Open KON Dissertation FINAL.Pdf

The Pennsylvania State University

The Graduate School

College of Agricultural Sciences

EVALUATION OF CHEMICAL AND THERMAL BLOSSOM THINNING

STRATEGIES FOR APPLE

A Dissertation in

Horticulture

Thomas M. Kon

 2016 Thomas M. Kon

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December 2016

The dissertation of Thomas M. Kon was reviewed and approved* by the following:

James R. Schupp Professor of Pomology Dissertation Advisor Chair of Committee

Robert M. Crassweller Professor of Horticulture

Paul Heinemann Professor and Head of Agricultural and Biological Engineering

Richard Marini Professor of Horticulture

Erin L. Connolly Professor of Plant Science Head of the Department of Plant Science

*Signatures are on file in the Graduate School

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ABSTRACT

Since 1989, approximately 150 compounds and multiple mechanical devices were evaluated as apple blossom thinners. Despite these efforts, blossom thinner adoption has been limited to a few apple producing regions or states. The purpose of this work was to: 1) compare the efficacy of promising chemical blossom thinners using a predictive model as a timing aid, and

2) evaluate the potential of short-duration thermal treatments as a blossom thinning strategy.

Using a predictive model as a timing aid, the efficacy of several promising chemical blossom thinners was evaluated at Penn State’s Fruit Research and Extension Center in Biglerville, PA.

Blossom thinner effects on pollen tube growth, fruit set, and yield responses were evaluated.

Calcium polysulfide and ammonium thiosulfate inhibited pollen tube growth in vivo and reduced initial fruit set. Endothal was a potent thinner, but was ineffective in reducing pollen tube growth and caused excessive leaf injury. When used as the sole method of crop load management, none of the chemistries evaluated over-thinned or increased fruit injury. However, endothal caused excessive thinning when evaluated as part of a commercial crop load management program. In a series of experiments, short duration forced heated air treatments (thermal shock; TS) were evaluated as a potential blossom thinning strategy. TS treatments were applied to solitary blossoms and spur leaf tissue with a variable temperature heat gun. At effective temperatures, TS reduced stigmatic receptivity and pollen tube growth in vivo when applied up to 24 h after the pollination event. At the range of temperatures evaluated, a minimum of a 2 s application was required to influence pollen tube growth in vivo. While our data shows that TS was effective in reducing pollen tube growth in vivo, the onset of visible injury to leaf tissue occurred at similar temperatures. Environmental conditions appeared to influence heat gun performance and TS treatment efficacy.

TABLE OF CONTENTS

List of Figures ...... vi

List of Tables ...... vii

Acknowledgements ...... ix

Chapter 1 Crop load management strategies for apple ...... 1

Abstract ...... 1 Introduction ...... 1 Advantages of reducing crop load early ...... 2 Disadvantages of reducing crop load early ...... 4 Pruning to reduce crop load ...... 5 Blossom thinners ...... 11 Chemical blossom thinners ...... 11 Finding an alternative to Elgetol: caustic products and photosynthetic inhibitors ...... 12 Alternatives to Elgetol: use of plant growth regulators ...... 17 Alternatives to Elgetol: screening efforts ...... 18 Timing of chemical blossom thinning applications ...... 27 Mechanical blossom thinning ...... 29 Post bloom thinners: status and new chemistries ...... 31 Post bloom thinners: advances in application timing ...... 33 Integrated crop load management strategies ...... 35 Conclusions ...... 36 Literature Cited ...... 40

Chapter 2 Evaluation of chemical blossom thinners using ‘Golden Delicious’ and ‘Gala’ pollen tube growth models as timing aids ...... 64

Introduction ...... 64 Materials and Methods ...... 68 ‘Golden Delicious’ ...... 68 ‘Gala’ ...... 73 Statistical analysis ...... 75 Results and Discussion ...... 75 ‘Golden Delicious’ ...... 75 ‘Gala’ ...... 87 Conclusions ...... 93 Literature Cited ...... 95

Chapter 3 Thermal shock temperature and timing effects on apple stigmatic receptivity, pollen tube growth, and leaf injury ...... 101

Introduction ...... 101 Materials and Methods ...... 104

Expt.1: TS effects on stigmatic receptivity of ‘Crimson Gala’ apple...... 104 Expt. 2: TS effects on pollen tube growth in vivo...... 106 Expt. 3: TS effects on visible spur leaf injury...... 109 Statistical analysis ...... 110 Results and Discussion ...... 110 Expt.1: TS effects on stigmatic receptivity of ‘Crimson Gala’ apple...... 110 Expt. 2: TS effects on pollen tube growth in vivo...... 115 Expt. 3: TS effects on visible spur leaf injury...... 121 Conclusions ...... 122 Literature Cited ...... 124

Chapter 4 Apple pollen tube growth and spur leaf injury in response to thermal shock temperature and duration...... 129

Introduction ...... 129 Materials and Methods ...... 130 Expt.1: Effects of TS temperature and treatment duration on stigmatic receptivity of ‘York’ apple...... 130 Expt. 2. Effects of TS temperature and treatment duration on visible spur leaf injury of ‘York’ apple...... 132 Statistical analysis ...... 134 Results and Discussion ...... 134 Expt.1: Effects of TS temperature and treatment duration on stigmatic receptivity of ‘York’ apple...... 134 Expt. 2. Effects of TS temperature and treatment duration on visible spur leaf injury of ‘York’ apple...... 141 Conclusions ...... 142 Literature Cited ...... 143

