagus has economic consequences (Elmer et al., 1996; Meyer et al., 2000). SLS in season is the source of primary infection on the spears the following spring. The consumer tolerance for blemished spears is low and the processing market that would normally accept low grade asparagus has moved outside of

Canada. Most growers in Ontario apply foliar fungicides on a 14-day interval to manage SLS in asparagus fern, but adequate control is difficult to achieve. In Ontario, growers are looking for management tools to improve control, such as the forecasting model, TOM-CAST. In the presented study, TOM-CAST was assessed on two cultivars over two years at one site and at a second site in two locations in 2013 for a total of six trials.

Two fungicides were compared at different application timings for their effect on SLS counts, sAUDPC and defoliation. In 2013, at Site 1, the fern in the untreated control began defoliating by 28 days after cladophyll emergence in ‘Guelph Millennium’ (data not shown). The

SLS counts in the untreated control at subsequent counts were then reduced due to the decline in fern tissue (Fig. 1). At the final evaluation, the 30 DSV treatment for both fungicides had the highest SLS even including the untreated control. The fungicides delayed defoliation, but did not reduce final disease severity.

The present study is the first to compare the effectiveness of contact and systemic fungicides for SLS control in asparagus using the TOM-CAST forecasting model. Fungicides differ in their intrinsic activity against pathogens, and the current study indicates fungicides also differ in their efficacy between cultivars. In ‘Jersey Giant’ at Site 1 and ‘Guelph Millennium’ at

Site 2, both fungicides provided similar control, but in ‘Guelph Millennium’ at Site 2, azoxystrobin/difenoconazole had lower SLS counts than chlorothalonil. The TOM-CAST forecasting model was simplified from the FAST model and tested to determine if conditions were ideal for pathogen development over the duration of the collection period (Madden et al., 1978;

Pitblado, 1992). Both FAST and TOM-CAST were developed and tested prior to the wide use of

QoI and DeMethylation Inhibitor (DMI) fungicides. In asparagus specifically, Meyer et al. (2000) compared just two contact fungicides since no systemic fungicides were registered in asparagus.

The models assume all fungicides are equal. Since fungicides are timed according to environmental parameters in TOM-CAST, often fungicides are applied post-infection. In the present study, when treatments were applied prior to SLS incidence on the branches, there were lower sAUDPC and final leaf spot counts than when treatments were applied after SLS symptoms were observed. Most fungicides, including those with curative activity, have increased efficacy

when applied preventively rather than curatively (Anesiadis et al., 2003; Keinath, 2000). Due to improved efficacy with preventive applications, and to prevent the development of fungicide resistance, TOM-CAST should be initiated prior to the observation of SLS symptoms in asparagus.

The effectiveness of TOM-CAST differed by cultivar, which is an important consideration before the wide adoption of TOM-CAST. From 1996 to 1998, TOM-CAST was tested in Michigan and demonstrated to be effective in the popular commercial cultivars of that time: Jersey Giant,

Jersey Knight and Viking KB3 (Meyer et al., 2000). However, the majority of fields in Ontario are now established with ‘Guelph Millennium’. Over the 2 years of the study, ‘Guelph Millennium’ was more susceptible to SLS than ‘Jersey Giant,’ which influenced the effectiveness of the forecasting model within each cultivar. Fungicides applied according to TOM-CAST at 20 DSV resulted in fewer lesions than the untreated control and were more effective than a 14-day calendar based timing in ‘Jersey Giant’. Whereas in ‘Guelph Millennium’, spraying according to TOM-

CAST was no more effective than the 14-day calendar treatment, despite having the same number of applications.

Often the forecasting treatments were applied after disease symptoms were already observed in ‘Guelph Millennium’, since the first application was made according accumulated

DSV following cladophyll emergence. In carrots, Rogers and Stevenson (2006) used a disease threshold to time the first application within a forecasting model, instead of crop phenology. The use of a disease threshold improved the effectiveness of a forecasting model regardless of cultivar susceptibility. Since asparagus cultivars differ in their susceptibility, applications initiated based on crop phenology (cladophyll emergence) is likely not appropriate.

Forecasting models need to be effective in multiple environments. In pears, BSPcast was developed to control brown spot, caused by S. vesicarium. BSPcast uses leaf wetness and air

temperature during leaf wetness periods, similar to TOM-CAST (Llorente et al., 2000). Llorente et al. (2001) assessed a modified version of BSPcast that included relative humidity during periods without leaf wetness, however, the modification did not improve efficacy. In asparagus, Granke and Hausbeck (2010) suggested that a model to predict disease on the fern should include temperature, leaf wetness, vapour pressure deficit and rainfall. Since TOM-CAST is a simplification of the FAST model, the re-inclusion of environmental factors to the model, such as

VPD and rainfall, could improve the efficacy in asparagus. Also, it is important to consider that

TOM-CAST was developed for use in an annual crop not a perennial crop. As a result, TOM-

CAST does not account for the continuous addition of inoculum year over year. In the presented study, TOM-CAST was not effective at multiple field sites, and other epidemiological factors that affect S. vesicarium in asparagus should be considered.

Forecasting models need to be validated locally, with relevant cultivars, for asparagus.

Even though TOM-CAST is used in all cultivars in Michigan, it was not effective in ‘Guelph

Millennium’. In ‘Jersey Giant’, growers can improve control of SLS with a 20 DSV application schedule without increasing the number of sprays compared to a 14-day treatment. However, the results in ‘Guelph Millennium’ differed between sites, and ‘Jersey Giant’ was only tested at one site. In ‘Guelph Millennium’, at Site 1, a 15 DSV application schedule only maintained similar levels of SLS compared to the 14-day treatment, but increased the number of fungicide sprays.

Also of concern, at Site 2, none of the treatments, whether TOM-CAST or calendar-based sprays, provided > 50 % control of SLS, nor did the disease progress differ greatly from the untreated control. Prior to the adoption of TOM-CAST, Michigan growers were applying fungicides every

7 to 10 days, and TOM-CAST improved disease control while reducing the number of sprays

(Meyer et al. 2000). In Ontario, however, due to differences in susceptibility among cultivars,

modifications to the TOM-CAST forecasting model are needed before it is used by commercial growers.

CHAPTER FIVE

THE EFFECT OF FERTILIZER, IRRIGATION AND FUNGICIDES ON FOLIAR

DISEASE PROGRESS AND FERN HEALTH IN ASPARAGUS (ASPARAGUS

OFFICINALIS L.)

5.1 Introduction

Stemphylium leaf spot of asparagus is caused by the fungus Stemphylium vesicarium

(Wallr.) E.G. Simmons (teleomorph Pleospora herbarum) (Falloon, 1982; Lacy, 1982). Asparagus is a perennial crop, and emerging spears in the spring are harvested for a period of six to eight weeks. Following harvest, the spears are allowed to grow into ferns, with modified photosynthetic stem tissue, called cladophylls. Stemphylium vesicarium infects the stems, branches and cladophylls of asparagus, and pseudothecia develop on the dead fern over the winter. Stemphylium leaf spot symptoms, including purple to brown coloured spots, can be observed on the stems, ferns and cladophylls of asparagus (Falloon et al., 1987; Lacy, 1982). In cases of high severity, the cladophylls, defoliate which consequently reduces the amount of photosynthate returning to the crown (Elmer et al., 1996). Since asparagus is a perennial crop, growers expect the field to remain in production for greater than 15 years. If the fern defoliates prematurely, production is decreased in subsequent seasons (Meyer et al., 2000). Management of Stemphylium leaf spot is essential for asparagus production to remain sustainable in Ontario.

Fungicides are applied every 14 days to control foliar disease in asparagus, in Ontario and other regions. Regardless of the number of foliar applications, growers are not attaining very good control of Stemphylium leaf spot. Forecasting models have been tested in asparagus to predict infection periods and time fungicide sprays accordingly (Eichhorn et al. 2009; Hausbeck et al.

2008; Meyer et al. 2000). Specifically, growers in Michigan have adopted the forecasting model

TOM-CAST for use in asparagus. TOM-CAST uses the hours of leaf wetness in a 24-hour period and the average temperature during the periods of leaf wetness to calculate a disease severity value

(DSV). A fungicide application is recommended once the field has accumulated 15 DSV

(Hausbeck et al., 2008). Use of the TOM-CAST program in Michigan reduced fungicide sprays by 60% compared to a calendar-based program, without compromising fern health (Meyer et al.,

2000). At the time, only contact fungicides were registered for control of Stemphylium leaf spot.

Currently there are site-specific fungicides registered for use in asparagus, including strobilurins

(Quinone Outside Inhibitors) and DeMethylation Inhibitor fungicides. A robust resistance management strategy includes rotating site-specific fungicides with contact fungicides. Foliar programs alternating fungicides with different modes of action within a forecasting model have not been tested in asparagus in Ontario.

Nutrient deficiency is a form of abiotic stress that can increase disease susceptibility.

Nitrogen is the most extensively studied nutrient in plant disease research and has been attributed to both increasing and decreasing the disease severity in several crops (Jensen & Munk, 1997;

Mascagni et al., 1997; Tompkins et al., 1992) and necrotrophs (Howard et al., 1994; Leitch &

Jenkins, 1995; Long et al., 2000; Simon et al., 2003; Talukder et al., 2005). When nitrogen was applied in excess, the severity of foliar disease increased due to the high production of lush susceptible host tissue (Long et al., 2000; Tompkins et al., 1992). Also, the dense canopy created an environment that promotes leaf wetness (Leitch & Jenkins, 1995). When nitrogen was applied to cereals late in the season, the disease severity was higher than that in the control (Neumann et al., 2004). Furthermore, the practice of applying nitrogen fertilizer at a single time in the crop production system often results in rapid growth of foliage (Neumann et al., 2004). However,

asparagus growers are advised to apply nitrogen fertilizer following harvest to promote such rapid plant growth. Consequently, growers have been experimenting with the use of foliar fertilizer applied throughout the season, with varying reported success. In general, a balanced fertility program decreases disease by increasing plant resistance, enabling the plant to grow out of the disease, increasing plant tolerance, or inhibiting pathogen activity (Huber & Watson, 1974).

However, since S. vesicarium is a foliar pathogen in asparagus, it is unknown if the application of nitrogen would increase or decrease Stemphylium leaf spot and premature defoliation.

Moisture stress results in poor plant growth, and prolonged periods of drought can have a significant impact on perennial crops. In Ontario, asparagus is grown on light soil types, such as sand or sandy loam which provides good drainage and reduces infection caused by root rots, such as Phytophthora and Fusarium (Elmer et al., 1996). However, during periods of drought, asparagus grown on light soil types are most affected, and the fern defoliates prematurely and senesces prior to dormancy (Panka & Rolbiecki, 2008). Premature senescence weakens the crown year over year, due to a lack of carbohydrates returning to the crown (Elmer et al., 1996; Hausbeck et al., 2008; Meyer et al., 2000). This process results in the early decline of asparagus fields. The impact of S. vesicarium infection of asparagus, in addition to moisture stress, is relatively unknown. Asparagus growers have adopted the use of irrigation to prevent moisture stress during periods of drought. In dense foliar canopies, however, overhead irrigation is often attributed to a rise in foliar disease (Rotem & Palti, 1969). The impact of overhead irrigation on S. vesicarium infection of asparagus, specifically, is unknown.

Asparagus growers in Ontario are not attaining adequate control of Stemphylium leaf spot with their current management program. Unlike soil-borne disease in asparagus, cultural control methods such as irrigation and nutrient management have not been studied extensively in foliar

disease of asparagus. Also, it is not known if the contribution of moisture stress, poor nutrition and

S. vesicarium infection is additive in asparagus. Since asparagus is a perennial crop, likely an integrated strategy that includes both cultural and chemical control methods will be required to increase efficacy and prevent early decline. The objectives of this research were to determine the effect of irrigation, nitrogen fertilizer and fungicide application on Stemphylium leaf spot control, premature defoliation and early senescence.

5.2 Materials and Methods

5.2.1 Site selection and description

Six field trials were conducted in commercial asparagus fields in 2015 and 2016 (Table

5.1). Due to the differences observed in 2012 and 2013, five field sites were selected. Four non- irrigated field trials were conducted in 2015 at sites 1 and 2 and in 2016 at sites 1 and 3. Two irrigated field trials were conducted in 2016 at sites 4 and 5. One of the field sites (site 1) is the same used in CHAPTER FOUR (site 2). The fields were established from either seed or 1-year- old crowns and first harvested either 3 or 5 years after planting (Table 5.2). The asparagus plants in each field were spaced 15 cm apart within row, and the rows were spaced 1.2 m apart.

Table 5.1. Soil description at five field sites where fertilizer and foliar fungicide treatments were compared to control Stemphylium vesicarium in asparagus, 2015-16. Sand Silt Clay CEC Site Location Soil type pH (%) (%) (%) (meg/100 g) 1 Elgin County loamy sand 80.1 14.6 5.3 5.8 6.1 2 Norfolk County loamy sand 78.1 16.6 5.3 6.5 4.9 3 Elgin County sandy 88.1 7.6 4.3 6.8 5.9 4 Elgin County loamy sand 86.1 9.6 4.3 7.8 5.6 5 Norfolk County loamy sand 86.1 9.6 4.3 6.5 6.1

Table 5.2. Location and planting description of asparagus field sites where fertilizer and fungicide treatments were compared for control of Stemphylium vesicarium, 2015-16. Establishment Year First full season Site Location Previous crop method established of harvest 1 Elgin County seed 2004 2009 cucumber 2 Norfolk County 1-year-old crowns 2009 2012 rye 3 Elgin County 1-year-old crowns 2008 2011 field corn 4 Elgin County 1-year-old crowns 2008 2011 field corn 5 Norfolk County 1-year-old crowns 2004 2007 rye

5.2.2 Field trial design and treatment application

Field trials were established immediately following the final harvest of spears. The trials were arranged in a split-plot design, with the whole-plot factor being fertilizer treatment (four levels) and sub-plot factor being fungicide treatment (three levels). Fertilizer treatments were no fertilizer, 571 kg granular NK21 (Agronomy Company of Canada, Thorndale, ON) per ha (120 kg actual N and 120 g P2O5), 571 kg NK21 + 130 kg granular urea (60 kg actual N) per ha, and 571 kg NK21 per hectare + 20 L of liquid foliar fertilizer, Alpine SRN (7.2 kg actual N) (Nachurs

Alpine Solutions, Marion, OH). In commercial production fields, the recommended rate of actual

N is 120 kg/ha. A foliar fertilizer treatment was included to test new recommendations from a retail. The granular fertilizer was applied by hand immediately following harvest. The Alpine SRN was applied 28 days after cladophyll emergence (DACE) with a compressed CO2-powered backpack sprayer and three-nozzle hand-boom equipped with flat-fan nozzles (TeeJet XR11005,

TeeJet Technologies, Glendale Heights, IL) operated at 241 kpa and calibrated to deliver 400 L/ha.

Whole-plot factors were blocked and repeated four times.

Fungicide treatments were untreated, chlorothalonil (Bravo 500, Syngenta Canada Inc.,

Guelph, ON) alternated with azoxystrobin/difenoconazole (Quadris Top, Syngenta Canada Inc.,

Guelph, ON) applied according to the TOM-CAST forecasting model at 15 DSV and at 30 DSV.

Hourly temperature and leaf wetness were measured from sensors (HOBO USB Micro Station

Data Logger-H21-USB, Onset Computer Corporation, Bourne, MA) placed at mid-fern height to calculate daily DSV. Subsequent sprays were applied after the accumulation of either 15 or 30

DSV since the last fungicide application in the respective treatment. Chlorothalonil was applied at

1700 g ai/ha and azoxystrobin/difenoconazole was applied at 244 g ai/ha (150 g ai/ha azoxystrobin

+ 94 g ai/ha difenoconazole). Fungicide treatments were applied with a compressed CO2-powered backpack sprayer and three-nozzle hand-boom equipped with flat-fan nozzles (TeeJet XR11005) operated at 241 kpa and calibrated to deliver 400 L/ha. Fungicide treatments were initiated once the cladophylls had fully emerged. An experimental unit was one 6-m-long row and each fungicide treatment was replicated four times, in a randomized complete block design within each whole- plot fertilizer treatment Individual treatment plots were separated by an untreated buffer row.

Blocks were separated by 2 m of untreated fern.

In the irrigated field trials, both sites were irrigated with 1.5 million L of water per hectare from June to August. At Site 4, the water was applied through permanent drip irrigation placed at planting in the furrow, whereas at Site 5, the water was applied with an overhead irrigation gun.

5.2.3 Assessments and data analysis

The asparagus fern was assessed every 14 days following cladophyll emergence for symptoms of Stemphylium leaf spot. The number of Stemphylium leaf spot lesions on ten branches per experimental unit were assessed using a 0 to 5 scale modified from Falloon et al. (1987): 0 to

5, 0 = no lesions, 1 = 1 to 20 lesions, 2 = 21 to 50 lesions, 3 = 51 to 90 lesions, 4 = 91 to 150 lesions, and 5 > 150 lesions. A disease severity index (DSI) was calculated using the following formula: DSI = [∑[(class no.)(no. of branches in each class)]]/[(total no. of branches per

sample)(no. classes - 1)] x 100 (Kobriger & Hagedorn, 1983). The area under the disease progress curve (AUDPC) was calculated from the DSI using the following equation: AUDPC = Σ [((Yi +

Yi-1) (Xi – Xi-1))/2] where Yi is the DSI at day Xi and Yi-1 is the DSI at day Xi-1 (Shaner & Finney,

1977). The AUDPC was calculated both at 14 days after the last application (DALA) or 56 days after cladophyll emergence (DACE) and upon conclusion of the assessments at 98 DACE to account for the residual effect of the fungicide (14 DALA) and fertilizer treatments (98 DACE).

The AUDPC were standardized (sAUDPC) according to the methods described by Simko &

Piepho (2013). The defoliation (% of cladophylls dropped from branches) and fern yellowing (% area of yellow fern) of each experimental unit was visually estimated.

In 2016, the Normalized difference vegetative index (NDVI) for each experimental unit was measured using a Crop Circle ACS-430 (Holland Scientific, Lincoln, NE) the same weeks the other disease assessments were made. NDVI quantifies green vegetation by measuring the different between near-infrared and red light. The instrument was held 30 cm above the canopy height and walked the length of the plot. Weedy and empty sections were excluded. Also in 2016, at 42 DACE, fern tissue was collected from the no fertilizer and 180 kg N fertilizer plots that were not treated with foliar fungicide. The tissue were analyzed by for concentration (%) of N, P, K, Ca and Mg (Agri-Food Laboratories in Guelph, ON, Canada).

The field trials were separated by fields with irrigation and fields without irrigation and then analyzed as a split-plot factorial design, with the whole-plot factor of fertilizer and sub-plot factor of fungicide treatment. Parameters were subjected to ANOVA. The restricted maximum likelihood (REML) covariance parameter estimates were used to analyze parameters. Variance was partitioned into random effects (block(location), block × fertilizer) and fixed effects (location, fertilizer, fungicide treatment, location × fertilizer, location × fungicide treatment, fertilizer ×

fungicide treatment, and location × fertilizer × fungicide. For those parameters that were measured every 14 days, the repeated measures fixed effects (DACE, location × DACE, fertilizer × DACE, fungicide treatment × DACE, location × fertilizer × DACE, location × fungicide treatment ×

DACE, fertilizer × fungicide treatment × DACE, and location × fertilizer × fungicide treatment ×

DACE) were also included in the ANOVA.

Shapiro-Wilk statistic. Homogeneity and the distribution of error were tested using Levene’s test and by visual assessment of the residual plots, respectively. The parameters were either square- root or log + 1 transformed to improve the fit to a normal distribution if required. Non-transformed means and standard errors are presented. Correlation between DSI and parametric and non- parametric data were determined using the PROC CORR procedure.

5.3 Results

5.3.1 Disease severity and progression

The number of applications made on a 15 DSV schedule varied at each site and year (Table

5.3), and the cumulative DSV recorded at each site did not correspond to the final disease assessments in the untreated controls (Fig. 5.1). At cladophyll emergence, SLS was present at Site

3, 4 and 5, but not until 14 and 28 DACE at Site 1 and 2, respectively (Fig. 5.1). As a result, fungicides were applied preventively at Site 1 and 2, but curatively at Site 3, 4 and 5. The highest cumulative DSV were recorded at Site 1 in 2015 which also had the lowest final DSI in the

untreated control. The lowest cumulative DSV was recorded at Site 2, however, this site had the highest final DSI in the untreated controls.

Table 5.3. Location, soil type, irrigation, number of applications and cumulative DSV recorded at trial sites. CEC No. of Sand/Silt/ Cumulative Site Year Soil type pH (meg/ Irrigation Applications Clay (%) DSV 100g) 15 DSV 30 DSV 1 2015 loamy sand 5.5 78.1/16.6/5.3 9.4 none 9 5 120 2 2015 loamy sand 4.9 78.1/16.6/5.3 6.7 none 4 2 45 1 2016 loamy sand 6.1 80.1/14.6/5.3 5.8 none 8 4 105 3 2016 sand 5.9 88.1/7.6/4.3 6.8 none 5 3 60 4 2016 loamy sand 5.6 86.1/9.6/4.3 7.8 drip 5 3 60 5 2016 loamy sand 6.1 86.1/9.6/4.3 3.6 overhead 6 3 75

Figure 5.1 Disease severity index observed in the untreated control in six field trials conducted in 2015 and 2016. Error bars are the standard error, n=16.

In both the irrigated and non-irrigated fields, the interaction between location, fungicide treatment and DACE was significant in the repeated measures ANOVA (Table A4.1 and Table

A4.6). Locations were analyzed separately, and the fixed effect of fungicide treatment × DACE was significant at all six field sites (Table A4.2, Table A4.3, Table A4.4, Table A4.5, Table A4.7 and Table A4.9). At most assessments beyond 42 DACE, the 15 DSV timing had a lower SLS DSI than the untreated control.

An interaction for DSI between fertilizer and fungicide treatment was only observed at Site

4. The untreated fungicide plots that were treated with liquid foliar fertilizer at 28 DACE had a higher DSI over time than the 15 DSV treatment with the foliar fertilizer (Table 5.4). The untreated fungicide plots that were treated with foliar fertilizer also had a higher DSI than 15 DSV treatment with no fertilizer and the 30 DSV treatment with 180 kg N fertilizer.

Table 5.4. Interaction in disease severity index (DSI) between fertilizer and fungicide timing treatments at one site, irrigated with drip irrigation, 2016. Fertilizer DSI Fungicide Timing (0-100) No Fertilizer Untreated 35 bc1 15 DSV 27 ab 30 DSV 31 abc 120 kg N + 120 kg P2O5 Untreated 30 abc 15 DSV 31 abc 30 DSV 29 abc 180 kg N + 120 kg P2O5 Untreated 30 abc 15 DSV 30 abc 30 DSV 26 ab 120 kg N + 7.2 kg foliar N Untreated 37 c 15 DSV 25 a 30 DSV 30 abc Standard error 1.9 P-value 0.0246 1Means within a column which share a letter in common are not significantly different, Tukey’s HSD (P≤0.05), n=4.

In the irrigated fields, the interaction between location, fertilizer and fungicide treatment was significant for both the sAUDPC calculated 14 days after the last application and the sAUDPC calculated 98 days after cladophyll emergence (Table A4.9, Table A4.10). The locations were then analyzed separately (Table A4.13, Table A4.14, Table A4.19 and Table A4.20). In the non-

irrigated fields, there was no interaction between location, fertilizer and fungicide treatment, but there was an interaction between location and fungicide treatment for both sAUDPC (Table A4.11 and Table A4.12). The locations were then analyzed separately (Table A4.15, Table A4.16, Table

A4.17, Table A4.18, Table A4.21, Table A4.22, Table A4.23 and Table A4.24). The effect of fungicide was significant at five locations, and sAUDPC differed among fungicide treatments

(Table 5.5 & 5.6). The 15 DSV treatment had lower sAUDPC than the untreated control. The 30

DSV treatment also had lower sAUDPC than the untreated control at five of six sites. At one site,

Site 5, the 30 DSV treatment had a lower sAUDPC than the untreated control at 14 DALA (56

DACE), but not by 98 DACE. Fertilizer treatment did not affect sAUDPC.

Table 5.5 Standardized area under the disease progress curve (sAUDPC) of Stemphylium leaf spot calculated at 14 days after the last fungicide application on asparagus treated with chlorothalonil and azoxystrobin + difenoconazole according to the forecasting model TOM- CAST at either 15 or 30 DSV1. Non-irrigated2 Irrigated1 Treatment 2015 2016 2016 Site 1 Site 2 Site 1 Site 3 Site 5 Untreated 5 b2 22 b 15 b 25 b 19 c 15 DSV 2 a 11 a 8 a 17 a 11 a 30 DSV 2 a 14 a 9 a 19 a 15 b Standard error 0.4 1.3 0.7 1.0 0.7 P-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 1An interaction between fertilizer and fungicide was observed at Site 4 in 2016 and the main effect of fungicide was not summarized (P=0.0349). 2Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=16.

Table 5.6 Standardized area under the disease progress curve (sAUDPC) of Stemphylium leaf spot calculated at 98 days after cladophyll emergence on asparagus treated with chlorothalonil and azoxystrobin + difenoconazole according to the forecasting model TOM-CAST at either 15 or 30 DSV1. Non-irrigated Irrigated Treatment 2015 2016 2016 Site 1 Site 2 Site 1 Site 3 Site 5 Untreated 16 b2 35 b 28 b 25 b 31 b 15 DSV 5 a 18 a 18 a 17 a 21 a 30 DSV 7 a 22 a 22 a 19 a 29 b Standard error 0.83 1.30 0.81 1.01 0.95 P-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 1An interaction between fertilizer and fungicide was observed at Site 4 (P=0.0135). 2Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05.

An interaction for sAUDPC at 14 DALA and 98 DACE between fungicide and fertilizer treatment was observed at Site 4. In the no fertilizer, 180 kg fertilizer and foliar fertilizer plots, the untreated control had greater sAUDPC than either the 15 or 30 DSV fungicide treatment at 14

DALA (data not shown). By 98 DACE, few differences were observed, and only the 15 and 30

DSV treatment plots treated with foliar fertilizer had a lower sAUDPC than the untreated control.

5.3.2 Fern yellowing

The first incidence and severity (%) of fern yellowing differed among the field sites and years. In the non-irrigated fields, fern yellowing was first observed earlier after cladophyll emergence at Sites 2 and 3 than at Site 1 in the untreated controls (Fig. 5.3). The final fern yellowing, however, was > 80% at three of the four sites. At the fourth site, Site 1 in 2016, the final fern yellowing was < 40 %. In the irrigated fields, fern yellowing was first observed earlier at the site with overhead irrigation (Site 5) than at the site with drip irrigation (Site 4). The final fern yellowing was between 60 and 80 % at both sites.

Figure 5.3 Fern yellowing (%) observed in the untreated control in six field trials conducted in 2015 and 2016. Error bars are the standard error of the mean, n=16.

Similar to the DSI, in both the irrigated and non-irrigated fields, the interaction between location, fungicide treatment and DACE was significant in the repeated measures ANOVA for yellowing (Table A4.24 and Table A4.25). Locations were analyzed separately, and the fixed effect of fungicide treatment × DACE was significant at all six field sites (Table A4.26, Table

A4.27, Table A4.28, Table A4.29, Table A4.30 and Table A4.31). The fixed effects of fertilizer, fertilizer × fungicide treatment, fertilizer × DACE, fungicide treatment × DACE, and fertilizer × fungicide treatment × DACE were not significant. In the non-irrigated fields, as most assessments beyond 42 DACE, the 15 DSV fungicide treatment had less fern yellowing than the untreated control. In the irrigated fields, consistent fern yellowing was not observed in the untreated controls

until after 70 DACE. The 15 DSV fungicide treatment again had less fern yellowing than the untreated control.

Figure 5.4 Fern yellowing (%) observed in six field trials treated with fungicides according to the TOM-CAST forecasting model (* 15DSV < 30 DSV and untreated, ** 15 and 30 DSV < untreated, *** 15 DSV < 30 DSV < untreated, **** 15 DSV = 30 DSV and 30 DSV = untreated, ***** 15 DSV = untreated and 30 DSV = untreated but 15 DSV ≠ 30 DSV). Error bars are the standard error of the mean, n=16.

5.3.3 Defoliation

The progression of percent defoliation differed among the field sites and years. In the non- irrigated fields, two of the four sites had nearly 100 % defoliation in the untreated control by the final assessment (Fig. 5.5). At the remaining two sites, the final defoliation was < 80 %. In the irrigated fields, similar to fern yellowing, defoliation was observed earlier in the overhead irrigated field than in the drip irrigated field.

Figure 5.5 Cladophyll defoliation (%) observed in the untreated control in six field trials conducted in 2015 and 2016. Error bars are the standard error of the mean, n=16.

In both the irrigated and non-irrigated fields, the interaction between location, fungicide treatment and DACE was significant in the repeated measures ANOVA (Table A4.32 and Table

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A4.33). Locations were analyzed separately, and the fixed effect of fungicide treatment × DACE was significant at five of six field sites (Table A4.34, Table A4.35, Table A4.36, Table A4.37,

Table A4.38 and Table A4.39). The fixed effects of fertilizer, fertilizer × fungicide treatment, fertilizer × DACE, fungicide treatment × DACE, and fertilizer × fungicide treatment × DACE were not significant at most locations. Once defoliation in the untreated control was above 20 %, the 15 DSV fungicide treatment had less defoliation than the untreated control (Fig. 5.6).

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At only one location, Site 1 in 2016, an interaction was observed between DACE and fertilizer. Generally, the treatments that included an application of a fertilizer had less defoliation than the plots with no fertilizer (data not shown).

5.3.4 Correlation analysis

In the non-irrigated fields, DSI was correlated with fern yellowing (r= 0.74, P<0.0001) and defoliation (r= 0.78, P<0.0001). Fern yellowing and defoliation (r= 0.89, P<0.0001) were also positively correlated. NDVI measurements were negatively correlated with DSI (r= -0.65,

P<0.0001), fern yellowing (r= -0.68, P<0.0001) and defoliation (r= -0.67, P<0.0001). No differences were observed in NDVI among the factors or treatments over time at any site. In the irrigated fields, DSI and fern yellowing (r= 0.85, P<0.0001), DSI and defoliation (r= 0.83,

P<0.0001) and fern yellowing and defoliation (r= 0.97, P<0.0001) were positively correlated.

NDVI measurements were negatively correlated with DSI (r= -0.60, P<0.0001), fern yellowing

(r= -0.70, P<0.0001) and defoliation (r= -0.68, P<0.0001). No differences in NDVI were observed among the factors or treatments over time at either site.

The data were pooled between irrigated and non-irrigated plots to determine correlations between non-parametric agronomic treatments and assessments of disease. The total number of fungicide applications, number of azoxystrobin/difenoconazole applications and number of chlorothalonil applications were negatively correlated to DSI, fern yellowing, defoliation and sAUDPC at both 14 days after the last application and 98 DACE (Table 5.7). The sand (%) was correlated with both DSI and sAUDPC 14 DALA and 98 DACE. The amount of nitrogen or potassium fertilizer applied was not correlated to any of the disease assessments.

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Table 5.7 Spearman-rank correlation coefficient between disease response and agronomic treatments or characteristics. Spearman-rank correlation coefficient (ρ) DSI yellowing defoliation sAUDPC 14 days after last application # applications ...... -0.72 * -0.61 * -0.58 * -0.54 * # azoxystrobin/difenoconazole applications ...... -0.70 * -0.60 * -0.55 * -0.57 * # chlorothalonil applications ...... -0.71 * -0.61 * -0.59 * -0.51 * % sand ...... 0.16 ** 0.08 -0.00 0.57 * Nitrogen (kg/ha) ...... -0.03 -0.08 -0.10 -0.02 Potassium (kg/ha) ...... -0.05 -0.02 -0.00 -0.03 98 days after cladophyll emergence # applications ...... -0.43 * -0.21 ** -0.45 * -0.55 * # azoxystrobin/difenoconazole applications ...... -0.47 * -0.28 * -0.46 * -0.58 * # chlorothalonil applications ...... -0.39 * -0.16 ** -0.42 * -0.50 * % sand ...... 0.61 * 0.11 0.38 0.65 * Nitrogen (kg/ha) ...... -0.02 -0.08 -0.10 -0.03 Potassium (kg/ha) ...... -0.04 -0.05 -0.10 -0.04 *Significant at P<0.0001 **Significant at P<0.05

Phosphorus concentration in the leaf tissues was negatively correlated with defoliation and positive correlated with NDVI (Table 5.8). Calcium concentration was positive correlated with NDVI. Nitrogen and potassium concentration was not correlated with any of the disease assessments and was not different between no fertilizer and 180 kg N treatments.

Table 5.8 Pearson correlation coefficients between disease assessments and nutrient concentration measured at 42 days after cladophyll emergence from plots treated with 0 or 180 g N per ha. Pearson correlation coefficient Nutrient DSI yellowing defoliation NDVI N -0.14 0.02 -0.06 0.36 P -0.12 -0.20 -0.35 * 0.64 * K 0.11 0.22 -0.05 0.22 Ca 0.13 0.25 0.05 0.40 * Mg 0.18 0.32 0.33 0.18 *Significant at P<0.05

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5.4 Discussion

The economic effect of foliar disease on asparagus has been well established (Elmer et al.,

1996; Meyer et al., 2000), yet growers in Ontario are not able to adequately control Stemphylium leaf spot with the available chemical management tools. Since asparagus is a perennial vegetable, improved management of Stemphylium leaf spot would reduce premature defoliation and prevent early decline (Elmer et al., 1996). Cultural methods, such as irrigation and nutrient management, have been studied at length for control of root and crown rot in asparagus. This is the first study where both irrigation and fertility treatments had been assessed to determine the effects on foliar disease and plant health indicators such as defoliation and fern yellowing.

Fungicide programs applied according to TOM-CAST at 15 DSV had fewer symptoms of

Stemphylium leaf spot than the untreated control. Greater control of Stemphylium leaf spot was observed in 2015 than in 2016. In CHAPTER FOUR, TOM-CAST provided inconsistent control of Stemphylium leaf spot when tested in ‘Guelph Millennium’ and ‘Jersey Giant’. In the current study, the first fungicide application was made at cladophyll emergence. In the previous work, fungicide applications were initiated once TOM-CAST had accumulated the prescribed DSV following cladophyll emergence, as recommended in Michigan. The delay in fungicide application often resulted in curative applications and poor control of Stemphylium leaf spot. Despite the change in application timing, in 2016, the fungicide programs were still applied curatively. Most fungicides, including those with curative activity, have greater efficacy when applied preventively rather than curatively (Anesiadis et al., 2003; Keinath, 2000). In asparagus, with the limited curative effect of the fungicides available, it is difficult to develop a management system reliant on crop phenology. Presumably, the crop is accumulating DSV immediately following harvest completion, but DSV were not calculated until cladophyll emergence. Additional research is

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required to determine the optimal timing to initiate the TOM-CAST forecasting model in asparagus.

The disease severity and progress in the untreated plots differed among the field sites.

Specifically, at Site 1, severe defoliation was not observed in either the treated or untreated plots until the crop approached dormancy. A similar result was observed at Site 1 in 2012 and 2013 in

CHAPTER FOUR. Despite the accumulation of high DSV, Site 1 had less Stemphylium leaf spot, fern yellowing and premature defoliation than the other field sites in all 4 years. In 2012 and 2013, the grower at Site 1 used a different fertility program, with higher inputs, than the growers at the other field sites. In 2015 and 2016, the fertility programs were the same at all sites, yet fertility had only a limited effect on Stemphylium leaf spot. Rarely was an effect observed from the various fertilizer treatments, and seldom was an interaction observed among the fertilizer and fungicide factors and irrigation. Also, DSI, fern yellowing, defoliation and sAUDPC were not correlated with the quantity of nitrogen or potassium applied. Generally, the effect of nutrients on plant disease is understood, however, there are contradictory results among different plant pathogen systems (Walters & Bingham, 2007). Since asparagus is a perennial crop, the effect of polyetic epidemics in combination with abiotic factors that affect plant growth and physiology is not well understood or studied. Further research should investigate the interaction of seasonal environmental differences and abiotic stress on disease epidemics in asparagus.

In the irrigated fields, low fern yellowing and defoliation was observed up to fern dormancy regardless of the foliar fungicide program. Specifically, in the drip irrigation site, no differences in fern yellowing and defoliation were observed between the fungicide treatments and the untreated control. Irrigation can either increase or decrease the susceptibility of the host to plant pathogen infection (Palti & Shoham, 1983; Rotem & Palti, 1969). Specifically, the incidence of

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foliar disease was reduced by additional irrigation in sesame, sorghum, walnuts, tobacco, coffee and rice (Brooks et al., 2010; Culp & Thomas, 1964; Edmunds et al., 1964; Foot et al., 1955; de

Paiva Custodio et al., 2014; Rotem et al., 1968). Plants can have increased susceptibility to pathogen attack in drought conditions (Rotem & Palti, 1969) and low soil moisture in asparagus can lead to premature defoliation and fern yellowing prior to dormancy. The pooled sand (%) measured at each site was positively correlated with both DSI and sAUDPC, regardless of irrigation. Interestingly, even though irrigation appears to delay defoliation and fern yellowing, the fields with irrigation did not have less Stemphylium leaf spot or lower sAUDPC than the fields without irrigation. Depending on the cultivar, Panka & Rolbiecki (2008) found that drip irrigation either increased or decreased Stemphylium leaf spot in asparagus, but generally, Stemphylium leaf spot was reduced by irrigation. Increased canopy density prolongs leaf wetness, which creates conditions ideal for disease development (Walters & Bingham, 2007). Often foliar or overhead irrigation results in enhanced disease severity, especially for those diseases dependent on leaf wetness, such as Stemphylium spp. (Rotem & Palti, 1969). Unexpectedly, no differences in

Stemphylium leaf spot were observed between the site irrigated with drip irrigation and the site irrigated with overhead irrigation. Overhead irrigation is easy to implement in a perennial crop as drip irrigation needs to be laid at planting and maintained for the duration of the crop. However, before overhead irrigation is widely adopted, additional research is required to confirm the results since each irrigation method was tested at just a single field site.

Since little remains of the processing market in Ontario, growers need to produce asparagus spears with few blemishes in order to sell to the fresh market. The current control of Stemphylium leaf spot is not adequate with the typical calendar-based contact fungicide program. The present study demonstrated that a fungicide program which included contact and systemic fungicides

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applied according to TOM-CAST at 15 DSV provided good control of Stemphylium leaf spot. The greatest control was achieved when fungicide applications were initiated prior to disease symptoms. These results indicate the model could be further refined to include a threshold to initiate applications. The various fertilizer programs did not affect the development of

Stemphylium leaf spot. Sites which were irrigated had a delay in defoliation and fern yellowing, although irrigation had no effect on Stemphylium leaf spot incidence or severity. Further research is warranted to investigate the effect of both drip and overheard irrigation on Stemphylium leaf spot and premature defoliation.

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CHAPTER SIX

DEVELOPMENT AND MANAGEMENT OF PLEOSPORA HERBARUM IN DORMANT

ASPARAGUS (ASPARAGUS OFFICINALIS L.)

6.1 Introduction

Stemphylium vesicarium (Wallr.) E.G. Simmons (teleomorph Pleospora herbarum), is the primary foliar pathogen that reduces yield and marketability of asparagus in Ontario. Although not formally reported, purple spot and Stemphylium leaf spot caused by S. vesicarium have been recognized as diseases of asparagus in Canada since production switched to no-tillage practices in the 1990s (K. Wall, personal communication). Growers adopted the practice of no-tillage to reduce wind-erosion and to reduce mechanical damage to the crowns (Lacy, 1982; Putnam & Lacy,

1977). However, the pseudothecia of P. herbarum overwinter and mature on the standing dormant fern (Hausbeck et al., 2008; Kelly & Bai, 1999).

In the spring, during harvest, ascospores of P. herbarum are ejected from mature pseudothecia following a rain or wetting event (Atanasoff, 1919; Hausbeck et al., 1999). Infection on the spears occurs through open stomata or through artificial wounds (Falloon et al., 1987;

Johnson & Lunden, 1986), resulting in distinct purple spot lesions. When damage is severe, the spears are graded into a low market class, which reduces the value of the harvested crop.

Unfortunately, the application of fungicides during this period would not be effective due to the rapid growth of asparagus spears.

Control of purple spot on asparagus spears relies on management of foliar infection in the fern of the previous season (Hausbeck et al., 2008). However, even if Stemphylium leaf spot is

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well managed in season, purple spot is still prevalent the following spring in Ontario (Asparagus

Farmers of Ontario, personal communication).

The maturation of pseudothecia on pear and garlic is influenced by both RH and temperature (Llorente & Montesinos, 2004; Prados-Ligero et al., 1998). In P. allii, specifically, at a high RH of 98%, the optimal temperature for the development of pseudothecia was found to be

10 to 15°C (Llorente & Montesinos). In garlic, the optimal temperature for P. allii pseudothecia development was 5 to 10°C at a saturated RH (Prados-Ligero et al., 1998). However, the maturation of pseudothecia on asparagus fern in Ontario would be delayed by winter.

Necrotrophic fungi survive and thrive on senescent or dormant plant tissue (Everts & Lacy,

1990; Rowell, 1953; Russell, 1966). In Ontario, as the fern approaches dormancy in the fall, the severity of Stemphylium leaf spot appears to increase. Ideally the fern could be removed prior to dormancy, however, this would reduce the health of the crown (Kelly & Bai, 1999). Dormancy in asparagus has been well characterized in the popular cultivar ‘Guelph Millennium’ (Kim & Wolyn,

2014; Kim & Wolyn, 2015; Landry & Wolyn, 2011; Landry & Wolyn, 2012; Panjtandoust &

Wolyn, 2016a; Panjtandoust & Wolyn, 2016b). Simple sugars and proline have cryoprotective properties that are associated with dormancy in asparagus (Landry & Wolyn, 2011). In other plant systems, proline stabilizes proteins during cold acclimation (Heber et al., 1971; Steponkus, 1984) and sucrose, specifically, acts as a water substitute during freezing (Caffrey et al., 1988). Fern yellowing, measured by chlorophyll concentration in fern, is also a sign of dormancy (Landry &

Wolyn, 2011). If dormancy affects the development of P. herbarum, compounds associated with dormancy should relate to the incidence and severity of pseudothecia on asparagus fern. The relationship between fern senescence and the occurrence of P. herbarum on asparagus fern, however, is not well understood.

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In a preliminary study, attached spears developed purple spot symptoms only when inoculated with pseudothecia collected in the spring rather than the fall (Trueman & Roddy, 2012).

Management of the primary inoculum prior to winter should reduce disease on asparagus spears the following spring. Sanitation is widely used to reduce primary inoculum in perennial fruit crops

(Burchill, 1968; Llorente et al., 2006; Llorente et al., 2010; Sutton et al., 2000). In a previous study, the burial of asparagus residue reduced purple spot infection on the spears the following season (Johnson, 1990). As well, Falloon et al. (1984) noted decreased disease on spears from fields where the residue was removed from the field. In other perennial cropping systems, such as tree fruit, the shredding of leaf litter increased the speed of decomposition of the host substrate and reduced the incidence of fruit infection (Llorente et al., 2006; Sutton et al., 2000). In pears specifically, leaf shredding provided similar control of Pleospora alli as compared to a standard fungicide program (Llorente et al., 2010). Other sanitation methods, such as the application of urea, increased the decomposition of leaf litter in both pears and apples (Burchill, 1968; Llorente et al., 2010). Also, the application of urea increased the breakdown of fern in non-temperate asparagus (Fantino et al., 1990), however, it is not known if the same effect would occur in temperate climates. Shredding dormant fern in the fall, rather than the spring, and applying foliar amendments may help reduce the primary inoculum in asparagus.

Little is known about the epidemiology of P. herbarum on senescing asparagus fern in temperate climates, despite being the primary source of inoculum for the spring crop. The objectives of this research were to study the effect of dormancy on Stemphylium leaf spot development, determine if dormancy parameters are appropriate tools to predict the production of pseudothecia, and to evaluate sanitation methods to reduce overwintering inoculum and control or reduce purple spot on asparagus spears.

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6.2 Materials and Methods

6.2.1 Pleospora herbarum maturity in fern

As described in CHAPTER TWO, a field trial was established in 2011 at Syngenta Canada

Inc. Honeywood Research Farm in Oxford County, ON on silt loam soil. Briefly, in total, six cultivars or lines were transplanted and maintained according to standard practice. Each cultivar was replicated five times in 6-m-long single row experimental units, and the experimental units were arranged in a randomized complete block design. For the purpose of the described experiment, only ‘Guelph Millennium’ and ‘Jersey Giant’ were assessed, and these cultivars were selected for their differing dormancy patterns (Landry & Wolyn, 2011; Landry & Wolyn, 2012;

Panjtandoust & Wolyn, 2016a; Panjtandoust & Wolyn, 2016b). Symptoms of dormancy, such as a reduction in chlorophyll in the fern, occurs earlier in ‘Guelph Millennium’ than in ‘Jersey Giant’ following a temperature change (Landry & Wolyn, 2011). The trial area was inoculated in the spring of 2012, 2013 and 2014 by spreading infected fern, collected from Ontario asparagus fields, evenly across plots. No fungicides were applied for the duration of the experiment. Every spring, the infected fern was chopped according to commercial practice, and the residue was left in the field to encourage infection on the new fern.

Hourly measurements of air temperature (ZW-005 Cable Temp/RH, Onset Computer

Corporation, Bourne, MA), relative humidity (ZW-005 Cable Temp/RH), and rainfall (HOBO

Rain Gauge Data Logger - RG3 (Onset Computer Corporation) were recorded using a HOBO USB

Micro Station Data Logger-H21-USB (Onset Computer Corporation).