Chapter 5 Summary and Conclusions……………………………………………………..145

LIST OF FIGURES

Figure 1-1. The chemical thinning triangle...... 34

Figure 2-1. Graphical representation of ‘Golden Delicious’ pollen tube growth model application timings, estimates of pollen tube growth rates, and temperature in 2014 and 2015...... 71

Figure 2-2. Graphical representation of ‘Gala’ pollen tube growth model application timing, estimates of pollen tube growth rates, and temperature in 2015 ...... 74

Figure 2-3. Leaf phytotoxicity observed on ‘Golden Delicious’ treated with 1.5 mL∙L-1 TR (Thin Rite; United Phosphorus, Inc., King of Prussia, PA) in 2014 ...... 81

Figure 2-4. Leaf phytotoxicity observed on ‘Buckeye Gala’ treated with 2% (v:v) ammonium thiosulfate in 2015 ...... 90

Figure 3-1. Effects of thermal shock temperature and timing on pollen density (A and B) and the number of pollen tubes that enter the style (C and D) in 2014 and 2015 ...... 118

Figure 3-2. Effects of thermal shock temperature on the average length of the longest pollen tube in 2014 ...... 120

Figure 3-3. Effect of thermal shock temperature on visible spur leaf injury in 2015 ...... 121

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LIST OF TABLES

Table 1-1. Summary of recent spur pruning (extinction) studies ...... 8

Table 1-2. List of compounds screened and/or evaluated for blossom thinning apple ...... 20

Table 1-3. A summary of several recent mechanical thinning studies ...... 31

Table 2-2. Comparison of blossom thinning treatments on phytotoxicity, fruit set, and crop density of 'Golden Delicious'/'Budagovski 9' apple trees in 2014 and 2015 ...... 80

Table 2-3. Comparison of blossom thinning treatments on fruit number, crop density, yield, and fruit weight of 'Golden Delicious'/'Budagovski 9' apple trees in 2014 and 2015 ...... 83

Table 2-4. Comparison of blossom thinning treatments on fruit russet, seed number, and return bloom of 'Golden Delicious'/'Budagovski 9' apple trees in 2014 and 2015 ...... 85

Table 2-6. Comparison of four blossom thinning treatments on phytotoxicity, crop density, and fruit set of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015 ...... 89

Table 2-7. Comparison of four blossom thinning treatments on fruit number, crop density, yield, and fruit weight of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015 ...... 91

Table 2-8. Comparison of four blossom thinning treatments on fruit russet, seed number, fruit LD ratio, and return bloom of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015 ...... 92

Table 3-1. Expt. 1: Descriptive statistics for thermal shock (TS) output temperatures applied to ‘Crimson Gala’ in 2014 and 2015 ...... 105

Table 3-2. Expt.2: Descriptive statistics for thermal shock treatments applied to ‘Crimson Gala’ in 2014 ...... 108

Table 3-3. Expt. 2: Descriptive statistics for thermal shock treatments applied to ‘Crimson Gala’ in 2015 ...... 108

Table 3-4. Expt. 3: Descriptive statistics for thermal shock output temperature applied to ‘Crimson Gala’ spur leaf tissue in 2015 ...... 109

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Table 3-6. Effects of thermal shock temperature on stylar browing rating, pollen tube growth on the stigma, pollen tube growth in the style, percentage of receptive stigmas, and percentage of stylets supporting pollen tube growth of 'Crimson Gala' in 2014 and 2015...... 113

Table 4-1. Expt. 1: Descriptive statistics for thermal shock (TS) output temperatures applied to ‘York’ blossoms in 2014 and 2015 ...... 131

Table 4-2. Expt. 2: Descriptive statistics for thermal shock (TS) output temperatures applied to ‘York’ spur leaves in 2014 and 2015 ...... 133

Table 4-3. Main effects and interactions of thermal shock temperature and duration on stylar browning rating, pollen density on the stigma, number of pollen tubes penetrating the style, length of the longest pollen tube, and the number of pollen tubes at the style base of 'York Imperial' in 2014 and 2015 ...... 135

Table 4-6. Effects thermal shock temperature and duration visible injury ratings of ‘York Imperial' spur leaves in 2014 and 2015 ...... 141

ACKNOWLEDGEMENTS

Firstly, I would like to extend my heartfelt appreciation to my advisor and great friend

Jim Schupp. I started my graduate education in 2009 with little experience in research and tree fruit production. With Jim’s guidance and encouragement, I discovered a truly rewarding vocation. I am so fortunate to have you as a mentor – thank you for believing in me.

My doctoral committee - Rob Crassweller, Paul Heinemann, and Rich Marini - provided excellent advice and suggestions that dramatically improved the quality of this work. I appreciate your service on my committee and critical review of my dissertation.

A special thanks to Melanie Schupp and Edwin Winzeler – I am grateful for your friendship and support. Melanie prepared blossoms for microscopic evaluation, played a key role in the execution of experiments, and gave me confidence when I needed it most. Edwin encouraged me to evaluate thermal shock, provided creative solutions and technical support, and taught me the joy of home brewing.

My time at Penn State’s Fruit Research and Extension Center (FREC) will always be special to me. I am indebted to the faculty, staff, and students at FREC. I am grateful for the support provided by the PA tree fruit industry and the State Horticultural Association of

Pennsylvania. A sincere thanks to our grower cooperator: Bruce Hollabaugh and Hollabaugh

Bros., Inc. Appreciation is extended to Keith Yoder, Greg Peck, and Leon Combs for allowing me to use the pollen tube growth model in this work. Leon demonstrated the microscopic and hand-pollination procedures utilized in this project.

A special thanks to my incredible wife, Renée, for her unwavering support and patience throughout my graduate education. She even helped me edit my dissertation during our long drives across the great state of Pennsylvania. I am thankful for the love and support of my family and friends throughout this endeavor.

Chapter 1

Crop load management strategies for apple

Abstract

There have been several reviews of apple crop load management practices (Batjer,

1965; Byers, 2003; Dennis, 2000; Forshey, 1986; Wertheim, 2000; Williams, 1979;

Williams 1994; Williams and Edgerton, 1981); however, few have focused on crop load management strategies prior to petal fall (Moran and Southwick, 2000). A significant research effort to screen and develop blossom thinning products has occurred since the loss of Elgetol in 1989. Since the most recent review on crop load management of apple, there are no new registered post bloom thinners in the US, however, some new thinning products and decision-making aids show promise. The bulk of this manuscript will review and catalogue blossom thinners of apple. Recent advances with post bloom thinners and decision-making aids will be discussed.

Introduction

In commercial apple production, the number of blossoms and/or fruit is manipulated annually. Crop load management (thinning), can influence yield, bearing habit, fruit storage potential, and profitability. Thinning is one of the most important annual management decisions in apple production, since orchard productivity and crop value is influenced for multiple production cycles.

Chemical thinning is the primary method of crop load management in modern fruit production and has reduced labor costs and stabilized production cycles (Williams

and Edgerton, 1981). However, two factors make continued thinning research relevant: 1) inconsistent results from year-to-year, and 2) regulations on chemistries that can be legally applied. While chemical thinning is routinely practiced by apple growers worldwide and will continue for the foreseeable future, there has been some adoption of labor intensive practices to ensure consistent annual production and adhere to regulations of specific markets, such as spur pruning and hand-thinning at bloom. Developing sustainable, effective and environmentally-friendly thinning products remain a research priority (Dennis, 2000).

Advantages of reducing crop load early

Early crop load reduction has the greatest potential to increase fruit size (Lakso et al., 1996). Delayed thinning results in carbohydrate allocation to fruit that will not be present at harvest, often resulting in smaller fruit size (McArtney et al., 1996; Quinlan and Preston, 1968). To illustrate this waste of resources, Bobb and Blake (1938) counted and weighed fruit that were hand-thinned from a mature solitary tree. A total of 4,575 fruit were removed; weighing 159 kg. The majority of apple fruit cell division occurs within the first four to six weeks after bloom (Denne, 1960; Goffinet et al., 1995, Lakso et al., 1996; Quinlan and Preston, 1968; Westwood et al., 1967). The expression of genes associated with cell proliferation was regulated by carbohydrate availability during early fruit growth (Dash et al., 2013; Malladi and Johnson, 2011). After reducing the number of fruit to one lateral fruit per cluster on ‘Golden Delicious’ 11 days after full bloom

(DAFB ; 5 mm fruit diameter), a rapid increase in fruit growth was observed and was associated with an increase in sorbitol and fructose concentrations in the fruit. Genes associated with fruit growth and cell production had up to a 5-fold increase in expression

in fruit on thinned trees (Dash et al., 2013). Cell number is a key factor in determining fruit size (Goffinet et al., 1995), although early thinning treatments resulted in a minor increase in cell expansion and related gene activity (Dash et al., 2013; Westwood et al.,

1967). Dash et al. (2013) suggested that early thinning effects on cell expansion may be cultivar dependent.

Additional effects of bloom thinning treatments on return bloom can be difficult to separate from responses due to reduced crop load since some thinning compounds promote flowering (Tromp, 2000). Bloom thinning promoted return bloom in the following season (Batjer, 1965; Bobb and Blake, 1938); however, this relationship has been inconsistent. Bloom thinning treatments reduced spur quality in the following season (McArtney et al., 1996). Tromp (2000) suggested that early thinning stimulated shoot growth and reduced return bloom. Long-term chemical blossom thinning trials in the Pacific Northwest did not consistently increase return bloom. In numerous trials over a 10-year period, a significant increase in return bloom was observed in 7% to 24% of trials, pending upon chemistry (Schmidt et al., 2011).

There is some evidence that flower bud cold hardiness can be increased by bloom thinning. Edgerton (1948) observed that chemically thinned peach trees had twice as many viable peach flower buds after a winter injury event of 3 ˚C. Blossom thinned peach trees had greater cold tolerance in the following year compared to trees thinned at

38 d after bloom (Byers and Marini, 1994). Fruit set was 400% greater on blossom thinned trees when compared with a control. While counterintuitive, bloom thinning may be of benefit to peach producing regions that are prone to spring freezes (Moran and

Southwick, 2000). We are aware of no such evaluation on apple. However, Roberts

(1952) observed that spur pruned ‘Golden Delicious’ had minimal winter injury while unpruned trees were winter killed.

Disadvantages of reducing crop load early

Apple growers in the majority of the United States do not have consistent, registered blossom thinning options. Though existing blossom thinners may reduce fruit set, there are negative consequences associated with chemical and mechanical blossom thinners, such as erratic responses from year-to-year (Byers, 1997; Webster and Spencer,

1999), a temporary reduction in photosynthesis (McArtney et al, 2006), and chemical or mechanical injury to vegetative structures (Byers, 1997; Kon et al., 2013). Spur leaves are important in promoting fruit growth, and injury to these tissues had negative impacts on fruit size, fruit set, and fruit mineral content (Ferree and Palmer, 1982). Early fruit development is almost entirely supported by spur leaves (Hansen, 1971). Spur leaf area was strongly related (r = 0.65) to cumulative productivity over a 17-year period of nine cultivars (Rom and Ferree, 1983), and yield increased linearly with light interception of primary spur leaves (Wünsche and Lakso, 2000). A key to developing effective blossom thinners for apple is to limit injury to these important vegetative tissues.