The fern were assessed weekly for disease symptoms in the fall of 2015 and 2016.

Stemphylium leaf spot was assessed on ten randomly selected branches per experimental unit using a 0 to 5 index scale modified from Falloon et al., 1987 (0 = no lesions, 1 = 1 to 20 lesions, 2 = 21

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to 50 lesions, 3 = 51 to 90 lesions, 4 = 90 to 200 lesions, and 5 > 200 lesions). The disease severity index was calculated from the Stemphylium leaf spot assessments using the following formula:

DSI = [∑[(class no.)(no. of branches in each class)]]/[(total no. of branches per sample)(no. classes

- 1)] x 100 (Kobriger & Hagedorn, 1983). The presence of pseudothecia on both the branches and the lower 30 cm of stems was assessed on ten randomly selected ferns per experimental unit using the same index scale described above, however, the number of pseudothecia were estimated rather than the number of lesions. The DSI formula was used to convert these values to a 1 to 100 index scale (Kobriger & Hagedorn, 1983). The area under the disease progress curve (AUDPC) was calculated (Shaner & Finney, 1977) and standardized (sAUDPC) (Simko & Piepho, 2012) to describe the cumulative disease severity throughout the duration of the experiment. The area of fern that had yellowed (% area) and the defoliation (% cladophyll drop) per experimental unit were visually estimated. Finally, ten branches per plot were collected weekly, chopped and homogenized into a single mixture per experimental unit. From the mixture, a sample of fern was packed into 15 ml conical tubes, flash frozen, lyophilized and stored at room temperature until further use. Chlorophyll concentration (mg/g dry weight) was measured from the fern samples as described by Landry & Wolyn (2011), with the change to dry fern instead of fresh fern tissue.

Briefly, tissue was extracted with 99% methanol at 65ºC for 10 min, then cooled and stored at 4°C overnight. The supernatant was collected after centrifugation, and measured with a spectrophotometer (Ultraspec 2100 pro ultraviolet/Visible; Biochrom, Cambridge, UK) at both

652 and 665 nm. If the measured absorbance was outside of the acceptable range (Lichtenthaler,

2001), the solution was diluted to reduce the absorbance or the volume of methanol was decreased to increase the absorbance. The total chlorophyll concentration was calculated by using the

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following formula described by Lichtenthaler, 1987: mg chlorophyll/ml of extract = (1.44)(A665nm)

+ (24.93)(A652nm).

Pseudothecia were collected upon conclusion of the field trials for an assessment of maturity. Twenty stems were randomly collected from each plot of ‘Guelph Millennium’ and

‘Jersey Giant’. In the controlled environment facility at Syngenta Canada, Inc., 12-week-old seedlings of ‘Guelph Millennium’ were inoculated with a conidial suspension (5.0 x 105 conidia ml-1), 50 psuedothecia from either ‘Guelph Millennium’ or ‘Jersey Giant’ field plots, or not inoculated. The conidial suspension was prepared as described in CHAPTER TWO. Briefly, 10 drops of sterile water were placed around the outer edge of 7 day-old cultures, and a sterile inoculation rod was used to gently mat down the mycelia. Plates were then placed under UV light at 20 ± 2°C for 12 hours, followed by darkness for another 12 hours to induce the production of conidia. Conidial inoculum was prepared by flooding actively sporulating cultures with sterile distilled water and dislodging the conidia with a sterile inoculation rod or microscope slide. A drop of the surfactant Tween 20 (J.T Baker Inc., Philipsburg, NJ, USA) was added to every 10 ml of conidial suspension for improved spore wetting. The concentration of the conidial suspension was estimated using a Neubauer hemacytometer and adjusted accordingly. The suspension was composed of two isolates collected from asparagus fern in Ontario (OA46 and OA48). The pseudothecia were still attached to small pieces of the fern residue which helped maintain moisture.

Each experimental unit was one seedling wrapped in a cellophane tube to prevent cross- contamination. Experimental units arranged in a randomized complete block design were replicated five times and the experiment was repeated in 2016. During the first week after inoculation, the plants were misted daily and bagged to promote conditions ideal for disease development. In addition, the seedlings were watered daily to promote the release of mature

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ascospores. The chamber was held at 15ºC with 16 h days and 8 h night for the duration of the experiment. The number of lesions on the seedlings was counted weekly, and the Stemphylium leaf spot incidence was calculated. Data were collected up to 70 days after inoculation, and the

AUDPC and sAUDPC were calculated from lesion counts.

6.2.2 Pleospora herbarum development in seedlings - outdoor experiment

Seeds of ‘Guelph Millennium’ and ‘Jersey Giant’ were planted in 128-cell plug trays and the plugs were transplanted 6 weeks later into 50-cell trays filled with Sunshine mix # 5 (Sun Gro

Horticulture, MA, United States). After 6 weeks, plugs were transplanted into 4-inch pots. The seedlings were grown in a greenhouse equipped with supplemental lighting (1000W HPS lighting,

190 to 230 μmol m−2s−1) with 16 h days (25°C) and 8 h nights (20°C) from Sept 3 up until the different experiments commenced.

The first experiment was conducted outdoors in the fall of 2016. The experiment was conducted as a split-plot design with cultivars, Jersey Giant and Guelph Millennium, as whole- plots and the repeated measure of sampling dates as subplots. The whole-plots were replicated five times and subplots were replicated three times. The treatments were arranged in a randomized complete block design, and the study was replicated at two locations at the research station at

Syngenta Canada, Inc. The seedlings were moved outdoors from the greenhouse on Oct 4 and placed onto wooden pallets. The seedlings inoculated were Oct 11 with a conidial suspension as described above including the concentration and selected isolates. Following inoculation, the seedlings were bagged for 2 days to maintain relative humidity and leaf moisture.

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Seedlings were assessed for symptoms of disease weekly. Stemphylium leaf spot was assessed by counting the number of lesions on each seedling. The number of pseudothecia were also counted on each seedling. The AUDPC was calculated as described.

Subplots were destructively sampled 10 days after inoculation and then every 21 days until

74 days for a total of four sampling periods. Metabolites associated with dormancy: chlorophyll, proline and sucrose, were measured from the fern and crown at each sampling date. The fern were separated from the crowns, bulked per subplot, packed into 50 ml conical tubes, flash frozen, lyophilized and stored at room temperature until further use. Both the rhizomes and storage roots were bulked per subplot and placed into 50-ml polypropylene conical tubes as described by Landry

& Wolyn, 2012. Crown tissue was flash frozen, lyophilized, ground to pass a 60-mesh screen, then stored at -80°C until further use.

Fern chlorophyll concentration was determined as described above. Sucrose was extracted from 0.1 g of dry crown tissue using a commercially available kit, K-SUCGL (Megazyme

International, Bray, Ireland). Briefly, the homogenized mixture was extracted with water at 60°C.

D-glucose was measured by adding 0.2 ml of extract to 0.2 ml of acetate buffer. D-Glucose and sucrose was measured by adding 0.2 ml of extract to 0.2 ml of beta-fructosidase. The original extract was subsampled and incubated at 50°C for 20 min, and 3.0 ml of GOPOD reagent was added to the tubes. The solutions were incubated for another 20 min at 50°C. The absorbance was measured with a spectrophotometer (Ultraspec 2100 pro ultraviolet/Visible spectrophotometer) at

510 nm. The Mega-Calc software tool (Megazyme International) was used to calculate the amount of D-glucose + sucrose and D-glucose per g dry weight of crown. The amount of sucrose per g dry weight of crown was calculated by subtracting the two values. Proline concentration was determined as described by Patton et al., 2007. Briefly, 0.3 g of dry crown tissue was mixed with

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1 ml of ethanol solution (90%). Samples were heated to 70°C for 20 min, cooled on a shaker for

15 min and centrifuged for 10. The resulting pellet was rinsed twice with ethanol. Centrifugation and rinsing was repeated two additional times. A 100 µl aliquote of the supernatant was added to a borosilicate glass tube with 1 ml of acid ninhydrin, 1 ml of glacial acetic acid. The tubes were vortexed briefly and heated to 90°C for 1 h, and then immediately placed into an ice bath to stop the reaction. Toluene was added to the tubes and vortexed again briefly. The non-aqueous phase was pipetted into a quartz cuvette, and absorbance was measured at 520 nm (Ultraspec 2100 pro ultraviolet/Visible spectrophotometer). A standard curve was created by spiking a control solution with a known quantity of proline (0, 5, 10, 25, 50 and 100 mg/g). The standard curve was then used to calculate the mg/g of proline per g dry weight of crown tissue. The ninhydrin assay extracts all amino acids, not just proline, and the values obtained through spectrophotometry may not be exclusive to proline (Kalsoom et al., 2016).

6.2.2 Pleospora herbarum development in seedlings - controlled environment

Seeds of ‘Guelph Millennium’ were planted in 128-cell plug trays and the plugs were transplanted 6 weeks later into 50-cell trays filled with Sunshine mix # 5 (Sun Gro Horticulture,

MA, United States). After 6 weeks, plugs were transplanted into 4-inch pots. The seedlings were grown in a greenhouse equipped with supplemental lighting (1000W HPS lighting, 190 to 230

μmol m−2s−1) with 16 h days (25°C) and 8 h nights (20°C) from Oct 28 up until the experiment commenced Nov 25.

The seedlings were moved from the greenhouse to the controlled environment chambers on Nov 25. Once moved, the seedlings were inoculated with a conidial suspension as described.

The suspension was composed of two isolates collected from asparagus fern in Ontario (OA46 and

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OA48). Following inoculation, the seedlings were bagged for 2 days to maintain relative humidity.

Temperature outside the bag in the chamber was maintained at 20ºC.

The experiment was conducted as a split-block design. The whole block factor was temperature treatment with two levels, 7ºC and 15ºC in separate chambers with supplemental lighting (Sylvania PENTRON 4100K 39W, 190 to 230 μmol m−2s−1) with 16 h days and 8 h nights modified from the methods described by Landry & Wolyn, 2012. The temperature in the chamber was reduced 7 days after inoculation. The sub-plot factor were sampling date with four levels: 0,

21, 42 and 63 days after temperature induction. Non-inoculated control plants were included at the

0 and 63 day level. There were three plants per sampling date in each experimental unit, and experimental units were replicated four times in each temperature. Plots were arranged in a randomized complete block design. Hourly measurements of air temperature and relative humidity were recorded using a HOBO External Temp/RH Data Logger (Onset Computer Corporation,

Bourne, MA). The experiment was repeated simultaneously in different chambers.

Seedlings were assessed for symptoms of disease every 21 days in the controlled environment experiment. Stemphylium leaf spot was assessed by counting the number of lesions on each seedling. The number of pseudothecia were also counted on each seedling. Metabolites associated with dormancy, chlorophyll, proline and sucrose, were measured from the fern and crown also at the designated days after temperature induction. The metabolites were measured according to the methods described above.

6.2.3 Sanitation field trials

Field trials were initiated in the fall of 2012 and 2013 in two commercial asparagus fields.

The first site (42.612645 -80.659944) was located in Norfolk County, ON, with loamy sand soil

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(pH 4.9, 2.2 % OM, 10.4 CEC), and the field was established in 2009 with one-year-old crowns of ‘Guelph Millennium’ spaced 23 cm apart within the row and 1.2 m spacing between rows. The first full year of harvest was in 2013. In both seasons, a cover crop of rye was established at the end of August. The second site (42.693437 -80.898697) was located in Elgin County, ON, with loamy sand soil (pH 5.8, 2.5 % OM, 5.8 CEC), and the field was established in 2004 from seeds of ‘Guelph Millennium’ with a final stand of crowns spaced 23 cm apart with the row and 1.2 m spacing between rows. The first full year of harvest was in 2009.

The field experiments were arranged in a split-plot design, and whole-plot treatments were mowing time and sub-plot treatments were nitrogen and fungicide treatments. Each subplot had two levels, for a total of eight treatments. Mowing time levels were fall and spring. The fern was mowed with a standard deck mower according to commercial practice. The whole-plot contained seven 70-m-long rows and were replicated three times arranged in a randomized complete block design. There were two subplot treatments: fungicide treatment and fertilizer treatment. The fungicide factor had two levels: copper hydroxide (Kocide 2000 53.8DF at 5.98 g ai/ha, E.I. du

Pont Canada Company Agricultural Products, Mississauga, ON) and untreated. The fertilizer factor had two levels: UAN 28% (39.2 % ai v/v) and no fertilizer. Both the fertilizer and fungicide treatments were applied with a pressurized CO2-powered backpack sprayer equipped with TeeJet

XR11002 nozzles operated at 240 kPa and calibrated to deliver 200 L/ha. Fungicide and fertilizer treatments were applied a day after the fall mowing to either mowed or standing fern. Each experimental unit consisted of seven 15-m-long rows. Treatments were replicated three times and arranged in a randomized complete block design within the whole plot.

In the spring, ten spears were hand-harvested from the middle 10 m of the center three rows in each experimental unit. Spears were harvested six times and evaluated for purple spot according

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to the following scale of 0 to 4, where 0 = no lesions, 1 = 1 to 20 lesions, 2 = 21 to 50 lesions, 3 =

21 to 50 lesions, and 4 > 90 lesions (Falloon et al., 1987). The spears were assessed twice: at- harvest and 3 days following harvest to determine the impact of latent infection. Data from all six harvests were combined, and the disease incidence and DSI was calculated.

6.2.4 Data analysis

Statistical analyses were conducted using SAS v.9.4 (SAS Institute Inc., Cary, NC). Data were analyzed with an analysis of variance (ANOVA) in PROC MIXED. The restricted maximum likelihood (REML) covariance parameter estimates were used to analyze disease and metabolite parameters among factors. Parameters were subjected to ANOVA. Depending on the experiment, variance was partitioned into random effects (block, block × temperature) and fixed effects

(cultivar, inoculation, sampling date, cultivar × sampling date, temperature, temperature × sampling date). In the experiments with both ‘Jersey Giant’ and ‘Guelph Millennium’, the data were pooled between the cultivars if no effect × cultivar interaction was observed. The non- inoculated control was excluded from the ANOVA. The Shapiro-Wilk test was used to test the normality of residuals and distribution of error was determined by residual plots. The data were checked for outliers using Lund’s test. Means were separated using a Tukey’s HSD (P≤0.05).

Pearson’s correlations were estimated using the PROC CORR procedure in SAS.

6.3 Results

6.3.1 Pleospora herbarum maturity in fern

The random effects of year and the interaction of year by cultivar or sampling date were significant for each parameter, and data were not pooled over years. An interaction between

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cultivar and sampling date was not observed for most parameters, and data were often pooled between the cultivars. The statistical significance of fixed effects, cultivar and sampling date, and their interaction, are summarized for all parameters (Table 6.1).

Table 6.1 Summary of significant fixed effects P ≤ 0.05 as determined from the restricted maximum likelihood (REML) covariance parameter estimates for asparagus cultivars Jersey Giant and Guelph Millennium sampled or assessed weekly from mid-October to mid-December in 2015 and 2016, n=5. 2015 2016 Cultivar x Cultivar x Parameter Sampling Sampling Cultivar sampling Cultivar sampling date date date date Lesion incidence (branches) 0.1372 <0.0001 0.2216 0.2089 <0.0001 0.4205 Lesion severity (branches) 0.2722 <0.0001 0.5650 0.0344 <0.0001 0.7977 Pseudothecia incidence (stems) 0.0860 <0.0001 0.4050 0.5423 <0.0001 0.3695 Pseudothecia severity (stems) 0.0212 <0.0001 0.5668 0.2767 <0.0001 0.3872 Pseudothecia incidence (branches) 0.2085 <0.0001 0.5994 0.5002 <0.0001 0.4476 Pseudothecia severity (branches) 0.1550 <0.0001 0.3519 0.8444 <0.0001 0.8646 Fern yellowing <0.0001 <0.0001 <0.0001 0.0198 <0.0001 0.7271 Fern defoliation 0.1392 <0.0001 0.1731 0.0237 <0.0001 0.0692 Chlorophyll 0.6292 <0.0001 0.6603 0.0752 <0.0001 0.7931

In both 2015 and 2016, the Syngenta Canada Inc. research station consistently experienced daily minimum air temperatures below 10ºC from October to December (Fig. 6.1A and Fig. 6.2A).

The average daily temperature during the sampling period was 6ºC in both years as well. The relative humidity was between 60 and 99 % (Fig. 6.1B and Fig. 6.2B). More rainfall occurred from

October to December in 2015 (154 mm) than in 2016 (248 mm) (Fig. 6.1C and 6.2C).

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Figure 6.1 Air temperature (A), relative humidity (B), and rainfall (C) recorded at the Syngenta Canada Inc. research station in Oxford County, ON, in 2015.

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Figure 6.2 Air temperature (A), relative humidity (B), and rainfall (C) recorded at the Syngenta Canada Inc. research station in Oxford County, ON, in 2016.

Stemphylium leaf spot lesions were present throughout the duration of the sampling period, and pseudothecia first appeared on the stems by mid-November in both 2015 and 2016 (Fig. 6.3A and 6.4A). Pseudothecia appeared on the branches 3 weeks following the observation on the stems

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in 2015, and after 1 week in 2016. In 2016, however, the pseudothecia appeared on the branches

1 week following the observation on the stems.

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Defoliation was evident by early November in 2015, and mid-October in 2016 (Fig. 6.3C and 6.4C). In both years, the cladophylls were completely defoliated by the end of November. In

2015, the final pseudothecia severity on either the stems or branches was below 20 DSI, but in

2016, the severity was above 40 DSI. Throughout the sampling period, fern yellowing was consistently lower in ‘Jersey Giant’ than ‘Guelph Millennium’ (Fig. 6.3C and 6.3C), however the chlorophyll concentration in the fern did not differ between the cultivars (Fig. 6.3D and 6.4D).

The incidence and severity of both Stemphylium leaf spot and pseudothecia were positively correlated with fern yellowing and cladophyll defoliation, and negatively correlated with chlorophyll in both years (Table 6.2). Stemphylium leaf spot incidence was highly correlated with chlorophyll concentration in 2015, but weakly correlated in 2016. In both years, SLS incidence was positively correlated with fern yellowing and defoliation. In 2016, pseudothecia incidence and severity were negatively correlated with chlorophyll concentration, whereas in 2015, only the pseudothecia incidence in the stems was correlated.

Table 6.2 Correlations (multivariate) between disease and dormancy parameters collected from field trials in 2015 and 2016, n=5. Fern Chlorophyll Disease parameter Defoliation yellowing concentration 2015 SLS incidence 0.69 ** 0.61 ** -0.82 ** Pseudothecia incidence (stems) 0.60 ** 0.67 ** -0.34 * Pseudothecia severity (stems) 0.56 ** 0.62 ** -0.30 Pseudothecia incidence (branches) 0.40 ** 0.43 ** -0.08 Pseudothecia severity (branches) 0.38 * 0.41 ** -0.09 2016 SLS incidence 0.35 * 0.30 * -0.23 * SLS severity 0.62 ** 0.55 ** -0.44 ** Pseudothecia incidence (stems) 0.69 ** 0.72 ** -0.61 ** Pseudothecia severity (stems) 0.65 ** 0.68 ** -0.58 ** Pseudothecia incidence (branches) 0.54 ** 0.55 ** -0.44 ** Pseudothecia severity (branches) 0.47 ** 0.47 ** -0.38 * *P ≤ 0.05 **P ≤ 0.0001

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The fixed effect of inoculum was significant for all parameters (Table A5.1, Table A5.2 and Table A5.3). In the pseudothecia maturity experiment, lesions were observed on the seedlings by 42 days after inoculation in both the seedlings inoculated with a conidial suspension and with the pseudothecia collected from ‘Guelph Millennium’ in both years (Table 6.3). In ‘Jersey Giant’, the first lesions were observed earlier in 2016 (42 DAI) than in 2015 (63 DAI). The seedlings inoculated with pseudothecia, regardless of cultivar source, had few lesions, lower lesion incidence and a lower sAUDPC than the plants inoculated with a conidial suspension.

Table 6.3 Final lesion count, lesion incidence and sAUDPC recorded on asparagus seedlings 70 days after inoculation (DAI) with either a conidial suspension or pseudothecia collected from two cultivars at the end of two seasons, 2015 and 2016. DAI symptoms Lesion count Lesion Inoculum source observed (lesions/ incidence sAUDPC 2015 2016 seedling) (%) non-inoculated - - 0.0 - 0 - 0.0 - conidial suspension 42 42 6.3 a1 100 a 2.1 a pseudothecia ‘Guelph Millennium’ 42 42 1.4 b 40 b 0.6 b pseudothecia ‘Jersey Giant’ 63 42 0.3 b 30 b 0.1 b Standard error - - 0.68 0.1 0.25 P-value - - <0.0001 0.0019 <0.0001 1Means in a column followed by the same letter do not differ using Tukey’s HSD at P ≤ 0.05, n=8.

6.3.2 Pleospora herbarum development in seedlings - outdoor experiment

In the outdoor experiment, the random effects of repetition and the interaction of repetition by cultivar or sampling date were not significant for each parameter (Table 6.4), and data were combined between repetitions. There was no interaction between cultivar and sampling date for any parameter, and data were pooled between the cultivars.

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Table 6.4 Summary of significant fixed effects P ≤ 0.05 as determined from the restricted maximum likelihood (REML) covariance parameter estimate. Data from both repetitions and bothasparagus cultivars Jersey Giant and Guelph Millennium were pooled. Variables were sampled or assessed weekly in asparagus seedlings from mid-October to mid-December in 2016, n=20. Parameter Cultivar Sampling date Cultivar x sampling date Lesion count 0.2409 <0.0001 0.7426 Pseudothecia count 0.0340 <0.0001 0.0876 Chlorophyll <0.0001 <0.0001 0.1038 Proline 0.0013 <0.0001 0.0848 Sucrose 0.3799 <0.0001 0.5194

In 2016, the minimum air temperature was consistently below 10ºC from mid-October, when the seedlings were inoculated, through the duration of the experiment (Fig. 6.5A). The relative humidity was between 70 and 99 % (Fig. 6.5B). Cumulative rainfall from October to

December was 102 mm for the duration of the experiment (Fig. 6.5C).

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Assessment date was significant for all parameters (Table A5.4, Table A5.5, Table A5.6,

Table A5.9 and Table A5.10). An interaction between date and repetition was observed in lesion counts (Table A5.7 and Table A5.8). The chlorophyll in the fern declined over time (Fig. 6.6A),

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whereas the number of lesions and pseudothecia increased on the seedlings with time (Fig. 6.6B).

Stemphylium leaf spot lesions were observed by 32 days after inoculation, and pseudothecia were evident by 53 days after inoculation (Fig. 6.6B). Proline and sucrose increased over time in the rhizome and storage root (Fig. 6.6C and 6.6D). The chlorophyll concentration in the fern was not measurable by the final sampling date, as all of the fern was necrotic.

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Both lesion and pseudothecia counts were positively correlated with proline and sucrose concentration in the storage roots and rhizomes, but negatively correlated with chlorophyll concentration (Table 6.5).

Table 6.5 Correlations between disease and dormancy parameters collected in seedling fern (chlorophyll) and crowns (proline and sucrose) of ‘Jersey Giant’ and ‘Guelph Millennium’ asparagus grown in pots outdoors from mid-October to mid-December in 2016, n=20. Data was pooled between repetitions and varieties. Chlorophyll Proline Sucrose Disease parameter concentration concentration concentration r P-value r P-value r P-value Stemphylium leaf spot counts -0.64 <0.0001 0.59 <0.0001 0.76 <0.0001 Pseudothecia counts -0.51 0.0012 0.39 0.0004 0.45 <0.0001

6.3.3 Pleospora herbarum development in seedlings - controlled environment trials

In the controlled environment experiment, the random effects of repetition and the interaction of repetition by temperature or sampling date were not significant for any parameter, except sucrose, and data were combined between repetitions (Table A5.13, Table A5.14, Table

A5.15, Table A5.16, Table A5.17 and Table A5.18). An interaction between temperature and sampling date was observed for all parameters.

Chlorophyll concentration decreased in the fern of the plants exposed to 7ºC, whereas these dormancy parameters remained stable at 15ºC (Fig. 6.7A). More lesions were observed in the seedlings incubated at 15°C than those at 7ºC (Fig. 6.7B). Proline and sucrose concentration increased in the crowns of the plants exposed to 7ºC, whereas these dormancy parameters remained stable at 15ºC (Fig. 6.7A, 6.7C and 6.7D). No pseudothecia developed on any seedlings, regardless of dormancy. No differences in the dormancy parameters were observed between the inoculated and non-inoculated control at either temperature (data not shown).

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Chlorophyll concentration was negatively correlated with lesion counts at both 7ºC and

15°C (Table 6.6). Proline concentration were related to lesion counts at 7ºC, but not 15°C. Sucrose levels were not correlated with lesion counts

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Table 6.6 Correlations between disease and dormancy parameters collected from seedlings and crowns of ‘Guelph Millennium’ in a controlled environment with two temperatures, n=8. Data was pooled between repetitions. Chlorophyll Proline Sucrose Disease parameter concentration concentration concentration r P-value r P-value r P-value 7ºC Stemphylium leaf spot counts -0.50 0.0034 0.37 0.0371 0.20 0.2690 Pseudothecia counts ------15ºC Stemphylium leaf spot counts -0.42 0.0180 -0.15 0.4230 0.10 0.5872 Pseudothecia counts ------

6.3.4 Sanitation field trial

No interaction among location, year, fertilizer, fungicide or fertilizer by fungicide was observed, and data were combined among locations and years (Table A5.19, Table A5.20, Table

A5.21 and Table A5.22). Disease incidence and severity at harvest was lower when fern residue was chopped in the fall than when the residue was chopped prior to harvest (Fig. 6.8). Purple spot incidence and severity increased on the spears following 3 days of storage, demonstrating the presence of latent infections. There was no difference in incidence related to the time of fern chopping, but severity was lowest in stored spears from the treatment where the fern was chopped in the fall. No interaction among the factors was observed, and no significant effects of copper or nitrogen was observed for either disease incidence or severity at harvest or for latent infections.

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6.4 Discussion

The epidemiology of P. herbarum on asparagus fern is not well understood, despite the fact that the ascospores are the primary for the spring crop. This is the first research to document the development of P. herbarum pseudothecia in asparagus, even though the development in other crops has been studied in detail. The development of pseudothecia increased with the decrease in concentrations of proline and chlorophyll in outdoor experiments but, there was no relationship between those compounds and the development of pseudothecia in controlled environment trials.

Fall mowing reduced purple spot incidence and severity, but copper application and nitrogen did not control the spring inoculum.

The ascospores from pseudothecia collected in the fall from ‘Guelph Millennium’ fern were able to infect asparagus seedlings after 42 days of incubation at 15ºC. These results are

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contrary to those observed by Trueman & Roddy (2012) in preliminary experiments. Trueman &

Roddy (2012) found pseudothecia on fern residue, collected in late fall and held below 0ºC, were unable to produce ascospores that could infect asparagus seedlings the following spring. The optimal temperatures for the maturation of pseudothecia of P. allii are between 10 and 15ºC, and the cumulative degree days (CDD) required for maturation was 750 (Llorente & Montesinos,

2004). In 2015, 133 CDD accumulated in the field from the time pseudothecia were first observed to when the residue were collected (base = 0ºC). In 2016, however, only 48 CDD accumulated. In the growth chamber, 630 CDD accumulated. Considering the time required from ascospore release to disease symptoms, the pseudothecia of P. herbarum from asparagus accumulated fewer CDD to mature than that described for P. allii. However, the CDD for P. allii was calculated from pseudothecia growth on artificial media. Since the P. herbarum pseudothecia were produced on host substrate, it is difficult to make a direct comparison. Similar to P. allii, further studies using controlled temperature intervals are warranted to confirm the CDD required for the pseudothecia of P. herbarum to reach maturity.

In both the mature fern and seedling experiments in the natural environment, disease parameters were correlated with the measured parameter of dormancy, chlorophyll concentration.

As mentioned, the methods used to determine the concentration of proline likely extracted all of the amino acids (Kalsoom et al., 2016). However, in the mature seedling trial, pseudothecia did not develop consistently on either the stem or branches while measurable amounts of chlorophyll were still present in the fern. Subsequently, the correlation between chlorophyll concentration and pseudothecia formation on mature fern was weak. The presented results indicate that chlorophyll concentration cannot be used as a measure to predict the development of pseudothecia on asparagus fern.

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Since no pseudothecia developed in the controlled environment experiments, it is not possible to determine if the correlations between disease and dormancy parameters observed in the field were caused by dormancy. In the seedling experiment in the natural environment, pseudothecia were observed as early as 32 days after inoculation. The controlled environment experiments were conducted for 70 days and pseudothecia never developed. Relative humidity is a limiting factor for pseudothecia development (Gadoury et al., 1984; James & Sutton, 1982;

Llorente & Montesinos, 2004; O’Leary & Sutton, 1986; Prados-Ligero et al., 1998; Trapero-Casas

& Kaiser, 1992). In the growth chamber, the seedlings were bagged for 14 days following inoculation to maintain saturated conditions and the humidity was set for greater than 95 % for the remainder of the experiment. However, in both the 7ºC and 15ºC treatment chambers, the relative humidity frequently dropped below 95 % once the bags were removed. Even though the temperature was conducive to the development of pseudothecia, the relative humidity was not optimal for the duration of the experiment in the growth chambers. Further studies are warranted with improved control of the relative humidity to conclusively determine if pseudothecia production is directly related to dormancy in asparagus.

Neither of the chemical and fertilizer amendments tested, copper and urea, reduced purple spot incidence or severity, despite wide use of these sanitation methods in both pears and apples

(Burchill, 1968; Llorente et al., 2010). Even though Fantino et al. (1990) found urea increased the breakdown of fern in tropical asparagus, its application did not reduce disease in the presented study.

Chopping of fern in the fall reduced purple spot incidence and severity on the spears when compared to chopping in the spring. These results are similar to those observed in other perennial cropping systems (Llorente et al., 2006; Sutton et al., 2000), however, the amount of purple spot

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on the spears was still high. The timing of chopping did not provide a practical level of control when tested in a single season. Even though the timing of fern chopping produced promising results, additional studies with multiple seasons should be conducted. In addition, the impact of fern chopping prior to dormancy on crown health should be investigated.

It was clear that ascospores from pseudothecia were able to infect asparagus seedlings after fewer cumulative degree days (CCD) than what was established for pears. These results indicate the CCD required for pseudothecia maturity in P. herbarum may be shorter than that of P. allii.

Since pseudothecia did not develop in the controlled environment experiment, it is not possible to determine if sexual reproduction of P. herbarum is related to dormancy in asparagus. In perennial crop systems, it is well understood that control of the primary inoculum reduces disease in the crop. Even though the least purple spot occurred in plots chopped in the fall, the timing of chopping did not provide a practical level of control when tested in a single season, and additional experiments conducted over multiple seasons should be conducted. In addition, the impact of fern chopping prior to dormancy on crown health should be investigated in order to determine the long term effect on crown health.

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CHAPTER SEVEN - GENERAL DISCUSSION

Foliar diseases limit asparagus production in temperate climates including the primary growing region in Canada, Ontario. Asparagus hectares are growing, and most of the production is sold into the fresh-market. In order to meet the required quality grade, spears must be free of blemishes or damage. Asparagus is established 3 years before the first year of harvest, which means growers maintain the field free of weeds, diseases and pests. Those 3 years are spent building a large healthy crown, that when managed properly, will permit subsequent harvests for up to 20 years after planting.

Abiotic and biotic factors affect the long-term viability of an asparagus field. Foliar diseases, such as Stemphylium leaf spot and rust, contribute to asparagus decline, which is a reduction in yield observed year over year. In Ontario, very little research has been done on management of these two diseases, despite the fact that both Stemphylium leaf spot and rust have been affecting fields for decades. Also, most of the management tools were validated on the commercially relevant cultivars of the time, not on those of today or the future. Even though the economic damage caused by the foliar diseases is well understood, most recently growers in

Ontario have seen a rise in purple spot on the spears during harvest, especially on the high yielding

‘Guelph Millennium’. Since the overwintering inoculum contributes to the disease on the spears in the following spring, control of purple spot needs to be planned and executed the previous season. Effective management tools for both Stemphylium vesicarium and Puccinia asparagi must be validated in current and future cultivars appropriate for Ontario asparagus production. Since few studies have ever been conducted in Ontario on foliar disease in asparagus, this research was performed with the intention of investigating the epidemiology of S. vesicarium and determining effective management tools for control of these devastating foliar diseases.

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The presented research is the first study in asparagus and pear to determine the pathogenicity of S. vesicarium isolates collected in Canada. All isolates tested from both asparagus and onions were pathogenic to asparagus, however, the aggressiveness of the isolates of S. vesicarium varied. Although not always statistically significant, the isolates collected from onion appeared to be less aggressive on asparagus spears than those collected from asparagus.

Interestingly, onion and asparagus isolates were only able to infect artificially wounded pears.

These results could explain why S. vesicarium is prevalent across Ontario, yet has never been reported in Ontario pear orchards. Clearly, there are differences in the virulence factors among the isolates. It is not clear, however, if the virulence factors are separated by geography or host crop.

Virulence factors of S. vesicarium, have never been studied in asparagus, and further research could assist selection for resistance to purple spot.

Stemphylium vesicarium was identified only by conidial morphology, and no molecular techniques were utilized. The genus and species of the isolates should be confirmed using typical techniques as prescribed by Inderbitzin et al. (2009): sequencing of rDNA-ITS, EF-1α, GPD and vmaA-vpsA. This information could be compared to the data collected in the European population that infects predominantly pear.

Stemphylium vesicarium did not produce disease symptoms on rye, however, S. vesicarium was re-isolated from the inoculated dead rye leaves. Similar to studies in pear orchards, these results indicate that S. vesicarium is able to survive as a saprophyte on dead non-host tissue. It is unknown, if these isolates are able to re-infect asparagus tissue, but regardless growers should reconsider their choice of cover crop. The potential for S. vesicarium to survive on living or senesced weed species and cover crops should be assessed as to further understand the etiology of

Stemphylium leaf spot in asparagus.

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Although the asparagus lines and cultivars differed in their susceptibility to purple spot, all had more disease than what is commercially acceptable. In the detached spear assay, ‘Guelph

Eclipse’ had a lower sAUDPC and high final DSI, which indicates a longer latent period than other asparagus cultivars. If a long latency period is also observed in the fern, ‘Guelph Eclipse’ could be considered tolerant. In order to confirm tolerance, the impact on yield in subsequent seasons would need to be assessed.

The asparagus lines UG010, ‘Guelph Equinox’ and UG023 were identified as slow-rusting phenotypes, similar to ‘Jersey Giant’. UG023 also had the greatest retention of cladophylls.

UG010, ‘Guelph Equinox’ and UG023 are all promising additions to an integrated rust management program.

In the field trials, purple spot and Stemphylium leaf spot were negatively correlated at the

Simcoe site and positively correlated at the Plattsville site. There was asparagus rust at the Simcoe site, which influenced the results of the Stemphylium leaf spot severity in the fern. Both

Stemphylium leaf spot DSI and sAUDPC were negatively correlated with asparagus rust DSI and sAUDPC. These results demonstrate it is difficult to screen for resistance when multiple pathogens are present. As well, the results confirm the importance of site selection for plant breeding research.

Disappointingly, none of the fungicides tested provided >70 % control in the field with high rust pressure. In the field with lower rust pressure, several fungicides provided >70 % control, including the registered fungicides metiram and tebuconazole. Fungicides were applied on a 14- day interval, however, in periods of high disease pressure, or in young asparagus fields, a 7- to 10- day application interval is required to obtain adequate control (Hausbeck et al., 2008). Of concern, though, is only half of the registered fungicides recommend an interval shorter than 14 days: metiram, myclobutanil, and trifloxystrobin.

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This study confirms that ‘Guelph Millennium’ is more susceptible to Stemphylium leaf spot than ‘Jersey Giant’. Ultimately, the difference in susceptibility subsequently reduced the effectiveness of the TOM-CAST forecasting model within each cultivar. TOM-CAST treatments were not initiated until the described DSV had accumulated at each site following cladophyll emergence, in accordance to the methods described by Meyer et al. (2000). However, since the cultivars differed in susceptibility, the forecasting treatments were often applied after disease symptoms were observed. In subsequent studies, to account for this, TOM-CAST sprays were first applied at cladophyll emergence. Consistent control was observed when fungicide applications were initiated at cladophyll emergence regardless of the treatments being applied every 14 days or according to TOM-CAST. Growers in Ontario with fields planted to ‘Guelph Millennium’ should initiate fungicide applications earlier than in fields planted to less susceptible cultivars, such as

‘Jersey Giant’.

Over 4 years and 10 field sites, TOM-CAST at 15 and 20 DSV provided more consistent control of Stemphylium leaf spot than TOM-CAST at 30 DSV, regardless of site or fertility management program. However, the effectiveness of the TOM-CAST forecasting model varied by site. Specifically, the same site over 4 years, Site 2 in CHAPTER 4 and Site 1 in CHAPTER 5, had accumulated the highest DSV in the season, but did not have the greatest amount of

Stemphylium leaf spot in the untreated control. Similar observations were made in subsequent experiments with controlled fertility. TOM-CAST was developed for use in an annual crop, not a perennial crop. Forecasting models should be effective across locations, and TOM-CAST should be modified to improve the effectiveness of the model in asparagus. The re-inclusion of other environmental factors to TOM-CAST from the original FAST model could improve the efficacy in asparagus, as suggested by Granke and Hausbeck (2010).

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All of the field locations were managed by different growers. TOM-CAST does not account for the continuous addition of inoculum year over year, and one grower in particular, Site

2 in CHAPTER FOUR and Site 1 in CHAPTER FIVE, incorporated the fern residue in an attempt to reduce the amount of overwintering inoculum. The differences in inoculum management could have further impacted the effectiveness of TOM-CAST. In addition, the closest asparagus field to this field site is 15 km away, whereas the other sites were located close together. As identified in the pathogenicity experiments, the aggressiveness of S. vesicarium differs among isolates. Field results indicate that the population at this site is less aggressive than the populations at the other sites. In order to confirm this observation, controlled experiments with attached seedlings would need to be conducted. Improved understanding of the aggressiveness of S. vesicarium and the subsequent effect on forecasting models, could improve the usefulness of TOM-CAST over multiple sites.

The presented research is the first to assess irrigation for its impact on foliar disease in asparagus. In irrigated fields, fern yellowing and defoliation was delayed until 70 days after cladophyll emergence, even in the plots that were not treated with fungicides. Interestingly, even though irrigation appears to have delayed defoliation and fern yellowing, the fields with irrigation did not have less Stemphylium leaf spot or lower sAUDPC than the fields without irrigation.

Unexpectedly, no differences in Stemphylium leaf spot was observed between the site irrigated with drip irrigation and the site irrigated with overhead irrigation. Overhead irrigation is easier to implement in a perennial crop as drip irrigation needs to be laid at planting and maintained for the duration of the crop. However, before overhead irrigation is widely adopted, the results should be confirmed since each irrigation method was tested at a single field site.

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This study is also the first to assess fertilizer for control of foliar disease in asparagus.

Unfortunately, fertilizer had a negligible effect on foliar disease. DSI, fern yellowing, defoliation and sAUDPC were not correlated with the quantity of nitrogen or potassium applied. Since asparagus is a perennial crop, it is possible the effect of fertilizer treatments in-season on foliar disease would not be observed until the following season. Similarly, the positive impact of fertilizer applications on yield is not observed until the following season. The effect of polyetic epidemics in combination with abiotic factors that affect plant growth and physiology is not well understood or studied in asparagus. The interaction between the effect of epidemics and abiotic stress in asparagus over multiple seasons should be investigated.

In perennial crop systems, it is well understood that control of the primary inoculum reduces disease in the crop. However, no research has been conducted that investigates the development of primary inoculum in asparagus. The presented research is the first attempt to investigate pseudothecia maturity. In the controlled environment studies, however, no pseudothecia developed on dormant fern. Even though the temperature was conducive to the development of pseudothecia, the relative humidity dropped below 90 % following the removal of the inoculation bags. Assuming the optimal relative humidity for pseudothecia production and maturity is similar between P. herbarum and P. allii, the relative humidity was not optimal for the duration of the experiment in the growth chambers. Further studies are warranted that control the relative humidity to conclusively determine if pseudothecia production is directly impacted by dormancy in asparagus.

Contrary to preliminary results, the pseudothecia collected in the fall were able to infect asparagus seedlings just 42 days after incubation at 15ºC. These results indicate the cumulative degree days (CCD) required for pseudothecia maturity in P. herbarum may be shorter than that of

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P. allii. However, since the P. herbarum pseudothecia were produced on host substrate, it is difficult to make a direct comparison. Controlled environment experiments should be conducted to confirm the CDD required for the pseudothecia of P. herbarum to reach maturity on asparagus fern. The CDD would be useful to determine if and when sanitation methods are necessary in the fall.

Very little research has been conducted that identifies effective tools to control the primary inoculum in asparagus. This study is the first to test sanitation methods in temperate asparagus.

Spears had lower incidence of purple spot when fern was chopped in the fall instead of the spring.

These results are similar to those observed in other perennial cropping systems, but the overall amount of purple spot on the spears was high. The timing of chopping did not provide a practical level of control when tested in a single season, and additional experiments conducted over multiple seasons should be conducted. In addition, the impact of fern chopping prior to dormancy on crown health should be investigated in order to determine the long term effect on crown health.

The results of these series of studies have major implications for growers and have contributed to a greater understanding of the epidemiology of S. vesicarium. The discovery that

Stemphylium can colonize rye has potentially major repercussions for growers. Alternative cover crops should be considered. Also the timing of the herbicide burndown should perhaps be made in the fall. Since it is difficult to get adequate coverage of full asparagus fern, fungicides should be applied early in the developing fern. Fungicide use should be prescribed based on cultivar susceptibility. Specifically, in ‘Guelph Millennium’, intensive management of Stemphylium leaf spot is required. Fall mowing is a practical way to reduce primary inoculum in the spring. Even though the reductions were not likely of economic significance, moving the mowing timing does not change costs to the grower. The impact of mowing fern in the fall on crown health, is not yet

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known. The isolate virulence differed by the initial host crop. In pears specifically, wounding was required for infection to occur. It appears as though the population of Stemphylium in Canada causes a similar disease reaction to the isolates from parsley in California, but differ from European pear isolates. In the field, the severity of pseudothecia on fern and seedlings were correlated to dormancy metabolites. In the controlled environment, SLS was correlated to dormancy metabolites, however, no pseudothecia developed. The relative humidity in the controlled environment was likely not adequate for pseudothecia to develop, but it does not appear as though the occurrence of pseudothecia is related to fern senescence. Improved understanding of the environmental factors that contribute to pseudothecia maturity would enable growers to best time fall management strategies. The presented study has contributed to the body of knowledge for the epidemiology of S. vesicarium in asparagus, and also validated and tested new tools for control of foliar disease in asparagus. The hectares planted to asparagus in Ontario are increasing year over year, yet the area of suitable land for asparagus production is limited. This greater understanding of both the pathogen and management of foliar disease will hopefully contribute to the continued growth of asparagus production in Ontario.