With early thinning strategies, growers have limited knowledge of crop potential for the season. Adverse weather conditions during bloom can impact pollinator activity, floral longevity, and floral viability (i.e freeze or frost injury). Additionally, poor drying conditions during bloom can increase leaf phytotoxicity and/or fruit russet following application of blossom thinners. Application windows for blossom thinners are short- lived and difficult to predict, making application to large acreage difficult (Byers, 2003;

Moran and Southwick, 2000).

Pruning to reduce crop load

Annual dormant pruning is an important practice to improve light distribution within the canopy early in the season (Ferree and Schupp, 2003; Forshey et al., 1992).

Pruning, the intentional removal of plant parts for horticultural purposes, can reduce the number of reproductive buds on the tree and subsequent crop potential. Pruning is done for several reasons, and a key, yet indirect motive is to increase fruit size (Ferree and

Schupp, 2003). The practice of pruning has been referred to as an art and a science; however, pruning decisions can drastically alter the physiological state of apple trees

(Forshey and Elfving, 1989). Ferree and Schupp (2003) and Forshey et al. (1992) reviewed apple pruning practices.

Spur pruning was reserved initially for spur-type cultivars and old trees that were

“spur bound” (Type I growth habit, according to Lespinasse, 1977). Spur-type cultivars have fewer shoots and less shoot length per tree when compared to standard trees of the same variety (Curry and Looney, 1986). Removal of ~ 1/4 - 1/3 of spurs was recommended to increase fruit size and improve shoot growth on spur bound trees

(Ferree and Schupp, 2003; Forshey et al., 1992). However, the fruit size response of spur bound apple trees to spur pruning was inconsistent, as some studies suggested an improvement in fruit size (Barritt et al., 1987; Roberts, 1952), while others did not

(Ferree and Forshey, 1988; Marini and Sowers, 1991). Spur pruning tended to reduce yield and crop load (Ferree and Forshey, 1988; Ferree et al., 1990; Marini and Sowers,

1991; Rom, 1992). When comparing several pruning methods on mature spur-bound

‘Delicious’ trees, spur pruned trees (30% of each spur complex removed) had the greatest crop value (Ferree et al., 1990).

A number of factors can influence spur quality, including: cultivar, growth habit, light, canopy position, and wood age (Barritt et al., 1987; Robinson et al., 1983; Rom and

Ferree, 1986; Rom and Barritt, 1990). Several authors observed that fruit size declined as wood age increased (Denne, 1960; Rom and Barritt, 1990). However, Robinson et al.

(1983) determined that fruit size was strongly correlated with light environment, and explained a larger proportion of the variation observed in fruit size than spur age. As stated by Rom (1992), “Pruning to improve light penetration in the tree may improve spur and fruit quality, but spur pruning without improving light interception by spurs has no demonstrated advantage.”

The majority of spur pruning research in the 1980’s and 1990’s was conducted on mature spur-type trees grafted onto vigorous rootstocks. Forshey et al. (1992) suggested that, “Pruning may contribute to annual production, but it is a compliment to, rather than a substitute for, effective fruit thinning.” Little emphasis on utilizing pruning as a principal means of apple crop load management occurred until recently. Given the widespread adoption of pruning and training systems that result in narrow canopies with good light distribution, several researchers revisited pruning as a crop load management strategy.

In an attempt to determine if annual bearing was related to tree growth habit, fruiting and flowering cycles were observed on unpruned branches (Lauri et al., 1995). In annual cultivars with Type IV growth habits, a propensity for apple spurs to die was observed. The natural habit of spur death (spur extinction) corresponded with annual bearing habits, and led researchers to attempt to simulate bud senescence via physical removal of flowering spurs (Lauri and Térouanne, 1999). Lauri et al. (2004) determined

that artificial spur extinction (ASE; spur pruning) was an effective means to increase fruit size and mitigate biennial bearing in the Centrifugal training system (Lauri et al., 2011), which relies primarily on tree training, and minimal limb pruning. Table 1 summarizes the outcomes of recent ASE research.

8 Table 1-1. Summary of recent spur pruning (extinction) studies. Floral bud Increased Increased Increased Training Hand- Laterals Reduced Increased Reduced Author(s) Cultivar(s) densities fruit return shoot system thinned modified crop load fruit size yield evaluated set/spur bloom Growth Breen et al., 3 and 5 buds/cm2 ‘Gala' Tall Spindle Yesz Yes Yes NAy NA NA NA NA 2014 LCA Breen et al., 2, 4, and 6 ‘Royal Gala' Tall Spindle Yes Yes Yes No Yes No NA NA 2015 buds/cm2 LCA Lauri et al., 2 and 4 buds/cm2 ‘Galaxy' Centrifugal Yes Yes* NA NA Yes No No Yes 2004 LCA ~ 40, 60, and 80 Central Nichols et al., ‘Honeycrisp' buds/m-3 canopy Leader and NA No NA Yes Yes NA NA Yesw 2011 volume Vertical Axis Robinson et ‘Gala' and ~ 1/3 of spurs 2 of 3 2 of 3 Tall Spindle No* No No Yes No NA al., 2014 'Honeycrisp' removed studies studies Tustin et al., 2 to 6 buds/cm2 Yes, in off ‘Scifresh' Tall Spindle Yes Yes Yes NA NA NA NA 2012 LCA year van Hooijdonk 1 of 3 2 of 3 1 of 3 2 of 3 ‘Scilate' 5 buds/cm2 LCA Tall Spindle Yes Yes Yes Yes et al., 2014 years years years years z“Yes” indicates a significant (P>.05) difference reported between the control and a mechanical thinning treatment or significant relationship. “No” indicates that response variable was tested, but there was no difference between the control and any of the mechanical thinning treatments listed or no relationship. yNA indicates the response variable was not evaluated. xEffects of bud density modification of one-year-old wood was evaluated in a separate study. wTreatment effects were not consistent across all years and/or indices of shoot growth.

ASE is a selective process, as buds that are small or located on the underside of limbs are removed preferentially. Spurs on pendant wood produced smaller fruit when compared to spurs in vertical or horizontal positions, due to poor light conditions (Tustin et al., 1988). On horizontal limbs, spurs oriented vertically downward had fewer and smaller leaves and small fruit size (Rom, 1992). In recent ASE research, the majority of lateral buds are removed, leaving buds 5 to 10 cm apart (Breen et al., 2015; Tustin et al.,

2012; van Hoodijonk et al., 2014). Terminal buds and spurs on two-year-old wood produced larger fruit when compared to lateral buds of multiple cultivars, and this was related to higher spur leaf area (Volz et al., 1994). Terminal buds and spurs located in light rich environments are selected and retained with ASE.

Tustin et al. (2012) suggested that ASE could be utilized as the principal method of crop load management in the vertical axis system on the biennial cultivar ‘Scifresh’.

As the level of spur bud removal increased, there was an increase in the number of fruit at persisting spurs. The authors suggested that carbohydrate availability increased with increasing spur removal (Tustin et al., 2012). While increasing of the number of fruit that set at a given spur may be undesirable, there is a greater probability that spurs will carry a fruit with ASE. Reducing the number of fruiting sites to a specified number may lead to more predictable yield outcomes and simplified targets for supplemental chemical and/or hand-thinning treatments (Breen et al., 2015; Tustin et al., 2012; van Hoodijonk et al.,

2014).

Return bloom of tree fruit crops is often expressed as the number of blossom clusters per unit limb cross-sectional area (Lombard et al., 1988). Since a significant proportion of spurs is removed (>30%) with ASE treatments, return bloom is generally

not increased from year-to-year (Table 1). However, multi-year studies indicate that fluctuation in yield observed with biennial cultivars was moderated with ASE (Nichols et al., 2011; Tustin et al., 2012; van Hoodijonk et al., 2014). Since apple flowers are produced in a mixed bud, ASE also reduces potential spur leaf area early in the season.

Breen et al. (2015) compared a range of ASE and blossom cluster removal treatments on

‘Gala’. While blossom cluster-thinned trees had presumably greater spur leaf area when compared to ASE, fruit weight was not different between the two treatments.

Similar ASE studies were conducted on several cultivars, different rootstocks and climatic conditions (Breen et al., 2014). ASE effects on fruit quality were variable from year-to-year (Breen et al., 2015; van Hoodijonk et al., 2014). Fruit dry matter content, a fruit quality metric that is positively related to consumer preference (Palmer et al., 2010), was improved in 2 of 3 years of an ASE trial on ‘Scilate’ (van Hoodijonk et al., 2014), but was not improved in ‘Gala’ (Breen et al., 2015). In conjunction with hand-thinning,

ASE was proposed as a primary method of crop load management in New Zealand.

Robinson et al. (2014b) compared ASE to heading cuts on ‘Gala’ and

‘Honeycrisp’ trees that had already been subjected to renewal pruning strategies for Tall

Spindle (Robinson et al., 2006). Heading cuts removed one-year-old wood in an effort to remove lateral buds, rather than focus on the removal of spurs. ASE and heading cuts improved fruit size when compared to trees that only received renewal pruning treatments. Few differences were apparent between spur pruning and heading cuts. The proportion of spurs removed was lower in this trial (~33% spurs removed) when compared to those previously cited (>50%). While promising, ASE is a labor intensive

and expensive procedure. Efforts to mechanize ASE are underway and initial results suggest some promise (Kon et al., 2015).

Blossom thinners

There are two strategies to reduce apple crop load at bloom: 1) use of chemical blossom thinners, and 2) use of mechanical blossom thinners. Chemical blossom thinners range widely in their mode of action, including: prevention of anthesis, inhibition of pollen germination, films/coatings to create a barrier on the stigma, inhibition of pollen tube growth, desiccation/mortality of stylar tissue, and/or reduced photosynthesis (Miller and Tworkoski, 2010; Rom and McFerson, 2004). Conversely, mechanical blossom thinners appear to have a single mode of action: the physical removal of reproductive structures (Kon et al., 2013), though some suggest that additional thinning responses are derived from wound induced ethylene production (Dorogoni et al., 2008; Kong et al.,

2009).

Chemical blossom thinners

In the 1930’s and 1940’s, a surge of research occurred to find chemical products to eliminate or reduce apple fruit set (for a historical review, see Dennis, 2000). Elgetol

(dinitro-ortho-cresol; DNOC) was identified from this research, and became the primary blossom thinning product from 1949 to 1989 in the West. Elgetol had multiple modes of action and was used as a fungicide to control apple scab. Elgetol had pollenicidal effects in vitro (MacDaniels and Hilldebrand; 1939), and in vivo (Embree and Foster, 1999).