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APPENDIX 1: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER TWO

Table A1.1 REML covariance parameter estimates for disease severity index (0-100) among five cultivars inoculated with seven isolates of Stemphylium vesicarium. The experiment was repeated. First Experiment Covariance parameters Estimate SE Rep -3.6023 5.2329 Block(Rep) 2.0813 5.6518 Rep*cultivar -3.8667 6.408 Rep*isolate 26.8826 25.0526 Rep*isolate*cultivar 11.7881 23.8760 Residual 262.38 26.4701 Effect Num DF Den DF F Value Pr > F Cultivar 4 4 10.58 0.0211 Isolate 6 6 6.27 0.0210 Cultivar*isolate 24 24 1.15 0.3708

Table A1.2 REML covariance parameter estimates for sAUDPC among five cultivars inoculated with seven isolates of Stemphylium vesicarium. The experiment was repeated. First Experiment Covariance parameters Estimate SE Rep 932.02 1433.77 Block(Rep) 97.7662 76.8863 Rep*cultivar 145.95 135.69 Rep*isolate 144.06 118.07 Rep*isolate*cultivar -22.4838 92.2603 Residual 1223.68 123.71 Effect Num DF Den DF F Value Pr > F Cultivar 4 4 2.75 0.1754 Isolate 6 6 6.91 0.0166 Cultivar*isolate 24 24 1.21 0.3217

Table A1.3 REML covariance parameter estimates for disease incidence among five cultivars inoculated with seven isolates of Stemphylium vesicarium. The experiment was repeated. First Experiment Covariance parameters Estimate SE Rep 0.3761 1.5963 Block(rep) 0.1786 0.3656 Rep*cultivar -1.0142 1.0198 Rep*isolate 2.8868 3.5263 Rep*isolate*cultivar 11.0846 4.3582 Residual 15.5681 1.5656 Effect Num DF Den DF F Value Pr > F Cultivar 4 4 3.12 0.1480 Isolate 6 6 0.90 0.5513 Cultivar*isolate 24 24 0.76 0.7499

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Table A1.4 REML covariance parameter estimates for disease severity index (0-100) among six cultivars and two isolate of Stemphylium vesicarium. The experiment was repeated. Second Experiment. Covariance parameters Estimate SE Repetition -92.2488 133.77 Block(Repetition) 23.2389 19.0242 Repetition*Cultivar 43.0944 33.0336 Repetition*Isolate 184.60 265.26 Repetition*Isolate*Cultivar -14.6426 10.8621 Residual 296.06 30.7959 Effect Num DF Den DF F Value Pr > F Cultivar 5 5 0.46 0.7913 Isolate 1 1 3.79 0.3020 Cultivar*Isolate 5 5 1.17 0.4319

Table A1.5 REML covariance parameter estimates for sAUDPC among six cultivars and two isolate of Stemphylium vesicarium. The experiment was repeated. Second Experiment. Covariance parameters Estimate SE Rep 0 . Block(rep) 38.5783 40.4623 Rep*cultivar 39.9932 51.7961 Rep*isolate 545.05 560.37 Rep*isolate*cultivar 0 . Residual 816.22 82.9961 Effect Num DF Den DF F Value Pr > F Cultivar 5 5 0.79 0.5985 Isolate 1 1 6.15 0.2440 Cultivar*isolate 5 5 0.63 0.6889

Table A1.6 REML covariance parameter estimates for disease incidence among six cultivars and two isolate of Stemphylium vesicarium. The experiment was repeated. Second Experiment. Covariance parameters Estimate SE Rep 12.9226 50.9029 Block(rep) 148.90 70.2383 Rep*cultivar -8.2458 73.5111 Rep*isolate -14.3394 31.5370 Rep*isolate*cultivar 70.6985 111.69 Residual 742.10 83.2142 Effect Num DF Den DF F Value Pr > F Cultivar 5 5 0.62 0.6907 Isolate 1 1 0.33 0.6672 Cultivar*isolate 5 5 0.83 0.5775

164

Table A1.7 REML covariance parameter estimates for disease incidence among three isolates of Stemphylium vesicarium inoculated onto pear fruit. First Repetition. Covariance parameter Estimate SE Block -451E-19 0.1925 Residual 0.06667 0.03333 Effect Num DF Den DF F Value Pr > F Isolate 2 8 21.00 0.0007

Table A1.8 REML covariance parameter estimates for lesion size among three isolates of Stemphylium vesicarium inoculated onto pear fruit. First Repetition. Covariance parameter Estimate SE Block 6.4 14.6494 Residual 37.1333 18.5667 Effect Num DF Den DF F Value Pr > F Isolate 2 8 2.46 0.1467

Table A1.9 REML covariance parameter estimates AUDPC among three isolates of Stemphylium vesicarium inoculated onto pear fruit. First Repetition. Covariance parameter Estimate SE Block 124.65 290.03 Residual 739.80 369.90 Effect Num DF Den DF F Value Pr > F Isolate 2 8 2.50 0.1438

Table A1.10 REML covariance parameter estimates for disease incidence among five isolates of Stemphylium vesicarium inoculated onto pear fruit. Second Repetition. Covariance parameter Estimate SE Block 0.075 0.07126 Residual 0.1250 0.04419 Effect Num DF Den DF F Value Pr > F Isolate 4 16 4.00 0.0195

Table A1.11 REML covariance parameter estimates for lesion size among five isolates of Stemphylium vesicarium inoculated onto pear fruit. Second Repetition. Covariance parameter Estimate SE Block 0.4600 1.4776 Residual 7.4600 2.6375 Effect Num DF Den DF F Value Pr > F Isolate 4 16 43.24 <0.0001

165

Table A1.12 REML covariance parameter estimates AUDPC among five isolates of Stemphylium vesicarium inoculated onto pear fruit. Second Repetition. Covariance parameter Estimate SE Block -35.83 19.5201 Residual 243.85 86.2140 Effect Num DF Den DF F Value Pr > F Isolate 4 16 46.10 <0.0001

Table A1.13 REML covariance parameter estimates for lesion size among three isolates of Stemphylium vesicarium inoculated onto pear fruit. Third Repetition. Covariance parameter Estimate SE Block -12.3167 9.5724 Residual 52.85 26.4250 Effect Num DF Den DF F Value Pr > F Isolate 2 8 3.24 0.0931

Table A1.14 REML covariance parameter estimates for AUDPC among three isolates of Stemphylium vesicarium inoculated onto pear fruit. Third Repetition. Covariance parameter Estimate SE Block -189.46 233.79 Residual 1143.19 571.59 Effect Num DF Den DF F Value Pr > F Isolate 2 8 2.78 0.1211

Table A1.15 REML covariance parameter estimates for disease incidence among six cultivars infected with Stemphylium vesicarium at two locations, 2014-16. Covariance Parameter Estimate SE Block(location) 3.1858 3.6339 Residual 72.2948 8.7670 Effect Num DF Den DF F Value Pr > F Year 2 136 21.56 <0.0001 Location 1 8 220.24 <0.0001 Year*location 2 136 48.69 <0.0001 Cultivar 5 136 10.96 <0.0001 Year*cultivar 10 136 1.36 0.2070 Location*cultivar 5 136 14.69 <0.0001 Year*location*cultivar 10 136 0.55 0.8493

166

Table A1.16 REML covariance parameter estimates for disease severity index (0-100) among six cultivars infected with Stemphylium vesicarium at two locations, 2014-16. Covariance Parameter Estimate SE Block(location) -0.3000 0.8166 Residual 33.6417 4.0797 Effect Num DF Den DF F Value Pr > F Year 2 136 5.00 0.0080 Location 1 8 298.09 <0.0001 Year*location 2 136 10.55 <0.0001 Cultivar 5 136 5.16 0.0002 Year*cultivar 10 136 0.90 0.5335 Location*cultivar 5 136 8.92 <0.0001 Year*location*cultivar 10 136 0.33 0.9720

Table A1.17 REML covariance parameter estimates for disease incidence among six cultivars infected with Stemphylium vesicarium at Simcoe Research Station, Simcoe, Ontario, 2014-16. Covariance Parameter Estimate SE Block 5.5147 6.1580 Residual 56.8853 9.7557 Effect Num DF Den DF F Value Pr > F Year 2 68 66.64 <0.0001 Cultivar 5 68 30.32 <0.0001 Year*cultivar 10 68 1.94 0.0545

Table A1.18 REML covariance parameter estimates for disease severity index (0-100) among six cultivars infected with Stemphylium vesicarium at Simcoe Research Station, Simcoe, Ontario, 2014-16. Covariance Parameter Estimate SE Block 1.5588 1.7773 Residual 16.9967 2.9149 Effect Num DF Den DF F Value Pr > F Year 2 68 28.97 <0.0001 Cultivar 5 68 22.50 <0.0001 Year*cultivar 10 68 2.13 0.0337

Table A1.19 REML covariance parameter estimates for disease severity index (0-100) among six cultivars infected with Stemphylium vesicarium at Simcoe Research Station, Simcoe, Ontario, 2014. Covariance Parameter Estimate SE Block -0.1467 3.5802 Residual 8.4633 9.0009 Effect Num DF Den DF F Value Pr > F Cultivar 5 20 8.82 0.0002

167

Table A1.20 REML covariance parameter estimates for disease severity index (0-100) among six cultivars infected with Stemphylium vesicarium at Simcoe Research Station, Simcoe, Ontario, 2015. Covariance Parameter Estimate SE Block 1.0300 2.9118 Residual 17.2867 5.4665 Effect Num DF Den DF F Value Pr > F Cultivar 5 20 3.71 0.0155

Table A1.21 REML covariance parameter estimates for disease severity index (0-100) among six cultivars infected with Stemphylium vesicarium at Simcoe Research Station, Simcoe, Ontario, 2016. Covariance Parameter Estimate SE Block 4.0700 3.4727 Residual 4.9633 1.5693 Effect Num DF Den DF F Value Pr > F Cultivar 5 20 28.17 <0.0001

Table A1.22 REML covariance parameter estimates for disease incidence among six cultivars infected with Stemphylium vesicarium at Honeywood Research Facility, Plattsville Ontario, 2014-16. Covariance Parameter Estimate SE Block 0.8569 4.1365 Residual 87.7042 15.0412 Effect Num DF Den DF F Value Pr > F Year 2 68 14.68 <0.0001 Cultivar 5 68 1.48 0.2077 Year*cultivar 10 68 0.32 0.9742

Table A1.23 REML covariance parameter estimates for disease severity index (0-100) among six cultivars infected with Stemphylium vesicarium at Honeywood Research Facility, Plattsville, Ontario, 2014-16. Covariance Parameter Estimate SE Block -2.1588 0.6566 Residual 50.2866 8.6241 Effect Num DF Den DF F Value Pr > F Year 2 68 0.61 0.5441 Cultivar 5 68 1.81 0.1220 Year*cultivar 10 68 0.10 0.9997

168

Table A1.24 REML covariance parameter estimates for Stemphylium leaf spot incidence (%) among six cultivars at two locations in both 2015 and 2016. Covariance parameter Estimate SE Block(location) 5.6439 10.2677 Residual 173.11 26.0927 Effect Num DF Den DF F Value Pr > F Year 1 88 13.02 0.0005 Location 1 8 26.80 0.0008 Year*location 1 88 724.72 <0.0001 Cultivar 5 88 6.54 <0.0001 Year*cultivar 5 88 5.93 <0.0001 Location*cultivar 5 88 8.68 <0.0001 Year*location*cultivar 5 88 8.68 <0.0001

Table A1.25 REML covariance parameter estimates for Stemphylium leaf spot severity (0-100) among six cultivars at two locations in both 2015 and 2016. Covariance parameter Estimate SE Block(location) -0.1909 3.0446 Residual 72.0576 10.8631 Effect Num DF Den DF F Value Pr > F Year 1 88 3.75 0.0561 Location 1 8 29.86 0.0006 Year*location 1 88 359.86 <0.0001 Cultivar 5 88 2.95 0.0166 Year*cultivar 5 88 0.48 0.7939 Location*cultivar 5 88 2.29 0.0521 Year*location*cultivar 5 88 0.15 0.9792

Table A1.26 REML covariance parameter estimates for Stemphylium leaf spot standardized Area Under the Disease Progress Curve among six cultivars at two locations in both 2015 and 2016. Covariance parameter Estimate SE Block(location) 0.06765 0.1350 Residual 2.3488 0.3541 Effect Num DF Den DF F Value Pr > F Year 1 88 0.03 0.8530 Location 1 8 202.19 <0.0001 Year*location 1 88 169.54 <0.0001 Cultivar 5 88 3.15 0.0117 Year*cultivar 5 88 0.79 0.5638 Location*cultivar 5 88 2.74 0.0239 Year*location*cultivar 5 88 1.45 0.2152

169

Table A1.27 REML covariance parameter estimates for Stemphylium leaf spot standardized Area Under the Disease Progress Curve among six cultivars at Honeywood Research Facility in Plattsville, Ontario, in both 2015 and 2016. Covariance parameter Estimate SE Block(location) 0.09913 0.1968 Residual 2.0898 0.4455 Effect Num DF Den DF F Value Pr > F Year 1 44 98.02 <0.0001 Cultivar 5 44 0.27 0.9288 Year*cultivar 5 44 0.27 0.9287

Table A1.28 REML covariance parameter estimates for Stemphylium leaf spot standardized Area Under the Disease Progress Curve among six cultivars at Simcoe Research Station in Simcoe, Ontario, in both 2015 and 2016. Covariance parameter Estimate SE Block(location) 0.03617 0.1851 Residual 2.6079 0.5560 Effect Num DF Den DF F Value Pr > F Year 1 44 74.18 <0.0001 Cultivar 5 44 5.09 0.0009 Year*cultivar 5 44 1.80 0.1331

Table A1.29 Daily average, high and low temperature (ºC), relative humidity (%) and rainfall (mm) recorded at Honeywood Research Facility, Plattsville, ON, from 2013 to 2016. Precipitation Relative humidity (%) Temperature (°C) Date (mm) min max average min max average 01.05.2013 0.0 43 90 69 10.6 20.4 14.9 02.05.2013 0.0 27 72 47 11.3 23.6 17.2 03.05.2013 0.0 25 59 44 11.8 22.8 16.9 04.05.2013 0.0 22 70 45 10.8 23.6 16.7 05.05.2013 0.0 28 66 46 10.9 21.2 15.7 06.05.2013 0.0 17 59 39 9.6 22.8 16.7 07.05.2013 0.0 27 56 37 9.7 22.8 16.1 08.05.2013 0.0 39 66 51 11.5 24.0 18.1 09.05.2013 0.0 24 95 64 13.3 23.7 17.4 10.05.2013 4.6 34 96 71 12.9 25.4 18.2 11.05.2013 4.2 72 100 91 7.4 20.8 13.8 12.05.2013 1.4 64 96 85 2.9 11.3 7.4 13.05.2013 0.0 38 93 72 0.7 5.3 2.7 14.05.2013 0.0 43 82 66 0.1 8.2 3.8 15.05.2013 0.2 35 91 68 1.6 10.1 6.8 16.05.2013 0.0 24 80 51 7.4 22.9 15.7 17.05.2013 0.0 37 84 58 8.6 21.2 15.1 18.05.2013 0.0 31 53 43 4.6 16.8 12.1 19.05.2013 0.0 37 72 55 11.9 19.8 15.3

170

20.05.2013 0.0 48 98 73 11.1 26.9 18.7 21.05.2013 7.4 58 100 86 13.6 28.5 21.1 22.05.2013 1.8 53 99 85 15.2 27.4 20.1 23.05.2013 0.0 63 93 84 15.3 26.5 19.9 24.05.2013 2.2 54 92 76 3.6 18.3 12.2 25.05.2013 0.0 41 93 69 1.6 9.3 4.8 26.05.2013 0.0 31 88 58 0.3 14.2 7.5 27.05.2013 0.0 21 87 48 1.7 17.0 10.1 28.05.2013 17.2 31 100 82 3.0 20.4 13.4 29.05.2013 7.0 75 100 92 9.3 15.6 12.3 30.05.2013 0.2 56 97 79 15.6 25.2 20.7 31.05.2013 0.4 58 90 75 17.7 28.2 23.1 01.06.2013 2.6 77 100 90 18.3 27.7 22.2 02.06.2013 21.0 77 100 92 18.2 23.6 20.7 03.06.2013 0.0 55 92 76 6.6 20.7 15.8 04.06.2013 0.0 36 90 67 5.7 14.2 10.0 05.06.2013 0.0 39 71 53 5.1 18.7 12.4 06.06.2013 1.2 45 96 71 10.2 19.3 14.4 07.06.2013 2.6 81 100 93 10.8 13.2 11.9 08.06.2013 0.0 66 100 87 11.2 15.1 12.9 09.06.2013 0.0 43 100 75 11.9 19.1 15.1 10.06.2013 4.8 59 100 85 8.9 23.4 17.0 11.06.2013 4.8 69 100 91 14.2 18.9 16.4 12.06.2013 1.6 62 100 84 15.8 22.1 18.1 13.06.2013 14.6 60 99 85 14.0 23.6 18.8 14.06.2013 0.0 58 94 77 13.8 22.5 17.2 15.06.2013 0.0 43 96 71 11.9 22.0 17.3 16.06.2013 7.6 53 100 83 8.7 22.9 17.2 17.06.2013 0.0 53 98 77 15.9 22.9 19.0 18.06.2013 0.0 41 92 71 13.9 23.4 19.1 19.06.2013 0.0 37 71 53 11.4 21.2 16.3 20.06.2013 0.0 33 81 54 10.1 21.6 15.9 21.06.2013 0.0 36 83 58 10.2 24.9 18.3 22.06.2013 0.2 50 84 68 12.8 27.2 20.7 23.06.2013 0.4 47 100 81 16.8 28.8 22.8 24.06.2013 0.0 55 91 74 19.6 30.4 24.8 25.06.2013 2.4 62 100 82 20.5 29.7 25.0 26.06.2013 0.0 50 100 80 19.4 26.6 22.5 27.06.2013 0.4 60 98 84 18.6 28.8 23.4 28.06.2013 15.4 81 100 94 19.0 26.4 21.4 29.06.2013 0.0 60 100 85 16.7 21.1 18.5 30.06.2013 0.0 57 97 77 15.3 22.8 18.8 01.07.2013 0.0 53 83 65 14.3 24.2 19.2 02.07.2013 0.0 63 85 74 15.3 20.9 18.2

171

03.07.2013 0.0 60 98 85 14.8 23.1 18.4 04.07.2013 3.8 67 100 86 15.8 28.1 21.6 05.07.2013 10.6 74 100 92 19.6 26.2 22.5 06.07.2013 0.0 66 100 86 18.8 24.4 20.9 07.07.2013 7.2 71 100 91 17.4 27.3 22.7 08.07.2013 0.0 69 100 90 20.1 25.4 22.0 09.07.2013 3.8 74 100 94 18.5 28.0 22.2 10.07.2013 3.8 63 100 89 19.4 26.9 22.3 11.07.2013 0.0 54 100 80 14.7 28.9 22.3 12.07.2013 0.0 56 94 77 12.8 24.1 18.3 13.07.2013 0.0 56 94 74 14.0 25.7 19.8 14.07.2013 0.0 53 94 76 14.8 26.9 21.4 15.07.2013 0.0 49 100 73 17.5 30.6 24.5 16.07.2013 0.0 51 97 72 19.6 31.6 26.2 17.07.2013 0.0 61 100 81 20.9 32.2 27.1 18.07.2013 0.0 55 100 79 22.1 31.9 26.9 19.07.2013 17.2 53 100 81 21.8 32.4 27.4 20.07.2013 11.2 49 100 80 19.8 32.3 25.9 21.07.2013 0.0 51 91 70 17.5 25.9 22.2 22.07.2013 0.0 65 97 82 14.6 25.4 19.6 23.07.2013 1.2 73 100 88 14.7 24.6 19.4 24.07.2013 0.0 44 88 69 14.4 24.8 18.5 25.07.2013 0.0 40 95 68 11.6 21.1 15.8 26.07.2013 0.0 45 93 65 9.9 24.1 17.2 27.07.2013 4.2 60 97 88 10.9 24.5 18.5 28.07.2013 13.8 56 100 85 15.2 21.0 17.9 29.07.2013 0.6 68 100 88 11.5 18.7 14.7 30.07.2013 0.0 57 100 82 11.2 19.8 14.6 31.07.2013 7.2 61 100 87 11.4 23.7 17.7 01.08.2013 19.8 57 100 86 13.3 22.9 17.4 02.08.2013 2.7 55 100 81 14.2 23.1 18.6 03.08.2013 14.5 55 100 83 12.6 24.1 17.8 04.08.2013 0.0 53 98 79 12.8 22.4 17.2 05.08.2013 0.0 49 100 76 10.9 20.6 15.7 06.08.2013 0.0 56 94 78 8.8 21.1 15.7 07.08.2013 2.6 77 100 86 13.4 24.4 18.4 08.08.2013 0.0 67 100 85 16.9 25.5 20.8 09.08.2013 0.0 52 88 71 17.1 25.7 21.1 10.08.2013 0.0 50 96 74 15.6 24.3 19.3 11.08.2013 0.0 56 98 78 11.2 21.3 16.1 12.08.2013 0.0 72 100 88 9.1 22.2 16.7 13.08.2013 1.2 66 100 86 12.4 22.6 17.8 14.08.2013 0.0 51 95 77 10.6 17.3 14.5 15.08.2013 0.0 46 100 76 9.8 19.8 14.3

172

16.08.2013 0.0 44 94 72 7.8 20.7 14.8 17.08.2013 0.0 40 96 69 10.6 25.4 17.7 18.08.2013 0.0 47 97 71 13.0 26.0 19.1 19.08.2013 0.0 45 100 75 11.7 26.3 19.7 20.08.2013 0.0 53 100 80 12.6 26.4 19.7 21.08.2013 0.0 54 100 79 14.2 27.1 20.7 22.08.2013 0.6 60 100 82 17.1 28.9 22.3 23.08.2013 0.0 45 97 71 17.9 27.2 21.5 24.08.2013 0.0 43 94 71 13.6 25.4 19.3 25.08.2013 0.0 46 100 76 12.3 25.1 18.6 26.08.2013 0.4 69 91 79 10.6 27.1 20.3 27.08.2013 0.2 67 100 91 19.1 26.3 22.1 28.08.2013 1.6 68 100 90 16.6 28.6 22.3 29.08.2013 0.0 51 100 80 20.3 28.1 23.0 30.08.2013 0.0 68 100 85 17.4 30.6 23.7 31.08.2013 14.4 69 100 89 17.2 27.2 22.3 01.09.2013 4.6 70 100 93 18.2 25.8 21.7 02.09.2013 0.6 74 100 93 18.1 25.9 20.9 03.09.2013 0.0 68 100 88 13.8 20.9 18.3 04.09.2013 0.0 55 100 83 11.5 19.2 15.0 05.09.2013 0.0 45 100 76 10.5 24.0 16.8 06.09.2013 0.0 35 100 70 7.2 17.0 12.8 07.09.2013 5.0 69 100 88 4.0 21.7 13.2 08.09.2013 0.0 45 100 77 12.1 21.6 16.6 09.09.2013 0.0 61 98 80 9.9 20.5 16.6 10.09.2013 0.2 44 100 81 8.8 22.7 16.0 11.09.2013 2.8 59 94 80 19.2 34.1 26.2 12.09.2013 24.4 62 100 90 18.9 30.8 23.2 13.09.2013 0.0 69 94 83 13.7 21.3 18.3 14.09.2013 0.0 47 100 77 5.9 13.9 10.1 15.09.2013 0.2 61 100 84 2.9 18.1 10.4 16.09.2013 0.6 52 100 84 8.4 17.7 12.4 17.09.2013 0.0 45 93 75 4.6 15.1 11.0 18.09.2013 0.0 43 100 78 3.9 17.8 10.3 19.09.2013 0.0 66 100 88 7.2 22.6 14.6 20.09.2013 0.2 88 100 97 11.4 21.4 16.5 21.09.2013 51.8 76 100 96 16.2 22.8 19.4 22.09.2013 0.0 61 100 83 9.2 18.4 14.4 23.09.2013 0.0 51 94 73 6.5 11.7 9.1 24.09.2013 0.0 37 100 72 6.6 16.3 10.9 25.09.2013 0.0 35 95 70 2.9 20.2 11.6 26.09.2013 0.0 34 95 67 6.8 22.1 13.8 27.09.2013 0.0 56 95 77 8.3 22.9 15.2 28.09.2013 0.0 52 100 85 8.7 21.2 14.2

173

29.09.2013 0.2 71 100 90 10.2 22.5 14.7 30.09.2013 1.2 68 100 92 9.9 20.9 15.7 01.10.2013 0.0 57 100 87 12.2 20.8 16.7 02.10.2013 0.0 31 100 78 10.4 23.9 17.2 03.10.2013 0.0 43 86 61 9.4 24.0 17.4 04.10.2013 13.4 70 100 93 7.7 21.6 14.9 05.10.2013 12.0 93 100 99 13.5 22.9 17.1 06.10.2013 1.8 83 100 95 13.1 15.3 14.1 07.10.2013 41.6 63 100 90 13.8 22.6 17.3 08.10.2013 0.2 44 100 80 8.5 15.8 11.2 09.10.2013 0.0 46 100 80 5.9 18.2 11.7 10.10.2013 0.0 41 100 80 7.1 20.2 12.7 11.10.2013 0.0 34 99 73 5.3 21.2 12.5 12.10.2013 0.0 51 86 63 6.7 21.6 13.4 13.10.2013 6.0 69 100 90 9.4 22.8 15.3 14.10.2013 0.0 56 100 83 7.2 17.9 14.3 15.10.2013 0.0 65 100 85 6.2 16.3 10.7 16.10.2013 3.6 52 100 88 6.3 18.6 12.6 17.10.2013 10.6 69 100 90 9.9 20.1 15.1 18.10.2013 0.2 54 100 86 8.8 14.2 10.7 19.10.2013 11.2 80 99 94 5.1 14.1 9.4 20.10.2013 7.2 52 100 85 3.6 9.0 6.1 21.10.2013 6.0 73 100 85 2.0 11.2 6.4 22.10.2013 3.4 64 100 79 5.6 13.3 10.2 23.10.2013 3.2 56 100 85 0.4 6.4 3.9 24.10.2013 15.6 75 100 96 -0.6 6.7 2.9 25.10.2013 0.6 67 100 92 -1.0 5.3 1.4 26.10.2013 18.4 67 100 85 0.9 6.7 3.2 27.10.2013 1.4 61 100 89 1.1 5.7 3.4 28.10.2013 0.0 36 93 72 1.6 6.4 3.7 29.10.2013 0.0 53 84 68 -0.2 7.8 3.7 30.10.2013 0.0 59 99 84 -2.3 7.3 2.9 31.10.2013 28.0 87 100 97 0.6 13.1 6.2 01.11.2013 10.8 75 100 88 5.5 15.8 12.6 02.11.2013 2.2 81 100 91 7.0 15.0 10.3 03.11.2013 0.0 44 89 70 0.9 7.2 5.2 04.11.2013 0.0 53 84 70 -3.4 2.9 -0.5 05.11.2013 0.0 63 92 75 -4.0 4.1 0.3 06.11.2013 9.8 69 100 79 -0.3 12.9 7.2 07.11.2013 5.6 66 99 87 5.7 13.4 10.3 08.11.2013 2.0 73 100 89 0.8 5.7 3.2 09.11.2013 0.0 66 88 78 -0.9 3.1 1.3 10.11.2013 0.4 72 91 80 -0.2 9.5 5.7 11.11.2013 3.4 80 99 89 1.1 6.9 3.9

174

12.11.2013 0.0 68 92 81 -3.1 4.2 0.3 13.11.2013 0.0 61 98 83 -5.6 -0.6 -3.7 14.11.2013 0.0 53 72 65 -5.7 2.1 -1.6 15.11.2013 0.0 47 74 61 -1.8 7.4 3.2 16.11.2013 0.0 71 84 75 1.7 9.7 5.0 17.11.2013 2.2 79 100 90 2.9 11.4 8.1 18.11.2013 6.6 68 96 80 8.7 15.6 12.9 19.11.2013 0.0 67 92 77 -0.1 8.7 3.6 20.11.2013 0.0 51 94 81 -4.8 1.6 -0.8 21.11.2013 0.0 73 100 82 -5.8 3.4 -1.5 22.11.2013 8.8 88 100 99 -1.3 5.4 2.9 23.11.2013 0.0 70 99 84 -2.4 6.5 3.1 24.11.2013 0.0 66 91 82 -10.7 -1.1 -5.3 25.11.2013 0.0 62 91 75 -12.5 -6.6 -9.3 26.11.2013 0.0 71 100 92 -9.7 -0.9 -3.9 27.11.2013 0.0 73 100 89 -2.7 -0.2 -1.6 28.11.2013 0.0 67 93 84 -7.6 -1.4 -4.2 29.11.2013 0.0 64 99 87 -9.8 -3.9 -6.8 30.11.2013 0.4 74 93 86 -11.1 -1.5 -6.8 01.12.2013 0.0 85 100 95 -7.2 1.9 -1.9 02.12.2013 0.0 73 100 94 -0.8 1.4 0.6 03.12.2013 0.0 63 99 85 -1.9 1.2 -0.2 04.12.2013 1.8 76 100 94 -5.6 3.0 -0.4 05.12.2013 3.0 80 100 93 0.2 5.2 2.1 06.12.2013 0.0 77 83 80 -0.3 12.4 6.1 07.12.2013 0.0 65 87 77 -4.2 -0.3 -2.7 08.12.2013 0.0 68 92 80 -7.8 -4.2 -6.0 09.12.2013 0.0 70 100 89 -9.7 -4.2 -6.6 10.12.2013 0.0 62 90 75 -6.8 -0.6 -3.4 11.12.2013 0.0 65 96 86 -11.2 -6.8 -8.7 12.12.2013 0.0 72 89 83 -14.7 -7.4 -10.1 13.12.2013 0.0 72 96 84 -15.2 -9.2 -11.9 14.12.2013 0.0 74 92 87 -12.3 -5.8 -8.9 15.12.2013 0.0 84 95 91 -13.9 -10.5 -12.1 16.12.2013 0.0 70 94 87 -12.3 -5.6 -9.3 17.12.2013 0.0 87 99 93 -15.3 -8.6 -12.1 18.12.2013 0.0 84 99 94 -13.4 -5.3 -7.7 19.12.2013 0.0 73 93 82 -6.6 -3.3 -4.5 20.12.2013 12.0 92 100 99 -4.1 1.1 -1.0 21.12.2013 24.4 100 100 100 -0.5 1.2 0.2 22.12.2013 19.8 93 100 99 -1.2 1.5 0.6 23.12.2013 1.0 85 100 97 -1.5 2.1 0.1 24.12.2013 0.0 75 98 89 -8.2 -0.1 -3.8 25.12.2013 0.0 88 98 93 -17.8 -6.1 -11.4

175

26.12.2013 0.0 83 100 96 -17.6 -5.9 -10.8 27.12.2013 0.0 78 99 90 -8.5 -3.2 -5.6 28.12.2013 8.4 79 98 88 -7.7 1.3 -3.6 29.12.2013 0.0 73 100 82 1.3 3.6 2.1 30.12.2013 0.0 62 97 81 -8.2 3.3 -0.1 31.12.2013 0.0 78 95 86 -11.7 -8.2 -10.5 01.01.2014 0.0 60 93 78 -11.7 -9.1 -10.6 02.01.2014 0.0 63 85 81 -15.4 -8.1 -12.2 03.01.2014 0.0 57 87 77 -18.0 -15.4 -16.7 04.01.2014 0.0 62 90 76 -23.1 -9.7 -17.6 05.01.2014 0.4 72 100 97 -9.7 -2.4 -5.1 06.01.2014 0.0 82 100 93 -3.8 -1.0 -2.3 07.01.2014 0.0 78 90 84 -23.9 -1.5 -11.8 08.01.2014 0.0 73 87 81 -26.4 -18.8 -22.3 09.01.2014 0.0 72 94 87 -18.8 -10.6 -14.3 10.01.2014 0.0 88 100 97 -16.1 -8.6 -11.7 11.01.2014 39.8 100 100 100 -8.9 1.9 -2.3 12.01.2014 0.0 76 100 90 -0.1 5.4 3.1 13.01.2014 0.0 68 97 80 -0.9 1.3 -0.2 14.01.2014 1.4 68 100 90 -0.6 5.6 3.4 15.01.2014 0.0 71 100 87 -2.4 2.9 -0.1 16.01.2014 0.0 73 100 88 -7.5 -0.3 -4.3 17.01.2014 2.2 73 100 92 -8.2 -3.9 -5.6 18.01.2014 0.0 80 99 90 -4.9 -0.3 -2.6 19.01.2014 0.0 87 97 92 -11.6 -4.9 -8.7 20.01.2014 0.0 70 98 85 -11.3 -3.9 -6.8 21.01.2014 0.0 59 85 72 -16.8 -3.3 -10.0 22.01.2014 0.0 67 88 79 -23.3 -16.6 -19.2 23.01.2014 0.0 70 92 84 -24.6 -13.6 -18.2 24.01.2014 0.2 66 90 83 -20.9 -14.3 -16.8 25.01.2014 0.0 71 98 91 -20.6 -9.7 -14.9 26.01.2014 0.0 72 93 82 -16.3 -5.4 -10.2 27.01.2014 0.0 67 99 86 -19.2 -5.8 -13.3 28.01.2014 0.0 68 87 78 -20.1 -6.4 -14.4 29.01.2014 0.0 74 85 79 -23.9 -14.6 -19.4 30.01.2014 0.0 54 90 72 -21.2 -12.6 -16.2 31.01.2014 1.4 50 96 85 -14.9 -4.6 -9.2 01.02.2014 0.0 90 100 99 -5.2 -1.5 -3.2 02.02.2014 0.0 91 100 98 -4.2 0.2 -1.7 03.02.2014 0.0 72 97 89 -11.1 -1.9 -6.3 04.02.2014 0.0 66 99 88 -17.0 -5.9 -11.8 05.02.2014 0.0 82 97 91 -18.9 -2.2 -10.9 06.02.2014 0.0 77 93 86 -17.3 -7.3 -9.8 07.02.2014 0.0 75 92 83 -18.7 -9.5 -12.3

176

08.02.2014 0.0 75 91 83 -19.0 -13.2 -15.4 09.02.2014 0.0 81 95 91 -21.7 -11.2 -15.8 10.02.2014 0.0 73 95 88 -16.4 -7.9 -12.8 11.02.2014 0.0 68 93 83 -16.2 -10.1 -13.4 12.02.2014 0.0 60 93 82 -22.2 -12.3 -15.1 13.02.2014 0.0 57 86 75 -21.4 -8.6 -15.2 14.02.2014 0.0 85 99 92 -15.4 -3.6 -9.1 15.02.2014 0.0 77 96 88 -7.3 -4.6 -6.0 16.02.2014 0.0 62 94 86 -16.1 -5.9 -10.7 17.02.2014 0.0 58 91 75 -18.9 -4.6 -13.6 18.02.2014 0.0 69 100 88 -21.7 -5.9 -12.6 19.02.2014 1.6 77 96 89 -6.9 -2.8 -5.1 20.02.2014 0.0 92 100 99 -4.7 2.1 -2.4 21.02.2014 24.2 70 100 90 -7.7 0.6 -2.7 22.02.2014 0.0 45 86 63 -2.2 2.6 0.8 23.02.2014 0.0 59 80 70 -3.7 1.3 -0.7 24.02.2014 0.0 73 93 81 -9.7 -3.7 -6.5 25.02.2014 0.0 72 94 82 -12.8 -8.1 -9.6 26.02.2014 0.0 59 95 78 -15.3 -9.2 -11.3 27.02.2014 0.0 63 89 77 -20.7 -11.9 -15.0 28.02.2014 0.0 51 89 75 -21.1 -12.7 -16.1 01.03.2014 0.0 59 95 72 -24.6 -12.2 -17.2 02.03.2014 0.0 54 90 75 -13.9 -4.7 -9.8 03.03.2014 0.0 52 90 73 -20.9 -13.2 -15.9 04.03.2014 0.0 64 88 80 -22.8 -15.5 -19.2 05.03.2014 1.0 57 94 81 -22.4 -9.8 -14.0 06.03.2014 0.0 52 93 73 -15.8 -7.1 -11.3 07.03.2014 0.0 55 91 72 -14.9 -5.9 -10.3 08.03.2014 0.0 67 97 80 -10.8 4.2 -2.9 09.03.2014 0.0 57 95 83 -11.5 -0.4 -4.2 10.03.2014 0.0 71 90 81 -14.0 -0.6 -6.8 11.03.2014 0.0 55 92 77 -0.6 5.5 2.3 12.03.2014 0.0 64 100 88 1.6 9.2 4.7 13.03.2014 0.0 64 78 70 -16.3 2.0 -6.0 14.03.2014 0.0 61 85 71 -20.4 -7.3 -13.9 15.03.2014 0.0 68 100 82 -7.3 7.1 1.4 16.03.2014 0.0 51 89 70 -10.1 3.5 -2.9 17.03.2014 0.0 55 76 67 -15.9 -10.1 -12.7 18.03.2014 0.0 56 91 76 -18.2 -4.8 -11.1 19.03.2014 1.0 72 100 87 -8.2 2.3 -2.4 20.03.2014 2.2 72 100 91 -1.9 5.0 1.9 21.03.2014 0.0 53 90 75 -3.2 0.7 -0.8 22.03.2014 0.0 69 100 86 -7.5 1.9 -2.6 23.03.2014 0.0 62 90 77 -8.1 -0.1 -2.2

177

24.03.2014 0.0 55 92 76 -13.0 -7.7 -10.3 25.03.2014 0.2 53 97 83 -13.5 -5.6 -9.3 26.03.2014 0.0 57 81 70 -12.4 -2.9 -7.8 27.03.2014 0.0 55 100 76 -16.1 -8.6 -11.7 28.03.2014 8.4 75 100 92 -12.4 2.5 -3.0 29.03.2014 0.0 84 98 91 -1.2 6.6 2.9 30.03.2014 0.0 52 86 68 -1.1 1.0 0.2 31.03.2014 0.0 35 84 60 -4.1 3.7 -0.1 01.04.2014 0.0 60 83 72 -2.8 10.1 3.6 02.04.2014 0.0 73 87 81 -0.1 11.6 3.9 03.04.2014 0.0 64 88 73 -2.0 2.8 0.3 04.04.2014 13.0 71 100 90 -3.7 2.0 -0.3 05.04.2014 1.8 68 100 83 -0.5 6.3 1.8 06.04.2014 0.0 45 91 72 -2.7 2.0 -0.6 07.04.2014 2.5 54 95 65 -2.9 10.0 3.6 08.04.2014 11.7 67 100 91 0.9 9.3 4.2 09.04.2014 0.0 48 97 75 0.1 10.1 4.1 10.04.2014 0.0 42 88 64 -0.7 8.1 3.7 11.04.2014 0.2 29 97 69 1.4 16.6 9.1 12.04.2014 0.2 37 100 74 3.4 14.9 8.9 13.04.2014 6.4 58 100 80 2.0 17.0 9.2 14.04.2014 2.4 57 96 78 4.1 20.8 13.2 15.04.2014 5.4 67 100 89 0.6 19.5 11.6 16.04.2014 5.6 48 93 73 -8.1 0.6 -3.4 17.04.2014 0.0 42 83 65 -9.1 2.8 -2.9 18.04.2014 0.0 63 92 74 -2.3 11.1 3.8 19.04.2014 0.0 36 98 72 0.0 10.3 6.0 20.04.2014 0.0 25 70 52 -1.8 12.6 4.9 21.04.2014 0.0 28 65 45 1.8 19.1 10.2 22.04.2014 3.2 41 100 82 6.1 22.7 14.9 23.04.2014 0.0 44 95 75 2.1 14.4 6.9 24.04.2014 0.0 25 79 53 -1.5 8.6 3.5 25.04.2014 6.0 39 96 55 -1.7 12.1 5.6 26.04.2014 1.0 77 100 92 1.9 10.3 5.8 27.04.2014 0.0 40 95 69 1.0 6.9 4.2 28.04.2014 0.0 40 75 57 -0.6 12.6 5.9 29.04.2014 34.0 47 100 83 2.7 11.0 7.6 30.04.2014 7.0 91 100 98 5.3 9.6 7.4 01.05.2014 3.6 61 100 89 6.1 7.9 7.2 02.05.2014 2.8 73 100 91 6.1 11.6 8.5 03.05.2014 6.0 79 100 94 5.2 10.8 7.6 04.05.2014 0.0 61 99 79 4.9 10.6 7.8 05.05.2014 0.0 46 92 73 1.7 10.3 6.3 06.05.2014 0.0 40 92 68 1.8 12.2 7.6

178

07.05.2014 0.0 41 67 53 3.2 15.4 9.9 08.05.2014 0.0 49 77 63 5.6 14.3 9.4 09.05.2014 0.0 49 84 67 6.9 20.9 13.3 10.05.2014 0.2 34 96 65 9.6 25.1 17.6 11.05.2014 0.0 31 86 52 10.1 18.9 14.9 12.05.2014 0.2 42 92 65 5.6 22.2 15.4 13.05.2014 7.6 57 100 89 12.7 23.0 17.8 14.05.2014 11.2 84 100 95 14.9 28.2 20.0 15.05.2014 9.8 86 100 98 11.6 18.1 15.0 16.05.2014 0.0 72 98 85 8.8 17.7 14.2 17.05.2014 0.0 43 98 77 2.4 8.8 5.9 18.05.2014 0.4 43 98 74 2.0 11.4 6.7 19.05.2014 0.0 40 82 63 3.2 16.8 10.1 20.05.2014 21.6 45 100 69 6.6 20.4 14.2 21.05.2014 1.8 65 100 89 10.2 15.2 11.8 22.05.2014 4.4 75 100 94 10.2 23.4 16.7 23.05.2014 0.0 86 98 92 7.8 16.2 12.0 24.05.2014 0.0 33 98 67 6.2 13.6 10.0 25.05.2014 0.0 34 90 56 7.3 22.2 16.0 26.05.2014 0.0 35 80 56 8.0 25.3 18.3 27.05.2014 0.0 52 86 71 12.4 27.6 21.0 28.05.2014 0.0 63 95 78 16.6 27.1 21.8 29.05.2014 0.0 48 97 75 10.6 21.4 16.2 30.05.2014 0.0 41 96 69 9.3 21.2 14.9 31.05.2014 0.0 25 71 49 10.5 24.9 18.2 01.06.2014 0.0 31 63 48 12.8 22.2 17.7 02.06.2014 0.0 48 89 71 11.5 26.4 19.4 03.06.2014 15.6 54 100 83 15.1 27.8 21.7 04.06.2014 0.0 42 88 65 12.9 26.2 20.3 05.06.2014 0.0 46 93 68 9.9 20.1 15.9 06.06.2014 0.0 35 95 64 8.1 19.5 13.7 07.06.2014 0.0 25 86 47 7.9 23.9 16.6 08.06.2014 3.8 45 94 70 7.3 25.6 18.3 09.06.2014 0.0 46 94 72 13.6 18.2 16.3 10.06.2014 0.0 40 88 61 12.9 24.9 19.2 11.06.2014 0.6 61 97 77 14.6 25.9 20.6 12.06.2014 4.8 68 100 87 14.9 24.3 19.8 13.06.2014 0.4 58 100 82 18.2 24.9 20.6 14.06.2014 0.0 58 92 73 10.2 20.6 16.5 15.06.2014 0.0 44 93 71 9.2 18.4 13.4 16.06.2014 0.0 35 89 68 9.2 24.4 16.3 17.06.2014 0.8 45 92 70 12.9 29.7 22.3 18.06.2014 1.8 58 95 83 17.5 29.7 22.8 19.06.2014 0.2 25 99 71 16.9 25.7 19.8

179

20.06.2014 0.0 26 70 49 13.6 25.3 20.1 21.06.2014 0.0 31 62 48 12.2 23.4 18.1 22.06.2014 0.0 35 78 52 14.9 24.7 19.3 23.06.2014 4.4 44 95 67 12.7 26.7 20.3 24.06.2014 12.8 76 100 94 15.5 26.9 20.1 25.06.2014 0.4 70 100 91 17.7 25.6 21.2 26.06.2014 2.0 58 100 85 17.6 24.2 20.3 27.06.2014 0.2 50 100 77 17.2 25.7 20.6 28.06.2014 0.0 43 86 68 14.8 27.3 21.4 29.06.2014 1.4 50 97 84 17.1 29.8 23.4 30.06.2014 0.0 64 100 86 19.2 28.2 23.1 01.07.2014 0.0 61 96 80 20.4 28.6 24.6 02.07.2014 12.2 57 100 84 19.8 27.0 23.9 03.07.2014 1.4 67 96 83 16.1 27.2 20.8 04.07.2014 0.0 44 100 74 11.2 19.1 15.6 05.07.2014 0.0 36 96 64 8.3 21.3 15.2 06.07.2014 0.0 48 94 68 8.4 25.3 18.1 07.07.2014 35.0 60 100 85 14.3 25.4 20.7 08.07.2014 20.2 74 100 94 18.8 26.7 22.0 09.07.2014 0.0 57 100 84 14.2 22.8 18.7 10.07.2014 0.0 57 92 75 11.8 20.1 16.3 11.07.2014 0.0 49 95 71 11.3 22.5 17.1 12.07.2014 0.0 53 98 74 11.6 25.5 19.3 13.07.2014 7.6 63 100 82 15.2 26.9 21.5 14.07.2014 0.0 66 90 79 17.1 26.0 22.3 15.07.2014 1.2 54 98 82 13.9 23.4 18.3 16.07.2014 0.0 72 100 88 12.4 21.6 17.1 17.07.2014 0.0 44 100 77 9.7 18.1 14.6 18.07.2014 0.0 44 100 71 8.7 21.2 15.8 19.07.2014 0.4 65 100 85 9.4 25.2 18.3 20.07.2014 0.4 68 100 91 15.2 20.4 17.4 21.07.2014 0.0 48 100 80 16.9 24.6 20.3 22.07.2014 0.0 48 100 75 14.4 27.8 21.4 23.07.2014 2.2 67 97 83 15.9 28.6 23.1 24.07.2014 0.0 45 96 71 12.4 23.1 18.6 25.07.2014 0.0 50 100 76 11.6 22.6 16.8 26.07.2014 0.6 52 93 77 8.9 23.3 16.7 27.07.2014 14.2 67 100 84 15.4 24.3 19.7 28.07.2014 28.2 65 100 90 16.0 25.9 20.6 29.07.2014 0.6 61 100 85 10.3 19.2 15.2 30.07.2014 1.8 55 100 87 9.7 18.9 14.7 31.07.2014 0.2 56 100 80 10.9 21.3 15.7 01.08.2014 0.2 59 100 83 10.7 22.9 17.1 02.08.2014 0.0 63 100 84 14.1 25.9 19.0