Elgetol applied 50 h after hand pollination eliminated fruit set and desiccated stigmatic papillae and stylar tissue (Watson, 1952). Leaf phytotoxicity and pedicel injury were commonly observed with Elgetol applications. When used as a broadleaf herbicide,

Elgetol inhibited electron transport to Photosystem II (van Rensen and Hobe, 1979). In the 1940’s, several trials were conducted in different geographical areas to determine appropriate rates, formulations, timings, and to evaluate its potential use for thinning stone fruit. Elgetol was notoriously susceptible to rewetting, which caused over-thinning and phytotoxicity (Williams, 1994). The frequently wet and cool springs prevalent in the

East were not favorable for Elgetol, and commercial use of the product was limited to western states and other semi-arid regions. The high expense of re-registration resulted in

Elgetol being removed from the market in 1989. Elgetol contained heavy metals (Dennis,

2000) and was toxic to honey bees (Goble and Patton, 1946). For a more detailed review of Elgetol as an apple blossom thinner, review Batjer (1965) and/or Williams and

Edgerton (1981).

Finding an alternative to Elgetol: caustic products and photosynthetic

inhibitors

After the registration of Elgetol was cancelled, a second wave of blossom thinning research occurred. This work was partially expedited due to previous evaluation of caustic blossom thinning products for peach, conducted in the 1980’s (Byers and

Lyons 1982, 1983, 1984, 1985). The majority of blossom thinning compounds utilized in commercial apple production were evaluated in this series of trials. In the early 1990’s, two products were registered for use as blossom thinners on apple: sulfcarbamide

[monocarbamide dihydrogen sulfate; Wilthin; D-88] and pelargonic acid [Thinex].

Sulfcarbamide, which is comprised of sulfuric acid and urea, was the first blossom thinning product to be registered after the withdrawal of Elgetol (Williams,

1994). Utilized as a foliage desiccant on vegetable and row crops, the product was

assumed to have caustic properties comparable to Elgetol. Early reports suggested that sulfcarbamide was equally effective in reducing fruit set and improving packout when applied at 70%-90% bloom (Williams, 1993). However, applications at 100% bloom, rates greater than 0.375% (v/v), and sprays within 2 weeks of a copper spray resulted in fruit marking and injury to spur leaf tissue (Williams, 1993). In a series of experiments,

Byers (1997) used high rates (5 to 15 ml L-1) and generally observed excessive russeting and/or leaf injury. Addition of a surfactant improved efficacy, reduced the incidence of fruit marking, and facilitated use of lower rates (0.25% v/v; Williams, 1994). Night applications with prolonged drying time increased fruit and leaf injury (Williams, 1994).

In the east, sulfcarbamide reduced crop load linearly as concentration increased, however, severe russeting occurred with the highest concentration (3.75 ml L-1) on ‘Delicious’ which reduced the grade of the fruit (Greene, 2004). In the UK, sulfcarbamide was only effective when higher than recommended rates (4000 mg L-1) were applied (Webster and

Spencer, 1999). Registration for sulfcarbamide was discontinued in 2006.

Pelargonic acid (Thinex) was registered as a blossom thinning agent in 1996.

Pelargonic acid was an active ingredient of commercially available, broad spectrum organic herbicides. Handling of pelargonic acid was cumbersome, as the solution was thick and difficult to tank mix (Fallahi and Greene, 2010). While early studies suggested some promise (Williams, 1994), excessive phytotoxicity, fruit marking, and a lack of efficacy (Byers, 1997; Greene, 2004; Webster and Spencer, 1999), led to cancellation of pelargonic acid as a registered blossom thinner in 2003.

The fertilizer ammonium thiosulfate (ATS) was shown to desiccate floral tissues of peach when applied during bloom (Byers and Lyons, 1985). Pollen tube growth was

inhibited in vitro and in vivo following ATS application (Embree and Foster, 1999; Myra et al., 2006). Schroder [2001, in Schroder and Bangerth (2006)] suggested the mode of action of ATS is a combination of damaged floral tissue and reduced photosynthesis.

When a 3.7 g∙l-1 solution of ATS was painted directly onto stamens and styles of field pollinated ‘Braeburn’ at full bloom, initial fruit set was reduced by 68%, while application to petals or leaves resulted in a minor, yet significant reduction in initial fruit set (Irving et al., 1989). The influence of drying time and ATS efficacy was evaluated, but the outcome remains unclear, since observations only included a visual evaluation of pistil injury (Janoudi and Flore, 2005). Apple blossoms that are open are susceptible to damage from ATS treatment, while blossoms at balloon stage did not exhibit damage even if petals were removed prior to ATS treatment (Janoudi and Flore, 2005). The authors speculated that unopened blossoms have higher levels of antioxidants when compared to open blossoms, but the potential of unopened ATS-treated blossoms to support pollen tube growth or set fruit was not evaluated. In a series of trials, ATS applications caused the greatest leaf injury, but the least fruit injury in a comparison of several blossom thinners (Byers, 1997). ATS is utilized as a commercial blossom thinner of apple in Europe, Australia, and New Zealand. US EPA regulations require that blossom thinning agents be registered as pesticides. Fertilizer applications as a blossom thinner are not permitted; however, some US apple growers apply ATS as a foliar fertilizer during bloom. Initial reports of another fertilizer, potassium thiosulfate

[dipotassium sulfurothioate] are promising (Bound and Wilson, 2004; Milic et al., 2011).