180

03.08.2014 0.0 50 95 76 14.8 25.9 19.1 04.08.2014 0.0 63 100 84 15.0 26.8 20.7 05.08.2014 3.2 62 100 88 15.3 26.9 20.6 06.08.2014 0.0 47 100 77 15.1 23.9 18.8 07.08.2014 0.0 47 100 77 13.5 24.1 18.5 08.08.2014 0.0 51 92 72 11.2 24.0 17.7 09.08.2014 0.0 49 94 71 12.4 23.9 18.1 10.08.2014 0.0 54 90 70 12.5 25.1 19.1 11.08.2014 0.8 67 100 85 14.3 26.3 20.2 12.08.2014 17.2 69 100 91 16.5 25.8 19.9 13.08.2014 0.2 66 100 89 15.8 23.9 19.7 14.08.2014 0.0 57 100 81 11.6 17.6 15.2 15.08.2014 0.0 50 97 76 9.9 15.9 12.6 16.08.2014 0.8 70 94 85 9.3 20.1 14.4 17.08.2014 0.0 61 100 87 10.7 19.1 15.4 18.08.2014 0.0 60 100 83 11.9 22.8 17.2 19.08.2014 0.0 62 100 83 11.6 22.3 16.2 20.08.2014 8.4 78 100 94 10.9 25.1 17.6 21.08.2014 0.2 66 100 89 15.4 24.3 19.3 22.08.2014 0.0 65 100 88 15.9 25.4 20.1 23.08.2014 0.0 78 100 92 15.8 26.3 20.4 24.08.2014 0.0 75 100 91 16.6 23.1 19.1 25.08.2014 0.0 50 100 80 16.1 23.0 19.2 26.08.2014 0.0 63 100 85 15.2 26.6 20.4 27.08.2014 0.0 62 100 81 16.7 29.2 22.8 28.08.2014 0.0 52 100 78 10.6 21.5 17.4 29.08.2014 0.0 55 100 79 8.2 21.1 14.8 30.08.2014 0.0 56 98 80 10.1 23.7 16.8 31.08.2014 0.0 63 97 80 15.2 28.9 22.3 01.09.2014 3.0 76 100 92 18.7 26.8 23.1 02.09.2014 8.4 83 100 97 18.4 25.7 22.1 03.09.2014 0.0 49 100 83 14.0 22.2 19.3 04.09.2014 0.0 53 100 82 12.7 26.4 19.3 05.09.2014 24.2 71 100 85 14.6 27.3 21.3 06.09.2014 27.8 58 100 88 18.6 28.8 22.7 07.09.2014 0.0 43 100 79 10.7 19.8 16.1 08.09.2014 0.0 53 100 80 8.8 21.9 14.9 09.09.2014 0.0 80 100 91 10.3 23.5 16.7 10.09.2014 44.8 88 100 98 14.6 21.4 17.8 11.09.2014 19.2 83 100 94 15.2 22.2 19.4 12.09.2014 0.0 70 100 87 9.6 22.1 13.4 13.09.2014 4.2 77 100 91 8.3 12.8 10.6 14.09.2014 0.0 54 100 85 6.3 13.5 9.7 15.09.2014 0.2 67 100 86 2.2 15.1 9.5

181

16.09.2014 0.2 53 100 84 6.9 16.6 11.8 17.09.2014 0.0 49 100 79 7.4 17.2 11.7 18.09.2014 0.0 55 100 82 6.1 18.8 12.2 19.09.2014 0.0 55 99 81 4.7 15.4 10.3 20.09.2014 0.0 64 100 87 3.9 16.1 9.4 21.09.2014 8.0 67 100 89 7.6 22.9 16.9 22.09.2014 0.0 61 96 81 8.6 20.4 16.0 23.09.2014 0.0 53 100 81 5.4 12.8 8.8 24.09.2014 0.0 51 100 81 5.6 20.2 13.1 25.09.2014 0.0 55 100 82 8.8 22.8 15.4 26.09.2014 0.0 46 100 76 10.8 22.3 15.8 27.09.2014 0.0 45 100 75 8.8 24.6 16.9 28.09.2014 0.0 44 98 74 10.4 25.9 17.9 29.09.2014 0.0 49 100 78 12.1 25.7 18.5 30.09.2014 13.2 76 100 96 11.2 24.0 17.3 01.10.2014 0.0 90 100 97 12.3 17.1 14.9 02.10.2014 0.0 62 100 91 13.3 16.2 14.6 03.10.2014 10.8 70 100 94 14.2 21.3 16.1 04.10.2014 9.0 62 100 90 11.9 21.8 16.1 05.10.2014 0.0 56 100 84 2.6 11.9 7.5 06.10.2014 2.0 60 100 88 2.1 11.2 6.7 07.10.2014 8.8 58 100 92 6.9 16.7 10.8 08.10.2014 0.2 54 100 83 7.7 15.3 11.1 09.10.2014 0.0 54 100 77 4.5 10.7 8.8 10.10.2014 0.0 56 98 81 1.7 12.8 6.9 11.10.2014 0.0 48 100 79 2.1 10.9 6.7 12.10.2014 0.0 48 100 75 0.4 12.4 5.8 13.10.2014 0.4 85 100 97 -0.5 13.8 7.1 14.10.2014 2.4 76 100 94 6.9 17.3 13.4 15.10.2014 11.8 82 100 96 14.1 20.9 17.6 16.10.2014 0.0 73 100 96 10.8 17.2 14.2 17.10.2014 0.0 84 100 95 10.7 17.8 13.0 18.10.2014 2.2 76 100 92 10.4 14.4 12.3 19.10.2014 0.0 61 95 77 3.6 10.4 6.6 20.10.2014 1.6 80 100 88 -1.1 8.9 4.2 21.10.2014 9.2 92 100 98 4.0 11.9 8.8 22.10.2014 0.0 59 100 81 5.0 9.3 7.1 23.10.2014 0.0 57 100 82 2.1 13.3 7.3 24.10.2014 0.0 50 100 87 1.8 15.3 7.7 25.10.2014 0.0 63 99 85 0.1 15.1 7.4 26.10.2014 0.0 68 91 76 6.9 15.6 10.4 27.10.2014 0.0 58 100 86 1.7 9.6 6.9 28.10.2014 4.4 76 100 89 -0.6 13.9 7.3 29.10.2014 0.0 67 100 82 7.8 19.6 13.8

182

30.10.2014 0.0 66 100 87 2.8 10.1 6.8 31.10.2014 6.0 88 100 97 0.9 7.7 4.4 01.11.2014 1.2 66 100 88 0.7 6.0 4.2 02.11.2014 0.0 49 95 76 -0.6 2.8 0.6 03.11.2014 0.0 37 90 65 -4.0 6.7 0.7 04.11.2014 6.4 53 100 71 0.3 12.7 7.4 05.11.2014 1.8 64 100 84 5.3 11.9 9.7 06.11.2014 0.0 77 100 92 2.4 8.6 5.1 07.11.2014 1.2 62 100 84 2.3 9.9 5.4 08.11.2014 5.0 76 100 91 -2.3 3.4 0.8 09.11.2014 0.8 72 100 90 -1.3 5.3 2.1 10.11.2014 0.0 53 95 78 -1.2 5.4 2.7 11.11.2014 0.0 55 95 77 2.7 12.7 6.2 12.11.2014 0.0 68 99 81 4.1 15.7 10.0 13.11.2014 0.0 71 97 83 -1.4 4.5 0.4 14.11.2014 0.4 81 99 90 -5.4 -1.0 -2.7 15.11.2014 0.0 62 100 87 -5.2 -0.7 -3.3 16.11.2014 0.0 72 88 82 -4.8 -1.0 -2.9 17.11.2014 0.0 83 100 98 -4.4 0.3 -2.1 18.11.2014 0.0 73 92 80 -9.6 -0.7 -3.4 19.11.2014 0.0 60 100 85 -11.2 -7.2 -9.2 20.11.2014 0.0 73 100 87 -13.7 -2.4 -7.3 21.11.2014 0.0 60 97 85 -11.1 -5.0 -7.1 22.11.2014 7.0 72 100 88 -13.0 -4.9 -9.1 23.11.2014 5.6 94 100 99 -9.4 5.1 0.8 24.11.2014 49.0 75 100 93 4.3 7.9 6.3 25.11.2014 0.0 78 90 83 1.4 13.9 8.5 26.11.2014 0.0 76 93 86 -1.1 1.5 -0.2 27.11.2014 0.4 81 100 95 -1.7 0.1 -1.1 28.11.2014 0.0 73 99 84 -6.4 0.1 -2.2 29.11.2014 0.4 75 100 88 -8.6 -5.3 -6.8 30.11.2014 0.0 77 98 89 -6.8 7.2 0.2 01.12.2014 0.0 65 98 82 1.2 12.1 8.7 02.12.2014 0.0 72 100 82 -9.1 1.2 -3.3 03.12.2014 0.0 78 100 96 -10.3 -1.3 -5.3 04.12.2014 0.0 66 87 77 -3.9 0.1 -1.0 05.12.2014 0.0 75 88 84 -7.0 -2.1 -4.4 06.12.2014 0.0 66 100 88 -5.2 1.6 -0.9 07.12.2014 0.0 48 87 70 -4.1 3.3 0.2 08.12.2014 0.0 53 88 76 -6.9 -1.3 -4.3 09.12.2014 0.0 65 100 90 -5.1 2.7 -0.9 10.12.2014 0.0 78 100 90 -0.3 3.2 1.6 11.12.2014 0.0 52 97 83 -4.8 -0.3 -3.3 12.12.2014 0.0 75 100 84 -3.8 0.6 -1.7

183

13.12.2014 0.0 100 100 100 -4.9 3.1 -0.6 14.12.2014 0.2 99 100 100 -1.4 2.7 0.1 15.12.2014 0.0 100 100 100 1.6 3.0 2.2 16.12.2014 5.2 100 100 100 1.7 4.2 2.9 17.12.2014 1.2 94 100 99 0.9 4.9 2.7 18.12.2014 0.0 88 100 96 -2.6 4.9 0.1 19.12.2014 0.0 76 97 91 -5.8 -1.9 -2.8 20.12.2014 0.0 79 100 93 -7.3 -3.7 -5.8 21.12.2014 0.0 71 97 87 -7.9 -1.3 -4.6 22.12.2014 0.0 80 93 88 -4.6 -0.7 -3.1 23.12.2014 0.0 93 100 99 -3.3 1.1 -1.3 24.12.2014 11.6 100 100 100 -1.0 4.2 2.2 25.12.2014 12.4 81 100 93 1.7 9.9 6.5 26.12.2014 0.0 86 100 93 0.3 2.9 2.0 27.12.2014 0.0 77 100 96 -1.1 5.0 1.8 28.12.2014 2.8 76 100 87 0.8 8.1 4.9 29.12.2014 0.0 56 89 76 -1.9 2.8 -0.1 30.12.2014 0.0 61 89 75 -5.5 -1.3 -3.6 31.12.2014 0.0 62 90 75 -9.0 -5.2 -6.9 01.01.2015 0.0 60 76 68 -10.6 -7.9 -9.2 02.01.2015 0.0 62 93 77 -8.8 -3.4 -6.1 03.01.2015 0.0 80 100 91 -8.1 -2.2 -4.8 04.01.2015 25.8 95 100 100 -6.8 0.2 -2.6 05.01.2015 0.0 79 100 88 -7.8 1.6 -1.2 06.01.2015 0.0 71 92 83 -15.1 -7.7 -13.4 07.01.2015 0.0 62 95 80 -14.4 -10.6 -12.2 08.01.2015 0.0 68 91 86 -19.4 -11.4 -15.9 09.01.2015 0.0 72 94 89 -16.6 -10.8 -13.7 10.01.2015 0.0 72 89 83 -15.8 -9.6 -11.9 11.01.2015 0.0 61 89 75 -17.9 -10.8 -14.7 12.01.2015 0.0 72 100 91 -10.8 -2.3 -5.1 13.01.2015 0.0 54 85 73 -15.8 -2.5 -6.1 14.01.2015 0.0 77 96 89 -22.4 -12.2 -16.8 15.01.2015 0.0 75 95 89 -17.9 -10.3 -14.7 16.01.2015 0.0 67 100 81 -16.1 -4.1 -9.7 17.01.2015 0.0 77 92 86 -18.3 -3.4 -9.8 18.01.2015 6.2 68 100 88 -19.8 4.9 -6.7 19.01.2015 0.0 83 99 90 -1.6 4.4 0.8 20.01.2015 0.0 54 96 82 -10.3 -1.6 -4.9 21.01.2015 0.0 61 86 75 -13.8 -4.6 -9.8 22.01.2015 0.0 74 99 89 -15.8 -4.3 -8.8 23.01.2015 0.0 81 96 87 -10.8 -3.5 -6.3 24.01.2015 0.0 95 100 99 -6.4 -2.2 -4.7 25.01.2015 0.0 48 100 78 -5.3 -0.3 -3.3

184

26.01.2015 0.0 59 89 77 -14.1 -3.7 -10.3 27.01.2015 0.0 59 81 71 -14.0 -8.3 -11.1 28.01.2015 0.0 29 93 71 -15.3 -8.1 -11.4 29.01.2015 0.0 44 100 79 -16.9 -2.8 -10.1 30.01.2015 0.0 74 100 86 -7.0 -1.6 -3.7 31.01.2015 0.0 74 92 82 -15.1 -2.7 -11.6 01.02.2015 0.0 80 100 88 -13.3 -3.9 -7.7 02.02.2015 0.0 69 94 86 -14.1 -3.9 -9.5 03.02.2015 0.0 75 92 83 -18.4 -11.9 -14.8 04.02.2015 0.0 83 100 97 -18.1 -6.7 -11.5 05.02.2015 0.0 54 95 77 -11.6 -2.5 -5.6 06.02.2015 0.0 74 90 80 -17.7 -11.1 -14.4 07.02.2015 0.4 77 95 86 -14.9 -6.7 -9.8 08.02.2015 0.0 86 100 98 -6.7 -1.0 -3.6 09.02.2015 0.0 81 96 91 -13.7 -4.5 -9.2 10.02.2015 3.0 56 97 85 -13.9 -9.3 -11.6 11.02.2015 0.0 82 100 94 -10.9 -3.0 -8.9 12.02.2015 0.4 74 100 86 -9.9 -3.2 -5.9 13.02.2015 0.0 63 90 81 -23.9 -4.9 -15.7 14.02.2015 0.0 73 97 88 -24.3 -9.8 -16.3 15.02.2015 0.0 58 82 72 -23.2 -7.8 -13.3 16.02.2015 0.0 57 87 74 -27.3 -21.7 -24.7 17.02.2015 0.0 39 89 68 -29.6 -14.2 -20.8 18.02.2015 0.6 71 98 90 -20.8 -10.4 -14.9 19.02.2015 0.0 56 92 80 -19.3 -8.3 -12.9 20.02.2015 0.0 46 86 74 -25.9 -17.6 -20.9 21.02.2015 0.0 74 97 84 -27.7 -17.0 -21.7 22.02.2015 0.0 77 98 91 -18.7 -7.1 -11.9 23.02.2015 0.0 67 92 80 -18.7 -6.1 -12.7 24.02.2015 0.0 72 94 80 -24.9 -18.3 -21.3 25.02.2015 0.0 64 96 80 -22.3 -9.4 -14.4 26.02.2015 0.0 54 88 74 -18.1 -8.2 -14.5 27.02.2015 0.0 63 90 79 -19.7 -12.2 -15.6 28.02.2015 0.0 52 91 76 -23.3 -13.3 -17.9 01.03.2015 0.2 74 91 83 -20.7 -7.1 -14.1 02.03.2015 0.0 66 97 85 -14.6 -5.2 -9.6 03.03.2015 0.0 72 100 94 -14.1 -5.5 -8.6 04.03.2015 0.0 72 100 86 -14.1 -0.1 -7.1 05.03.2015 0.0 63 88 75 -13.4 -1.1 -4.6 06.03.2015 0.0 65 92 81 -19.3 -12.3 -16.1 07.03.2015 0.0 70 95 80 -20.3 -7.1 -12.5 08.03.2015 1.6 62 96 82 -8.0 -0.8 -4.3 09.03.2015 0.6 58 98 80 -5.6 1.3 -1.9 10.03.2015 0.0 72 100 89 -4.1 4.8 0.3

185

11.03.2015 0.0 69 100 88 -4.3 6.0 0.9 12.03.2015 0.0 26 84 63 -1.2 6.7 2.4 13.03.2015 0.0 41 79 58 -4.8 3.6 -1.1 14.03.2015 0.2 78 100 94 -4.4 4.5 0.1 15.03.2015 0.0 81 98 88 0.1 3.6 2.1 16.03.2015 1.0 77 100 92 -2.1 3.2 -0.2 17.03.2015 0.2 43 100 81 -1.8 10.9 3.8 18.03.2015 0.0 58 86 71 -4.7 8.6 -0.1 19.03.2015 0.0 39 89 64 -7.8 0.3 -3.8 20.03.2015 0.0 40 75 58 -9.2 2.2 -3.3 21.03.2015 7.0 56 100 86 -2.9 6.6 1.7 22.03.2015 0.0 58 86 72 -6.6 8.5 2.0 23.03.2015 0.0 39 90 65 -9.9 -4.7 -7.2 24.03.2015 0.0 43 72 60 -11.1 -2.9 -7.0 25.03.2015 4.0 62 100 86 -9.5 2.7 -3.7 26.03.2015 0.0 83 100 92 -4.7 9.7 1.1 27.03.2015 0.0 75 98 87 -0.7 6.4 1.7 28.03.2015 0.0 52 77 64 -9.7 -0.7 -5.7 29.03.2015 0.0 43 94 70 -11.9 -3.4 -7.6 30.03.2015 2.2 61 100 87 -9.9 3.1 -2.0 31.03.2015 0.0 55 93 80 -2.2 3.6 1.4 01.04.2015 0.0 35 99 71 -3.1 5.2 0.7 02.04.2015 2.6 39 100 82 -3.4 7.6 1.9 03.04.2015 4.8 73 100 97 -0.9 15.6 8.4 04.04.2015 0.0 55 100 83 -0.6 11.2 7.3 05.04.2015 3.4 85 100 94 -2.4 2.5 -0.6 06.04.2015 0.0 65 100 87 -3.9 0.2 -1.1 07.04.2015 0.0 42 91 74 -1.4 9.6 2.9 08.04.2015 20.4 48 100 81 0.7 5.3 2.7 09.04.2015 5.8 100 100 100 -0.3 2.9 0.8 10.04.2015 21.2 73 100 89 0.6 14.6 3.7 11.04.2015 0.0 54 91 76 2.3 15.6 9.3 12.04.2015 0.0 33 90 66 0.8 8.6 4.4 13.04.2015 9.2 42 100 64 1.6 17.1 9.4 14.04.2015 0.0 28 91 65 5.3 21.2 11.9 15.04.2015 0.0 28 73 42 3.7 14.8 9.4 16.04.2015 0.0 28 98 55 2.3 15.8 9.4 17.04.2015 3.4 38 100 78 3.5 18.9 9.3 18.04.2015 0.0 22 94 59 8.4 19.4 13.3 19.04.2015 0.0 35 72 58 6.3 17.6 12.1 20.04.2015 26.8 53 100 87 3.4 9.6 6.7 21.04.2015 1.0 52 96 77 5.7 16.7 10.9 22.04.2015 0.2 61 93 80 2.9 9.0 5.9 23.04.2015 0.0 67 100 83 -1.5 4.9 2.0

186

24.04.2015 0.0 49 90 72 -2.9 -0.4 -1.3 25.04.2015 0.0 43 91 64 -2.4 4.5 0.6 26.04.2015 0.0 53 81 65 -2.9 10.7 4.2 27.04.2015 0.0 64 98 80 0.9 10.7 5.9 28.04.2015 0.0 54 98 77 1.9 9.6 5.5 29.04.2015 0.0 43 94 70 0.7 15.8 8.6 30.04.2015 0.0 45 99 73 2.6 17.9 10.2 01.05.2015 0.0 34 94 63 2.3 18.2 10.8 02.05.2015 0.0 32 90 57 5.5 20.3 13.8 03.05.2015 0.0 30 82 54 5.0 21.9 14.2 04.05.2015 0.2 40 75 61 7.7 24.9 16.9 05.05.2015 0.0 43 91 62 11.6 19.6 16.1 06.05.2015 0.0 30 100 70 7.6 16.1 12.2 07.05.2015 0.0 40 69 52 8.7 21.8 15.3 08.05.2015 0.0 37 82 61 9.7 27.3 19.2 09.05.2015 0.0 40 95 65 13.9 29.9 22.0 10.05.2015 0.2 53 100 83 15.0 28.6 22.2 11.05.2015 0.4 47 100 83 16.4 27.8 21.3 12.05.2015 0.4 58 96 79 13.7 26.8 19.7 13.05.2015 1.0 48 100 78 5.9 18.3 12.3 14.05.2015 0.0 29 87 54 3.6 12.6 7.5 15.05.2015 0.0 40 75 62 1.0 18.3 10.8 16.05.2015 0.0 56 99 78 9.3 17.6 13.7 17.05.2015 0.0 63 93 76 12.9 23.4 18.3 18.05.2015 0.0 53 100 78 12.4 25.6 18.3 19.05.2015 0.0 67 96 79 15.1 27.3 21.7 20.05.2015 0.0 31 87 64 3.3 18.0 10.7 21.05.2015 0.0 23 62 42 2.2 14.1 7.5 22.05.2015 0.0 21 71 48 6.8 17.8 12.6 23.05.2015 0.0 23 82 45 2.4 13.2 8.7 24.05.2015 0.0 28 53 39 -0.9 21.4 11.4 25.05.2015 0.0 34 71 58 8.8 26.7 18.8 26.05.2015 0.0 55 94 74 15.6 25.7 21.3 27.05.2015 2.6 59 99 83 17.4 26.7 22.3 28.05.2015 0.0 46 100 76 16.1 25.9 20.2 29.05.2015 0.0 42 84 65 12.2 25.7 18.7 30.05.2015 41.0 63 100 85 14.0 27.6 20.8 31.05.2015 32.2 86 100 97 11.2 26.7 19.8 01.06.2015 0.0 71 94 81 6.5 11.2 7.9 02.06.2015 0.0 51 100 78 5.7 15.2 10.9 03.06.2015 0.0 48 95 69 7.0 20.2 14.1 04.06.2015 0.0 59 92 76 8.7 19.8 14.5 05.06.2015 0.0 61 100 84 9.8 23.7 17.2 06.06.2015 0.0 29 92 68 13.7 25.2 18.9

187

07.06.2015 0.0 33 74 59 10.3 19.9 14.8 08.06.2015 59.2 65 100 92 8.5 23.6 17.3 09.06.2015 8.2 67 100 90 15.9 21.6 18.4 10.06.2015 0.0 61 100 83 13.6 20.2 17.1 11.06.2015 0.0 56 97 79 12.3 28.2 18.9 12.06.2015 9.8 73 100 91 11.1 22.5 16.9 13.06.2015 0.0 67 100 90 14.3 20.1 16.3 14.06.2015 8.2 76 100 94 12.9 22.9 17.3 15.06.2015 1.6 67 100 90 15.7 23.2 19.1 16.06.2015 0.2 56 100 83 18.1 26.3 22.2 17.06.2015 0.0 67 92 78 13.8 23.7 20.2 18.06.2015 0.0 67 94 79 12.8 22.6 17.6 19.06.2015 2.4 50 100 83 13.8 24.8 19.0 20.06.2015 0.0 58 88 77 13.5 23.1 17.3 21.06.2015 0.0 61 100 83 10.9 24.2 18.1 22.06.2015 4.6 52 100 77 16.8 24.0 20.6 23.06.2015 9.8 72 100 88 14.3 26.9 20.7 24.06.2015 0.0 44 100 77 12.7 23.1 18.8 25.06.2015 0.0 41 79 64 10.1 24.3 17.9 26.06.2015 0.0 53 100 79 14.8 25.8 19.4 27.06.2015 12.6 54 100 77 14.2 22.3 18.1 28.06.2015 9.2 86 100 97 13.2 17.5 15.1 29.06.2015 7.8 63 100 87 11.8 15.5 13.7 30.06.2015 0.4 72 100 92 12.1 24.0 17.6 01.07.2015 0.0 78 100 91 14.4 19.1 16.8 02.07.2015 0.0 47 100 77 12.8 20.4 16.9 03.07.2015 0.0 41 91 62 10.8 21.4 16.4 04.07.2015 0.0 43 94 68 8.7 23.1 16.8 05.07.2015 0.0 54 99 75 10.6 24.8 18.2 06.07.2015 0.0 48 96 75 13.7 26.9 21.0 07.07.2015 21.8 60 100 89 15.9 28.0 21.8 08.07.2015 0.0 60 100 80 15.1 24.9 19.3 09.07.2015 0.0 58 99 82 13.0 19.6 16.4 10.07.2015 0.0 47 100 76 13.3 23.2 17.6 11.07.2015 0.0 51 100 75 11.3 25.3 18.9 12.07.2015 0.0 55 97 74 12.6 27.4 20.9 13.07.2015 0.0 57 100 77 15.6 26.8 21.2 14.07.2015 9.6 73 100 95 14.6 27.1 21.7 15.07.2015 0.0 49 94 74 17.8 23.7 20.2 16.07.2015 0.0 52 100 74 11.1 22.1 16.7 17.07.2015 8.0 70 100 91 10.0 24.0 17.6 18.07.2015 6.6 65 100 89 14.4 23.8 19.2 19.07.2015 3.4 67 99 87 18.6 29.9 24.1 20.07.2015 0.0 58 100 82 18.8 28.7 23.9

188

21.07.2015 0.0 53 97 74 14.6 26.6 21.3 22.07.2015 0.0 51 94 71 16.7 23.1 19.9 23.07.2015 0.0 44 94 68 11.5 22.2 17.3 24.07.2015 0.0 39 100 70 11.9 24.8 18.4 25.07.2015 0.0 56 100 78 10.2 27.2 20.1 26.07.2015 0.0 50 100 80 17.4 28.8 22.7 27.07.2015 0.0 45 91 70 14.2 29.4 22.3 28.07.2015 0.0 43 97 69 17.9 30.9 24.4 29.07.2015 0.0 46 100 74 17.2 31.0 24.7 30.07.2015 0.0 44 95 70 18.1 31.1 24.9 31.07.2015 0.0 48 100 73 16.5 26.8 22.3 01.08.2015 0.2 50 100 79 14.2 27.5 20.8 02.08.2015 0.0 44 100 75 13.6 25.0 19.4 03.08.2015 13.0 51 100 82 13.5 27.2 20.7 04.08.2015 3.6 61 100 83 16.1 24.3 19.2 05.08.2015 0.0 59 100 83 15.2 23.1 18.4 06.08.2015 0.0 49 100 79 11.7 21.0 16.2 07.08.2015 0.0 49 93 71 9.4 24.2 17.4 08.08.2015 0.0 63 94 78 13.1 24.6 18.8 09.08.2015 0.0 53 100 78 14.5 20.9 17.4 10.08.2015 16.6 64 100 89 13.1 24.9 18.8 11.08.2015 0.2 72 100 91 14.2 20.7 17.1 12.08.2015 0.0 52 100 83 14.7 22.4 18.5 13.08.2015 0.0 59 100 79 10.6 21.4 16.7 14.08.2015 12.2 74 100 93 8.9 26.0 18.7 15.08.2015 0.0 61 100 87 18.2 26.1 21.2 16.08.2015 0.0 53 100 81 18.0 28.3 22.7 17.08.2015 0.0 53 100 84 16.4 29.3 22.9 18.08.2015 0.0 72 100 90 19.1 30.3 24.3 19.08.2015 0.0 64 100 89 19.2 26.0 22.2 20.08.2015 8.8 63 100 87 18.1 28.1 23.0 21.08.2015 0.0 60 93 78 15.2 23.6 20.7 22.08.2015 0.0 52 100 76 12.8 21.4 16.8 23.08.2015 0.0 51 100 79 11.2 25.6 18.8 24.08.2015 1.2 47 100 78 13.7 24.9 19.2 25.08.2015 0.0 65 96 82 13.5 22.4 17.8 26.08.2015 0.2 79 100 91 10.8 17.8 14.5 27.08.2015 0.0 67 100 87 12.3 17.9 14.7 28.08.2015 0.0 55 100 83 11.6 19.8 15.2 29.08.2015 0.0 58 100 87 8.9 22.8 16.2 30.08.2015 3.4 64 100 87 13.7 24.5 18.6 31.08.2015 0.0 65 100 88 16.7 26.7 21.0 01.09.2015 0.0 59 100 86 16.9 27.0 21.4 02.09.2015 2.2 57 100 86 14.7 28.9 21.4

189

03.09.2015 2.0 53 100 90 18.5 30.9 23.9 04.09.2015 6.4 58 100 89 18.1 30.8 22.3 05.09.2015 0.0 47 100 75 17.0 26.2 21.4 06.09.2015 0.0 62 100 84 17.4 29.1 22.8 07.09.2015 0.0 53 100 82 17.9 29.2 23.5 08.09.2015 7.4 65 100 89 19.3 31.6 24.8 09.09.2015 1.2 63 100 85 20.3 29.4 24.1 10.09.2015 0.0 38 95 67 12.8 24.2 19.8 11.09.2015 1.0 55 95 80 11.4 23.7 17.4 12.09.2015 6.0 61 100 85 11.4 21.2 15.4 13.09.2015 0.0 62 96 78 9.9 17.7 13.4 14.09.2015 0.0 45 100 78 6.7 15.7 11.3 15.09.2015 0.0 46 100 76 5.4 22.8 14.3 16.09.2015 0.0 50 93 68 12.6 27.4 19.2 17.09.2015 0.0 45 86 67 13.9 27.7 20.3 18.09.2015 0.2 53 100 82 13.7 27.6 20.1 19.09.2015 7.6 77 100 94 14.1 25.1 18.4 20.09.2015 0.0 44 100 79 10.1 23.0 16.7 21.09.2015 0.0 50 88 71 7.4 19.6 13.6 22.09.2015 0.0 52 100 83 9.4 20.1 14.2 23.09.2015 0.0 30 100 72 9.3 22.6 15.5 24.09.2015 0.0 43 98 72 9.1 25.8 17.6 25.09.2015 0.0 63 95 81 13.9 25.3 18.7 26.09.2015 0.0 65 94 82 13.7 22.5 16.9 27.09.2015 0.0 62 100 86 10.8 21.7 15.6 28.09.2015 0.8 79 100 93 11.4 22.6 16.9 29.09.2015 4.6 83 100 97 16.2 21.0 18.5 30.09.2015 0.0 51 95 78 13.3 21.6 17.7 01.10.2015 0.0 40 83 64 7.6 17.6 12.4 02.10.2015 0.0 43 72 60 3.9 14.1 8.7 03.10.2015 3.8 49 97 69 4.9 11.7 7.9 04.10.2015 0.8 75 100 92 5.6 9.5 7.4 05.10.2015 0.0 74 100 90 7.8 16.1 10.8 06.10.2015 0.0 79 100 92 10.4 18.1 13.7 07.10.2015 0.0 63 100 88 9.9 16.2 13.7 08.10.2015 0.0 60 100 85 7.5 19.8 13.2 09.10.2015 11.2 83 100 95 5.9 18.6 11.3 10.10.2015 0.0 53 100 85 4.2 14.7 10.9 11.10.2015 0.0 39 95 75 3.3 15.9 9.4 12.10.2015 0.0 50 97 71 7.7 22.0 14.9 13.10.2015 0.2 55 98 78 12.1 21.6 17.3 14.10.2015 0.0 62 98 86 9.4 17.1 12.7 15.10.2015 8.9 40 100 82 3.6 11.3 8.2 16.10.2015 10.5 74 100 93 2.1 17.1 8.4

190

17.10.2015 0.0 58 100 85 0.8 10.6 5.4 18.10.2015 1.0 53 100 83 -2.6 4.9 0.9 19.10.2015 0.0 48 95 74 -3.1 5.4 1.1 20.10.2015 0.0 50 87 60 -3.3 13.8 7.4 21.10.2015 2.6 70 100 90 10.9 17.2 12.9 22.10.2015 0.0 59 90 71 10.7 16.6 13.4 23.10.2015 0.0 46 97 76 4.3 16.4 11.8 24.10.2015 5.6 77 100 91 -0.6 10.9 5.1 25.10.2015 14.2 62 100 86 5.9 16.6 10.1 26.10.2015 0.0 57 100 85 0.9 12.1 8.1 27.10.2015 0.0 61 93 81 1.7 12.2 6.1 28.10.2015 40.0 80 100 96 3.4 10.7 6.7 29.10.2015 1.8 60 100 81 7.1 15.2 11.2 30.10.2015 0.0 61 100 84 3.8 13.1 6.6 31.10.2015 0.0 70 99 89 0.1 8.5 4.7 01.11.2015 11.4 64 100 88 0.1 9.2 5.3 02.11.2015 0.0 62 100 86 4.4 14.2 9.9 03.11.2015 0.0 45 95 73 2.7 16.1 9.0 04.11.2015 0.0 46 100 78 8.4 21.2 14.1 05.11.2015 0.0 60 100 83 8.4 22.1 14.3 06.11.2015 7.6 74 100 90 10.3 22.0 16.1 07.11.2015 0.0 60 94 81 7.8 17.0 13.1 08.11.2015 0.2 60 100 84 1.7 9.7 6.2 09.11.2015 0.0 55 93 77 0.2 8.9 3.8 10.11.2015 7.0 71 100 96 -1.1 11.8 5.1 11.11.2015 4.8 63 100 91 2.6 7.9 6.2 12.11.2015 3.8 80 100 96 3.8 12.3 7.9 13.11.2015 2.8 79 98 91 5.2 11.6 7.4 14.11.2015 1.2 77 100 89 0.9 7.6 3.6 15.11.2015 0.0 65 89 79 0.4 4.2 2.0 16.11.2015 0.0 45 92 74 2.6 13.3 7.3 17.11.2015 0.0 67 93 82 4.5 17.1 8.4 18.11.2015 0.0 66 93 82 2.8 8.7 5.9 19.11.2015 4.8 67 100 84 8.6 15.1 12.2 20.11.2015 0.0 50 77 62 1.7 14.7 10.3 21.11.2015 0.4 65 100 84 0.0 4.4 1.7 22.11.2015 0.2 84 100 95 -0.7 2.4 0.6 23.11.2015 0.0 60 100 88 -10.7 0.1 -3.7 24.11.2015 5.8 86 100 96 -10.7 0.5 -4.1 25.11.2015 1.4 68 100 91 -2.3 2.6 0.0 26.11.2015 0.0 69 88 76 -3.5 8.9 2.7 27.11.2015 12.2 61 100 81 3.9 14.0 10.2 28.11.2015 0.8 83 98 92 0.6 13.9 7.9 29.11.2015 0.0 75 100 93 -4.6 1.0 -1.0

191

30.11.2015 0.0 71 100 88 -6.6 2.4 -1.9 01.12.2015 1.0 93 100 99 -1.6 4.1 0.2 02.12.2015 0.8 66 100 93 0.6 7.9 4.7 03.12.2015 0.0 82 100 93 0.2 6.0 2.5 04.12.2015 0.0 83 92 88 0.6 4.8 2.7 05.12.2015 0.0 89 100 98 -1.0 3.4 2.1 06.12.2015 0.0 86 100 99 -1.6 5.4 1.7 07.12.2015 0.0 95 100 100 -0.9 7.1 1.8 08.12.2015 0.0 95 100 99 -1.1 3.1 1.1 09.12.2015 0.0 78 100 95 -0.4 4.5 2.5 10.12.2015 0.0 86 100 96 2.3 9.8 6.1 11.12.2015 0.0 62 93 83 0.7 10.6 6.6 12.12.2015 0.0 81 100 93 2.1 10.9 7.1 13.12.2015 1.8 86 100 96 0.6 7.8 5.0 14.12.2015 8.0 90 100 99 4.8 6.5 5.6 15.12.2015 0.2 89 100 94 6.5 13.7 9.7 16.12.2015 0.4 92 100 98 1.2 7.4 4.6 17.12.2015 1.4 75 100 92 0.0 5.4 2.6 18.12.2015 0.0 70 90 78 -0.3 6.8 3.5 19.12.2015 0.0 70 98 82 -4.3 0.0 -1.4 20.12.2015 0.0 67 94 80 -5.1 -3.0 -4.1 21.12.2015 17.6 62 100 86 -7.1 3.6 -0.8 22.12.2015 4.2 95 100 99 3.1 8.8 5.4 23.12.2015 0.0 100 100 100 2.9 11.2 8.2 24.12.2015 1.4 59 100 83 3.4 14.7 9.1 25.12.2015 0.0 69 100 87 3.1 15.2 8.5 26.12.2015 0.0 85 99 92 -0.4 8.2 4.3 27.12.2015 16.6 83 100 96 -1.8 3.4 1.3 28.12.2015 0.0 72 100 83 -0.8 3.7 1.7 29.12.2015 29.4 95 100 99 -6.6 -0.6 -4.1 30.12.2015 0.0 90 100 97 -0.6 3.8 1.4 31.12.2015 0.0 88 100 95 -0.7 1.1 0.1 01.01.2016 0.0 83 100 90 -2.1 -0.6 -1.2 02.01.2016 0.0 81 93 88 -5.1 -2.1 -3.8 03.01.2016 0.0 77 94 88 -4.2 -1.6 -2.7 04.01.2016 0.0 55 86 73 -9.3 -0.3 -3.6 05.01.2016 0.0 71 93 85 -16.9 -9.0 -13.6 06.01.2016 0.0 56 99 83 -17.6 -3.8 -10.5 07.01.2016 0.0 71 100 88 -9.0 0.6 -4.1 08.01.2016 0.0 66 100 86 -5.9 4.3 -1.7 09.01.2016 6.4 86 100 99 -2.4 2.7 0.0 10.01.2016 15.2 91 100 99 1.3 5.5 4.2 11.01.2016 0.0 67 98 86 -8.9 4.8 -0.5 12.01.2016 0.0 83 100 94 -12.6 -8.9 -10.8

192

13.01.2016 0.0 77 98 88 -11.7 -4.1 -7.8 14.01.2016 0.0 83 97 91 -12.6 -9.7 -10.6 15.01.2016 4.0 83 100 95 -10.1 -3.7 -6.3 16.01.2016 7.8 82 100 95 -3.7 3.6 0.7 17.01.2016 0.0 79 96 85 -4.4 2.2 -2.3 18.01.2016 0.0 73 97 90 -12.9 -3.2 -7.7 19.01.2016 0.0 83 95 91 -11.9 -9.5 -10.7 20.01.2016 0.0 74 97 86 -17.7 -7.2 -10.6 21.01.2016 0.0 88 100 95 -12.3 -7.0 -8.8 22.01.2016 0.0 68 98 79 -11.2 -4.7 -6.6 23.01.2016 0.0 66 91 81 -10.8 -5.1 -7.6 24.01.2016 0.0 77 100 93 -13.3 -6.5 -9.9 25.01.2016 0.2 64 96 84 -11.0 -1.4 -5.2 26.01.2016 0.2 57 100 87 -2.2 3.7 0.0 27.01.2016 0.0 67 100 86 -0.9 4.4 1.8 28.01.2016 0.0 80 100 90 -3.6 -0.9 -2.3 29.01.2016 0.0 84 100 90 -3.9 0.1 -1.6 30.01.2016 0.0 75 96 87 -9.7 -2.3 -7.4 31.01.2016 4.0 79 100 93 -9.0 3.8 -0.5 01.02.2016 1.8 76 100 92 1.0 8.5 4.8 02.02.2016 0.0 82 100 95 -1.2 5.3 1.6 03.02.2016 10.6 50 100 90 -3.7 2.1 -0.4 04.02.2016 0.0 68 100 83 0.7 14.1 6.6 05.02.2016 0.0 66 100 85 -2.8 2.1 -0.8 06.02.2016 0.0 61 98 83 -5.4 -0.1 -2.2 07.02.2016 0.0 66 100 85 -2.2 1.5 -0.8 08.02.2016 1.0 71 100 84 -3.4 5.3 1.2 09.02.2016 1.2 89 100 98 -0.2 4.8 1.9 10.02.2016 0.0 78 100 95 -3.4 0.3 -1.2 11.02.2016 0.0 71 95 85 -14.1 -3.4 -7.8 12.02.2016 0.0 72 95 85 -19.9 -11.4 -14.9 13.02.2016 0.0 66 93 74 -16.1 -6.8 -11.1 14.02.2016 0.0 61 90 75 -23.6 -16.1 -20.1 15.02.2016 0.0 74 100 89 -25.0 -11.2 -17.8 16.02.2016 0.2 84 100 97 -15.9 -2.0 -6.9 17.02.2016 0.0 69 100 89 -3.7 -1.1 -2.2 18.02.2016 0.0 61 97 81 -11.5 -2.9 -6.2 19.02.2016 3.2 55 87 72 -16.0 -4.5 -10.1 20.02.2016 4.6 64 99 79 -8.5 8.4 1.5 21.02.2016 0.0 72 100 88 0.3 9.8 5.1 22.02.2016 0.0 55 79 67 -1.6 4.1 1.4 23.02.2016 0.0 58 87 76 -4.1 -0.1 -2.0 24.02.2016 9.4 74 100 93 -5.5 1.7 -2.2 25.02.2016 5.0 78 100 95 -2.7 0.7 -0.4

193

26.02.2016 0.0 69 88 80 -6.2 0.3 -2.6 27.02.2016 1.4 74 92 82 -10.8 -4.4 -7.2 28.02.2016 0.0 64 94 82 -7.2 3.3 -0.1 29.02.2016 5.8 62 100 86 1.4 12.4 7.2 01.03.2016 0.0 69 99 83 -6.6 9.2 -1.5 02.03.2016 0.0 73 98 85 -10.7 -5.7 -7.3 03.03.2016 2.6 59 94 79 -11.9 -6.3 -8.7 04.03.2016 0.4 53 100 85 -9.5 -2.8 -6.4 05.03.2016 0.0 74 97 88 -7.4 1.1 -4.2 06.03.2016 0.0 63 100 89 -6.6 -1.1 -3.8 07.03.2016 0.0 68 95 86 -10.4 1.3 -3.1 08.03.2016 0.0 62 94 81 0.8 10.7 5.6 09.03.2016 0.0 67 92 79 5.7 15.9 10.2 10.03.2016 3.4 92 100 99 8.9 16.3 13.1 11.03.2016 0.0 66 100 91 2.6 12.4 9.3 12.03.2016 0.0 45 100 82 -1.2 7.9 2.6 13.03.2016 0.0 53 89 74 -2.4 14.1 5.9 14.03.2016 8.6 67 100 95 3.1 8.0 5.7 15.03.2016 4.6 93 100 99 2.6 11.0 7.2 16.03.2016 6.4 66 100 94 5.0 8.8 7.1 17.03.2016 0.0 57 82 71 4.4 12.7 7.2 18.03.2016 0.0 57 95 84 2.2 8.9 5.6 19.03.2016 0.0 35 83 57 -3.7 5.0 1.5 20.03.2016 0.0 42 85 68 -5.9 2.9 -2.2 21.03.2016 0.0 57 95 75 -5.8 4.5 -1.2 22.03.2016 1.2 61 94 79 -3.3 1.3 -1.2 23.03.2016 4.6 70 100 95 -3.9 8.6 3.1 24.03.2016 9.9 100 100 100 -0.3 5.8 2.2 25.03.2016 10.5 84 100 93 -1.4 0.3 -0.4 26.03.2016 14.2 74 100 92 -2.9 1.9 -0.7 27.03.2016 0.0 52 100 86 -3.3 5.7 0.3 28.03.2016 23.8 74 100 92 -1.6 16.3 6.1 29.03.2016 0.0 46 98 73 0.9 9.4 4.8 30.03.2016 0.0 44 97 72 -1.3 6.2 1.7 31.03.2016 27.2 51 100 88 -3.3 14.2 6.4 01.04.2016 12.4 71 100 89 10.2 14.7 11.8 02.04.2016 0.0 74 100 91 1.4 12.1 5.6 03.04.2016 0.0 62 100 84 -5.4 2.4 -0.6 04.04.2016 0.0 48 100 82 -8.2 -3.9 -5.7 05.04.2016 4.6 31 80 60 -12.2 -3.6 -6.3 06.04.2016 5.0 66 100 85 -13.2 0.7 -5.8 07.04.2016 18.8 88 100 97 -5.7 6.9 -0.1 08.04.2016 0.0 58 97 82 -2.7 6.7 1.7 09.04.2016 0.0 62 84 71 -4.2 1.7 -1.8

194

10.04.2016 0.0 61 100 82 -6.9 -3.1 -4.7 11.04.2016 26.4 93 100 100 -7.7 0.0 -3.8 12.04.2016 0.4 63 95 84 0.0 9.7 4.6 13.04.2016 0.0 41 100 72 -2.7 3.4 0.7 14.04.2016 0.0 26 77 58 -3.9 7.9 1.6 15.04.2016 0.0 29 62 45 -1.4 9.4 4.0 16.04.2016 0.0 32 55 44 1.9 16.8 8.9 17.04.2016 0.0 24 47 34 5.8 20.3 12.2 18.04.2016 0.0 30 81 46 6.9 23.8 15.9 19.04.2016 0.0 30 95 59 6.9 24.4 16.8 20.04.2016 0.0 30 64 48 4.4 16.2 10.4 21.04.2016 2.2 31 100 57 4.3 16.8 9.9 22.04.2016 3.8 79 100 94 6.2 14.8 11.4 23.04.2016 0.0 28 94 67 4.0 16.2 11.9 24.04.2016 0.0 35 63 48 1.7 12.5 6.7 25.04.2016 0.2 49 81 65 1.4 14.3 7.9 26.04.2016 18.2 76 100 93 3.4 10.0 5.9 27.04.2016 0.0 43 100 67 1.2 4.9 3.3 28.04.2016 0.0 43 63 52 0.5 11.9 5.9 29.04.2016 0.0 42 62 52 0.9 7.1 4.7 30.04.2016 0.0 49 66 58 4.4 10.6 7.1 01.05.2016 4.2 49 100 89 3.7 13.6 8.9 02.05.2016 1.0 63 100 88 5.9 9.3 7.6 03.05.2016 0.0 55 100 81 5.7 12.7 8.6 04.05.2016 0.0 51 97 76 5.2 14.4 9.7 05.05.2016 0.0 51 97 74 3.9 16.1 10.3 06.05.2016 0.0 28 79 54 6.5 15.7 11.4 07.05.2016 0.2 31 82 60 7.9 21.3 15.3 08.05.2016 0.0 48 100 76 5.7 18.8 12.1 09.05.2016 0.0 36 85 60 2.2 13.7 8.6 10.05.2016 0.0 34 65 48 1.7 14.5 8.8 11.05.2016 0.0 30 54 42 3.9 15.6 10.0 12.05.2016 0.0 31 75 53 7.2 20.4 14.0 13.05.2016 8.0 65 100 85 11.5 23.9 17.9 14.05.2016 6.8 68 100 87 11.9 17.7 14.9 15.05.2016 0.0 53 88 73 2.3 12.2 7.5 16.05.2016 0.2 40 91 72 1.1 5.6 3.1 17.05.2016 3.2 36 100 75 0.3 16.6 8.3 18.05.2016 0.0 43 71 57 6.0 16.3 11.1 19.05.2016 0.0 37 96 61 5.4 16.4 11.4 20.05.2016 0.0 32 68 50 5.2 18.9 13.2 21.05.2016 0.0 34 57 49 8.8 21.2 15.9 22.05.2016 0.0 42 87 64 11.0 21.8 16.2 23.05.2016 0.0 32 58 45 10.4 21.4 16.1

195

24.05.2016 0.0 24 73 47 10.8 25.2 18.6 25.05.2016 0.0 33 69 52 11.4 27.2 19.8 26.05.2016 3.2 45 94 73 13.9 28.1 21.6 27.05.2016 0.0 54 100 77 16.5 26.8 21.4 28.05.2016 0.0 47 93 71 19.2 28.8 23.6 29.05.2016 0.0 57 97 75 17.5 30.6 24.3 30.05.2016 14.6 49 100 80 15.9 27.5 22.3 31.05.2016 0.2 34 91 61 15.6 24.2 20.3 01.06.2016 0.0 43 82 64 13.9 24.9 19.7 02.06.2016 0.0 41 91 66 12.4 23.1 17.3 03.06.2016 0.0 40 89 62 14.9 27.1 21.0 04.06.2016 0.0 41 77 60 12.9 26.2 20.2 05.06.2016 16.6 51 100 90 14.4 26.3 20.0 06.06.2016 2.4 46 98 78 15.3 21.9 17.9 07.06.2016 0.4 79 100 93 14.3 24.1 17.2 08.06.2016 0.0 56 91 79 9.4 15.4 13.1 09.06.2016 0.0 36 89 63 6.4 13.4 9.5 10.06.2016 0.0 33 85 55 4.9 18.4 12.6 11.06.2016 0.0 43 96 61 7.7 24.2 16.6 12.06.2016 0.0 46 73 57 15.6 30.4 23.8 13.06.2016 0.0 54 95 71 11.4 19.1 15.3 14.06.2016 0.0 31 92 63 8.2 18.7 13.5 15.06.2016 13.2 37 100 61 9.1 24.1 16.7 16.06.2016 0.0 47 100 81 12.5 22.5 16.6 17.06.2016 0.0 29 71 49 14.6 23.6 19.4 18.06.2016 0.0 27 72 45 16.4 29.9 23.3 19.06.2016 0.0 34 73 53 15.3 29.9 23.4 20.06.2016 0.0 45 76 63 16.4 31.0 24.1 21.06.2016 0.0 36 74 52 16.3 31.3 24.8 22.06.2016 0.8 37 100 63 12.2 24.4 19.2 23.06.2016 0.0 30 73 56 12.6 23.6 18.6 24.06.2016 0.0 36 67 51 12.9 23.9 18.8 25.06.2016 0.0 33 84 54 13.7 27.3 21.0 26.06.2016 0.0 42 76 61 13.5 29.4 22.5 27.06.2016 0.4 35 100 73 17.2 29.4 23.9 28.06.2016 0.0 48 92 78 18.2 31.0 24.6 29.06.2016 0.0 36 95 68 11.7 19.7 15.8 30.06.2016 0.0 34 95 59 9.6 24.4 17.3 01.07.2016 4.0 37 100 75 8.1 26.7 18.7 02.07.2016 0.0 42 100 73 10.1 20.4 15.9 03.07.2016 0.0 35 91 61 8.6 23.3 17.2 04.07.2016 0.0 27 91 57 11.9 26.0 19.6 05.07.2016 0.0 34 82 63 11.7 27.4 21.2 06.07.2016 0.0 53 93 72 18.6 29.2 23.8

196

07.07.2016 0.0 53 100 81 18.4 29.8 24.1 08.07.2016 2.6 60 100 81 19.7 30.8 24.9 09.07.2016 3.0 77 100 95 17.7 29.1 23.1 10.07.2016 0.0 43 100 79 16.6 20.3 18.6 11.07.2016 0.0 46 75 62 14.6 27.3 21.2 12.07.2016 0.0 46 90 68 17.4 27.3 22.1 13.07.2016 0.0 42 93 69 17.9 30.9 24.9 14.07.2016 20.2 62 100 87 19.9 32.6 26.1 15.07.2016 1.6 71 100 88 19.9 27.7 23.0 16.07.2016 0.0 50 100 79 14.4 22.5 19.3 17.07.2016 0.0 51 99 72 13.7 22.8 17.6 18.07.2016 0.0 48 94 72 10.4 24.9 19.7 19.07.2016 0.0 43 84 61 15.9 26.4 23.3 20.07.2016 0.0 42 98 67 13.6 24.6 18.9 21.07.2016 0.0 49 96 75 10.5 27.5 19.4 22.07.2016 0.0 41 100 77 15.7 30.3 23.7 23.07.2016 0.0 39 97 64 21.2 31.5 26.1 24.07.2016 0.0 47 83 71 17.3 31.2 24.7 25.07.2016 22.8 56 100 88 18.9 27.3 23.1 26.07.2016 0.0 54 100 78 19.1 28.4 24.1 27.07.2016 0.0 49 100 75 15.2 27.9 21.8 28.07.2016 5.8 63 100 81 17.2 30.4 24.1 29.07.2016 0.0 65 100 81 19.2 27.9 23.1 30.07.2016 2.4 67 100 83 18.6 26.1 21.8 31.07.2016 0.0 68 97 84 16.3 24.1 19.9 01.08.2016 0.0 63 100 84 18.2 24.6 20.6 02.08.2016 0.0 43 99 71 16.6 27.0 21.8 03.08.2016 0.0 42 87 64 17.3 29.1 23.4 04.08.2016 0.0 43 98 69 17.2 30.7 24.1 05.08.2016 0.0 52 100 88 17.8 31.1 24.9 06.08.2016 0.0 52 100 77 20.2 29.4 24.2 07.08.2016 0.0 42 100 74 14.7 26.6 20.7 08.08.2016 0.0 35 100 66 14.3 26.9 20.1 09.08.2016 0.0 40 74 55 12.9 27.7 21.0 10.08.2016 0.0 41 99 69 16.4 30.1 23.6 11.08.2016 0.0 45 100 83 18.5 32.6 25.7 12.08.2016 0.0 58 100 87 19.2 29.9 24.9 13.08.2016 21.6 81 100 98 22.5 32.0 26.4 14.08.2016 0.2 71 100 90 21.7 27.1 23.9 15.08.2016 0.0 72 100 92 18.3 25.7 21.8 16.08.2016 19.2 83 100 97 16.4 26.3 21.2 17.08.2016 4.6 70 100 93 16.5 25.0 21.3 18.08.2016 0.4 59 100 88 14.6 26.6 19.8 19.08.2016 0.0 63 100 87 18.3 27.8 22.0

197

20.08.2016 0.0 64 100 86 15.8 27.7 22.4 21.08.2016 12.6 65 100 88 20.0 29.0 24.3 22.08.2016 0.0 55 100 83 15.1 23.1 19.7 23.08.2016 0.0 58 100 80 11.2 22.2 16.8 24.08.2016 0.0 57 100 81 13.4 25.4 19.4 25.08.2016 82.2 67 100 96 15.2 26.4 21.4 26.08.2016 0.0 55 100 86 19.1 26.7 22.5 27.08.2016 0.8 65 100 82 17.2 26.4 21.8 28.08.2016 0.4 74 100 91 16.2 26.9 20.8 29.08.2016 0.0 54 100 84 17.1 27.1 21.8 30.08.2016 0.0 69 100 86 14.4 26.2 20.2 31.08.2016 0.0 50 100 86 14.5 26.7 21.1 01.09.2016 0.0 59 100 83 14.0 25.9 20.6 02.09.2016 0.0 52 99 78 13.1 22.1 17.0 03.09.2016 0.0 58 100 80 12.1 22.5 16.8 04.09.2016 0.0 47 100 74 11.1 23.2 16.9 05.09.2016 0.0 49 100 79 10.5 25.3 18.0 06.09.2016 0.0 63 100 84 13.2 27.3 20.5 07.09.2016 0.0 67 100 89 17.6 30.7 23.9 08.09.2016 10.2 80 100 95 20.7 31.1 25.2 09.09.2016 0.0 54 100 85 20.2 27.8 23.7 10.09.2016 3.0 73 100 92 18.4 27.0 21.9 11.09.2016 0.0 53 100 81 17.2 26.0 20.8 12.09.2016 0.0 55 100 79 11.3 20.7 16.4 13.09.2016 0.0 52 100 76 8.6 23.0 16.0 14.09.2016 0.0 52 100 81 13.1 25.9 19.6 15.09.2016 0.0 52 100 81 11.1 21.4 17.3 16.09.2016 0.0 50 100 77 10.1 20.7 14.7 17.09.2016 11.8 72 100 98 9.9 24.3 16.9 18.09.2016 0.0 60 100 87 15.8 21.3 19.1 19.09.2016 0.0 48 100 79 16.1 24.7 20.3 20.09.2016 0.0 38 100 76 13.5 27.1 20.0 21.09.2016 0.0 39 87 61 13.2 26.7 20.1 22.09.2016 0.0 55 100 73 11.2 26.4 18.7 23.09.2016 0.0 64 100 93 13.5 27.9 21.1 24.09.2016 0.0 39 100 75 9.9 20.2 16.1 25.09.2016 0.0 49 92 72 8.1 18.7 12.7 26.09.2016 7.0 70 100 89 7.8 19.1 12.7 27.09.2016 0.0 37 93 69 10.6 17.2 13.2 28.09.2016 0.4 52 90 72 8.8 19.8 14.0 29.09.2016 4.8 82 100 95 11.4 20.5 15.9 30.09.2016 3.2 64 100 85 13.0 16.4 14.5 01.10.2016 11.2 83 100 98 12.3 18.1 14.6 02.10.2016 7.2 84 100 99 11.8 13.9 12.9

198

03.10.2016 0.0 79 100 95 12.9 17.8 14.3 04.10.2016 0.0 71 100 92 12.9 17.7 14.5 05.10.2016 0.0 69 100 91 13.2 20.4 16.0 06.10.2016 0.0 66 100 89 12.3 22.2 16.4 07.10.2016 0.0 50 100 86 13.1 24.8 18.2 08.10.2016 1.8 60 100 83 13.1 23.1 17.3 09.10.2016 0.0 51 100 81 5.3 16.7 11.8 10.10.2016 0.0 40 100 75 2.3 13.2 7.6 11.10.2016 0.0 56 100 79 0.3 14.2 7.1 12.10.2016 0.0 60 100 81 4.5 16.6 10.4 13.10.2016 2.6 55 100 79 8.1 21.8 15.5 14.10.2016 0.0 52 100 78 2.7 18.8 9.7 15.10.2016 0.0 64 100 87 2.5 15.0 8.7 16.10.2016 1.6 87 100 94 5.2 20.5 13.2 17.10.2016 0.0 75 100 94 16.6 19.8 17.9 18.10.2016 1.4 76 98 84 16.4 23.3 19.4 19.10.2016 0.0 51 100 87 11.5 23.1 19.4 20.10.2016 7.8 75 100 98 8.9 20.0 13.7 21.10.2016 4.4 82 100 96 10.3 13.0 11.7 22.10.2016 0.0 69 100 88 3.4 10.3 7.7 23.10.2016 0.0 66 100 87 0.9 7.3 4.1 24.10.2016 0.0 60 94 78 2.2 13.6 9.1 25.10.2016 0.0 60 97 78 3.7 9.7 7.1 26.10.2016 0.0 55 93 78 -1.3 7.9 3.9 27.10.2016 8.2 71 100 97 -2.3 4.0 1.3 28.10.2016 0.0 64 100 90 -0.2 4.1 1.9 29.10.2016 0.0 86 100 92 -2.6 9.8 3.8 30.10.2016 1.4 85 100 97 6.0 18.4 13.7 31.10.2016 0.0 66 100 89 0.9 9.8 6.1 01.11.2016 0.0 76 98 90 -1.1 8.8 3.6 02.11.2016 7.2 72 100 90 2.2 19.9 12.1 03.11.2016 19.0 74 100 94 12.5 18.1 15.4 04.11.2016 0.0 72 100 92 5.5 12.9 10.3 05.11.2016 0.0 80 100 95 1.9 10.2 5.8 06.11.2016 0.0 40 100 78 3.7 14.1 8.2 07.11.2016 0.0 56 100 82 5.3 15.3 9.3 08.11.2016 1.4 68 100 86 4.7 17.9 10.4 09.11.2016 0.0 73 100 91 6.2 15.3 9.7 10.11.2016 0.0 52 100 85 -0.5 9.2 4.3 11.11.2016 0.0 61 87 73 -0.4 13.8 7.3 12.11.2016 0.0 57 96 79 -2.4 10.3 4.1 13.11.2016 0.0 55 95 80 -4.1 7.8 2.1 14.11.2016 0.0 53 91 73 0.1 10.8 4.7 15.11.2016 0.0 60 100 87 1.5 13.3 6.1

199

16.11.2016 0.2 81 100 94 2.4 13.4 7.1 17.11.2016 0.4 75 100 94 3.6 10.4 6.9 18.11.2016 0.0 59 100 87 -1.9 12.2 5.3 19.11.2016 13.4 68 100 85 5.0 18.6 12.0 20.11.2016 0.0 83 100 92 -1.1 14.0 5.1 21.11.2016 0.0 76 96 80 -2.3 -0.2 -1.4 22.11.2016 0.0 76 91 80 -2.6 -0.4 -1.5 23.11.2016 0.0 91 100 95 -3.9 0.4 -1.5 24.11.2016 7.6 100 100 100 -5.3 -0.2 -2.2 25.11.2016 1.8 95 100 99 -0.2 4.0 1.9 26.11.2016 4.8 87 100 98 1.0 5.3 3.7 27.11.2016 1.8 72 100 95 0.4 5.0 2.2 28.11.2016 0.0 80 100 95 0.2 7.6 2.6 29.11.2016 5.6 78 100 93 0.2 7.1 4.0 30.11.2016 0.8 93 100 99 5.2 12.3 8.8 01.12.2016 3.8 88 100 95 4.6 12.0 8.8 02.12.2016 0.0 81 100 94 1.8 4.6 3.0 03.12.2016 1.6 92 100 98 1.6 3.2 2.3 04.12.2016 0.0 98 100 100 1.1 3.3 2.1 05.12.2016 4.2 87 100 96 -0.2 1.9 0.9 06.12.2016 0.8 86 100 96 -0.6 3.3 1.2 07.12.2016 1.2 76 100 89 -1.7 2.9 0.4 08.12.2016 0.0 76 98 88 -1.4 1.8 0.1 09.12.2016 0.0 86 100 95 -4.0 -1.4 -2.8 10.12.2016 0.0 75 100 94 -7.3 -3.3 -5.4 11.12.2016 0.0 90 100 98 -8.0 -4.6 -6.0 12.12.2016 0.4 87 100 97 -8.9 -0.6 -4.9 13.12.2016 0.0 85 98 91 -3.3 0.3 -1.5 14.12.2016 0.0 72 92 82 -10.2 -2.9 -5.2 15.12.2016 0.0 92 99 97 -13.9 -7.9 -10.4 16.12.2016 0.0 77 97 91 -13.4 -10.9 -12.2 17.12.2016 0.0 93 100 99 -15.9 -8.2 -11.3 18.12.2016 0.0 75 100 91 -8.2 -1.6 -4.0 19.12.2016 0.0 64 90 78 -11.7 -3.6 -7.5 20.12.2016 0.0 49 93 75 -15.4 -9.5 -12.2 21.12.2016 0.0 68 98 88 -13.2 -1.1 -6.4 22.12.2016 4.6 89 100 96 -9.6 -1.2 -4.9 23.12.2016 0.0 75 100 90 -3.3 2.0 -0.2 24.12.2016 9.8 98 100 100 -5.0 0.3 -1.7 25.12.2016 0.0 79 100 91 -0.1 1.6 1.2 26.12.2016 13.9 91 100 97 -3.4 1.5 -0.5 27.12.2016 2.5 77 100 87 -3.3 8.8 3.3 28.12.2016 0.0 76 91 84 -4.2 5.1 -1.5 29.12.2016 0.0 83 100 96 -4.2 -1.2 -3.0

200

30.12.2016 0.0 81 100 94 -2.0 0.2 -0.4 31.12.2016 0.2 73 100 86 -4.6 -2.0 -3.4

Table A1.30 Daily average, high and low temperature (ºC), relative humidity (%) and rainfall (mm) recorded at the Simcoe Research Station, University of Guelph, Simcoe, ON, from 2013 to 2016. Precipitation Relative humidity (%) Temperature (°C) Date (mm) min max average min max average 01.05.2013 0 38 94 67 9.4 23.7 16.6 02.05.2013 0 19 72 46 11.2 23.5 16.5 03.05.2013 0 29 69 48 8.7 24 16.3 04.05.2013 0 19 80 50 9.1 21.8 14.9 05.05.2013 0 24 89 53 5.9 23.7 15.7 06.05.2013 0 13 72 43 7.4 22.9 15.4 07.05.2013 0 32 71 51 8.3 24.9 17.3 08.05.2013 2 45 100 72 12.1 22.6 16.3 09.05.2013 0.4 25 100 73 13.3 25.2 17.8 10.05.2013 1.4 60 97 81 10.9 22.5 16 11.05.2013 0.2 64 100 86 5.6 13.6 9 12.05.2013 3 51 92 73 2 7.3 4.4 13.05.2013 0 32 80 66 1.7 9.3 4.5 14.05.2013 0.2 46 86 71 3 10.4 7.2 15.05.2013 0.4 29 84 58 7.9 26.2 16.9 16.05.2013 0 20 77 45 11.3 24.5 18.1 17.05.2013 0 40 76 57 7 18.4 12.7 18.05.2013 0 41 60 52 12.3 20.3 14.7 19.05.2013 0 57 89 72 9.9 25.5 17.6 20.05.2013 0.2 54 100 79 13.2 27.2 20.5 21.05.2013 0 38 98 72 14.6 28.4 22 22.05.2013 0 43 83 64 18.1 28.7 23 23.05.2013 4.8 63 94 80 5.1 22.4 15.8 24.05.2013 0.6 44 93 70 3.7 10.9 6.4 25.05.2013 0 29 85 56 2.2 16.7 9.4 26.05.2013 0 21 76 47 3.9 19.2 11.9 27.05.2013 0 23 96 55 2 19.8 12.1 28.05.2013 57.4 69 100 96 9.9 17 13.2 29.05.2013 10.2 62 100 78 15.3 26.3 21.2 30.05.2013 0 53 97 74 16.9 29 22.7 31.05.2013 18.2 47 100 79 18.1 29.4 22.3 01.06.2013 6 67 100 84 16.7 26.1 21.5 02.06.2013 8.8 67 100 85 11.1 21.7 18.1 03.06.2013 0 45 93 68 7 16.7 11.4 04.06.2013 0 45 96 67 7.6 18.3 13.1 05.06.2013 0 43 83 56 9.9 19.6 14.5 06.06.2013 4.2 53 100 88 10.3 13.3 11.4

201

07.06.2013 0 82 100 93 11 15.4 13.2 08.06.2013 0.4 68 99 87 12.6 18.6 15 09.06.2013 0 36 100 69 9.6 23.3 17.3 10.06.2013 36.8 72 100 93 15.1 19.8 16.7 11.06.2013 2.2 71 100 87 15.2 22.8 18.5 12.06.2013 8.8 51 100 78 15.2 22.6 18.5 13.06.2013 16.4 65 100 86 13.3 22.1 17.1 14.06.2013 0 49 99 76 13.9 23.7 18.5 15.06.2013 0 45 99 69 10.4 23 17.8 16.06.2013 12 59 98 80 16.5 24.5 19.8 17.06.2013 0 40 95 66 15.5 26 21.2 18.06.2013 0 45 87 68 13.8 20.7 17.1 19.06.2013 0 37 91 59 10.7 19.9 15.5 20.06.2013 0 32 96 62 8.7 22.6 16.9 21.06.2013 0 33 97 63 10.4 24.9 18.7 22.06.2013 0 57 89 69 17.2 29.2 23.9 23.06.2013 0.2 43 95 69 19.8 29.6 25.3 24.06.2013 0 49 84 67 21.6 29.1 25.4 25.06.2013 0.8 65 94 79 19.5 26.6 23 26.06.2013 0 55 97 78 19.4 28.5 23.5 27.06.2013 70 77 100 96 18 25.3 19.7 28.06.2013 20.2 83 100 97 17 21 18.4 29.06.2013 2 60 100 85 16.6 24.8 19.5 30.06.2013 0 50 93 76 15 24.1 19.3 01.07.2013 0 61 87 75 15.9 20.6 18 02.07.2013 0 68 93 79 15.1 23.1 18.6 03.07.2013 6.2 58 100 85 15.9 28.7 22 04.07.2013 1.4 68 100 89 19.8 26.8 22.4 05.07.2013 1 69 100 86 20 25.5 22.5 06.07.2013 0.8 57 99 83 18.2 27.7 23.1 07.07.2013 9.8 73 100 89 20 25.3 22.3 08.07.2013 0 68 99 85 19.9 27.7 23.2 09.07.2013 0.8 70 100 92 20.4 27.3 22.6 10.07.2013 1 67 99 85 18.6 29 23.6 11.07.2013 0 47 94 74 14 24.6 19.5 12.07.2013 0 62 94 77 14.8 24.9 20.2 13.07.2013 0 52 100 77 15 27.8 21.9 14.07.2013 0 54 100 78 18.2 30.1 24.1 15.07.2013 0 55 99 78 20.3 30.5 25.8 16.07.2013 0 47 100 77 20.7 32.1 26.7 17.07.2013 0 52 99 78 23.4 33.4 28 18.07.2013 0 47 94 73 23.8 33.2 28.1 19.07.2013 12 59 100 76 20.6 31.1 27.8 20.07.2013 11.8 43 100 79 19.4 27.6 23

202

21.07.2013 0 57 95 72 16.8 25.1 20.9 22.07.2013 0 61 98 83 15.3 25.9 20.4 23.07.2013 0 64 100 84 14.9 26.9 20.2 24.07.2013 0 42 97 71 11.9 22 16.4 25.07.2013 0 48 98 74 10.4 22.9 17 26.07.2013 0 41 95 69 11.1 23.6 18.2 27.07.2013 5.8 77 97 89 15 22.5 19 28.07.2013 1.8 49 96 77 12.8 20.1 16.5 29.07.2013 0.2 61 92 79 12.9 21 16.6 30.07.2013 0 48 97 74 12.6 25 18.7 31.07.2013 0 47 100 77 12.7 24.2 19.1 01.08.2013 14.4 53 100 83 16.5 24.4 20.1 02.08.2013 0 48 100 79 13.8 24.1 19.1 03.08.2013 0 44 100 73 14.3 23.7 18.9 04.08.2013 0 45 97 72 11.6 22.9 16.9 05.08.2013 0 44 98 74 9 21.2 15.6 06.08.2013 0 45 97 76 11.7 24.7 19 07.08.2013 0.4 74 98 86 16.8 25.2 21.1 08.08.2013 33.6 69 100 88 18.3 26.8 22.2 09.08.2013 9.6 41 100 74 15 25.2 20.2 10.08.2013 0 44 92 69 13.6 23.1 18.2 11.08.2013 0 49 100 74 9.1 22.3 17 12.08.2013 0.8 74 99 87 12.8 22.6 18.5 13.08.2013 5.2 56 100 81 11.5 18.8 16.4 14.08.2013 0 44 95 72 10.1 20.9 15 15.08.2013 0 46 87 70 10.1 21.7 16.2 16.08.2013 0 48 91 74 11.8 23.4 18 17.08.2013 0 39 100 72 11 25.2 18.9 18.08.2013 0 47 100 76 12.4 25.1 19 19.08.2013 0 48 100 77 14.1 26.3 20.1 20.08.2013 0 48 100 77 16.3 28.2 22 21.08.2013 0 46 100 73 17.4 28.9 23.1 22.08.2013 0 55 98 77 18.2 28.1 23.2 23.08.2013 0 38 97 69 13.4 25.4 19.8 24.08.2013 0 49 95 72 12 23.2 17.9 25.08.2013 0 43 100 72 10.8 26.8 20.2 26.08.2013 14.6 63 100 81 20 27.4 23.4 27.08.2013 1.2 65 100 91 19.4 28.8 23.3 28.08.2013 5.6 67 100 89 20.5 27 23 29.08.2013 0 53 99 82 18.4 29.7 23.5 30.08.2013 1.4 62 100 81 17.7 27.4 23.3 31.08.2013 0 63 99 85 19.5 27.2 22.9 01.09.2013 9.4 74 100 94 19 25.3 21 02.09.2013 0.2 63 100 88 14 24.2 19.8

203

03.09.2013 0.6 65 99 87 12.7 19.2 15.7 04.09.2013 0.4 48 94 76 12.8 24.5 18.7 05.09.2013 0 34 99 69 8.3 19.8 14.7 06.09.2013 0 49 100 76 3.8 20.9 12.8 07.09.2013 6.2 74 100 90 12.2 20.7 17.5 08.09.2013 0 41 99 73 12.3 21.5 18.1 09.09.2013 0.4 68 100 84 8.4 21.6 15.8 10.09.2013 3.8 64 97 78 20.6 29.9 25.8 11.09.2013 10.4 61 100 80 19.8 29.1 25 12.09.2013 0.4 54 100 86 14.1 22.8 19.6 13.09.2013 0 65 96 78 8.8 14.3 11.8 14.09.2013 0 39 98 72 3.8 18.7 11.5 15.09.2013 1.8 62 96 83 8.3 18.8 14 16.09.2013 0 51 100 82 6.4 16.3 12.1 17.09.2013 0 38 98 74 4.3 17.7 10.7 18.09.2013 0.2 47 100 80 4.8 20.3 13.4 19.09.2013 0 69 100 94 12.4 22.7 16.5 20.09.2013 2.6 82 100 94 15.4 23.9 19.7 21.09.2013 71.6 81 100 97 10.5 20 15.6 22.09.2013 0 60 94 77 7.9 12.8 10.7 23.09.2013 0 44 93 73 6.9 17 11.1 24.09.2013 0 38 100 80 3.8 19.5 10.8 25.09.2013 0 38 100 70 6.2 21.9 13.5 26.09.2013 0 40 99 68 7.9 21.8 14.9 27.09.2013 0 56 99 82 9.6 20.6 14.3 28.09.2013 0 53 100 80 10.1 21.5 15.6 29.09.2013 0 69 98 81 12.6 20.7 16.9 30.09.2013 1.8 68 100 93 14.3 21.1 17.3 01.10.2013 0.2 59 100 86 11.3 22.8 17.4 02.10.2013 0 33 97 70 13.2 25.3 19 03.10.2013 7.6 37 100 66 7.7 22.1 15.3 04.10.2013 13.4 75 100 96 15 23.8 18.3 05.10.2013 5.4 91 100 98 14 16.5 15.2 06.10.2013 2 67 100 91 14.3 25.4 18.7 07.10.2013 23.4 49 100 84 9.2 18.5 12.6 08.10.2013 0.2 45 100 83 5.6 18.5 11.6 09.10.2013 0.2 48 100 79 6.3 18.1 12.5 10.10.2013 0.2 49 100 87 5.8 19.6 11.5 11.10.2013 0.4 36 100 79 8.6 21.3 13.5 12.10.2013 0 50 98 78 8.8 22.9 15.2 13.10.2013 4.4 78 100 89 11.2 18 16 14.10.2013 0 52 100 80 6.4 16.8 11.3 15.10.2013 0 75 100 91 6.8 17.7 12.6 16.10.2013 12 56 100 88 10.3 19 16.2

204

17.10.2013 13.4 64 100 88 8.9 15.5 11.4 18.10.2013 0 50 98 76 5.8 15 10.7 19.10.2013 7.8 74 100 88 4.7 11.1 7.7 20.10.2013 0 49 95 79 3.4 12.9 7.8 21.10.2013 12.6 65 100 79 8.8 15.2 12.1 22.10.2013 0 49 93 67 1.9 8.2 5.4 23.10.2013 0 51 93 77 0.6 8.2 4.3 24.10.2013 1.2 53 98 82 -0.3 7.5 2.9 25.10.2013 0 54 98 80 1.6 8.7 4.3 26.10.2013 7.2 56 95 78 4.4 7.7 6 27.10.2013 0.2 50 99 78 1.1 7.9 4.7 28.10.2013 0 45 83 66 1.7 8.4 5.4 29.10.2013 0 46 91 71 -1 8.8 3.6 30.10.2013 0.2 62 97 83 1.6 13 6.9 31.10.2013 32.6 85 100 96 5.8 17 13.2 01.11.2013 7 68 100 82 7.9 15.5 11.3 02.11.2013 5.4 72 100 92 2.5 7.9 5.8 03.11.2013 0 43 84 66 -2.5 3.5 0 04.11.2013 0 51 75 62 -2.6 4.9 1.4 05.11.2013 0 56 85 70 2.6 13.3 8.2 06.11.2013 17 60 99 75 8.1 15 11.6 07.11.2013 1 68 94 79 2.3 9.4 4.5 08.11.2013 0 60 94 75 -0.3 5.1 2.4 09.11.2013 0 54 84 69 0.3 11.8 6.7 10.11.2013 0.2 55 82 69 2.1 9.5 5.6 11.11.2013 5.4 72 97 83 -1.5 7.9 2.3 12.11.2013 0 59 90 76 -4.8 1.1 -2.3 13.11.2013 0 47 92 69 -4 3.6 0.1 14.11.2013 0 43 65 56 2.9 8.4 5.8 15.11.2013 0 46 76 57 6.2 10.6 7.8 16.11.2013 0 63 96 79 3.2 11.8 8.6 17.11.2013 8.4 70 99 85 11.2 15.7 13.1 18.11.2013 0.2 56 82 68 1.4 11 5.7 19.11.2013 0 54 88 72 -3.4 3.3 0.5 20.11.2013 0 42 90 74 -4.4 5.4 -0.5 21.11.2013 5 58 100 81 0.4 6.2 4.2 22.11.2013 3 74 100 93 -1.5 7.4 4.7 23.11.2013 0 53 91 77 -9.1 -1.4 -4.1 24.11.2013 0 57 85 72 -11.3 -5.3 -8.3 25.11.2013 0 52 97 71 -8.5 1.3 -2.6 26.11.2013 1.4 91 100 97 -1.2 0.3 -0.6 27.11.2013 3 64 100 85 -7.8 -0.6 -3.3 28.11.2013 0 53 97 81 -8.6 -3.1 -6.1 29.11.2013 4.2 64 98 86 -15.1 -2.1 -7.5

205

30.11.2013 0 67 91 79 -8.4 2.2 -1.5 01.12.2013 0.2 81 95 89 0.3 4.3 2 02.12.2013 0 71 100 88 -2.9 1.9 0.3 03.12.2013 1 58 99 83 -5.9 3.4 -0.8 04.12.2013 1.2 83 98 93 0 6.8 2.7 05.12.2013 0 57 95 74 2.2 14.4 9.2 06.12.2013 0 63 76 71 -3.1 2 -1.1 07.12.2013 0 57 81 70 -8.6 -3.2 -5.3 08.12.2013 0 65 91 76 -10.3 -1.8 -5.9 09.12.2013 2.2 72 100 87 -4.5 1.3 -1.1 10.12.2013 0 55 89 69 -9.2 -4.5 -6.5 11.12.2013 0 64 93 77 -11.9 -5.7 -7.7 12.12.2013 0 59 93 73 -14.5 -7.5 -10.8 13.12.2013 0 72 90 83 -10.3 -4.1 -7.3 14.12.2013 0 88 96 92 -11.1 -7.2 -9.6 15.12.2013 0 66 95 84 -11.6 -3.6 -7.9 16.12.2013 1 55 95 74 -14.9 -6 -11 17.12.2013 0 77 95 90 -12.2 -2.8 -6.4 18.12.2013 1.4 73 94 83 -5.9 -0.8 -3.4 19.12.2013 0.4 66 93 78 -3.4 3.9 1 20.12.2013 12.2 91 100 99 0.3 5.6 2.7 21.12.2013 25.2 100 100 100 -0.5 9.3 3.3 22.12.2013 29.4 85 100 96 -1.2 6.8 1.2 23.12.2013 0 73 97 85 -4.2 2 -0.9 24.12.2013 0 57 96 83 -14.6 -4 -8 25.12.2013 0 73 96 85 -17.9 -3.1 -9.3 26.12.2013 1.4 66 99 84 -8.4 -1.4 -4.1 27.12.2013 1.8 69 99 88 -7.2 2.4 -2.3 28.12.2013 1.2 59 84 74 1.9 6.7 3.9 29.12.2013 0 49 95 70 -4.8 5.9 2.7 30.12.2013 0 61 82 70 -9.4 -5 -7.9 31.12.2013 0 59 95 75 -10.8 -7.6 -8.8 01.01.2014 0 56 90 74.9 -14.3 -8.3 -10.5 02.01.2014 0 63 87 82.5 -17.8 -14.3 -15.8 03.01.2014 0 46 88 70.9 -22.1 -9.7 -16.5 04.01.2014 0 47 75 60.8 -9.7 0.3 -4 05.01.2014 12 64 100 89.3 -2.2 0.6 -0.8 06.01.2014 5 74 100 84.9 -20.9 0.1 -10 07.01.2014 0 71 85 77.6 -22.9 -16.1 -19.1 08.01.2014 0 67 90 79.2 -16.2 -8.3 -11.2 09.01.2014 1.4 62 93 83.1 -17.4 -6.8 -10.6 10.01.2014 2.8 92 100 97.7 -7.7 2.9 -0.9 11.01.2014 14.2 84 100 97.7 1.1 7.7 4.3 12.01.2014 0 76 91 83.7 -0.3 1.5 0.4

206

13.01.2014 2 61 99 80 0.7 6.7 4.2 14.01.2014 0 66 100 87.6 -3.1 4.9 1 15.01.2014 0 64 96 79.7 -4.6 1 -2.6 16.01.2014 0 66 97 80.6 -5.6 -2 -4 17.01.2014 0 53 99 82.9 -2.9 1.4 -1.3 18.01.2014 0 77 98 86.9 -11.3 -1.9 -8.4 19.01.2014 0 79 94 86.1 -9.6 -2.5 -5.8 20.01.2014 0 61 90 74.3 -12.6 -1.4 -6.4 21.01.2014 0 45 81 62.4 -20.3 -12.6 -16.5 22.01.2014 0 57 89 76.8 -22.4 -11.9 -16.8 23.01.2014 0 65 92 79.6 -20.2 -10.5 -14.1 24.01.2014 0 57 92 75.8 -19.5 -9.4 -13.9 25.01.2014 0 59 96 86.5 -14.7 -4 -8.8 26.01.2014 0 66 92 79.9 -19 -3.6 -12.4 27.01.2014 0 51 96 73.1 -19.5 -2.8 -12.1 28.01.2014 0 66 85 73 -21.5 -13.8 -17.4 29.01.2014 0 60 83 73.8 -17.9 -10.9 -13.6 30.01.2014 0 40 88 61.3 -14.5 -2.3 -8.5 31.01.2014 0 62 91 74.7 -4 1.6 -0.9 01.02.2014 16.6 90 100 97.6 -3.6 3.1 -0.7 02.02.2014 0.4 72 100 87.8 -8.7 0.3 -3.6 03.02.2014 0.2 57 95 78.7 -14.1 -4.2 -8.7 04.02.2014 0 69 95 83.9 -16.2 -4.5 -9.7 05.02.2014 0 76 95 88.1 -14.1 -4.7 -7.4 06.02.2014 0 63 92 82 -16.4 -7.7 -11 07.02.2014 0 74 86 78.5 -15 -11.6 -13.4 08.02.2014 0 67 93 81 -16.6 -9 -12.5 09.02.2014 1.4 72 95 90 -13.6 -6.1 -10.7 10.02.2014 0 58 93 80.6 -15.3 -7.5 -11.8 11.02.2014 0 54 92 75.2 -19.8 -9.8 -13.1 12.02.2014 0.4 50 92 76.2 -21.8 -7.4 -15.2 13.02.2014 0 55 96 75.9 -17.4 -3.5 -9.1 14.02.2014 0.2 70 98 89.6 -7 -2.1 -4.8 15.02.2014 0 63 94 80.8 -14.8 -4.9 -8.4 16.02.2014 2.8 66 94 83.3 -17.3 -6.2 -12.4 17.02.2014 0 54 97 69.4 -22.3 -3.1 -12.8 18.02.2014 0 62 98 85.4 -6.2 -1.8 -4.1 19.02.2014 0.2 57 97 75.2 -6.1 5.6 1.3 20.02.2014 16.2 74 100 94.3 -4.3 1.9 -0.6 21.02.2014 11.6 56 100 78.2 0.3 6.3 2.1 22.02.2014 0 42 68 58.4 0.1 2.3 1.2 23.02.2014 0 47 80 62 -6.4 0.1 -2.3 24.02.2014 0 61 92 77.1 -8.6 -6.3 -7.3 25.02.2014 0 57 89 75.4 -11.2 -6.7 -9

207

26.02.2014 0 46 87 65.5 -16.4 -9.3 -12.7 27.02.2014 0 45 86 65.6 -18.3 -9.6 -13.3 28.02.2014 0 44 85 64.5 -23.3 -11.7 -16.2 01.03.2014 0 42 91 72.2 -14.1 -0.3 -7.1 02.03.2014 0 38 91 65.4 -17.7 -10.9 -13.3 03.03.2014 0.2 41 87 64.2 -21 -13.4 -17.2 04.03.2014 0 60 87 75.2 -21 -8.4 -13 05.03.2014 0 53 94 78.4 -13.1 -6.2 -9.7 06.03.2014 0 36 90 63.1 -15.1 -4.9 -9.8 07.03.2014 0.6 51 92 70.6 -12.3 3.2 -4.3 08.03.2014 0 65 86 75.1 -8.3 0.6 -1.7 09.03.2014 0 57 96 74.7 -11.5 0.2 -5.5 10.03.2014 0 64 82 73.1 0.3 6.8 3.4 11.03.2014 0.2 57 91 75.4 1.8 9.2 5.2 12.03.2014 3.2 66 100 91.5 -14.7 3.4 -4.1 13.03.2014 0 51 75 65 -18.7 -7.3 -13.1 14.03.2014 0.2 44 74 60.8 -7.1 10.3 2.4 15.03.2014 0.2 54 93 72.7 -7.2 5.2 0.1 16.03.2014 0 48 82 62.8 -11.1 -7.3 -9.4 17.03.2014 0 55 83 66.6 -15 -4 -9 18.03.2014 0 51 86 69 -7.4 5.3 -1.8 19.03.2014 7.6 71 100 87.6 -1.5 6.5 3.3 20.03.2014 0.6 67 97 80.6 0 4 1 21.03.2014 0 55 79 70.1 -4.4 1.8 -1.1 22.03.2014 0.8 66 94 78.7 -3.1 1.9 0.1 23.03.2014 0 55 82 67.6 -9.9 -3.4 -7.5 24.03.2014 0 50 84 68.9 -10 -3.9 -7.1 25.03.2014 0 45 97 73 -10.6 -0.4 -5.5 26.03.2014 0 48 80 60.9 -12.6 -6.2 -9.2 27.03.2014 1 45 90 68.8 -10.3 4.3 -1.6 28.03.2014 4 67 99 85.2 -1.2 10.3 4.8 29.03.2014 0 80 98 87.3 -1.6 1.9 0.4 30.03.2014 0 43 93 61.5 -1.8 7.1 2.1 31.03.2014 0 42 76 61.1 -1.9 8.1 3.3 01.04.2014 0 40 75 58.5 2.1 16.3 7 02.04.2014 0 50 82 68 -1 9.2 3.2 03.04.2014 0 61 80 68.1 -2.5 2.8 0.6 04.04.2014 8 69 100 92.4 0.1 10.4 3.3 05.04.2014 0.2 55 93 72.8 -1.1 4.1 1 06.04.2014 0 19 97 64 -3.2 10.2 3.6 07.04.2014 12.8 41 100 74.6 0.1 10.5 4.5 08.04.2014 9 45 100 79 1.3 12.5 5.9 09.04.2014 0 30 93 63 0.2 10.5 5.5 10.04.2014 3.6 33 97 59.5 2.4 17.7 10

208

11.04.2014 0 43 100 73.2 3.8 13.5 9.2 12.04.2014 3 34 96 72 2.9 17 9.5 13.04.2014 0.6 55 96 72 5.2 22.7 14.5 14.04.2014 15.6 48 100 79.3 4.2 19.8 12.9 15.04.2014 15.6 65 100 83.7 -5 4.1 -1.2 16.04.2014 0.6 49 91 68.9 -7.3 1.5 -2.5 17.04.2014 0 32 83 58.5 -2.1 13.5 4.4 18.04.2014 0 50 87 65.2 1.5 15.1 8.1 19.04.2014 0 38 90 63.6 -0.8 11.5 5.6 20.04.2014 0 21 72 49.7 1.9 19.4 9.6 21.04.2014 0 23 74 46.5 5.6 23.1 14.3 22.04.2014 4.6 43 100 79.8 4 16.1 9.3 23.04.2014 0 30 85 62.1 1.4 10.8 5.5 24.04.2014 0 23 69 45 -1.3 10.8 4.9 25.04.2014 11 31 99 63.8 3.1 14.2 6.8 26.04.2014 0 63 99 82.4 3.4 9 5.9 27.04.2014 0 52 90 69.5 -0.6 10.1 5.3 28.04.2014 0 40 78 55 2.7 12.3 8.2 29.04.2014 26.2 52 100 80.1 7.9 12.4 9.5 30.04.2014 15.6 80 100 95.3 6.7 10.2 8.7 01.05.2014 4 53 100 75.8 8.2 12.8 10.3 02.05.2014 0.8 70 97 81.9 6.2 12 8.7 03.05.2014 3.8 66 92 82.9 7.2 12.4 9 04.05.2014 0 44 88 66.4 3.7 12.6 8 05.05.2014 0 43 88 67.9 2.2 12.6 7.8 06.05.2014 0 36 93 61.6 4.5 15.8 10.3 07.05.2014 0 42 71 58.2 4.7 15.3 9.7 08.05.2014 1 55 92 72.3 6.8 22.2 13.3 09.05.2014 1 40 90 70.4 9.5 26.9 17.7 10.05.2014 0.2 48 91 65.4 11.1 18.8 15 11.05.2014 0 22 95 59.9 5.2 22.8 14.2 12.05.2014 0.6 69 94 80.3 9.9 22.9 16.7 13.05.2014 5.8 50 100 83.5 15 29 20.8 14.05.2014 32.4 73 100 94.4 14.7 22.6 17.3 15.05.2014 14 84 100 94.1 9.9 18.1 14.8 16.05.2014 1 62 100 79.8 4.2 10.4 8 17.05.2014 0 56 97 80.7 2.5 11.1 6.7 18.05.2014 0 39 100 63.5 3.7 15.5 10.4 19.05.2014 0 33 76 50 7.7 20.6 14.4 20.05.2014 5.4 45 97 74.1 9.2 15.2 11.7 21.05.2014 12.2 64 100 88.9 9.9 23.9 16.1 22.05.2014 10.4 71 100 88.1 10.5 17.8 14.1 23.05.2014 0 75 92 84.8 6.9 15 11 24.05.2014 0 29 91 62.7 8.8 23.9 16.1

209

25.05.2014 0 30 94 55.1 8.3 25.5 18.1 26.05.2014 0 35 66 49 14.2 27.7 21.3 27.05.2014 2.8 52 96 71.8 18.7 27.6 22.3 28.05.2014 0 58 97 76.6 12.5 20.8 17.5 29.05.2014 0 53 92 73.7 10.4 19.7 15 30.05.2014 0 41 97 72.1 10.2 24 16.9 31.05.2014 0 26 92 50.7 12.6 23.1 17.9 01.06.2014 0 30 74 50.3 10.6 26.6 18.9 02.06.2014 0.2 60 84 73.7 15.5 26.4 22.1 03.06.2014 15.2 42 100 78.1 14.8 26.5 21.4 04.06.2014 0 50 95 71.1 11.9 20.5 16.8 05.06.2014 0 39 89 63.1 9.4 21.3 15.1 06.06.2014 0 35 93 61.9 8.6 24.2 16.6 07.06.2014 0 33 91 55.8 9.2 23.8 18.2 08.06.2014 9.2 66 97 82.9 13.6 18.5 15.6 09.06.2014 0 55 92 73.2 14.5 23.7 18.9 10.06.2014 0 47 92 69.8 14.6 25.2 20 11.06.2014 8 63 100 84.5 15.7 25.5 19.9 12.06.2014 0 63 100 84.1 18 25.3 21.2 13.06.2014 1.4 54 95 76.5 12.2 21.8 18.4 14.06.2014 0 48 82 65.2 10.5 20 14.9 15.06.2014 0 32 99 71.6 8.4 22.9 16 16.06.2014 0.6 48 96 71.3 12.2 29.4 21.9 17.06.2014 0 44 99 73.2 17.1 30.5 23.9 18.06.2014 2 46 98 79.8 15.6 27.5 20.7 19.06.2014 0.2 31 98 66.4 14.3 24.4 19.9 20.06.2014 0 34 83 53.1 11.7 22.8 17.7 21.06.2014 0 26 69 46.6 13.3 24.5 18.9 22.06.2014 0 26 87 51.4 12.5 26 20.2 23.06.2014 0 56 95 70.3 13.8 27.3 21.1 24.06.2014 3.6 60 99 83.2 19.7 28.3 23.1 25.06.2014 0.6 71 98 88 19.4 24.3 21.5 26.06.2014 7.4 60 100 88.9 17.2 25.6 20.3 27.06.2014 0.2 37 100 70.9 14.8 27.3 21.7 28.06.2014 0 42 97 72.1 15.9 29.8 23.3 29.06.2014 6.4 63 99 83.2 20.3 28.6 24.1 30.06.2014 0 63 99 83.5 20.6 29.2 24.7 01.07.2014 0 53 89 72.5 22.2 28.1 25.2 02.07.2014 0 44 96 70.4 19 27.8 23.6 03.07.2014 8.4 56 100 81.2 13.7 23.5 17.9 04.07.2014 0 34 97 64.3 9.9 22.9 16.6 05.07.2014 0 30 81 54.6 11 25.8 18.9 06.07.2014 0 41 92 65.2 15.7 25.9 21.5 07.07.2014 30.2 73 100 87.8 18.9 26 22.1