Of all blossom thinners evaluated since the loss of Elgetol, perhaps the most consistent blossom thinning programs include the fungicide lime sulfur (calcium

polysulfide; LS) (Schmidt et al., 2011). Coincidentally, LS was one of the first chemical constituents with recognized activity to inhibit fruit set of apple (Bagenal et al.,1925), but has only recently been utilized for the purpose of crop load management. LS increased the number of blossoms with <10 pollen tubes and effectively reduced fruit set of open pollinated ‘Braeburn’ (McArtney et al., 2006). Oil is often used to increase the efficacy and consistency of LS thinning programs (Schupp et al., 2005). A 2% LS + 2% fish oil

(FO) application prevented pollen tubes from reaching the base of the style when applied within 24 h of the pollination event, but later applications (48 h) did not influence the number of pollen tubes that reached the base of the style (Yoder et al., 2009). When compared to caustic products that merely damage the pistil, LS has an advantage since the window of application is larger and the potential number of applications is reduced

(Schmidt and Elfving, 2007). There was an inverse relationship between LS concentration and pollen tubes per flower (McArtney et al., 2006). FO did not influence the number of blossoms with limited pollen tube number, but reduced fruit set and net photosynthesis (Pn) by 10% (McArtney et al., 2006). Petroleum based oils are effective replacements for FO in LS blossom thinning programs, but surfactants were an inadequate alternative (Schupp et al., 2006).

Reduced Pn as a result of a LS program was confirmed in partial leaf (Noordijk and Schupp, 2003; McArtney et al., 2006), leaf (Hoffman, 1935), and whole tree experiments (Lombardini et al., 2003 Whiting, 2007). In the Pacific Northwest, this stress was reported to last between 4-10 d (Schmidt and Elfving, 2007), but spur leaf Pn was reduced for more than 57 d in New Zealand (McArtney et al., 2006). Spur leaf area, shoot growth, and trunk growth was not influenced by a whole season LS fungicide program,

but Pn was depressed (Palmer et al., 2003). LS fungicide programs reduced crop load, but fruit size at harvest did not increase. A lag in fruit growth was observed when using LS as a blossom thinner (Schmidt and Elfving, 2007), and is likely attributed to reduced Pn.

Lag phase occurrence is likely related to environmental conditions, and was not observed in fruit growth rates of two cultivars in British Columbia (Hampson and Bedford, 2011).

Leaf anatomy was altered in one year of a two-year study in New York, as LS + FO treated spur leaves had smaller epidermal and palisade cells, and thinner spongy mesophyll cells which contributed to lower photosynthetic capacity (Schupp et al., 2006).

Lack of a subsequent increase in fruit size may be partially attributed to the reduced photosynthetic capacity of spur leaves with LS thinning and fungicide programs. While

LS is a registered organic fungicide that is available to apple growers, legal use of LS for blossom thinning is restricted to certain geographical areas. In the US, use of LS in thinning programs is permitted in Washington, though the user must sign a waiver. Use of LS as a blossom thinner in all other states is prohibited. Antibacterial properties of LS

+ FO blossom thinning programs were partially effective in controlling fire blight without additional inputs of an antibiotic (Johnson and Temple, 2013). Sulfur based products, such as LS and ATS are lethal to mites and had negative effects on beneficial mite populations (Beers et al., 2009).

An aquatic herbicide, endothal [7,oxybicylo(2,2,2)heptane-2-3 dicarboxylic acid;

ThinRite; endothallic acid; endothall], has been evaluated as an apple blossom thinner since 1993, and was registered for use in Washington in 2014. Endothal was assumed to be a desiccant, but was found to kill pollen tubes that had partially traversed the style and also showed pollenicidal properties (Embree and Foster, 1999). Initial testing in

Washington, New Zealand, and Australia suggested that endothal was a promising blossom thinner, but phytotoxicity was especially severe on ‘Golden Delicious’

(Williams et al., 1995). Ferree and Schmid (2000) applied a range of concentrations in

Ohio and observed reduced fruit set in two years of a three-year evaluation with no increase in leaf or fruit injury. Crop load declined linearly with increasing concentrations of endothal, and 1.25 to 1.5 ml L-1 were identified as optimal rates for ‘Delicious’ (Bound and Jones, 1997). Differences in water volume applied did not influence efficacy of endothal treatments (Byers, 1997). Two applications of endothal during bloom reduced fruit set and improved fruit size when compared to a single application (Greene, 2004;

Williams, 1995), though this relationship was not consistent in a series of experiments in

Virginia (Byers, 1997).

Alternatives to Elgetol: use of plant growth regulators

NAA (1-naphthaleneacetic acid) was initially evaluated during bloom as a chemical thinner of apple (Burkholder and McCown, 1941), but NAA is primarily used post bloom at 7 to 16 mm fruit diameter. However, NAA thinned ‘Cox’s Orange Pippin’ at bloom, pink, full bloom, and petal fall bud stages (Irving et. al, 1989). NAA applications can reduce fruit growth rates (Lakso et al., 1996), which may be of value with large fruited cultivars. Use of NAA on summer ripening cultivars caused undesirable side effects, including foliar injury, fruit growth inhibition, and premature fruit ripening (Byers, 2003). NAD (naphthaleneacetamide) was developed as a milder auxin-based thinner for use on summer ripening cultivars. There is renewed interest in use of NAD as a bloom thinner in the eastern US (Greene et al., 2015). Greene et al.