210

08.07.2014 28 72 100 91.3 15.9 23 20.4 09.07.2014 0 50 98 77.1 13.7 22 18.1 10.07.2014 0 50 97 72.1 10.9 22.6 17.5 11.07.2014 0 48 96 70 11.9 24.7 19.4 12.07.2014 0 45 100 72.3 13.3 27.3 21.5 13.07.2014 8.8 51 98 80.9 20 27.7 23.5 14.07.2014 13 68 100 88.4 15.5 23.6 19.3 15.07.2014 3.4 47 100 78.4 14.5 23.4 19.2 16.07.2014 0.4 55 94 78.9 12.4 20.5 16.2 17.07.2014 0 37 98 69.3 9.9 22.7 17.1 18.07.2014 0 48 100 75.4 11.1 23.9 18.4 19.07.2014 13.6 81 100 94.1 15.1 19.6 17.4 20.07.2014 0 65 100 88.5 17.8 24.4 20.6 21.07.2014 0.2 62 100 83.1 14.8 26 21.1 22.07.2014 0 51 100 77.7 14.7 28.9 22.5 23.07.2014 0.8 67 97 82.4 13.5 24.8 20.5 24.07.2014 0 38 94 68.3 12.4 22.6 17.2 25.07.2014 0 43 100 71.3 9.5 23.6 17.4 26.07.2014 0 55 91 72.9 15.8 25.1 21 27.07.2014 35.6 64 100 83.9 16.5 26.2 21.9 28.07.2014 14.8 62 100 86.6 11 20.3 16.2 29.07.2014 0 48 99 78.4 10 19.9 15.3 30.07.2014 1 55 100 80.6 13.2 22.1 17.5 31.07.2014 2 46 100 78.5 13 23.4 18.2 01.08.2014 0.4 53 100 80 14.5 26.6 20.7 02.08.2014 0 61 100 85.5 15.8 25.9 20.2 03.08.2014 16.8 66 100 88.2 16.8 25.5 20 04.08.2014 0.2 56 100 84.3 14.8 26.9 21 05.08.2014 8 69 100 90.3 15.5 22.8 18.7 06.08.2014 0.2 43 100 78.1 14.1 24.2 19.3 07.08.2014 0 49 94 74.6 12.3 24.3 18.3 08.08.2014 0 50 99 75.6 10.4 23.5 17.5 09.08.2014 0 44 92 69.8 13.2 25.3 19.5 10.08.2014 0 40 93 70.2 13.8 27.4 20.9 11.08.2014 10.4 66 100 85.3 16.7 25.7 20.3 12.08.2014 5.6 78 100 90 17.4 23.7 20.6 13.08.2014 0.2 62 98 83.7 12.5 19.5 16.3 14.08.2014 0 49 96 71.4 11.1 17.7 14.2 15.08.2014 0 43 86 66.2 11.1 21.1 15.6 16.08.2014 1.2 61 96 77.5 13.1 20.4 17.2 17.08.2014 0 58 100 85.2 11.7 23 17.8 18.08.2014 0 57 100 81.7 11 22.9 16.8 19.08.2014 7.2 56 100 83.2 10.7 24.4 17.6 20.08.2014 0.4 77 100 93 16.7 23.9 20.5

211

21.08.2014 0.2 63 100 85.9 17.5 26.3 21.4 22.08.2014 0 63 100 87.2 16.3 26.1 20.5 23.08.2014 0 84 100 94.4 16.9 21.3 18.9 24.08.2014 0 60 100 85.4 16.1 24.8 19.6 25.08.2014 0 57 100 83.4 14.3 25.3 19.7 26.08.2014 0 59 100 80.6 17.6 28.4 23.2 27.08.2014 0 48 98 73.4 12.9 24.4 20.3 28.08.2014 0 43 98 73.4 9.6 22.2 15.6 29.08.2014 0 38 92 71.5 9.9 22.9 17 30.08.2014 1.2 57 98 77.7 14.4 29.2 22.2 31.08.2014 0 68 100 81.7 19.1 26 23.7 01.09.2014 5.4 70 100 89.8 18.4 26.6 22.7 02.09.2014 6.6 82 100 95.9 17.3 23.8 21 03.09.2014 0.4 51 100 83 14.9 26 20 04.09.2014 0.2 46 100 78.6 16.3 27.9 22.1 05.09.2014 38 64 100 79.6 18.9 29.9 24.5 06.09.2014 4.4 55 100 85.4 11.5 21.6 17.4 07.09.2014 0 41 99 77.3 10.2 22.2 15.4 08.09.2014 0.2 47 100 79.7 9.3 23.4 16.9 09.09.2014 0 70 100 81.2 17.1 23.3 19.8 10.09.2014 21.6 81 100 93.6 16.4 23.7 20.3 11.09.2014 1.6 73 100 85 10.4 22.7 15.9 12.09.2014 0 64 96 82 7.7 13.5 10.9 13.09.2014 6.8 63 100 86.1 7.1 15.7 10.6 14.09.2014 0 46 100 77.4 3.3 15.8 10.4 15.09.2014 3.6 56 100 85 7.2 17.6 12.5 16.09.2014 0.2 44 100 78.8 9 19.2 13.4 17.09.2014 0 47 100 75.9 7.6 19.9 13.1 18.09.2014 0 58 100 84.6 7 17.3 11.1 19.09.2014 0 41 99 77.8 5.3 16.5 10.5 20.09.2014 0 61 100 82 7.8 24 18.8 21.09.2014 5.8 51 100 80.9 10 22.9 18.2 22.09.2014 0 54 92 73.9 6.4 13.7 10 23.09.2014 0 47 98 74.5 6.3 20.9 13.3 24.09.2014 0 47 100 78.8 7.1 22.3 15.1 25.09.2014 0 52 100 81.7 8.6 21.2 15.6 26.09.2014 0 46 100 77.6 8.5 23.1 15.8 27.09.2014 0 37 100 77 9.1 25.3 16.7 28.09.2014 0 48 100 81.8 10.7 25.4 17.4 29.09.2014 0 44 100 80.4 10.3 24.7 17.7 30.09.2014 18.2 87 100 97.4 12.7 17.8 15.2 01.10.2014 0 74 100 92.2 14.1 19.2 15.6 02.10.2014 0 67 100 89.3 14.4 20.7 16.6 03.10.2014 12.2 71 100 89.1 14 22 17.6

212

04.10.2014 9.6 68 100 90.4 4.3 13.8 8.1 05.10.2014 0.2 53 99 76.4 2.3 12.9 7.7 06.10.2014 1.6 63 100 96 8.2 15.7 12.6 07.10.2014 32.6 100 100 100 10.5 20 12.7 08.10.2014 0.8 100 100 100 7.8 29.7 14 09.10.2014 0 100 100 100 2.9 14.4 8.2 10.10.2014 0 100 100 100 2.5 13.5 7.4 11.10.2014 0 100 100 100 0.5 14 7 12.10.2014 0 100 100 100 -0.9 14 7.1 13.10.2014 11.2 100 100 100 6.4 17.1 14 14.10.2014 2.6 100 100 100 15 20.5 18.3 15.10.2014 6 100 100 100 12.2 18.1 14.5 16.10.2014 5.8 100 100 100 11.1 18.9 13.7 17.10.2014 0.4 100 100 100 11 15.4 13.3 18.10.2014 0.6 100 100 100 3 12.7 8.4 19.10.2014 0 100 100 100 0.4 9.6 5 20.10.2014 2 76 100 92.1 4.4 12.8 9.6 21.10.2014 2.2 86 100 96 5.9 9.9 8.3 22.10.2014 0 57 95 76.6 4 13.3 8 23.10.2014 0 46 98 75.4 3.3 17 9.5 24.10.2014 0.2 47 100 86.3 1.2 15.4 7.6 25.10.2014 0 51 98 78.3 8.5 17.2 12.6 26.10.2014 0 52 95 70.8 1.8 12.6 8.2 27.10.2014 0.4 55 100 81.6 -0.7 13.7 7.6 28.10.2014 0.6 66 96 81.7 11.8 21.4 16.6 29.10.2014 0 46 86 68.2 6.3 13.6 9 30.10.2014 0 56 99 80.9 2 9.1 5.5 31.10.2014 8.6 87 100 96.3 3.2 6.7 5.3 01.11.2014 0 64 97 81.1 0.5 2.9 1.5 02.11.2014 0 38 88 65.9 -3.2 7.4 1.8 03.11.2014 0 42 64 52.8 2.1 12.5 9 04.11.2014 8.6 45 99 67.7 10 13.9 12.2 05.11.2014 0 56 100 78.1 3.2 11 7.1 06.11.2014 1.6 72 100 91.3 2.5 9.8 6.2 07.11.2014 0.4 54 95 74.6 -1.8 5 2 08.11.2014 1.8 69 97 84 -2.1 6.3 3.5 09.11.2014 0 58 100 74.5 -1.1 8.1 4.5 10.11.2014 0 53 86 68.6 4 12.1 7.6 11.11.2014 3.6 45 99 68.7 6.9 17 12.4 12.11.2014 0.4 60 97 68.4 -0.3 8.2 2 13.11.2014 0 62 81 72.7 -3.7 0.1 -1.2 14.11.2014 0.2 76 95 85.1 -4.7 0 -2.4 15.11.2014 0.4 50 90 71.9 -2.6 0.7 -1 16.11.2014 0.2 65 100 90.7 -3.1 -1.4 -2

213

17.11.2014 3.4 70 100 92.6 -7.5 0 -2.1 18.11.2014 0 59 81 70.6 -10.5 -6.2 -8.2 19.11.2014 0 71 100 87.4 -11.7 -1.1 -6.4 20.11.2014 0.2 52 91 73.8 -9.6 -2.2 -5.2 21.11.2014 0 43 90 71.3 -10.9 -3.7 -7.6 22.11.2014 5.6 64 100 85.8 -4.9 6.4 1.8 23.11.2014 9 68 100 86.5 6.1 10.4 7.7 24.11.2014 19.6 68 100 83.7 3.8 16.1 9.8 25.11.2014 0 66 80 73.9 -0.3 3.9 1.4 26.11.2014 0 68 94 79.5 -1.1 1.3 -0.2 27.11.2014 0.2 70 97 88.7 -4.9 1.6 -1.2 28.11.2014 0 59 83 70.4 -7.6 -3.7 -5.2 29.11.2014 0 62 96 76.3 -5.2 8 1.9 30.11.2014 3.4 69 100 81.6 5.7 14.2 11.2 01.12.2014 0 50 93 70.9 -6.1 5 -1.1 02.12.2014 0.6 67 100 82.2 -8.2 0.9 -4.3 03.12.2014 0.4 62 100 84 -1.6 1.5 0.7 04.12.2014 0 54 80 70.8 -4.7 0.3 -2.8 05.12.2014 0 77 100 86.6 -4.1 2.9 -0.3 06.12.2014 0 63 100 83.7 -2.1 4.8 1.4 07.12.2014 0 60 84 73.1 -5.5 -1.3 -3.5 08.12.2014 0 45 85 70.2 -5.6 4.2 -0.6 09.12.2014 0.6 81 100 93 1.1 4.7 2.4 10.12.2014 0 69 94 83 -3.9 1.4 -1.7 11.12.2014 1.4 50 99 80.4 -3.4 1.2 -1.3 12.12.2014 1.8 69 100 82.7 -2.1 3.8 0.5 13.12.2014 0 92 100 97.4 -1.3 2.8 0.2 14.12.2014 0.4 92 100 98.1 2.3 3.6 2.8 15.12.2014 0.2 100 100 100 1.8 4.1 3.2 16.12.2014 4 100 100 100 1.8 6.3 4 17.12.2014 1.4 93 100 99 -2.5 5.2 0.7 18.12.2014 0 95 100 99.1 -2.7 -0.7 -1.8 19.12.2014 0 74 90 81.9 -5.6 -1.5 -3.6 20.12.2014 0 70 95 84.4 -6.8 -0.5 -3.6 21.12.2014 0 61 93 79 -3.6 0 -1.6 22.12.2014 0 72 91 82.5 -2.1 2.8 -0.2 23.12.2014 0.4 87 100 94.2 -0.7 6.2 3 24.12.2014 7.4 89 100 98.1 3.6 11 7.7 25.12.2014 0.6 73 94 84.2 2.1 4.3 3.2 26.12.2014 0 68 93 82.4 1.2 7 3.7 27.12.2014 2.2 55 99 83.7 3.9 10.3 6.7 28.12.2014 4.6 62 100 81.1 -2.3 6.9 1.2 29.12.2014 0 55 93 78.4 -6.6 -1.7 -3.5 30.12.2014 0 52 86 68.3 -7.7 -3.2 -5.9

214

31.12.2014 0 47 75 62.2 -11.5 -6.5 -8.3 01.01.2015 0 51 72 63.5 -7.6 -1.7 -4.1 02.01.2015 0 46 80 63.2 -5.7 -0.1 -2.5 03.01.2015 17.8 73 100 91.8 -5.4 0.3 -2.2 04.01.2015 27.2 71 100 94.6 -5.6 5.8 0.8 05.01.2015 0 56 86 70.2 -14 -5.8 -11.9 06.01.2015 0 59 90 73.8 -13.3 -8.8 -11 07.01.2015 0 45 91 66.1 -17.5 -10 -14.2 08.01.2015 0 61 95 76.2 -16.4 -8.9 -12.4 09.01.2015 0 50 95 70.4 -14.6 -8.1 -10.1 10.01.2015 0 60 83 71.5 -15.9 -7.4 -12.1 11.01.2015 0 58 99 70 -7.3 -1.3 -3.7 12.01.2015 2.6 71 100 88 -12.3 -1.2 -3.9 13.01.2015 0 68 88 77 -21.3 -11.7 -15.9 14.01.2015 0 57 94 81.5 -22 -6 -13.9 15.01.2015 0 56 94 81.4 -15.6 -2.9 -8.5 16.01.2015 0 55 90 72.8 -16.9 -2.8 -7.6 17.01.2015 0 57 92 77.6 -18.8 6 -5.8 18.01.2015 2.2 54 98 84.7 -0.4 6.4 2.3 19.01.2015 0 65 96 79 -6.3 -0.3 -2.5 20.01.2015 0 68 91 80.3 -10.2 -4.5 -7.1 21.01.2015 0 55 95 76.8 -11.7 -3.1 -7.1 22.01.2015 0.2 52 92 79.6 -5.6 -1.7 -4.1 23.01.2015 0 73 98 82.2 -5.5 -2.3 -3.8 24.01.2015 0 90 98 94.8 -5.7 0 -2.8 25.01.2015 0 51 84 66.7 -11.6 -0.1 -7.2 26.01.2015 0 61 90 77.9 -11.6 -6.4 -8.8 27.01.2015 0 71 87 78.8 -13.7 -6.7 -10.4 28.01.2015 0 35 92 72.3 -15.6 -2.8 -9.9 29.01.2015 0 56 100 81.1 -6.7 0.1 -2.6 30.01.2015 0 57 91 76 -16.7 -1 -9.9 31.01.2015 0.2 70 90 79.6 -13.1 -2.6 -7.5 01.02.2015 0 75 93 88.6 -10.2 -2.7 -6.8 02.02.2015 0 61 93 79 -15.9 -9 -12.4 03.02.2015 0 69 95 82.2 -15.8 -5.6 -9.7 04.02.2015 1.4 70 99 90.9 -8.1 -0.2 -3.7 05.02.2015 0 37 86 67.9 -15.8 -8.3 -12.3 06.02.2015 0 62 86 73.9 -13.9 -4.7 -8.4 07.02.2015 0 64 91 79 -4.6 1.5 -1.7 08.02.2015 0 89 98 93.8 -10.3 -1.7 -6 09.02.2015 0 78 92 84.3 -13.1 -9.7 -11.2 10.02.2015 1.4 62 91 82.3 -10.9 -4.1 -8.6 11.02.2015 0 77 99 90.9 -8.1 -1.6 -4.7 12.02.2015 0 58 91 73.1 -19.5 -3.3 -12.1

215

13.02.2015 0 58 92 76.4 -19.7 -9.7 -14.7 14.02.2015 0 58 95 78.2 -20.6 -6.1 -10.8 15.02.2015 0 49 74 58.7 -26.2 -19.9 -22.8 16.02.2015 0 41 85 67 -29.2 -14.1 -21.4 17.02.2015 0 32 84 64.9 -24.1 -10.1 -15.5 18.02.2015 0 69 94 83.7 -15.9 -8.9 -11 19.02.2015 0 43 83 68.6 -23.4 -16 -18.8 20.02.2015 0 34 78 61.6 -24.4 -15.2 -19.9 21.02.2015 0.6 59 97 83.1 -14.4 -5.8 -9 22.02.2015 3.4 65 97 85.7 -16.1 -4.7 -10.5 23.02.2015 0 47 85 67 -23 -15.9 -19.3 24.02.2015 0 40 90 69 -21.4 -8.1 -14.1 25.02.2015 0 53 90 68.8 -16.2 -6.1 -11.9 26.02.2015 0 58 76 70.9 -16.7 -11.3 -14.2 27.02.2015 0 49 91 73.2 -22 -10.5 -15.7 28.02.2015 0 42 92 73.7 -17.9 -6.2 -12.9 01.03.2015 1.6 77 94 87.4 -13.5 -4.3 -8.3 02.03.2015 0.2 53 93 76 -12.4 -3 -7.3 03.03.2015 5.8 89 100 94.5 -14.6 1.7 -6.4 04.03.2015 0 55 89 69.8 -8.7 1.2 -1.3 05.03.2015 0 50 88 64.4 -17.4 -9 -13.4 06.03.2015 0 53 92 70.4 -19.5 -6.3 -11.4 07.03.2015 0 59 91 76.2 -7.4 -0.2 -3.4 08.03.2015 0 51 86 71.8 -2.6 3.9 -0.1 09.03.2015 0 44 98 71.6 -3.9 6.1 0.8 10.03.2015 0 64 100 86.1 -4.2 7.8 1.3 11.03.2015 0 53 100 78.2 1.8 5.5 3.5 12.03.2015 0 43 77 58 -3.9 2.1 -0.5 13.03.2015 0 46 99 57.8 -3.6 5.4 0.7 14.03.2015 0.2 77 100 92.9 1.9 5.2 3.6 15.03.2015 0 73 95 81.3 -1.4 2.8 0.8 16.03.2015 1.4 67 100 85.7 -1.1 8.9 3.6 17.03.2015 0.4 36 100 70.7 -3.1 4.4 1 18.03.2015 0 37 74 57.5 -5.7 2.6 -1.7 19.03.2015 0 27 91 59.5 -7.4 1.1 -2.6 20.03.2015 0 40 82 61 -1.8 8.6 2 21.03.2015 1.2 44 99 78.8 -3.8 4.2 1.7 22.03.2015 0 46 75 61.7 -8.2 -2.9 -5.4 23.03.2015 0 39 84 62.1 -8.4 -3.6 -6.1 24.03.2015 0 50 76 64.7 -8.6 2.6 -3.5 25.03.2015 2.6 69 100 85.8 -4.1 7.5 1.6 26.03.2015 15.2 77 100 88.9 0 5.5 2.2 27.03.2015 0 65 91 76.5 -8.7 1 -3.6 28.03.2015 0 36 77 54.9 -10.7 -2.2 -6.6

216

29.03.2015 0 44 89 64.4 -9.4 4.2 -1.9 30.03.2015 3 57 96 75.8 -0.2 6.2 3.2 31.03.2015 1 50 99 76.6 -2.1 6.2 1.1 01.04.2015 0 53 97 75.6 -2 4.8 0.7 02.04.2015 9.6 33 100 78.3 -0.9 14.8 7.4 03.04.2015 3.8 75 100 95.3 3.5 12.3 7.5 04.04.2015 0 39 87 69.4 -1.3 5.2 1 05.04.2015 0.8 84 99 90.8 -1.6 1.6 -0.1 06.04.2015 0 63 96 83.2 0.1 10.6 3.6 07.04.2015 1 41 92 71.5 1 6.8 3.4 08.04.2015 11.2 66 100 95.1 0.1 2.3 1.3 09.04.2015 23 100 100 100 0.7 14.3 4.6 10.04.2015 1.4 65 100 82.6 3.9 15.2 10.4 11.04.2015 0 45 91 67.1 0.9 10.2 5.3 12.04.2015 0 28 84 60.7 1.9 17 9 13.04.2015 2.8 42 96 63 4.4 24.3 13.4 14.04.2015 0 24 93 55.9 3.9 15.8 10.1 15.04.2015 0 27 65 41.5 3 15.1 9.3 16.04.2015 4 28 100 62.9 3.2 16.9 9.3 17.04.2015 0 36 100 78.3 9 21 13.5 18.04.2015 0 19 90 52.5 8.7 20.5 14.2 19.04.2015 4.6 50 98 63 3.7 11.1 7.5 20.04.2015 22.2 66 100 87.3 6.5 17.9 11.5 21.04.2015 0.8 41 89 66.4 4.7 10.9 7.7 22.04.2015 2.8 57 98 78.3 -0.5 6.1 3.1 23.04.2015 0.4 63 92 75.2 -1.4 0.6 -0.2 24.04.2015 2.4 39 84 62.9 -2 6 1.6 25.04.2015 0 44 81 64 -2.4 8.2 3.1 26.04.2015 0 42 72 60.6 2 11.5 6.7 27.04.2015 0 64 92 74.1 3.7 10.3 6.6 28.04.2015 0 44 90 67.2 1.9 17.9 10.1 29.04.2015 0 41 89 64.2 3.7 17.3 11.5 30.04.2015 0.8 39 95 72.2 3.8 18.4 10.6 01.05.2015 0 32 86 58.6 6.8 20.2 13.5 02.05.2015 0 27 90 54.1 5.1 21.3 14.2 03.05.2015 0 25 80 47.7 6.9 24.4 16.7 04.05.2015 0.4 36 97 62.1 11.9 24.4 17.3 05.05.2015 2.6 42 97 72.8 8.3 17.2 12.6 06.05.2015 0 41 100 65.9 10.3 21.2 15.4 07.05.2015 0 46 100 64.9 10.7 23.2 16.9 08.05.2015 0 31 100 67.4 11.3 30.2 20.2 09.05.2015 0 32 95 59.9 13.7 29.7 22.5 10.05.2015 0 47 97 75.6 15.4 28.2 21.2 11.05.2015 9.8 40 100 81 14.5 28.4 20.3

217

12.05.2015 0.2 62 95 78.1 10.1 18.3 13.6 13.05.2015 0 49 87 71 4.8 12.6 8.3 14.05.2015 0 24 83 57 2.7 17.5 10 15.05.2015 0 59 96 70.6 8.6 19.1 13.7 16.05.2015 0 56 100 81 11.3 23.3 17.9 17.05.2015 0 62 100 83.2 14 25.5 19 18.05.2015 0 53 100 79.1 16 27.1 22 19.05.2015 0 47 97 70.3 7.3 19.2 14 20.05.2015 1.6 28 79 54.4 3.3 14.7 8.6 21.05.2015 0 30 69 45.2 7.5 17.3 11.9 22.05.2015 0 15 71 42.9 5.1 16.5 10.9 23.05.2015 0 20 84 40.8 -0.8 19.8 11.1 24.05.2015 0 28 70 40.7 9.9 26.3 19.2 25.05.2015 0 46 84 59.9 14.6 27 21.4 26.05.2015 0 52 90 71.7 17.3 27.5 22.1 27.05.2015 0 45 96 79.2 16.1 27.2 20.8 28.05.2015 0.8 49 96 71.4 13.2 23.4 18.6 29.05.2015 0 45 89 67.6 13 27.3 20.1 30.05.2015 10.4 56 100 74.4 15.7 28.1 23 31.05.2015 54 91.3 99.7 98.3 5.6 15.8 8 01.06.2015 0 73.2 99.3 85.1 6.4 14.7 10.4 02.06.2015 0 45 97 73 10.3 18.5 15.3 03.06.2015 0 41 98 69.4 7.6 21.6 14.5 04.06.2015 0 64 100 82.9 7.8 22.3 15.7 05.06.2015 9.8 57 100 84.8 14.4 26.2 19.4 06.06.2015 0 31 91 64.8 10.1 17.9 14.8 07.06.2015 3.2 55 99 69.6 7.2 24.4 16.4 08.06.2015 14.8 81 100 90.1 17.4 20.9 19.4 09.06.2015 16 60 100 86 14.9 21.7 18 10.06.2015 0 57 100 78 13 26.4 19.8 11.06.2015 0 55 95 75.5 12.9 22.3 18 12.06.2015 2.6 68 97 86.4 15.6 25 19.1 13.06.2015 0 69 98 86.4 14.8 22.4 17.4 14.06.2015 29 83 100 96.5 16.3 24.6 19.7 15.06.2015 0.2 66 100 87.1 19.7 27.9 23.2 16.06.2015 1.4 43 100 76 15.5 25.9 21.8 17.06.2015 0 58 97 77.7 13.9 22.2 18 18.06.2015 0.6 72 98 84.5 13.8 25.4 19.4 19.06.2015 6.2 66 100 87.3 12.8 21.3 17.5 20.06.2015 0 68 100 87.3 10.9 22.4 17.6 21.06.2015 0 52 100 77.5 17.4 26.2 21.7 22.06.2015 3 52 100 79.8 14.4 25.8 20.8 23.06.2015 17 63 100 81 15.2 24.7 20.6 24.06.2015 0 37 98 68.6 12.2 25.8 18.9

218

25.06.2015 0 33 89 63.4 13.9 23.7 19 26.06.2015 0 37 99 70.1 13.7 22.6 18.1 27.06.2015 53.8 59 100 86.2 13.1 16.8 15.3 28.06.2015 7 88 100 96.9 11.3 16.2 13.6 29.06.2015 0 48 100 78.3 13.1 23.8 18.3 30.06.2015 0.4 68 100 86.9 14.9 23.2 18.5 01.07.2015 0 64 100 83.1 14.9 22.2 18.5 02.07.2015 0 42 100 76.8 11.4 22.5 16.8 03.07.2015 0 38 100 67.9 9.9 23 17 04.07.2015 0 37 92 65.6 10.9 25.5 18.8 05.07.2015 0 43 100 73.6 14.2 26.9 20.6 06.07.2015 0 65 100 82.3 15.4 27.1 21.5 07.07.2015 1.2 74 100 90.1 16.7 26.2 21.1 08.07.2015 0 54 95 75.3 13.1 20.4 16.9 09.07.2015 10.8 56 100 89.1 13.5 24.3 17.2 10.07.2015 0 50 99 73.8 12.3 24.7 19.2 11.07.2015 0.2 35 98 67.6 14.3 27 21.1 12.07.2015 0 47 92 69.8 15.2 26.2 21.1 13.07.2015 0.2 54 100 77 15.1 27.8 22 14.07.2015 28.4 87 100 98.1 18.6 23.2 20.6 15.07.2015 0 45 100 71.3 12.9 22.3 17.6 16.07.2015 0 44 100 76.8 10.5 23 17.4 17.07.2015 2 76 97 86.8 15.2 25.1 20 18.07.2015 1.8 62 100 85.9 21.3 30.3 24.7 19.07.2015 1.2 74 99 84.7 21.1 27.7 24.3 20.07.2015 0.2 44 100 72.6 16.1 27.1 22.4 21.07.2015 0 38 88 61.1 16.8 25.6 22.1 22.07.2015 0 37 84 59.2 13.5 24.4 19.1 23.07.2015 0 30 95 62.5 11.7 26.9 20 24.07.2015 0 34 95 61.9 12.8 26.5 20.7 25.07.2015 0 48 86 67.6 18.7 29 23.8 26.07.2015 0 49 100 74.9 16.4 28.6 23 27.07.2015 0 43 100 71.2 16.6 29.8 23.9 28.07.2015 0 38 96 65.6 17.3 30.9 24.9 29.07.2015 0 39 97 69.3 17.3 31.1 24.7 30.07.2015 0 32 93 58.9 18.5 29 24.4 31.07.2015 0 36 90 66.6 15.9 28.5 22.5 01.08.2015 0.4 43 93 71.9 15.3 25.7 20.4 02.08.2015 8.6 40 100 67.4 15.2 27.6 22.4 03.08.2015 10 50 100 82.5 16.1 25.2 20.1 04.08.2015 1.8 45 96 76.9 15.5 24.6 19.7 05.08.2015 0.8 45 100 74.7 13.4 22.8 18 06.08.2015 0 45 100 75.9 10.1 23.3 16.8 07.08.2015 0 45 94 71.6 13.6 25.2 19.2

219

08.08.2015 0 53 84 72.1 15 23.3 18.5 09.08.2015 0 42 95 70.3 13.5 25.9 19.5 10.08.2015 54.2 65 100 88.8 14.4 25.8 18.6 11.08.2015 0 61 100 82.4 16.3 24.6 19.8 12.08.2015 0 43 100 74.5 13.4 23.2 18 13.08.2015 0 46 94 71.8 10.5 24.9 19 14.08.2015 1.4 72 96 83.3 21 25.3 22.7 15.08.2015 0 61 100 83.4 18.6 27.2 22.9 16.08.2015 0 52 98 76.7 16.9 28.3 22.9 17.08.2015 0 59 99 80.7 19.2 29.5 24.6 18.08.2015 0.4 78 100 94.3 19.2 25.2 22 19.08.2015 4 64 100 88.1 18.2 28.4 22.9 20.08.2015 17.4 60 100 81.5 18.1 23.5 21.7 21.08.2015 0 46 97 68.8 12.6 22.8 17.9 22.08.2015 0 58 100 81.7 10.6 23.5 17.6 23.08.2015 0 48 100 76 12.3 25 18.8 24.08.2015 0.4 47 97 72.2 14.6 22.1 19.2 25.08.2015 0 54 83 70.9 13.6 19.5 16.2 26.08.2015 0 64 98 81.8 12.2 19.6 15.7 27.08.2015 0 61 99 82.7 13.2 20.1 16.4 28.08.2015 0 56 100 82.7 9.5 22.4 16.4 29.08.2015 1.2 54 100 84.8 12.6 25.1 18.5 30.08.2015 0.4 64 100 89 17.2 26.4 20.9 31.08.2015 0 54 100 85.3 18.5 28.3 22.7 01.09.2015 2.8 57 100 85 15.3 29 22.3 02.09.2015 0 57 100 83.3 18.8 29.9 24.2 03.09.2015 0 62 100 86.4 18.6 29.5 23 04.09.2015 0 53 100 81.6 17.8 28.7 22 05.09.2015 0 51 100 76.9 16.3 29.2 22.7 06.09.2015 0 46 100 80.3 18 30.5 24 07.09.2015 0 50 99 74.1 19.9 31.1 25.7 08.09.2015 0 52 100 80.4 20 29.9 24.8 09.09.2015 0 62 100 76.9 15.4 25.9 22.3 10.09.2015 0 43 89 66.8 11.3 23.3 17.7 11.09.2015 0 50 100 86.9 10 23.7 15.6 12.09.2015 0 73 100 94.9 10.4 15.3 12.6 13.09.2015 0 63 96 78 8.5 15.3 11.9 14.09.2015 0 40 95 70.9 7.6 22 15 15.09.2015 6.2 43 100 71.1 14.2 25.6 19.9 16.09.2015 0 43 100 74 12.9 27.1 20.1 17.09.2015 0 42 100 75.1 12.3 27.3 19.8 18.09.2015 0 48 100 81.6 15.1 25.5 19.7 19.09.2015 3.8 72 100 90 11.4 22.8 18 20.09.2015 0.2 39 100 71.3 8.2 19.3 13.7

220

21.09.2015 0 35 88 65.6 9.1 21.2 14.6 22.09.2015 0 56 100 85.4 8.4 22.4 14.7 23.09.2015 0.2 43 100 78.3 8.9 23.8 16 24.09.2015 0 46 97 76.3 12 25.7 18.1 25.09.2015 0 48 93 77.5 13.2 24.3 17.4 26.09.2015 0 61 100 78.6 12.5 22.2 16.1 27.09.2015 0 60 100 78.1 12.1 20.9 17.2 28.09.2015 0.4 82 100 94.8 16.6 20.8 18.5 29.09.2015 4.8 89 100 97.7 13.6 20.8 18 30.09.2015 0 57 91 75 9.7 17 13.4 01.10.2015 0 50 84 68.1 6 12.8 9.3 02.10.2015 0 54 85 68.6 4.4 11.4 8.3 03.10.2015 7.8 58 100 79 6.1 8.9 7.5 04.10.2015 0 68 99 88.4 7.2 17 11.2 05.10.2015 0 64 100 88.2 9.6 19.4 13.6 06.10.2015 0 83 100 92.9 10.9 16.4 13.9 07.10.2015 0 53 100 83.5 9.2 21.4 14.4 08.10.2015 6.2 64 100 86.2 5.4 17.5 11.7 09.10.2015 11.2 74 100 90 6.6 16.3 13 10.10.2015 0.2 50 100 81.3 4.1 16.3 9.7 11.10.2015 0 47 99 70.1 9.1 18.9 14.9 12.10.2015 0 44 92 64.4 12.2 21.4 17.4 13.10.2015 0 55 84 68.8 11 18.9 14.7 14.10.2015 0 58 99 81.6 3.9 12.1 9.4 15.10.2015 0 47 99 72.1 4 17.4 10.6 16.10.2015 1 57 98 81.1 3.7 13.2 7.1 17.10.2015 0 52 96 81 0 6.4 2.6 18.10.2015 0 46 100 74.8 -2.9 6.6 2.2 19.10.2015 0 37 92 63.6 -3.3 14.7 7.6 20.10.2015 1.4 44 96 61.8 11.6 18.8 14.8 21.10.2015 0.4 49 100 74.6 11.7 17.7 14.8 22.10.2015 0 47 85 64.4 6 18.7 14.5 23.10.2015 0 47 91 74.7 0.5 11.4 6.1 24.10.2015 12.4 79 100 88.5 5.8 16.3 10.8 25.10.2015 0.2 49 98 78.6 3.2 15.5 9.9 26.10.2015 0 59 100 82.3 0.9 11.2 6.2 27.10.2015 0 50 88 74.5 3.6 11.5 7.2 28.10.2015 67.8 79 100 97.3 8.1 15.9 12.2 29.10.2015 0.8 48 94 67.3 5.7 14 8.2 30.10.2015 0.2 51 98 78.9 0.4 10 6.1 31.10.2015 0.6 63 100 82.2 -1.1 10.4 6.1 01.11.2015 2.8 66 100 83 6.6 14.3 11.4 02.11.2015 0.2 60 100 85.1 1.9 14.4 9.1 03.11.2015 0 37 100 74.6 8.6 22.9 14.5

221

04.11.2015 0 45 100 80.8 6.8 21 13.3 05.11.2015 0.2 63 100 85.6 12.6 23.2 16.7 06.11.2015 6 61 99 81 9.4 17.4 14.9 07.11.2015 0.6 46 87 71.1 3.1 11.3 7.7 08.11.2015 0 49 92 74 0.7 10 4.4 09.11.2015 0 47 99 77.5 -2.2 12.5 5 10.11.2015 17.6 95 100 99.7 2.8 8.7 6.7 11.11.2015 0.2 56 100 89.2 4.6 12.9 8.4 12.11.2015 1.6 72 99 84 6.8 12.1 9.1 13.11.2015 0.4 59 96 74.6 2.1 8.6 5.4 14.11.2015 0 65 92 77.3 1 4.8 2.7 15.11.2015 0 53 82 63.9 4 14.2 10.4 16.11.2015 0 36 100 69.2 4.8 17.3 9.9 17.11.2015 0 56 100 74.1 2.5 11.6 6.8 18.11.2015 0 65 86 75.6 11 15.3 13.1 19.11.2015 4.2 39 97 70.6 3.9 15.4 12.1 20.11.2015 0 35 69 50.7 0.1 6 3.3 21.11.2015 15 54 100 87 -0.1 5.2 2.4 22.11.2015 0 64 96 79.4 -5.2 3.4 -0.8 23.11.2015 0.6 49 99 76.7 -7 1.8 -1.9 24.11.2015 0 71 100 85.9 -2.8 5 0.9 25.11.2015 0.2 62 100 83.7 -2.5 9 3.6 26.11.2015 0 52 71 60.3 7 14.6 11.3 27.11.2015 8.2 58 99 81.2 1.2 14.6 10 28.11.2015 1 83 100 93.1 -3 1.6 -0.1 29.11.2015 0.2 73 100 88.7 -5.5 1.6 -1.9 30.11.2015 0 60 89 79.8 -1.8 4.8 0.7 01.12.2015 1.2 86 100 95.7 1.3 9.4 5.9 02.12.2015 0 71 100 89.9 -0.1 7 3.8 03.12.2015 0 68 94 82.5 0.1 6.5 3.2 04.12.2015 0 69 95 79 1 4.6 3.1 05.12.2015 0 79 100 92.8 -0.9 5.1 2.4 06.12.2015 0 82 100 95.6 -0.8 6.2 2.6 07.12.2015 0.4 88 100 96.9 -2.9 2.8 0.5 08.12.2015 0 89 100 94.8 -0.5 5.8 3.1 09.12.2015 0 68 100 85.4 3.1 10.2 7.3 10.12.2015 0.2 78 100 88.7 3.6 10.5 7.9 11.12.2015 0 57 96 77.7 3.7 12.2 9 12.12.2015 0 76 98 86.2 2.6 9.3 6.3 13.12.2015 0 96 100 99.8 5.3 6.4 6 14.12.2015 0 75 100 91.6 5.5 15.1 9.9 15.12.2015 0 78 96 85.5 3.4 8 6.1 16.12.2015 0 85 100 92.9 0.9 7.8 4.2 17.12.2015 0 61 97 76.3 0.9 9.6 5.7

222

18.12.2015 0 49 91 64 -3.6 2.6 -0.3 19.12.2015 0 58 70 64.7 -5.5 -2.1 -3.3 20.12.2015 0 45 88 66.2 -6.7 5.3 -0.2 21.12.2015 0 47 100 80.8 4.7 9.8 6.4 22.12.2015 0 82 100 96.3 2.6 13.5 9.1 23.12.2015 0 84 100 94.8 3.3 15.5 9.7 24.12.2015 0 62 100 77.7 4.9 14.5 9.3 25.12.2015 0 66 100 85.5 1.3 8.7 5.5 26.12.2015 0.4 76 100 89.8 -1 4.1 2.1 27.12.2015 0.2 75 100 89.9 0.1 4.2 2.8 28.12.2015 0.4 68 100 82.8 -5.1 0.5 -3.2 29.12.2015 0.4 74 100 90.6 -1.7 6.3 2.6 30.12.2015 0 81 99 89.2 0.1 2.6 1.3 31.12.2015 0 70 88 81.2 -0.9 0.2 -0.3 01.01.2016 0.4 57 88 74 -3.8 -0.7 -2.2 02.01.2016 0 70 81 76.9 -3.4 1.6 -1 03.01.2016 0 62 91 74.6 -7.3 1.8 -1.5 04.01.2016 0 49 82 67.2 -15.3 -7.7 -11.5 05.01.2016 0 65 90 78.5 -16.1 -2.8 -9.3 06.01.2016 0 55 87 72.3 -5.6 3.3 -1.8 07.01.2016 0 53 98 79.5 -5.2 4.4 -0.7 08.01.2016 0 67 100 81.8 -3.1 4 0.2 09.01.2016 0 83 100 96.6 4.2 8.8 6.2 10.01.2016 1 63 100 89.6 -7.2 7.1 1.7 11.01.2016 0 53 93 67.6 -11.1 -7.4 -9.5 12.01.2016 1 66 100 87.1 -10.3 -2.6 -6.1 13.01.2016 0 53 95 74.6 -11.6 -7.4 -9.3 14.01.2016 0 76 99 87.9 -8.4 -3.1 -5.6 15.01.2016 0.4 67 100 89.1 -4.3 5.7 0.8 16.01.2016 1.2 63 100 82.4 -3.2 2 -1 17.01.2016 0 63 91 77.1 -12.4 -1.7 -5.9 18.01.2016 0 60 86 70.8 -12.1 -8.2 -10.7 19.01.2016 0 72 90 78.2 -12.8 -6.8 -9.4 20.01.2016 0 63 95 78.4 -10.6 -4.4 -7.5 21.01.2016 0 65 95 80.3 -11.1 -2.8 -6.2 22.01.2016 0 58 95 77.1 -10.6 -4.3 -6.9 23.01.2016 0 49 94 75.8 -13.3 -5.3 -8.2 24.01.2016 0 72 95 84.8 -14.3 -0.5 -5.8 25.01.2016 0.4 54 89 73.4 -2.6 3.3 -0.1 26.01.2016 2.2 74 93 84.6 0 4.4 1.6 27.01.2016 0 50 96 78 -2.2 0.9 -1 28.01.2016 0.8 66 96 82.8 -3.4 1.6 -0.7 29.01.2016 0 68 92 79.7 -9.4 -1 -4.8 30.01.2016 0 55 94 73.1 -9.1 7 0.9

223

31.01.2016 3.4 66 96 78.5 1.2 9.6 6.2 01.02.2016 0.2 62 98 83.1 -4.7 7.8 2.5 02.02.2016 4.4 65 100 89 -7 4.8 -1.2 03.02.2016 13.4 52 100 79.9 0.4 13.5 7.3 04.02.2016 0.4 56 97 81.3 -2.3 2.9 0.8 05.02.2016 0.2 57 98 79.9 -5.7 1.3 -1.9 06.02.2016 0 48 86 70.1 -1.2 2.8 0.3 07.02.2016 0 50 93 75.5 -2.8 6.6 1.6 08.02.2016 3.2 58 100 80.8 -0.3 6.5 2.7 09.02.2016 2 82 100 94.3 -2.9 0.6 -0.7 10.02.2016 0 59 95 83.1 -11.4 -2.7 -6.9 11.02.2016 0 60 90 72.1 -16.9 -9.4 -12.5 12.02.2016 0 62 93 73.6 -13.7 -4.7 -9.2 13.02.2016 0 50 89 65.2 -22.3 -11.9 -18.1 14.02.2016 0 58 87 73.6 -24.7 -7.6 -16.5 15.02.2016 0 70 99 89.2 -16.2 -1.1 -6.3 16.02.2016 2.2 77 100 91.1 -2.8 2 -1.3 17.02.2016 0.6 62 94 77.2 -13.3 -0.5 -4.4 18.02.2016 0.2 55 96 77.8 -22.8 -2.5 -12.2 19.02.2016 2.2 52 79 60.5 -12.8 10.1 2.3 20.02.2016 0.2 42 89 63.7 -1.3 13.9 7.2 21.02.2016 0 48 91 74.2 -2 6.4 2.2 22.02.2016 0 46 73 60.1 -4.7 0.8 -1.9 23.02.2016 0 54 88 74.1 -6.1 1.8 -2.3 24.02.2016 26.8 87 100 98.5 -1.6 1.5 0.4 25.02.2016 1 67 100 89 -3.7 1.6 -1.1 26.02.2016 0 61 90 73.3 -9.9 -2.5 -5.9 27.02.2016 0 60 85 69.3 -7.5 5 0.5 28.02.2016 0.4 36 73 54.2 3.3 16.2 9.7 29.02.2016 6 59 98 77.8 -6.3 10.6 0.9 01.03.2016 0 58 97 75.8 -8.7 -3.1 -5.1 02.03.2016 0 65 92 78.7 -10.1 -4 -7.2 03.03.2016 0 58 95 76.1 -10.9 -2.1 -5.3 04.03.2016 1.6 55 98 78.9 -10.3 1 -4 05.03.2016 0.4 61 91 78.2 -7.4 0.8 -2.6 06.03.2016 2.4 55 98 80 -11.3 2.7 -2.8 07.03.2016 0.8 54 85 68.3 0.3 12.6 6.9 08.03.2016 0 44 87 65.3 3.3 17.6 10.3 09.03.2016 0 49 92 63.6 5.4 18.7 14.3 10.03.2016 3.6 92 100 98 6.4 13.4 11 11.03.2016 0 80 100 94.4 -3.2 9.1 3 12.03.2016 0.2 51 100 95 -4.3 14.8 3.6 13.03.2016 1 72 100 90.7 3.8 8 5.7 14.03.2016 7 90 100 98.9 3.5 11.5 7.2

224

15.03.2016 6 86 100 98.6 5.1 9.6 7.8 16.03.2016 3 97 100 99.7 5.2 14.2 8.8 17.03.2016 0.8 46 71 55.5 4.3 11.3 7.4 18.03.2016 0 52 75 64.7 -2.5 8 3.6 19.03.2016 0 35 65 52.1 -6.9 4.1 -1.7 20.03.2016 0 38 78 59.4 -5.7 5.7 -0.9 21.03.2016 0 41 82 63.4 -4.2 5.6 -0.3 22.03.2016 0.6 42 86 67.3 -2.2 11.3 4.8 23.03.2016 3 43 95 80.3 0 10.4 4.2 24.03.2016 24.6 94 100 97.1 -0.3 6.1 1.1 25.03.2016 0.2 68 100 82.5 -4.4 6.6 1.1 26.03.2016 0 65 95 82.6 -3.8 9.7 1.9 27.03.2016 0 37 100 79.3 -2.5 19 6.3 28.03.2016 13.6 76 100 86.7 1.4 10.2 5.9 29.03.2016 0 37 90 64.6 -0.9 9.4 3.2 30.03.2016 0 39 99 67.9 -6.5 14.9 5.3 31.03.2016 42.4 41 98 84.2 8.9 15.9 12.1 01.04.2016 0.4 71 94 83.9 2 13.1 7.8 02.04.2016 0.6 62 95 84.5 -2.7 4 1.6 03.04.2016 0.2 53 96 79 -6.7 0.1 -3.9 04.04.2016 31 50 96 76.2 -9.3 -2.6 -4.6 05.04.2016 0.2 35 78 58.3 -12.1 1.9 -5.3 06.04.2016 4.8 59 91 75.1 -5.5 8.4 2.1 07.04.2016 6.6 73 96 85.9 -0.9 8.4 4.4 08.04.2016 0 53 86 69.1 -3.8 5.6 0.2 09.04.2016 0 47 81 60.6 -6.3 3.4 -2.1 10.04.2016 3.6 57 96 79.3 -10.6 0.6 -3.4 11.04.2016 12.8 72 99 94.2 0.2 10.4 6.5 12.04.2016 0 49 87 71 -5.2 7.1 1.8 13.04.2016 0 40 93 70.9 -6.1 9.7 1.3 14.04.2016 0 31 91 59.6 -1.6 12.5 4.6 15.04.2016 0 36 74 54.2 0.7 19.2 9 16.04.2016 0 31 62 45.1 3.9 22.1 12.2 17.04.2016 0 21 48 35.9 5 24.1 13.8 18.04.2016 0 28 76 46 2.2 25.5 14.5 19.04.2016 0 37 85 61.3 1.1 18.1 10.3 20.04.2016 0 15 93 51.9 -2.7 19.1 8.6 21.04.2016 0.4 39 90 62.6 5 17.4 11 22.04.2016 0 56 96 77.4 6.6 22.9 15.5 23.04.2016 0 27 87 62.5 1.8 15.8 7.9 24.04.2016 0 30 92 53.8 -2.7 14.7 6.5 25.04.2016 2.4 53 88 66.9 3.4 14.5 7.9 26.04.2016 6.2 82 97 91.4 -0.7 6 3.6 27.04.2016 0.2 33 100 64.3 -1.2 15.1 6.1

225

28.04.2016 0 43 64 53.8 1.9 10.9 5.7 29.04.2016 0 36 68 53.1 2.3 11.4 6.9 30.04.2016 0.6 43 86 60.2 -0.6 16.4 8.4 01.05.2016 8.6 88 100 96.6 6.8 10.7 8.3 02.05.2016 6 71 100 91.7 3.1 12.7 7.7 03.05.2016 0 59 100 79.6 1.3 17.2 8.9 04.05.2016 0 43 99 71.6 1 18.6 10.3 05.05.2016 0 49 96 75.5 6.3 18.4 11.4 06.05.2016 0 26 84 53.4 4.1 22.7 14.1 07.05.2016 0.4 39 83 63.6 3.9 20 12 08.05.2016 0 37 90 62.3 3 17.6 9.7 09.05.2016 0 35 77 58.5 0.2 17.3 9.1 10.05.2016 0 30 76 49.5 3.4 18.1 10.3 11.05.2016 0 34 61 46.5 7.4 22.5 14.5 12.05.2016 0 50 86 62.6 9.2 26.2 17.2 13.05.2016 17.4 63 100 81.3 11.8 20.3 15.8 14.05.2016 4.8 62 98 80.5 4.2 12 9.3 15.05.2016 0 41 75 61 2.5 8.7 5 16.05.2016 0 36 76 55.3 1.2 18.4 10.2 17.05.2016 0 54 88 72.4 6.8 18.2 11.4 18.05.2016 0 37 80 56.6 5.7 21.3 12 19.05.2016 0 36 95 60.4 2.1 21.2 12.4 20.05.2016 0 26 91 52.4 2.9 23.9 14.8 21.05.2016 0 42 84 56.6 11.9 23.7 15.6 22.05.2016 0 39 94 69.6 8.3 26.7 15.8 23.05.2016 0 32 78 51.3 6.4 29 18.2 24.05.2016 0 20 81 44.5 7.1 31.8 19.8 25.05.2016 0 30 75 47 14.2 32.1 22.7 26.05.2016 0 53 91 70.5 15.5 31 22.5 27.05.2016 0 47 92 69.9 16 32.3 24 28.05.2016 0 45 97 71.8 14.4 34.1 24 29.05.2016 1.8 53 99 75.5 16.6 31 23.5 30.05.2016 0 37 88 60.6 15.6 32 23.4 31.05.2016 0 26 85 52.1 13 33 22.5 01.06.2016 0 46 76 62.2 12.5 29.3 19.4 02.06.2016 4.4 50 99 72.5 14.2 32.4 21.5 03.06.2016 0 36 88 62.1 11.3 30.5 21.4 04.06.2016 3.8 30 92 63.7 12.4 30.1 20.8 05.06.2016 5.2 69 97 85.7 15.7 27 19.5 06.06.2016 1.2 41 91 66.2 13.9 28.1 20 07.06.2016 0.2 62 96 77.7 12.5 22.3 15.9 08.06.2016 0 48 80 65.9 6.3 17.6 12.3 09.06.2016 0 31 79 52.3 4.3 24.8 14.5 10.06.2016 0 27 89 48.6 5.2 26.5 16.7

226

11.06.2016 0 32 85 60.3 14.2 35.2 25.8 12.06.2016 0 35 70 50.2 11.3 25.5 18.7 13.06.2016 0 46 87 65.7 8 24.7 15.3 14.06.2016 0 36 86 61.8 7.8 25.9 16.5 15.06.2016 17 41 98 73.1 9.1 29.8 17 16.06.2016 1.4 49 99 80.6 13.9 26.3 19.3 17.06.2016 0 29 73 47.1 13.1 31.7 22.2 18.06.2016 0 29 84 49.6 11.4 33.6 22.9 19.06.2016 0 33 77 51.7 13.3 35.8 24.2 20.06.2016 0 53 72 61.7 18.7 33.4 26.2 21.06.2016 0 31 77 44.9 7.6 30.3 20.9 22.06.2016 0 32 84 53.6 13.4 30.7 21.1 23.06.2016 0 43 80 59.5 10 26.4 18.2 24.06.2016 0 28 84 51.6 9.2 31.4 20.7 25.06.2016 0 19 87 55.4 9.6 33 22 26.06.2016 0.2 47 94 65.4 14 34.7 25.1 27.06.2016 0.2 37 97 64.8 20.6 34.5 26.9 28.06.2016 0 50 83 69.5 14 27.4 19.4 29.06.2016 0 33 86 56.8 9.8 30.2 19.4 30.06.2016 0 25 85 54.3 5.4 30.4 19.2 01.07.2016 2.4 56 98 77.5 11.6 27.4 17.8 02.07.2016 0 34 85 61.5 9.4 30.3 19.3 03.07.2016 0 42 95 62.9 9.5 32.7 20.8 04.07.2016 0 31 94 56 9 31.2 22.1 05.07.2016 0 45 80 60.1 18.5 34.5 26 06.07.2016 0 49 95 71.8 18.1 34.6 25.9 07.07.2016 0 53 100 77.8 19.1 36.4 26.9 08.07.2016 3.2 57 98 77 19.5 32.3 25.1 09.07.2016 0.2 55 95 79.2 17 28.9 21.4 10.07.2016 0 42 96 72.4 14.5 31.4 22.8 11.07.2016 0 30 99 74 14.4 32.8 22.9 12.07.2016 0 41 94 66.4 17.4 35.7 26.6 13.07.2016 0 50 93 74.9 20.6 35.7 27.2 14.07.2016 17.8 64 100 82.4 20.4 29.2 23.9 15.07.2016 0 50 90 69.9 15.7 29.8 23.2 16.07.2016 0 49 95 73.5 12.6 27.9 20 17.07.2016 0 41 91 67.3 10.6 29.7 21.1 18.07.2016 0.2 43 98 66.9 18.8 32 25.7 19.07.2016 0 37 79 53.4 11.7 30.7 21.4 20.07.2016 0 41 88 62.6 9.7 30.9 20.8 21.07.2016 0 45 98 70.5 12.9 35.2 24.8 22.07.2016 0 31 90 71.9 22.7 37.3 28 23.07.2016 0 31 94 59.5 17.4 37.2 27.2 24.07.2016 0 61 96 75 15.2 31.6 23.4

227

25.07.2016 22.6 48 100 79.1 20.1 32.9 25.4 26.07.2016 0 48 91 72.9 15.8 32.6 23.5 27.07.2016 0 49 96 71 18.3 35.1 26 28.07.2016 0 58 94 75.6 17.7 31.2 24.7 29.07.2016 0 54 92 72.8 18.5 30.7 23.6 30.07.2016 0 60 91 74 18.9 31.1 22.7 31.07.2016 0.4 71 96 82.8 18.4 28.9 21.9 01.08.2016 0 56 99 79.7 16.5 31.6 23.1 02.08.2016 0 40 86 62.8 14.8 33.6 24.4 03.08.2016 0 43 90 64.8 13.5 35.2 24 04.08.2016 0 48 92 72.9 14.2 35.2 25.3 05.08.2016 0 53 94 76.3 21.2 34.7 27.7 06.08.2016 0 41 89 63.1 16.2 32.3 23.6 07.08.2016 0 38 86 60.4 15.7 31.4 22.9 08.08.2016 0 40 84 59.6 13.1 32.6 22.3 09.08.2016 0 48 92 65 13.2 33.9 24.6 10.08.2016 0 46 100 72.3 19.8 37.5 28.3 11.08.2016 0 54 100 83.8 21 37.6 27.6 12.08.2016 0 57 94 78.2 24 36.8 28.9 13.08.2016 9.2 67 99 85.5 23.7 32.2 26.9 14.08.2016 0.2 65 98 81.9 18.2 29.7 24 15.08.2016 2.8 79 99 91.2 17.2 27.8 21.8 16.08.2016 2.8 69 100 89.4 18 27.5 22.9 17.08.2016 0 63 100 85.2 14.4 29.6 21.9 18.08.2016 0 53 99 82.9 18.2 31.8 23.3 19.08.2016 0 60 100 83.1 16.3 31.8 23.9 20.08.2016 0 59 100 80.3 20.3 33.5 26.5 21.08.2016 0.6 50 93 71.8 16.4 28.8 23 22.08.2016 0 44 95 70.1 10.6 28 19 23.08.2016 0 48 95 70.7 10.5 30.7 20.1 24.08.2016 0.4 46 100 71.9 11.8 31.9 22.8 25.08.2016 8.2 64 100 84.6 21.1 33.5 25.6 26.08.2016 0.2 51 100 81.4 17.9 31.3 23.9 27.08.2016 0 36 97 72.1 15.6 33 23.1 28.08.2016 0 60 94 79.1 17.5 34.5 24.7 29.08.2016 0 39 96 74.5 13.9 32.4 21.9 30.08.2016 0 46 99 76.2 11.2 34.5 22.3 31.08.2016 1.6 36 96 76.2 15.3 30.4 23.1 01.09.2016 0 45 96 71.6 12 26.5 18.4 02.09.2016 0 43 91 67.8 11.9 27.5 18.4 03.09.2016 0 47 90 68.9 9.6 28.9 18 04.09.2016 0 39 97 67.3 8.2 31.5 18.7 05.09.2016 0 38 98 69.2 10.7 33.6 21.6 06.09.2016 0 45 95 76.4 14.9 37.7 25.3

228

07.09.2016 0 51 98 79.2 21.1 38.2 27.9 08.09.2016 0 70 98 85.7 21 32.1 26.1 09.09.2016 0 40 98 75.8 19 34.6 24.8 10.09.2016 11.6 66 99 89.8 15.9 31.4 21.6 11.09.2016 0 44 93 70.4 10.2 26 18.2 12.09.2016 0 45 99 73.7 5.9 26.7 16.1 13.09.2016 0 48 99 74 9 30 20 14.09.2016 4.6 51 98 76.3 11.3 23.9 18.6 15.09.2016 0 46 96 75 9.7 25.1 15.8 16.09.2016 0 35 89 68.9 9.5 28.2 18.7 17.09.2016 25.8 83 99 92.4 19.2 23.8 20.7 18.09.2016 0.2 49 100 76.4 16.8 29.1 22.8 19.09.2016 0 61 100 86.6 14.2 29.5 20.7 20.09.2016 0 40 100 73 12.2 30.9 21.2 21.09.2016 0 27 93 60.2 8.4 30 18.5 22.09.2016 0 43 97 73.2 11.4 32.6 21.3 23.09.2016 0 74 95 85.9 12.6 23.3 18.3 24.09.2016 0 40 95 66.8 6.5 21.5 13.6 25.09.2016 0 44 87 66.9 5.1 22.8 13.7 26.09.2016 16.4 70 99 86 7.8 18.4 13.4 27.09.2016 0 34 79 59.6 6.2 22.6 14.3 28.09.2016 0 50 94 72.5 7.6 24.1 15.7 29.09.2016 29 79 99 92.7 12.8 16.9 14.1 30.09.2016 11.8 71 99 83.7 11.6 17.7 14 01.10.2016 6.8 86 100 96.5 11.6 17 14 02.10.2016 32 94 100 99.6 12.9 19.2 14.5 03.10.2016 0.2 69 100 90.3 11.3 22.9 15.3 04.10.2016 0.2 60 100 90.4 9.7 23.3 15.9 05.10.2016 0 66 100 83.4 10.5 22.7 16.6 06.10.2016 0 62 96 79.9 13.7 25 18.3 07.10.2016 0 59 100 86.8 8.5 23.9 16.6 08.10.2016 0.4 43 98 75.5 5 18.9 14.1 09.10.2016 0 45 94 73.5 3.6 16.4 8.6 10.10.2016 0 42 96 74.3 0.5 16.9 6.7 11.10.2016 0 46 99 75.7 0.4 19.3 9 12.10.2016 0 53 100 75.3 5.3 23.4 15.5 13.10.2016 7.4 51 100 74.2 2.7 19.1 11.6 14.10.2016 0 51 98 78.2 -0.4 17.1 7.3 15.10.2016 0 63 98 81.9 1.9 21.1 12.9 16.10.2016 1 69 98 82.7 16.7 23.7 18.9 17.10.2016 0.2 67 100 86.1 16.9 25.1 20 18.10.2016 0 67 88 75.6 14 24.9 20.7 19.10.2016 0 51 99 86.2 8.7 22 14.1 20.10.2016 4.4 81 99 94.5 10.7 13.8 11.9

229

21.10.2016 2.2 79 98 91.2 3.7 10.6 8.7 22.10.2016 0 60 93 79.3 0.8 10.3 4.9 23.10.2016 1.4 61 96 80.6 3 15.1 9 24.10.2016 0 53 93 75.1 2.1 12.2 7.6 25.10.2016 0 52 92 71.5 -1.5 11.9 5.3 26.10.2016 3.6 51 95 75.1 -4.7 6.5 1.3 27.10.2016 15.6 86 100 95.9 -0.1 5.7 2.5 28.10.2016 0.2 60 98 83.3 -2.6 12.4 4.6 29.10.2016 0 76 91 82.3 8.5 19.5 14.6 30.10.2016 2.4 78 96 89.7 0.4 10 7.1 31.10.2016 0.2 66 100 85.9 -2.9 11 3.2 01.11.2016 0 67 98 82.1 -0.7 21.1 11.4 02.11.2016 16 70 100 89.3 10.6 21 15.2 03.11.2016 18.2 68 100 89.1 6 16.6 11.8 04.11.2016 0 61 100 84 0.2 12.4 6.3 05.11.2016 0 63 98 82.4 5.1 17.7 9.8 06.11.2016 0.2 56 99 83.1 1 15.7 7.4 07.11.2016 0.2 46 100 85.9 -0.3 20.5 7.3 08.11.2016 2.8 56 100 85.5 3.1 18.3 10.7 09.11.2016 0 64 95 82.9 -1.2 9.6 5.9 10.11.2016 0.2 53 98 73.4 -1.8 14.8 8 11.11.2016 0 52 88 66.4 -3.4 10.1 5.2 12.11.2016 0 55 85 70.9 -4.3 10.1 3.1 13.11.2016 0 50 73 64.5 4 13.1 7.9 14.11.2016 0 57 83 69.2 1.8 14.7 8.1 15.11.2016 0 57 99 87.6 -0.8 14.4 5.4 16.11.2016 3.4 71 100 90.8 2.7 13.7 7.2 17.11.2016 0.2 72 100 92.7 -1.7 12.7 5 18.11.2016 0.2 50 98 71.9 5.1 20.4 12.7 19.11.2016 7.6 53 98 79.5 -0.8 15.1 6.6 20.11.2016 0 65 91 78.2 -1.8 1.9 -0.3 21.11.2016 0 55 74 65.4 -1.7 2.3 -0.3 22.11.2016 0 61 89 71.6 -4.8 4.8 -0.2 23.11.2016 0 74 98 88.2 -7.3 1.1 -2 24.11.2016 3.8 93 100 97.3 -0.1 6.5 3 25.11.2016 0.8 77 99 90.8 2.9 6.5 4.9 26.11.2016 3.4 81 100 91.4 1.8 6.4 3.2 27.11.2016 0 67 97 85.5 -0.8 9.1 2.2 28.11.2016 1.2 63 98 81.1 -2.3 9.8 4.4 29.11.2016 5.8 70 100 86.3 3.4 13.7 8.7 30.11.2016 18.4 78 100 92.2 5.1 13.9 9.4 01.12.2016 0.2 74 94 80.8 2.2 7.5 4.2 02.12.2016 2.6 65 97 83.5 2.1 5.1 3.2 03.12.2016 0.4 84 97 92.1 1.4 4.4 2.7

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04.12.2016 2.8 74 96 89.5 0.9 3.6 2.3 05.12.2016 2.6 70 99 84.6 -1 5.3 2.3 06.12.2016 6.4 68 100 90.6 -2.2 4.7 0.9 07.12.2016 0 70 100 80.6 -0.9 4.2 1.3 08.12.2016 0 61 90 72 -3.3 -0.6 -1.8 09.12.2016 2.2 75 95 86.8 -7.8 1.8 -3.6 10.12.2016 0 57 92 74.5 -13 -3.1 -6.1 11.12.2016 0 81 100 92.1 -8.6 -0.2 -3.5 12.12.2016 11.2 70 100 87 -2.9 -0.1 -1.1 13.12.2016 0 66 93 80 -8.8 -0.6 -3.3 14.12.2016 0 57 91 71 -12.9 -4.1 -8.7 15.12.2016 0 53 76 66.1 -14.9 -11.2 -13.3

Table A1.31 Stemphylium vesicarium sample codes, collection year, host crop, location and GPS coordinates. Isolate Year Nearest town, County, Crop GPS Coordinates Code Collected Province OA01 2012 asparagus Port Burwell, Elgin, ON 42.623714° -80.722455° OA02 2012 asparagus Port Burwell, Elgin, ON 42.626159° -80.725263° OA03 2012 asparagus Hemlock, Norfolk, ON 42.606710° -80.678520° OA04 2012 asparagus Houghton Centre, Norfolk, ON 42.612266° -80.659826° OA05 2012 asparagus Houghton Centre, Norfolk, ON 42.612187° -80.645804° OA06 2012 asparagus Houghton Centre, Norfolk, ON 42.613547° -80.643056° OA07 2012 asparagus Fairground, Norfolk, ON 42.619312° -80.642409° OA08 2012 asparagus Fairground, Norfolk, ON 42.619223° -80.645628° OA09 2012 asparagus Fairground, Norfolk, ON 42.620734° -80.645656° OA10 2012 asparagus Fairground, Norfolk, ON 42.621890° -80.646772° OA11 2012 asparagus Fairground, Norfolk, ON 42.621407° -80.648026° OA12 2012 asparagus Fairground, Norfolk, ON 42.621876° -80.649158° OA13 2012 asparagus Fairground, Norfolk, ON 42.622118° -80.650284° OA14 2012 asparagus Fairground, Norfolk, ON 42.625508° -80.651557° OA15 2012 asparagus Fairground, Norfolk, ON 42.626592° -80.650278° OA16 2012 asparagus Fairground, Norfolk, ON 42.627085° -80.651736° OA17 2012 asparagus Fairground, Norfolk, ON 42.626531° -80.649058° OA18 2012 asparagus Fairground, Norfolk, ON 42.626035° -80.647857° OA19 2012 asparagus Fairground, Norfolk, ON 42.625453° -80.646636° OA20 2012 asparagus Fairground, Norfolk, ON 42.625067° -80.645525° OA21 2012 asparagus Froggetts Corners, Elgin, ON 42.723382° -80.784297° OA22 2012 asparagus Froggetts Corners, Elgin, ON 42.723232° -80.785848° OA23 2012 asparagus Calton, Elgin, ON 42.693757° -80.898652° OA24 2012 asparagus Calton, Elgin, ON 42.695813° -80.898946° OO25 2013 onion Holland Marsh, Simcoe, ON 44.052266° -79.607272° OO26 2013 onion Holland Marsh, Simcoe, ON 44.052514° -79.607783° OO27 2013 onion Holland Marsh, Simcoe, ON 44.052199° -79.607712° OO28 2013 onion Holland Marsh, Simcoe, ON 44.052781° -79.607112° OO29 2013 onion Holland Marsh, Simcoe, ON 44.052101° -79.606953°

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OO30 2013 onion Holland Marsh, Simcoe, ON 44.051473° -79.606869° OO31 2013 onion Holland Marsh, Simcoe, ON 44.051326° -79.606500° OO32 2013 onion Holland Marsh, Simcoe, ON 44.051125° -79.606854° NO33 2013 onion Nictaux, Annapolis, NS 44.939688° -65.020737° NO34 2013 onion Avonport, Kings, NS 45.085745° -64.275238° NO35 2013 onion Avonport, Kings, NS 45.085650° -64.245015° NO36 2013 onion Avonport, Kings, NS 45.092686° -64.240317° NO37 2013 onion Wallbrook, Kings, NS 45.070614° -64.297625° OO38 2013 onion Holland Marsh, York, ON 44.053444° -79.579387° OO39 2013 onion MCRS, York, ON 44.040568° -79.598182° OO40 2013 onion Holland Marsh, York, ON 44.043227° -79.598879° OO41 2013 onion Holland Marsh, Simcoe, ON 44.034396° -79.632802° OO42 2013 onion MCRS, York, ON 44.040751° -79.598189° OO43 2013 onion Bradford, Simcoe, ON 44.086800° -79.561776° OA46 2013 asparagus Gilbertville, Norfolk, ON 42.825295° -80.475641° OA47 2014 asparagus Florence, Chatham-Kent, ON 42.614740° -81.961257° OA48 2014 asparagus Harrow, Essex, ON 42.004415° -82.878202° OA49 2014 asparagus Harrow, Essex, ON 42.004334° -82.881810° OA50 2014 asparagus SRS, Norfolk, ON 42.852618° -80.269345° NA51 2014 asparagus Canning, Kings, NS 45.169411° -64.448570° NA52 2014 asparagus Sheffield Mills, Kings, NS 45.149598° -64.472788° OA53 2014 asparagus Winslow, Niagara, ON 43.043646° -79.581560° OO54 2014 onion SRS, Norfolk, ON 42.854295° -80.273194° OO55 2014 onion MCRS, York, ON 44.041101° -79.598457° OA56 2014 asparagus Houghton Centre, Norfolk, ON 42.610840° -80.659849° OA57 2014 asparagus Harrow, Essex, ON 42.004966° -82.876948° OA58 2014 asparagus Harrow, Essex, ON 42.005862° -82.877717° OA59 2014 asparagus Harrow, Essex, ON 42.004571° -82.883243° NA60 2014 asparagus Sheffield Mills, Kings, NS 45.150003° -64.470908° NA61 2014 asparagus Canning, Kings, NS 45.170843° -64.450093° NA62 2014 asparagus Sheffield Mills, Kings, NS 45.150659° -64.471176° OA63 2014 asparagus Houghton Centre, Norfolk, ON 42.611724° -80.658548° OA64 2014 asparagus Harrow, Essex, ON 42.005480° -82.883225° OA65 2014 asparagus Harrow, Essex, ON 42.005237° -82.881628° OA66 2015 asparagus Corinth, Elgin, ON 42.814643° -80.815074° OA67 2016 asparagus Calton, Elgin, ON 42.698593° -80.899013° OA68 2016 asparagus Port Burwell, Elgin, ON 42.624813° -80.722263° OA69 2015 onion Holland Marsh, York, ON 44.041880° -79.583832°

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Figure A1.1 Sampling sites located in Essex, Chatham-Kent, Elgin, Norfolk, Niagara, York and Simcoe Counties or Regions. Yellow and pink markers denote asparagus and onion sampling sites, respectively.

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Figure A1.2 Sampling sites located in Kings and Annapolis Counties in Nova Scotia. Blue and red markers denote onion and asparagus sampling sites, respectively.

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Figure A1.3 Culture shape and colour of Stemphylium vesicarium isolates collected in Ontario and Nova Scotia.

235

APPENDIX 2: SUPPLEMENTARY TABLES FOR CHAPTER THREE

Table A2.1 REML covariance parameter estimates for disease severity index (0-100) among six cultivars naturally infested with Stemphylium vesicarium, at Simcoe Research Station, Simcoe, Ontario, 2015-16. Covariance parameter Estimate SE Block -3.800 2.1492 Residual 74.3667 15.8550 Effect Num DF Den DF F Value Pr > F Year 1 44 211.74 <0.0001 Cultivar 5 44 4.61 0.0018 Year*Cultivar 5 44 0.36 0.8707

Table A2.2 REML covariance parameter estimates for sAUDPC among six cultivars naturally infested with Stemphylium vesicarium, at Simcoe Research Station, Simcoe, Ontario, 2015-16. Covariance parameter Estimate SE Block 0.03617 0.1851 Residual 2.6079 0.5560 Effect Num DF Den DF F Value Pr > F Year 1 44 74.18 <0.0001 Cultivar 5 44 5.09 0.0009 Year*Cultivar 5 44 1.80 0.1331

Table A2.3 REML covariance parameter estimates for disease severity index (0-100) among six cultivars naturally infested with Puccinia asparagi, at Simcoe Research Station, Simcoe, Ontario, 2015-16. Covariance parameter Estimate SE Block 9.3464 11.4849 Residual 80.1636 17.0909 Effect Num DF Den DF F Value Pr > F Year 1 44 167.66 <0.0001 Cultivar 5 44 2.93 0.0227 Year*Cultivar 5 44 1.62 0.1759

Table A2.4 REML covariance parameter estimates for sAUDPC among six cultivars naturally infested with Puccinia asparagi, at Simcoe Research Station, Simcoe, Ontario, 2015-16. Covariance parameter Estimate SE Block 1.0741 1.1844 Residual 7.0972 1.5131 Effect Num DF Den DF F Value Pr > F Year 1 44 234.33 <0.0001 Cultivar 5 44 13.60 <0.0001 Year*Cultivar 5 44 0.95 0.4573

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Table A2.5 REML covariance parameter estimates for fern yellowing among six cultivars, at Simcoe Research Station, Simcoe, Ontario, 2015-16. Covariance parameter Estimate SE Block -14.0758 17.2922 Residual 431.89 92.0798 Effect Num DF Den DF F Value Pr > F Year 1 44 0.54 0.4674 Cultivar 5 44 5.04 0.0010 Year*Cultivar 5 44 1.11 0.3674

Table A2.6 REML covariance parameter estimates for fern defoliation among six cultivars, at Simcoe Research Station, Simcoe, Ontario, 2015-16. Covariance parameter Estimate SE Block -10.8144 18.1358 Residual 411.44 87.7192 Effect Num DF Den DF F Value Pr > F Year 1 44 26.58 <0.0001 Cultivar 5 44 3.49 0.0096 Year*Cultivar 5 44 1.35 0.2629

Table A2.7 REML covariance parameter estimates for rust counts among 14 fungicide treatments in a 3-year-old field of asparagus, 27 June, 2011. Covariance parameter Estimate SE Block 655.91 856.31 Block*Trt 68.2424 1367.78 Residual 27526 2606.00 Effect Num DF Den DF F Value Pr > F Trt 13 39 3.37 0.0016

Table A2.8 REML covariance parameter estimates for rust counts among 14 fungicide treatments in a 3-year-old field of asparagus, 25 July, 2011. Covariance parameter Estimate SE Block 108.07 328.16 Block*Trt -1939.82 1068.95 Residual 29675 2810.45 Effect Num DF Den DF F Value Pr > F Trt 13 39 10.28 <0.0001

Table A2.9 REML covariance parameter estimates for rust sAUDPC among 14 fungicide treatments in a 3-year-old field of asparagus, 2011. Covariance parameter Estimate SE Block 275.77 284.95 Residual 1017.01 230.31 Effect Num DF Den DF F Value Pr > F Trt 13 39 8.47 <0.0001

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Table A2.10 REML covariance parameter estimates for rust counts among 14 fungicide treatments in a 4-year-old field of asparagus, 23 August, 2011. Covariance parameter Estimate SE Block 29.1600 41.4374 Block*Trt 91.1670 70.4928 Residual 1020.41 91.1421 Effect Num DF Den DF F Value Pr > F Trt 13 39 3.07 0.0034

Table A2.11 REML covariance parameter estimates for rust counts among 14 fungicide treatments in a 4-year-old field of asparagus, 20 September, 2011. Covariance parameter Estimate SE Block 51.3168 119.78 Block*Trt 906.20 298.84 Residual 1800.64 176.90 Effect Num DF Den DF F Value Pr > F Trt 13 39 2.35 0.0206

Table A2.12 REML covariance parameter estimates for rust sAUDPC among 14 fungicide treatments in a 4-year-old field of asparagus, 2011. Covariance parameter Estimate SE Block 7.9529 10.0892 Residual 59.6443 13.8542 Effect Num DF Den DF F Value Pr > F Trt 13 39 3.76 0.0007

Table A2.13 REML covariance parameter estimates for Stemphylium leaf spot counts among 14 fungicide treatments in a 4-year-old field of asparagus, 20 September, 2011. Covariance parameter Estimate SE Block -4.4893 180.10 Residual 8595.68 842.94 Effect Num DF Den DF F Value Pr > F Trt 13 39 2.28 0.0241

Table A2.14 REML covariance parameter estimates for defoliation among 14 fungicide treatments in a 4-year-old field of asparagus, 2011. Covariance parameter Estimate SE Block -17.1635 16.2645 Residual 479.65 110.98 Effect Num DF Den DF F Value Pr > F Trt 13 39 3.38 0.0017

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APPENDIX 3: SUPPLEMENTARY TABLES FOR CHAPTER FOUR

Table A3.1 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at six locations in southwestern Ontario, 2012-13. Covariance parameters Estimate SE block(location) 0 . block*location*trt 1278.20 171.83 Residual 2309.07 51.3796 Effect Num DF Den DF F Value Pr > F treatment 8 129.6 62.17 <0.0001 location 4 129.7 4.75 <0.0001 location*treatment 32 129.6 4.21 <0.0001

Table A3.2 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at Site 1, ‘Jersey Giant’, 2012. Covariance parameters Estimate SE block 0.006909 0.08140 block*trt 0.5901 0.2262 Residual 3.5446 0.1944 Effect Num DF Den DF F Value Pr > F trt 8 23.18 2.01 0.0902

Table A3.3 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at Site 1, ‘Jersey Giant’, 2013. Covariance parameters Estimate SE block -65.6433 34.7984 block*trt 737.70 241.34 Residual 1951.39 105.75 Effect Num DF Den DF F Value Pr > F trt 8 23.99 2.59 0.0341

Table A3.4 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at Site 1, ‘Guelph Millennium’, 2012. Covariance parameters Estimate SE block -0.1801 0.05341 block*trt 1.3749 0.4798 Residual 5.7261 0.3096 Effect Num DF Den DF F Value Pr > F trt 8 24 3.88 0.0047

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Table A3.5 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at Site 1, ‘Guelph Millennium’, 2013. Covariance parameters Estimate SE block -450.45 459.27 block*trt 7012.84 2308.22 Residual 10059 574.59 Effect Num DF Den DF F Value Pr > F trt 8 21.78 3.60 0.0082

Table A3.6 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at Site 2, ‘Guelph Millennium’, north, 2013. Covariance parameters Estimate SE block -20.2310 6.6895 block*trt 176.30 58.1012 Residual 500.59 27.0473 Effect Num DF Den DF F Value Pr > F trt 8 24.02 1.38 0.2567

Table A3.7 REML covariance parameter estimates for Stemphylium leaf spot counts among fungicide treatments, at Site 2, ‘Guelph Millennium’, south, 2013. Covariance parameters Estimate SE block -3.6063 2.5670 block*trt 43.3929 15.4387 Residual 199.73 10.8394 Effect Num DF Den DF F Value Pr > F trt 8 24 2.82 0.0232

Table A3.8 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at six locations in Southwestern Ontario, 2012-13. Covariance parameters Estimate SE Block(location) 0 . Location*fungicide*timing*block 1400.90 198.55 Residual 2300.29 54.3555 Effect Num DF Den DF F Value Pr > F Location 4 115 58.82 <0.0001 Fungicide 1 115.1 0.33 0.5653 Location*Fungicide 4 115 0.71 0.5879 Timing 3 115.1 10.86 <0.0001 Location*Timing 12 115 8.27 <0.0001 Fungicide*Timing 3 115.1 0.66 0.5804 Location*Timing*Fungicide 12 115 0.81 0.6364

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Table A3.9 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at Site 1, ‘Jersey Giant’, 2012. Covariance parameters Estimate SE Block(location) 0.02395 0.1075 Fungicide*timing*block 0.6316 0.2536 Residual 3.4453 0.2008 Effect Num DF Den DF F Value Pr > F Fungicide 1 20.3 0.10 0.7518 Timing 3 20.3 2.32 0.1053 Fungicide*Timing 3 20.3 1.38 0.2763

Table A3.10 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at Site 1, ‘Jersey Giant’, 2013. Covariance parameters Estimate SE Block(location) -67.9343 35.9056 Fungicide*timing*block 664.70 231.75 Residual 1717.07 98.6428 Effect Num DF Den DF F Value Pr > F Fungicide 1 21 0.16 0.6965 Timing 3 21 3.11 0.0484 Fungicide*Timing 3 21 0.32 0.8138

Table A3.11 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at Site 1, ‘Guelph Millennium’, 2012. Covariance parameters Estimate SE Block(location) -0.2205 0.07083 Fungicide*timing*block 1.5571 0.5644 Residual 5.4243 0.3111 Effect Num DF Den DF F Value Pr > F Fungicide 1 21 7.94 0.0103 Timing 3 21 0.65 0.5923 Fungicide*Timing 3 21 0.46 0.7112

Table A3.12 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at Site 1, ‘Guelph Millennium’, 2013. Covariance parameters Estimate SE Block(location) -0.00838 0.004083 Fungicide*timing*block 0.07829 0.02685 Residual 0.07016 0.004278 Effect Num DF Den DF F Value Pr > F Fungicide 1 20.01 1.54 0.2294 Timing 3 19.35 6.23 0.0039 Fungicide*Timing 3 19.31 0.80 0.5095

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Table A3.13 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at Site 2, ‘Guelph Millennium’, north, 2013. Covariance parameters Estimate SE Block(location) -0.01770 0.06307 Fungicide*timing*block 0.5934 0.2160 Residual 2.1349 0.1223 Effect Num DF Den DF F Value Pr > F Fungicide 1 21.02 6.06 0.0225 Timing 3 21.02 0.41 0.7464 Fungicide*Timing 3 21.02 0.43 0.7364

Table A3.14 REML covariance parameter estimates for Stemphylium leaf spot counts among two factors, application timing and fungicide, at Site 2, ‘Guelph Millennium’, south, 2013. Covariance parameters Estimate SE Block(location) 0.000508 0.000882 Fungicide*timing*block 0.002654 0.001372 Residual 0.03487 0.002009 Effect Num DF Den DF F Value Pr > F Fungicide 1 20.79 3.57 0.0728 Timing 3 20.79 0.38 0.7710 Fungicide*Timing 3 20.79 0.93 0.4444

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Table A3.15 REML covariance parameter estimates for Stemphylium leaf spot counts among three factors, cultivar, application timing and fungicide, at Site 1, 2012-13. Covariance parameters Estimate SE Block(year) 63.9212 51.0726 Cultivar*block 0 . Fungicide*block 60.1951 109.42 Timing*block 74.6512 138.83 Cultivar*Fungicide*Timing*block 784.07 224.23 Residual 3768.59 109.20 Effect Num DF Den DF F Value Pr > F Year 1 4.03 182.64 0.0002 Cultivar 1 31.24 58.95 <0.0001 Year*Cultivar 1 2406 402.15 <0.0001 Fungicide 1 5.313 0.83 0.4009 Year*Fungicide 1 2415 3.60 0.0579 Cultivar*Fungicide 1 31.27 1.00 0.3260 Year*Cultivar*Fungicide 1 2409 0.66 0.4150 Timing 3 11.06 10.48 0.0015 Year*Timing 3 2410 110.67 <0.0001 Cultivar*Timing 3 31.22 8.08 0.0004 Year*Cultivar*Timing 3 2408 60.99 <0.0001 Fungicide*Timing 3 31.22 0.54 0.6581 Year*Fungicide*Timing 3 2409 8.06 <0.0001 Cultivar*Fungicide*Timing 3 31.21 1.02 0.3954 Year*Cultivar*Timing*Fungicide 3 2407 12.00 <0.0001

Table A3.16 REML covariance parameter estimates for Stemphylium leaf spot counts among three factors, cultivar, application timing and fungicide, at Site 1, 2012. Covariance parameters Estimate SE Block 1.7141 4.8826 Cultivar*block -7.1916 4.5025 Fungicide*block -0.3745 12.3835 Timing*block -6.7725 5.0362 Cultivar*Fungicide*Timing*block 77.3824 23.7198 Residual 271.79 11.1098 Effect Num DF Den DF F Value Pr > F Cultivar 1 3.082 25.96 0.0137 Timing 3 8.822 1.07 0.4094 Cultivar*Timing 3 29.78 0.56 0.6427 Fungicide 1 2.816 9.21 0.0609 Cultivar*Fungicide 1 29.8 3.19 0.0843 Fungicide*Timing 3 29.78 0.93 0.4366 Cultivar*Fungicide*Timing 3 29.78 0.32 0.8100

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Table A3.17 REML covariance parameter estimates for Stemphylium leaf spot counts among three factors, cultivar, application timing and fungicide, at Site 1, 2013. Covariance parameters Estimate SE Block -440.90 540.80 Cultivar*block -307.29 181.25 Fungicide*block 467.27 697.62 Timing*block 720.17 984.85 Cultivar*Fungicide*Timing*block 3342.73 976.14 Residual 6078.43 254.17 Effect Num DF Den DF F Value Pr > F Cultivar 1 3.308 126.64 0.0009 Timing 3 9.327 7.82 0.0065 Cultivar*Timing 3 29.07 6.33 0.0019 Fungicide 1 2.993 0.14 0.7369 Cultivar*Fungicide 1 29.56 0.11 0.7404 Fungicide*Timing 3 29.07 0.49 0.6896 Cultivar*Fungicide*Timing 3 29.07 0.88 0.4618

Table A3.18 REML covariance parameter estimates for sAUDPC among fungicide treatments, at six locations, 2012-13. Covariance parameters Estimate SE Block(location) -16.8668 1.8695 Residual 303.7 33.6498 Effect Num DF Den DF F Value Pr > F Location 5 163.4 308.28 <0.0001 Treatment 8 163 7.96 <0.0001 Location*Treatment 32 163 4.90 <0.0001

Table A3.19 REML covariance parameter estimates for sAUDPC among fungicide treatments, at Site 1, ‘Jersey Giant’, 2012. Covariance parameters Estimate SE block -0.6925 0.5239 Residual 10.3471 3.0420 Effect Num DF Den DF F Value Pr > F Treatment 8 23.39 1.26 0.3126

Table A3.20 REML covariance parameter estimates for sAUDPC among fungicide treatments, at Site 1, ‘Jersey Giant’, 2013. Covariance Parameters Estimate SE block -6.1219 2.2506 Residual 62.9503 18.8492 Effect Num DF Den DF F Value Pr > F Treatment 8 22.78 3.31 0.0117

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Table A3.21 REML covariance parameter estimates for sAUDPC among fungicide treatments, at Site 1, ‘Guelph Millennium’, 2012. Covariance Parameters Estimate SE block -0.4557 1.2626 Residual 16.7031 4.8218 Effect Num DF Den DF F Value Pr > F Treatment 8 24 6.38 0.0002

Table A3.22 REML covariance parameter estimates for sAUDPC among fungicide treatments, at Site 1, ‘Guelph Millennium’, 2013. Covariance Parameters Estimate SE block -101.73 117.08 Residual 1811.19 556.26 Effect Num DF Den DF F Value Pr > F Treatment 8 21.72 4.05 0.0044

Table A3.23 REML covariance parameter estimates for sAUDPC among fungicide treatments, at Site 2, ‘Guelph Millennium’, north, 2013. Covariance Parameters Estimate SE block -0.5532 3.4061 Residual 39.7884 11.4859 Effect Num DF Den DF F Value Pr > F Treatment 8 24 0.56 0.7994

Table A3.24 REML covariance parameter estimates for sAUDPC among fungicide treatments, at Site 2, ‘Guelph Millennium’, south, 2013. Covariance Parameters Estimate SE block -2.8483 1.7016 Residual 38.5278 11.1220 Effect Num DF Den DF F Value Pr > F Treatment 8 24 1.15 0.3652

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Table A3.25 REML covariance parameter estimates for sAUDPC among three factors, cultivar, fungicide and application timing, at Site 1, 2012-13. Covariance parameters Estimate SE Block(year) -12.9185 . cultivar*block -13.6779 . fungicide*block -1.6165 . timing*block -2.0065 . Residual 375.36 . Effect Num DF Den DF F Value Pr > F Year 1 89 147.11 <0.0001 Cultivar 1 89 113.61 <0.0001 Year*Cultivar 1 89 108.92 <0.0001 Fungicide 1 89 0.39 0.5364 Year*Fungicide 1 89 0.52 0.4714 Cultivar*Fungicide 1 89 1.03 0.3127 Year*Cultivar*Fungicide 1 89 1.75 0.1888 Timing 3 89 13.55 <0.0001 Year*Timing 3 89 12.45 <0.0001 Cultivar*Timing 3 89 9.18 <0.0001 Year*Cultivar*Timing 3 89 9.09 <0.0001 Fungicide*Timing 3 89 1.31 0.2746 Year*Fungicide*Timing 3 89 1.79 0.1557 Cultivar*Fungicide*Timing 3 89 2.44 0.0694 Year*Cultivar*Timing*Fungicide 3 89 2.18 0.0964

Table A3.26 REML covariance parameter estimates for sAUDPC among three factors, cultivar, fungicide and application timing, at Site 1, 2012. Covariance Parameters Estimate SE block 0.5773 0.6182 block*cultivar -1.0961 0.6685 block*fungicide -1.4674 0.4895 block*timing -0.5377 1.6250 Residual 13.6639 3.5327 Effect Num DF Den DF F Value Pr > F Cultivar 1 3.052 1.40 0.3213 Fungicide 1 2.872 0.51 0.5292 Cultivar*Fungicide 1 30.13 1.53 0.2255 Timing 3 8.911 0.91 0.4727 Timing*Cultivar 3 30.13 0.04 0.9888 Timing*Fungicide 3 30.13 1.79 0.1701 Cultivar*Timing*Fungicide 3 30.13 0.63 0.6015

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Table A3.27 REML covariance parameter estimates for sAUDPC among three factors, cultivar, fungicide and application timing, at Site 1, 2013. Covariance Parameters Estimate SE block 0 . block*cultivar 0 . block*fungicide 0 . block*timing 0 . Residual 718.87 156.87 Effect Num DF Den DF F Value Pr > F Cultivar 1 42 101.88 <0.0001 Fungicide 1 42 0.41 0.5237 Cultivar*Fungicide 1 42 1.25 0.2693 Timing 3 42 11.89 <0.0001 Timing*Cultivar 3 42 8.35 0.0002 Timing*Fungicide 3 42 1.37 0.2664 Cultivar*Timing*Fungicide 3 42 2.07 0.1185

Table A3.28 REML covariance parameter estimates for defoliation among fungicide treatments, at two sites in Southwestern Ontario, 2013. Covariance parameters Estimate SE block(repetition) -9.6867 4.2460 Residual 284.57 40.3438 Effect Num DF Den DF F Value Pr > F Repetition 1 6.511 0.14 0.7202 Site 1 99.74 287.72 <0.0001 Repetition*Site 1 99.74 4.78 0.0311 Fungicide 8 100.1 21.83 <0.0001 Repetition*Fungicide 8 100.1 0.36 0.9400 Site*Fungicide 8 100.1 14.47 <0.0001 Site*Repetition*Fungicide 8 100.1 0.44 0.8969

Table A3.29 REML covariance parameter estimates for defoliation among fungicide treatments, at Site 1 in Southwestern Ontario, 2013. Covariance parameters Estimate SE block(repetition) 38.1042 35.8623 Residual 196.47 41.4400 Effect Num DF Den DF F Value Pr > F Repetition 1 5.931 1.66 0.2459 Fungicide 8 45.18 23.52 <0.0001 Repetition*Fungicide 8 45.18 0.55 0.8146

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Table A3.30 REML covariance parameter estimates for defoliation among fungicide treatments, at Site 2 in Southwestern Ontario, 2013. Covariance parameters Estimate SE block(repetition) 0.7234 21.6867 Residual 312.93 63.8774 Effect Num DF Den DF F Value Pr > F Repetition 1 6 1.74 0.2353 Fungicide 8 48 17.67 <0.0001 Repetition*Fungicide 8 48 0.35 0.9389

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APPENDIX 4: SUPPLEMENTARY TABLES FOR CHAPTER FIVE

Table A4.1 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) in four asparagus fields, southwestern Ontario, non-irrigated, 2015-16. Covariance parameters Estimate SE block(location) 1.2465 0.7885 block*fertilizer 0.7279 0.6119 Variance 0 . CS 0.05449 0.5004 Residual 46.4563 2.1170 Effect Num DF Den DF F Value Pr > F Location 1 10.43 235.98 <0.0001 Fertilizer 3 9.722 0.70 0.5751 Location*Fertilizer 3 963.1 2.14 0.0244 Fungicide 2 24 330.77 <0.0001 Location*Fungicide 2 963.1 11.56 <0.0001 Fertilizer*Fungicide 6 24 1.29 0.3004 Location*Fertilizer*Fungicide 6 963.1 1.76 0.0251 DAA1 6 963.1 868.45 <0.0001 Location*DAA1 6 963.1 48.46 <0.0001 Fertilizer*DAA1 18 963.1 0.72 0.7945 Location*Fertilizer*DAA1 18 963.1 0.66 0.9721 Fungicide*DAA1 12 963.1 23.74 <0.0001 Location*Fungicide*DAA1 12 963.1 3.83 <0.0001 Fertilizer*Fungicide*DAA1 36 963.1 0.64 0.9543 Location*Fertilizer*Fungicide*DAA1 36 963.1 0.66 0.9960

Table A4.2 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) at Site 1, non-irrigated, 2015. Covariance parameters Estimate SE block 0.6536 1.3954 block*fertilizer 0.4145 2.0684 CS 7.1634 2.9909 Residual 22.0106 2.1180 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.84 0.5056 Fungicide 2 24 50.37 <0.0001 Fertilizer*Fungicide 6 24 0.62 0.7096 DAA1 6 216 212.26 <0.0001 Fertilizer*DAA1 18 216 1.07 0.3795 Fungicide*DAA1 12 216 21.77 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.81 0.7725

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Table A4.3 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) at Site 1, non-irrigated, 2016. Covariance parameters Estimate SE block 2.0528 2.3636 block*fertilizer 0.8350 1.6657 CS 2.3411 2.1150 Residual 33.6561 3.2386 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.49 0.2832 Fungicide 2 24 54.70 <0.0001 Fertilizer*Fungicide 6 24 1.36 0.2705 DAA1 6 216 351.64 <0.0001 Fertilizer*DAA1 18 216 0.92 0.5552 Fungicide*DAA1 12 216 7.81 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.96 0.5341

Table A4.4 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) at Site 2, non-irrigated, 2015. Covariance parameters Estimate SE block -0.1416 2.8255 block*fertilizer 4.4415 6.2860 CS 14.5520 6.9591 Residual 65.4696 6.2998 Effect Num DF Den DF F Value Pr > F Fertilizer 3 12 0.73 0.5529 Fungicide 2 24 54.23 <0.0001 Fertilizer*Fungicide 6 24 0.80 0.5779 DAA1 6 216 216.91 <0.0001 Fertilizer*DAA1 18 216 0.56 0.9233 Fungicide*DAA1 12 216 10.41 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.58 0.9739

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Table A4.5 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) at Site 3, non-irrigated, 2016. Covariance parameters Estimate SE block -0.05580 0.4617 block*fertilizer -2.8569 1.7521 CS 10.2024 4.3376 Residual 33.1825 3.1930 Effect Num DF Den DF F Value Pr > F Fertilizer 3 36 0.20 0.8947 Fungicide 2 36 24.18 <0.0001 Fertilizer*Fungicide 6 36 0.72 0.6380 DAA1 6 216 493.97 <0.0001 Fertilizer*DAA1 18 216 1.02 0.4328 Fungicide*DAA1 12 216 6.44 <0.0001 Fertilizer*Fungicide*DAA1 36 216 1.02 0.4377

Table A4.6 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) in two asparagus fields, southwestern Ontario, irrigated, 2016. Covariance parameters Estimate SE block(location) 1.1856 1.2705 block*fertilizer 0.5125 1.4013 Variance -2.8399 2.9106 CS 3.3743 1.8532 Residual 47.2426 4.2299 Effect Num DF Den DF F Value Pr > F Location 1 3.79 15.42 0.0190 Fertilizer 3 8.674 0.68 0.5872 Location*Fertilizer 3 249.5 1.27 0.2835 Fungicide 2 24 31.58 <0.0001 Location*Fungicide 2 249.5 13.92 <0.0001 Fertilizer*Fungicide 6 24 2.77 0.0341 Location*Fertilizer*Fungicide 6 249.5 4.23 0.0004 DAA1 6 216 711.57 <0.0001 Location*DAA1 6 249.5 17.31 <0.0001 Fertilizer*DAA1 18 216 0.88 0.6051 Location*Fertilizer*DAA1 18 249.5 0.42 0.9826 Fungicide*DAA1 12 216 3.80 <0.0001 Location*Fungicide*DAA1 12 249.5 2.50 0.0040 Fertilizer*Fungicide*DAA1 36 216 0.73 0.8735 Location*Fertilizer*Fungicide*DAA1 36 249.5 0.84 0.7273

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Table A4.7 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) at Site 4, irrigated, 2016. Covariance parameters Estimate SE block 1.1316 1.8670 block*fertilizer -0.2796 2.4090 Variance 34.8234 3.4446 CS 8.5646 3.9792 Residual 0.9742 . Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.03 0.4234 Fungicide 2 24 8.14 0.0020 Fertilizer*Fungicide 6 24 3.01 0.0246 DAA1 6 216 488.96 <0.0001 Fertilizer*DAA1 18 216 0.99 0.4705 Fungicide*DAA1 12 216 1.87 0.0393 Fertilizer*Fungicide*DAA1 36 216 0.97 0.5175

Table A4.8 REML covariance parameter estimates for Stemphylium leaf spot DSI (0-100) at Site 5, irrigated, 2016. Covariance parameters Estimate SE block 1.7089 1.9955 block*fertilizer -2.2605 1.9717 Variance 44.8644 4.4113 CS 8.6482 4.4322 Residual 0.9794 . Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.72 0.5656 Fungicide 2 24 25.22 <0.0001 Fertilizer*Fungicide 6 24 1.49 0.2248 DAA1 6 216 281.15 <0.0001 Fertilizer*DAA1 18 216 0.46 0.9728 Fungicide*DAA1 12 216 4.57 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.77 0.8293

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Table A4.9 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application in two asparagus fields, southwestern Ontario, irrigated, 2015-16. Covariance parameters Estimate SE block(location) -0.3447 0.5368 block*fertilizer 0.3462 1.0733 Residual 13.5572 2.5235 Effect Num DF Den DF F Value Pr > F Location 1 4.677 14.11 0.0149 Fertilizer 3 12.89 0.47 0.7106 Location*Fertilizer 3 57.73 1.21 0.3146 Fungicide 2 57.73 24.87 <0.0001 Location*Fungicide 2 57.73 2.13 0.1280 Fertilizer*Fungicide 6 57.73 1.90 0.0956 Location*Fertilizer*Fungicide 6 57.73 2.58 0.0276

Table A4.10 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence in two asparagus fields, southwestern Ontario, irrigated, 2015-16. Covariance parameters Estimate SE block(location) 0.4642 1.3650 block*fertilizer 0.6077 1.7332 Residual 14.2848 2.6267 Effect Num DF Den DF F Value Pr > F Location 1 2.866 9.03 0.0609 Fertilizer 3 6.347 0.56 0.6604 Location*Fertilizer 3 59.15 0.71 0.5518 Fungicide 2 59.15 32.43 <0.0001 Location*Fungicide 2 59.15 7.58 0.0012 Fertilizer*Fungicide 6 59.15 3.04 0.0117 Location*Fertilizer*Fungicide 6 59.15 2.51 0.0311

Table A4.11 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application in four asparagus fields, southwestern Ontario, non-irrigated, 2015-16. Covariance parameters Estimate SE block(location) -0.07259 0.4704 block*fertilizer -0.1412 0.4532 Residual 13.7512 1.7231 Effect Num DF Den DF F Value Pr > F Location 3 10.78 204.73 <0.0001 Fertilizer 3 10.3 0.17 0.9171 Location*Fertilizer 9 127.4 0.96 0.4802 Fungicide 2 127.4 73.15 <0.0001 Location*Fungicide 6 127.4 2.96 0.0098 Fertilizer*Fungicide 6 127.4 0.61 0.7191 Location*Fertilizer*Fungicide 18 127.4 0.72 0.7826

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Table A4.12 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence in four asparagus fields, southwestern Ontario, non-irrigated, 2015-16. Covariance parameters Estimate SE block(location) 0.4412 0.7668 block*fertilizer 0.03609 0.6375 Residual 15.7379 1.9864 Effect Num DF Den DF F Value Pr > F Location 3 10.63 238.93 <0.0001 Fertilizer 3 9.345 0.78 0.5311 Location*Fertilizer 9 125.5 1.03 0.4206 Fungicide 2 125.5 153.78 <0.0001 Location*Fungicide 6 125.5 3.74 0.0019 Fertilizer*Fungicide 6 125.5 0.63 0.7043 Location*Fertilizer*Fungicide 18 125.5 0.80 0.7007

Table A4.13 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application at Site 4, irrigated, 2016. Covariance parameters Estimate SE block -0.2571 1.5067 block*fertilizer 1.2252 3.7786 Residual 17.7685 5.1293 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.99 0.4395 Fungicide 2 24 6.56 0.0053 Fertilizer*Fungicide 6 24 2.76 0.0349

Table A4.14 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application at Site 5, irrigated, 2016. Covariance parameters Estimate SE block -0.4730 0.3337 block*fertilizer -0.5748 1.5159 Residual 9.4453 2.7266 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.31 0.8156 Fungicide 2 24 26.41 <0.0001 Fertilizer*Fungicide 6 24 1.25 0.3170

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Table A4.15 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application at Site 1, non-irrigated, 2015. Covariance parameters Estimate SE block 0.1451 1.0919 block*fertilizer 0.8651 2.1963 Residual 9.9762 2.8799 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.90 0.4779 Fungicide 2 24 55.41 <0.0001 Fertilizer*Fungicide 6 24 0.73 0.6269

Table A4.16 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application at Site 1, non-irrigated, 2016. Covariance parameters Estimate SE block 0.03928 0.5505 block*fertilizer -0.7570 1.3523 Residual 8.9208 2.5752 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.39 0.3068 Fungicide 2 24 26.76 <0.0001 Fertilizer*Fungicide 6 24 1.02 0.4356

Table A4.17 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application at Site 2, non-irrigated, 2015. Covariance parameters Estimate SE block -0.6070 2.3185 block*fertilizer 3.5797 5.9389 Residual 24.0647 6.9469 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.73 0.5616 Fungicide 2 24 21.46 <0.0001 Fertilizer*Fungicide 6 24 0.50 0.8023

Table A4.18 REML covariance parameter estimates for sAUDPC calculated 14 days after the last application at Site 3, non-irrigated, 2016. Covariance parameters Estimate SE block -0.1848 0.8087 block*fertilizer -2.3380 2.6114 Residual 18.9252 5.4632 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.20 0.8967 Fungicide 2 24 16.62 <0.0001 Fertilizer*Fungicide 6 24 0.80 0.5768

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Table A4.19 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence at Site 4, irrigated, 2016. Covariance parameters Estimate SE block 0.9352 1.7771 block*fertilizer -0.8586 2.6573 Residual 16.2493 4.6908 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.09 0.4035 Fungicide 2 24 8.52 0.0016 Fertilizer*Fungicide 6 24 3.44 0.0135

Table A4.20 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence at Site 5, irrigated, 2016. Covariance parameters Estimate SE block 1.1722 1.5586 block*fertilizer -2.4847 2.0050 Residual 15.7842 4.5565 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.63 0.6123 Fungicide 2 24 27.44 <0.0001 Fertilizer*Fungicide 6 24 1.48 0.2280

Table A4.21 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence at Site 1, non-irrigated, 2015. Covariance parameters Estimate SE block 0.1451 1.0919 block*fertilizer 0.8651 2.1963 Residual 9.9762 2.8799 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.90 0.4779 Fungicide 2 24 55.41 <0.0001 Fertilizer*Fungicide 6 24 0.73 0.6269

Table A4.22 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence at Site 1, non-irrigated, 2016. Covariance parameters Estimate SE block 1.7043 2.1203 block*fertilizer 0.6720 1.7775 Residual 8.1386 2.3494 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.62 0.2517 Fungicide 2 24 53.82 <0.0001 Fertilizer*Fungicide 6 24 1.03 0.4290

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Table A4.23 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence at Site 2, non-irrigated, 2015. Covariance parameters Estimate SE block -0.2638 2.5598 block*fertilizer 3.7328 5.9443 Residual 23.7287 6.8499 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.87 0.4909 Fungicide 2 24 52.60 <0.0001 Fertilizer*Fungicide 6 24 0.87 0.5322

Table A4.24 REML covariance parameter estimates for sAUDPC calculated 98 days after cladophyll emergence at Site 3, non-irrigated, 2016. Covariance parameters Estimate SE block -0.04101 0.6484 block*fertilizer -3.7945 2.3534 Residual 20.0023 5.7742 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.35 0.7899 Fungicide 2 24 17.89 <0.0001 Fertilizer*Fungicide 6 24 0.56 0.7560

Table A4.24 REML covariance parameter estimates for fern yellowing in two asparagus fields, southwestern Ontario, irrigated, 2016. Covariance parameters Estimate SE block(location) 7.4001 4.9198 block*fertilizer 0 . CS 3.1795 2.2201 Residual 80.5984 5.2871 Effect Num DF Den DF F Value Pr > F Location 1 5.775 3.91 0.0973 Fertilizer 3 33.37 0.63 0.6022 Location*Fertilizer 3 464.8 0.99 0.3991 Fungicide 2 33.37 19.83 <0.0001 Location*Fungicide 2 464.8 10.61 <0.0001 Fertilizer*Fungicide 6 33.37 1.96 0.0999 Location*Fertilizer*Fungicide 6 464.8 1.62 0.1381 DAA1 6 464.8 783/19 <0.0001 Location*DAA1 6 464.8 47.23 <0.0001 Fertilizer*DAA1 18 464.8 0.55 0.9343 Location*Fertilizer*DAA1 18 464.8 0.32 0.9971 Fungicide*DAA1 12 464.8 3.83 <0.0001 Location*Fungicide*DAA1 12 464.8 3.20 0.0002 Fertilizer*Fungicide*DAA1 36 464.8 0.67 0.9261 Location*Fertilizer*Fungicide*DAA1 36 464.8 0.55 0.9859

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Table A4.25 REML covariance parameter estimates for fern yellowing in four asparagus fields, southwestern Ontario, non-irrigated, 2015-16. Covariance parameters Estimate SE block(location) 2.4319 1.7649 block*fertilizer 6.0225 3.4607 Variance -1.2888 2.7011 CS 1.4543 1.4663 Residual 102.87 5.3218 Effect Num DF Den DF F Value Pr > F Location 3 8.595 157.84 <0.0001 Fertilizer 3 10.01 1.21 0.3570 Location*Fertilizer 9 747.4 1.27 0.2497 Fungicide 2 24 168.77 <0.0001 Location*Fungicide 6 747.4 26.18 <0.0001 Fertilizer*Fungicide 6 24 0.90 0.5142 Location*Fertilizer*Fungicide 18 747.4 1.91 0.0126 DAA1 6 216 1342.26 <0.0001 Location*DAA1 6 747.4 98.25 <0.0001 Fertilizer*DAA1 18 216 1.90 0.0168 Location*Fertilizer*DAA1 18 747.4 1.01 0.4507 Fungicide*DAA1 12 216 22.82 <0.0001 Location*Fungicide*DAA1 36 747.4 6.62 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.77 0.8273 Location*Fertilizer*Fungicide*DAA1 108 747.4 0.93 0.6679

Table A4.26 REML covariance parameter estimates for fern yellowing at Site 4, irrigated, 2016. Covariance parameters Estimate SE block 0.1615 1.7008 block*fertilizer -0.3285 1.9557 CS 6.8989 3.2959 Residual 30.9607 2.9792 Effect Num DF Den DF F Value Pr > F Fertilizer 3 33 0.82 0.4914 Fungicide 2 33 10.21 0.0004 Fertilizer*Fungicide 6 33 0.52 0.7925 DAA1 6 216 1424.60 <0.0001 Fertilizer*DAA1 18 216 1.58 0.0661 Fungicide*DAA1 12 216 2.70 0.0021 Fertilizer*Fungicide*DAA1 36 216 0.75 0.8531

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Table A4.27 REML covariance parameter estimates for fern yellowing at Site 5, irrigated, 2016. Covariance parameters Estimate SE block 13.9973 12.1320 block*fertilizer -14.4132 5.3913 CS 38.4293 15.5049 Residual 105.33 10.1350 Effect Num DF Den DF F Value Pr > F Fertilizer 3 33 0.32 0.8082 Fungicide 2 33 8.73 0.0009 Fertilizer*Fungicide 6 33 1.15 0.3549 DAA1 6 216 216.70 <0.0001 Fertilizer*DAA1 18 216 0.20 0.9999 Fungicide*DAA1 12 216 4.59 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.72 0.8847

Table A4.28 REML covariance parameter estimates for fern yellowing at Site 1, non-irrigated, 2015. Covariance parameters Estimate SE block 1.8561 3.4435 block*fertilizer -3.3168 5.3624 CS 21.9204 10.4802 Residual 98.5572 9.4837 Effect Num DF Den DF F Value Pr > F Fertilizer 3 33 1.85 0.1567 Fungicide 2 33 16.73 <0.0001 Fertilizer*Fungicide 6 33 0.75 0.6103 DAA1 6 216 464.68 <0.0001 Fertilizer*DAA1 18 216 0.94 0.5336 Fungicide*DAA1 12 216 11.42 <0.0001 Fertilizer*Fungicide*DAA1 36 216 1.05 0.4013

Table A4.29 REML covariance parameter estimates for fern yellowing at Site 1, non-irrigated, 2016. Covariance parameters Estimate SE block 4.5340 6.1824 block*fertilizer 9.6671 5.4094 CS -2.6151 1.7008 Residual 55.2146 5.3130 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.71 0.5693 Fungicide 2 24 26.53 <0.0001 Fertilizer*Fungicide 6 24 2.79 0.0335 DAA1 6 216 129.16 <0.0001 Fertilizer*DAA1 18 216 1.18 0.2800 Fungicide*DAA1 12 216 3.90 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.87 0.6813

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Table A4.30 REML covariance parameter estimates for fern yellowing at Site 2, non-irrigated, 2015. Covariance parameters Estimate SE block -7.2043 5.1927 block*fertilizer 32.3984 18.8676 CS 2.4001 6.6588 Residual 137.98 13.2770 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.97 0.1888 Fungicide 2 24 81.34 <0.0001 Fertilizer*Fungicide 6 24 1.80 0.1424 DAA1 6 216 383.61 <0.0001 Fertilizer*DAA1 18 216 1.45 0.1129 Fungicide*DAA1 12 216 21.46 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.74 0.8556

Table A4.31 REML covariance parameter estimates for fern yellowing at Site 3, non-irrigated, 2016. Covariance parameters Estimate SE block 7.9514 8.7151 block*fertilizer 0.5087 5.7067 CS 20.2605 8.6675 Residual 67.1554 6.4620 Effect Num DF Den DF F Value Pr > F Fertilizer 3 36 0.46 0.7196 Fungicide 2 36 27.02 <0.0001 Fertilizer*Fungicide 6 36 0.14 0.9884 DAA1 6 216 1028.79 <0.0001 Fertilizer*DAA1 18 216 0.65 0.8564 Fungicide*DAA1 12 216 14.34 <0.0001 Fertilizer*Fungicide*DAA1 36 216 1.07 0.3670

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Table A4.32 REML covariance parameter estimates for fern defoliation in two asparagus fields, southwestern Ontario, irrigated, 2016. Covariance parameters Estimate SE block(location) 12.5108 8.3602 block*fertilizer 0 . CS 4.0716 3.6184 Residual 146.03 9.5791 Effect Num DF Den DF F Value Pr > F Location 1 5.811 9.29 0.0235 Fertilizer 3 33.32 0.57 0.6414 Location*Fertilizer 3 464.8 0.80 0.4946 Fungicide 2 33.32 20.40 <0.0001 Location*Fungicide 2 464.8 9.10 0.0001 Fertilizer*Fungicide 6 33.32 1.93 0.1042 Location*Fertilizer*Fungicide 6 464.8 2.26 0.0364 DAA1 6 464.8 858.46 <0.0001 Location*DAA1 6 464.8 67.65 <0.0001 Fertilizer*DAA1 18 464.8 0.36 0.9940 Location*Fertilizer*DAA1 18 464.8 0.25 0.9993 Fungicide*DAA1 12 464.8 3.93 <0.0001 Location*Fungicide*DAA1 12 464.8 2.36 0.0059 Fertilizer*Fungicide*DAA1 36 464.8 0.72 0.8915 Location*Fertilizer*Fungicide*DAA1 36 464.8 0.69 0.9180

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Table A4.33 REML covariance parameter estimates for fern defoliation in four asparagus fields, southwestern Ontario, non-irrigated, 2015-16. Covariance parameters Estimate SE block(location) 5.2883 3.2454 block*fertilizer 10.1337 5.7723 Variance 2.9013 4.0409 CS 3.0270 2.4797 Residual 138.87 7.1849 Effect Num DF Den DF F Value Pr > F Location 3 9.155 148.34 <0.001 Fertilizer 3 10.24 1.71 0.2259 Location*Fertilizer 9 747.1 2.90 0.0023 Fungicide 2 24 125.37 <0.001 Location*Fungicide 6 747.1 22.70 <0.001 Fertilizer*Fungicide 6 24 0.92 0.5001 Location*Fertilizer*Fungicide 18 747.1 1.72 0.0309 DAA1 6 216 860.12 <0.001 Location*DAA1 18 747.1 88.21 <0.001 Fertilizer*DAA1 18 216 1.74 0.0349 Location*Fertilizer*DAA1 54 747.1 0.98 0.5238 Fungicide*DAA1 12 216 21.45 <0.001 Location*Fungicide*DAA1 36 747.1 11.77 <0.001 Fertilizer*Fungicide*DAA1 36 216 0.53 0.9867 Location*Fertilizer*Fungicide*DAA1 108 747.1 0.67 0.9944

Table A4.34 REML covariance parameter estimates for fern defoliation at Site 4, irrigated, 2016. Covariance parameters Estimate SE block 1.1702 1.5561 block*fertilizer -0.1846 1.5611 CS 4.3184 2.5982 Residual 31.8727 3.0670 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.10 0.3986 Fungicide 2 24 10.21 0.0006 Fertilizer*Fungicide 6 24 0.80 0.5765 DAA1 6 216 2943.39 <0.0001 Fertilizer*DAA1 18 216 0.63 0.8764 Fungicide*DAA1 12 216 1.41 0.1643 Fertilizer*Fungicide*DAA1 36 216 0.44 0.9977

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Table A4.35 REML covariance parameter estimates for fern defoliation at Site 5, irrigated, 2016. Covariance parameters Estimate SE block 24.1657 22.0142 block*fertilizer -14.9749 9.1155 CS 44.4847 22.7219 Residual 234.03 22.5198 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 0.73 0.5618 Fungicide 2 24 8.87 0.0013 Fertilizer*Fungicide 6 24 1.23 0.3238 DAA1 6 216 177.01 <0.0001 Fertilizer*DAA1 18 216 0.29 0.9980 Fungicide*DAA1 12 216 3.73 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.81 0.7667

Table A4.36 REML covariance parameter estimates for fern defoliation at Site 1, non-irrigated, 2015. Covariance parameters Estimate SE block 0.03805 2.2943 block*fertilizer 4.6704 4.7545 CS 5.1447 4.3867 Residual 67.9216 6.5358 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.52 0.2751 Fungicide 2 24 41.06 <0.0001 Fertilizer*Fungicide 6 24 2.32 0.0660 DAA1 6 216 119.06 <0.0001 Fertilizer*DAA1 18 216 1.39 0.1391 Fungicide*DAA1 12 216 29.32 <0.0001 Fertilizer*Fungicide*DAA1 36 216 1.12 0.3031

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Table A4.37 REML covariance parameter estimates for fern defoliation at Site 1, non-irrigated, 2016. Covariance parameters Estimate SE block 8.3598 14.4865 block*fertilizer 28.7334 16.4240 CS -6.6738 5.6385 Residual 171.00 16.4541 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9 1.07 0.4091 Fungicide 2 24 19.04 <0.0001 Fertilizer*Fungicide 6 24 1.08 0.4031 DAA1 6 216 88.38 <0.0001 Fertilizer*DAA1 18 216 1.73 0.0366 Fungicide*DAA1 12 216 4.04 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.49 0.9944

Table A4.38 REML covariance parameter estimates for fern defoliation at Site 2, non-irrigated, 2015. Covariance parameters Estimate SE block 1.1346 11.5059 block*fertilizer 31.8798 21.7402 CS 19.2094 11.8623 Residual 148.87 14.3248 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9.01 1.97 0.1886 Fungicide 2 23.99 81.32 <0.0001 Fertilizer*Fungicide 6 23.99 1.80 0.1425 DAA1 6 216 383.62 <0.0001 Fertilizer*DAA1 18 216 1.45 0.1129 Fungicide*DAA1 12 216 21.46 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.74 0.8556

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Table A4.39 REML covariance parameter estimates for fern defoliation at Site 3, non-irrigated, 2016. Covariance parameters Estimate SE block 5.4309 7.6146 block*fertilizer -1.1887 8.2623 CS 29.1123 13.7979 Residual 128.06 12.3227 Effect Num DF Den DF F Value Pr > F Fertilizer 3 9.001 0.33 0.8028 Fungicide 2 24 27.92 <0.0001 Fertilizer*Fungicide 6 24 0.15 0.9872 DAA1 6 216 670.50 <0.0001 Fertilizer*DAA1 18 216 0.50 0.9574 Fungicide*DAA1 12 216 17.61 <0.0001 Fertilizer*Fungicide*DAA1 36 216 0.71 0.8854

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APPENDIX 5: SUPPLEMENTARY TABLES FOR CHAPTER SIX

Table A5.1 REML covariance parameter estimates for Stemphylium leaf spot counts on asparagus seedlings inoculated with a conidial suspension or residue infected with pseudothecia, 2015-16. Covariance parameters Estimate SE block(year) 1.1583 1.2351 Residual 3.5083 1.2404 Effect Num DF Den DF F Value Pr > F Inoculum 2 16 29.08 <0.0001 Year 1 8 0.31 0.5956 Year*Inoculum 2 16 1.49 0.2547

Table A5.2 REML covariance parameter estimates for sAUDPC on asparagus seedlings inoculated with a conidial suspension or residue infected with pseudothecia, 2015-16. Covariance parameters Estimate SE block(year) 0.02502 0.1329 Residual 0.6000 0.2121 Effect Num DF Den DF F Value Pr > F Inoculum 2 16 18.91 <0.0001 Year 1 8 0.71 0.4230 Year*Inoculum 2 16 2.30 0.1328

Table A5.3 REML covariance parameter estimates for disease incidence on asparagus seedlings inoculated with a conidial suspension or residue infected with pseudothecia, 2015-16. Covariance parameters Estimate SE block(year) 0.03333 0.04526 Residual 0.1500 0.05303 Effect Num DF Den DF F Value Pr > F Inoculum 2 16 9.56 0.0019 Year 1 8 0.13 0.7245 Year*Inoculum 2 16 0.22 0.8032

Table A5.4 REML covariance parameter estimates for chlorophyll concentration in asparagus seedlings, outdoor potted experiment, 2016. Covariance parameters Estimate SE block(location) -0.00746 0.009598 Residual 0.1166 0.02712 Effect Num DF Den DF F Value Pr > F Repetition 1 38.68 111.51 <0.0001 Cultivar 1 47 26.73 <0.0001 Repetition*Cultivar 1 47 2.65 0.1105 Date 2 47 271.83 <0.0001 Repetition*Date 2 47 2.43 0.0990 Cultivar*Date 2 47 2.20 0.1216 Repetition*Cultivar*Date 2 47 0.42 0.6563

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Table A5.5 REML covariance parameter estimates for proline concentration in asparagus seedlings, outdoor potted experiment, 2016. Covariance parameters Estimate SE block(repetition) 1.8479 1.1755 Residual 6.4381 1.2506 Effect Num DF Den DF F Value Pr > F Repetition 1 10.04 0.20 0.6657 Cultivar 1 3.629 26.80 0.0086 Repetition*Cultivar 1 53.01 6.69 0.0125 Date 2 53.01 17.56 <0.0001 Repetition*Date 2 53.01 0.46 0.7131 Cultivar*Date 2 53.01 2.24 0.0941 Repetition*Cultivar*Date 2 53.01 0.79 0.5043

Table A5.6 REML covariance parameter estimates for Stemphylium leaf spot lesions in asparagus seedlings, outdoor potted experiment, 2016. Covariance parameters Estimate SE block(repetition) 0.2036 0.1757 Residual 1.2952 0.2602 Effect Num DF Den DF F Value Pr > F Repetition 1 8.966 1.02 0.3381 Cultivar 1 4.576 2.56 0.1759 Repetition*Cultivar 1 49.86 4.28 0.0437 Date 2 49.84 49.69 <0.0001 Repetition*Date 2 49.84 3.04 0.0376 Cultivar*Date 2 49.84 0.44 0.7272 Repetition*Cultivar*Date 2 49.84 1.58 0.2067

Table A5.7 REML covariance parameter estimates for Stemphylium leaf spot lesions in asparagus seedlings, outdoor potted experiment, Repetition 1, 2016. Covariance parameters Estimate SE block 0.1208 0.1212 Residual 0.9679 0.2794 Effect Num DF Den DF F Value Pr > F Cultivar 1 4 1.42 0.2999 Date 3 24 23.87 <0.0001 Cultivar*Date 3 24 0.29 0.8291

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Table A5.8 REML covariance parameter estimates for Stemphylium leaf spot lesions in asparagus seedlings, outdoor potted experiment, Repetition 2, 2016. Covariance parameters Estimate SE block 0.3117 0.4352 Residual 1.5726 0.5609 Effect Num DF Den DF F Value Pr > F Cultivar 1 3.923 3.47 0.1374 Date 3 23.52 28.98 <0.0001 Cultivar*Date 3 23.52 23.52 0.2967 Table A5.9 REML covariance parameter estimates for pseudothecia on asparagus seedlings, outdoor potted experiment, 2016. Covariance parameters Estimate SE block(repetition) 0.5788 0.8153 Residual 13.4169 2.5850 Effect Num DF Den DF F Value Pr > F Repetition 1 13.98 3.73 0.0740 Cultivar 1 3.719 12.68 0.0266 Repetition*Cultivar 1 54.23 0.05 0.8200 Date 2 54.22 35.64 <0.0001 Repetition*Date 2 54.22 2.69 0.0550 Cultivar*Date 2 54.22 2.22 0.0959 Repetition*Cultivar*Date 2 54.22 0.20 0.8967

Table A5.10 REML covariance parameter estimates for sucrose concentration in asparagus seedlings, outdoor potted experiment, 2016. Covariance parameters Estimate SE block(repetition) 10.2550 17.9517 Residual 206.36 40.0593 Effect Num DF Den DF F Value Pr > F Repetition 1 8.044 6.87 0.0305 Cultivar 1 4.641 1.04 0.3585 Repetition*Cultivar 1 53.07 0.05 0.8186 Date 2 53.07 205.47 <0.0001 Repetition*Date 2 53.07 3.38 0.0247 Cultivar*Date 2 53.07 0.75 0.5276 Repetition*Cultivar*Date 2 53.07 1.03 0.3878

Table A5.11 REML covariance parameter estimates for sucrose concentration in asparagus seedlings, outdoor potted experiment, Repetition 1, 2016. Covariance parameters Estimate SE block 25.2502 42.4651 Residual 78.9676 22.7960 Effect Num DF Den DF F Value Pr > F Cultivar 1 8 0.19 0.6831 Date 3 24 241.27 <0.0001 Cultivar*Date 3 24 0.80 0.5079

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Table A5.12 REML covariance parameter estimates for sucrose concentration in asparagus seedlings, outdoor potted experiment, Repetition 2, 2016. Covariance parameters Estimate SE block 0 . Residual 331.46 95.6833 Effect Num DF Den DF F Value Pr > F Cultivar 1 8 1.02 0.3416 Date 3 24 72.55 <0.0001 Cultivar*Date 3 24 0.92 0.4477

Table A5.13 REML covariance parameter estimates for chlorophyll concentration in asparagus seedlings, controlled environment, 2016. Covariance parameters Estimate SE block(repetition) -0.2271 0.1618 Temperature*block -0.2668 0.1537 Date*block 0.1669 0.3453 Residual 3.9234 0.8599 Effect Num DF Den DF F Value Pr > F Repetition 1 5.059 0.00 0.9712 Temperature 1 4.609 183.48 <0.0001 Repetition*Temperature 1 41.64 1.55 0.2196 Date 3 16.39 19.51 <0.0001 Repetition*Date 3 41.64 1.39 0.2583 Temperature*Date 3 41.64 12.14 <0.0001 Repetition*Temperature*Date 3 41.64 0.70 0.5586

Table A5.14 REML covariance parameter estimates for proline concentration in asparagus seedlings, controlled environment, 2016. Covariance parameters Estimate SE block(repetition) 1.9607 2.0256 Temperature*block -0.7766 1.3403 Date*block -2.3365 1.6445 Residual 19.3914 4.8299 Effect Num DF Den DF F Value Pr > F Repetition 1 8.223 1.98 0.1961 Temperature 1 3.448 433.12 <0.0001 Repetition*Temperature 1 32.24 0.09 0.7674 Date 3 9.273 85.53 <0.0001 Repetition*Date 3 32.24 3.41 0.0291 Temperature*Date 3 32.24 39.01 <0.0001 Repetition*Temperature*Date 3 32.24 0.53 0.6632

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Table A5.15 REML covariance parameter estimates for sucrose concentration in asparagus seedlings, controlled environment, 2016. Covariance parameters Estimate SE block(repetition) 0 . Temperature*block 18.8580 23.0744 Date*block 0 . Residual 162.88 35.5440 Effect Num DF Den DF F Value Pr > F Repetition 1 42 1.08 0.3050 Temperature 1 6 34.77 0.0011 Repetition*Temperature 1 42 0.49 0.4861 Date 3 42 14.84 <0.0001 Repetition*Date 3 42 3.46 0.0245 Temperature*Date 3 42 5.59 0.0026 Repetition*Temperature*Date 3 42 3.22 0.0322

Table A5.16 REML covariance parameter estimates for sucrose concentration in asparagus seedlings, controlled environment, Repetition 1, 2016. Covariance parameters Estimate SE block -27.4730 27.4515 Temperature*block 35.4643 49.6211 Date*block 28.6386 42.2945 Residual 94.9708 44.7697 Effect Num DF Den DF F Value Pr > F Temperature 1 3 19.25 0.0219 Date 3 9 6.27 0.0138 Temperature*Date 3 9 5.60 0.0191

Table A5.17 REML covariance parameter estimates for sucrose concentration in asparagus seedlings, controlled environment, Repetition 2, 2016. Covariance parameters Estimate SE block -51.1564 38.7275 Temperature*block 33.7442 70.4463 Date*block 57.4185 85.2736 Residual 191.88 90.4509 Effect Num DF Den DF F Value Pr > F Temperature 1 3 19.68 0.0213 Date 3 9 6.61 0.119 Temperature*Date 3 9 4.70 0.0306

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Table A5.18 REML covariance parameter estimates for Stemphylium leaf spot counts on asparagus seedlings, controlled environment, 2016. Covariance parameters Estimate SE block(repetition) -1.2631 2.0325 Temperature*block -0.4057 2.3272 Date*block -0.3360 3.4287 Residual 28.1450 6.4803 Effect Num DF Den DF F Value Pr > F Repetition 1 2.485 4.91 0.1318 Temperature 1 3.111 16.61 0.0249 Repetition*Temperature 1 37.73 4.35 0.0439 Date 3 8.495 16.28 0.0007 Repetition*Date 3 37.73 2.59 0.0670 Temperature*Date 3 37.73 11.13 <0.0001 Repetition*Temperature*Date 3 37.73 2.75 0.0560

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Table A5.19 REML covariance parameter estimates for purple spot incidence on asparagus spears harvested from plots treated with three factors: mowing timing, fungicide and nitrogen, 2012-14. Covariance parameters Estimate SE block(location*year) -13.8158 . chopping*block -7.0661 . copper*block -5.0943 . fertilizer*block -3.7283 . Residual 1091.09 . Effect Num DF Den DF F Value Pr > F Location 1 352 1.95 0.1639 Year 1 352 24.35 <0.0001 Location*Year 1 352 14.19 0.0002 Chopping 1 352 6.02 0.0147 Location*Chopping 1 352 0.29 0.5918 Year*Chopping 1 352 1.09 0.2980 Location*Year*Chopping 1 352 0.39 0.5329 Copper 1 352 0.03 0.8540 Location*Copper 1 352 0.11 0.7386 Year*Copper 1 352 0.07 0.7958 Location*Year*Copper 1 352 0.12 0.7292 Chopping*Copper 1 352 0.00 0.9900 Location*Chopping*Copper 1 352 0.06 0.8078 Year*Chopping*Copper 1 352 0.08 0.7790 Location*Year*Chopping*Copper 1 352 0.15 0.6990 Fertilizer 1 352 0.18 0.6715 Location*Fertilizer 1 352 0.14 0.7129 Year*Fertilizer 1 352 0.12 0.7316 Location*Year*Fertilizer 1 352 0.06 0.8127 Chopping*Fertilizer 1 352 0.20 0.6557 Location*Chopping*Fertilizer 1 352 0.60 0.4376 Year*Chopping*Fertilizer 1 352 0.07 0.7910 Location*Year*Chopping*Fertilizer 1 352 0.00 0.9776 Copper*Fertilizer 1 352 0.03 0.8712 Location*Copper*Fertilizer 1 352 0.15 0.6944 Year*Copper*Fertilizer 1 352 0.03 0.8614 Location*Year*Copper*Fertilizer 1 352 0.00 0.9552 Chopping*Copper*Fertilizer 1 352 0.39 0.5309 Location*Chopping*Copper*Fertilizer 1 352 0.27 0.6026 Year*Chopping*Copper*Fertilizer 1 352 0.01 0.9032 Location*Year*Chopping*Copper*Fertilizer 1 352 0.07 0.7862

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Table A5.20 REML covariance parameter estimates for purple spot severity on asparagus spears harvested from plots treated with three factors: mowing timing, fungicide and nitrogen, 2012-14. Covariance parameters Estimate SE block(location*year) -1.0962 . chopping*block -1.2869 . copper*block -0.8320 . fertilizer*block -0.2545 . Residual 182.33 . Effect Num DF Den DF F Value Pr > F Location 1 352 4.12 0.0430 Year 1 352 40.84 <0.0001 Location*Year 1 352 25.95 <0.0001 Chopping 1 352 10.15 0.0016 Location*Chopping 1 352 0.00 0.9807 Year*Chopping 1 352 4.65 0.0318 Location*Year*Chopping 1 352 0.49 0.4838 Copper 1 352 0.03 0.8717 Location*Copper 1 352 0.09 0.7699 Year*Copper 1 352 0.04 0.8384 Location*Year*Copper 1 352 0.00 0.9605 Chopping*Copper 1 352 0.26 0.6101 Location*Chopping*Copper 1 352 0.00 0.9857 Year*Chopping*Copper 1 352 0.10 0.7538 Location*Year*Chopping*Copper 1 352 0.13 0.7234 Fertilizer 1 352 0.00 0.9620 Location*Fertilizer 1 352 0.32 0.5735 Year*Fertilizer 1 352 0.50 0.4799 Location*Year*Fertilizer 1 352 0.06 0.8006 Chopping*Fertilizer 1 352 0.40 0.5269 Location*Chopping*Fertilizer 1 352 0.35 0.5535 Year*Chopping*Fertilizer 1 352 0.12 0.7250 Location*Year*Chopping*Fertilizer 1 352 0.01 0.9335 Copper*Fertilizer 1 352 0.00 0.9537 Location*Copper*Fertilizer 1 352 0.01 0.9268 Year*Copper*Fertilizer 1 352 0.00 0.9873 Location*Year*Copper*Fertilizer 1 352 0.06 0.8090 Chopping*Copper*Fertilizer 1 352 0.22 0.6428 Location*Chopping*Copper*Fertilizer 1 352 0.53 0.4669 Year*Chopping*Copper*Fertilizer 1 352 0.10 0.7522 Location*Year*Chopping*Copper*Fertilizer 1 352 0.16 0.6859

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Table A5.21 REML covariance parameter estimates for purple spot incidence on asparagus spears harvested and stored for 3 days from plots treated with three factors: mowing timing, fungicide and nitrogen, 2012-14. Covariance parameters Estimate SE block(location*year) 5.3487 7.7242 chopping*block 0.1330 7.1587 copper*block -4.4879 4.1466 fertilizer*block -8.0822 1.0647 Residual 570.58 43.3393 Effect Num DF Den DF F Value Pr > F Location 1 352 8.13 0.0259 Year 1 352 24.56 <0.0001 Location*Year 1 352 15.82 <0.0001 Chopping 1 352 6.52 0.0791 Location*Chopping 1 352 0.51 0.4764 Year*Chopping 1 352 0.04 0.8341 Location*Year*Chopping 1 352 2.11 0.1474 Copper 1 352 1.52 0.3286 Location*Copper 1 352 0.01 0.9151 Year*Copper 1 352 0.02 0.8915 Location*Year*Copper 1 352 0.47 0.4924 Chopping*Copper 1 352 0.00 0.9804 Location*Chopping*Copper 1 352 0.19 0.6597 Year*Chopping*Copper 1 352 0.46 0.7980 Location*Year*Chopping*Copper 1 352 0.05 0.8305 Fertilizer 1 352 3.29 0.2090 Location*Fertilizer 1 352 0.11 0.7367 Year*Fertilizer 1 352 0.01 0.9213 Location*Year*Fertilizer 1 352 0.20 0.6517 Chopping*Fertilizer 1 352 0.36 0.5477 Location*Chopping*Fertilizer 1 352 0.04 0.8376 Year*Chopping*Fertilizer 1 352 0.16 0.6877 Location*Year*Chopping*Fertilizer 1 352 1.26 0.2618 Copper*Fertilizer 1 352 0.26 0.6089 Location*Copper*Fertilizer 1 352 0.09 0.7620 Year*Copper*Fertilizer 1 352 0.43 0.5133 Location*Year*Copper*Fertilizer 1 352 3.19 0.0750 Chopping*Copper*Fertilizer 1 352 3.06 0.0813 Location*Chopping*Copper*Fertilizer 1 352 0.73 0.3928 Year*Chopping*Copper*Fertilizer 1 352 0.28 0.5961 Location*Year*Chopping*Copper*Fertilizer 1 352 0.92 0.3376

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Table A5.22 REML covariance parameter estimates for purple spot severity on asparagus spears harvested from plots treated with three factors: mowing timing, fungicide and nitrogen, 2012-14. Covariance parameters Estimate SE block(location*year) 3.1919 2.0878 chopping*block -1.2896 1.0338 copper*block -0.7923 1.5406 fertilizer*block -2.2792 0.1745 Residual 146.43 11.1537 Effect Num DF Den DF F Value Pr > F Location 1 13.36 0.96 0.3454 Year 1 344.7 17.45 <0.0001 Location*Year 1 344.7 43.09 <0.0001 Chopping 1 1.913 27.44 0.0379 Location*Chopping 1 344.7 3.19 0.0751 Year*Chopping 1 344.7 0.00 0.9759 Location*Year*Chopping 1 344.7 0.18 0.6699 Copper 1 1.903 0.35 0.6184 Location*Copper 1 344.7 0.00 0.9961 Year*Copper 1 344.7 0.07 0.7844 Location*Year*Copper 1 344.7 0.63 0.4261 Chopping*Copper 1 344.7 0.74 0.3906 Location*Chopping*Copper 1 344.7 0.07 0.7933 Year*Chopping*Copper 1 344.7 1.13 0.2892 Location*Year*Chopping*Copper 1 344.7 0.15 0.6974 Fertilizer 1 1.999 5.96 0.1348 Location*Fertilizer 1 344.7 0.40 0.5273 Year*Fertilizer 1 344.7 0.40 0.7261 Location*Year*Fertilizer 1 344.7 0.23 0.6295 Chopping*Fertilizer 1 344.7 0.79 0.3751 Location*Chopping*Fertilizer 1 344.7 0.10 0.7468 Year*Chopping*Fertilizer 1 344.7 0.17 0.6823 Location*Year*Chopping*Fertilizer 1 344.7 0.26 0.6097 Copper*Fertilizer 1 344.7 0.25 0.6146 Location*Copper*Fertilizer 1 344.7 0.00 0.9479 Year*Copper*Fertilizer 1 344.7 0.34 0.5602 Location*Year*Copper*Fertilizer 1 344.7 0.92 0.3376 Chopping*Copper*Fertilizer 1 344.7 0.41 0.5227 Location*Chopping*Copper*Fertilizer 1 344.7 0.63 0.4291 Year*Chopping*Copper*Fertilizer 1 344.7 0.04 0.8421 Location*Year*Chopping*Copper*Fertilizer 1 344.7 0.76 0.3839

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