(2015) observed thinning activity at bloom and petal fall, with no observed dose

response. Additional evaluation of NAD as a bloom thinner on modern cultivars, such as

‘Ginger Gold’, ‘Honeycrisp’, and ‘Pink Lady’ is warranted, but effects on clustered fruit, fruit growth rate, development of pygmy fruit, and return bloom should be considered.

Six synthetic auxins were evaluated as bloom thinners in Japan and MCPB-ethyl

[ethyl 4-(4-chloro-2-methylphenoxy) butanoate] had promising results (Yokota et al.,

1995). NAA and MCPB-ethyl were both effective blossom thinners, and tended to thin clusters rather than blossoms or fruit within a cluster. Epinasty was observed at high concentrations (Guak et al., 2002). Despite reducing crop load in a dose dependent fashion, bloom-applied auxins depressed return bloom. The authors suggested that bloom applied auxins had negative effects on flower induction. MCPB-ethyl was an inconsistent blossom thinner in the two years of the study, and 1.0% ATS was considered a more beneficial treatment (Guak et al., 2002).

Ethephon [(2-chloroethyl) phosphonic acid] reduced crop load inconsistently

(Wertheim, 2000). Ethephon applied at bloom reduced crop load (Jones et al., 1993;

Meland and Kaiser, 2011), but cool temperatures may reduce efficacy since temperatures greater than 12 ˚C are required to break down ethephon to ethylene (Jones and Koen,

1985). Ethephon is a compound with localized activity, so sufficient coverage is essential for consistent results. Brushing a 0.35 g/L ethephon + 0.1% Regulaid solution on flower petals at pink bud stage or on spur leaves at full bloom reduced initial fruit set by 77%

(Irving et. al, 1989).

Alternatives to Elgetol: screening efforts

At least 150 different compounds have been evaluated as bloom thinners since

1989 (Table 2). With the exception of the aforementioned thinning products, none of

these products are registered for use as bloom thinners in the US. Screened products were segregated into one of the following categories: adjuvants, essential oils and extracts, fertilizers and salts, films and coatings, fungicides, herbicides and acids, and plant growth regulators.

Rom et al. (2004) identified characteristics of solutions that inhibited pollen germination in vitro and caused visible oxidation of styles. Complete inhibition of pollen tube growth in vitro was observed at pH <3 and >11 (Munzuroglu et al., 2003; Rom et al., 2004). As solution osmotic tension and electrical conductivity increased, pollen germination was reduced with complete inhibition occurring at -4.0 M Pa and 200 mV, respectively (Rom et al., 2004; Rom and McFerson, 2006).

Methods used to identify potential blossom thinning products are not standardized. Screening efforts evaluated product effects on pollen tube growth in vitro, pollen tube growth in vivo, stylar browning, and/or fruit set (Embree and Foster, 1999;

Myra et al., 2006; Rom et al., 2004; Rom and McFerson, 2006). Injury to vegetative tissues was quantified using a variety of methods, including visual evaluations, electrolyte leakage, or measuring leaf gas exchange (Embree and Foster, 1999; Myra et al., 2006; Rom et al., 2004; Rom and McFerson, 2006).

Table 1-2. List of compounds screened and/or evaluated for blossom thinning apple. Common and/or Chemical formula or description Author(s) chemical name Adjuvants Alkoxylated fatty alkylamine Armothin Basak, 2006b polymer Basic-H linear alcohol alkoxylates (28%) Embree and Foster, 1999 biodiesel derived from fish oil Myra et al., 2006 alkylarylpolyethoxy ethanol + Biofilm fatty acids + phosphatic Embree and Foster, 1999 acids+isopropanol Bound, 2006; Rom and McFerson; Canola oil unspecified formulation Stopar 2008 emulsifiable vegetable oil and Codacide Bound, 2006 and 2010 polyethyolated esters Dehydol TA 29 alkylpolyethylene glycol ether Kopcke et al., 2002 Dehydol TA 5 alkylpolyethylene glycol ether Kopcke et al., 2002 dormant/mineral oil unspecified formulation Alegre and Alins, 2007 fish emulsion unspecified formulation Bound, 2010 Byers and Wolf, 2003; Bound, 2006 fish oil unspecified formulation and 2010; McArtney et al., 2006; Bound, 2006 and 2010 ; Schmidt et mineral oil mineral oil al., 2011 Basak, 2006; Pfeiffer and Reuss, Olejan 85 EC; Telmion 85% rape/canola oil 2002 Alegre and Alins, 2007; Basak, olive oil unspecified formulation 2006; Belleggia et al., 2010; Pfeiffer and Reuss, 2002 PCC711 unspecified formulation Byers, 1997 PCC713 unspecified formulation Byers, 1997 PCC715 unspecified formulation Byers, 1997 polyalkyleneoxide modified Silwet 408 Bound and Klein, 2010 heptamethyltrisiloxane polyalkyleneoxide modified Silwett (L-77) Bound, 2006 and 2010 heptamethyltrisiloxane Rom and McFerson, 2006; Stopar, soya oil 96% soya oil + 4% emulsifiers 2008 Pfeiffer and Reuss, 2002; Stopar, sunflower oil unspecified formulation 2008 Tergitol; TMN-6; dodecyl ether of polyethylene Fallahi and Greene, 2010 Dupont WK glycol

Common and/or Chemical formula or description Author(s) chemical name Adjuvants (cont.) Triton B-1956; Latron Modified phyhalic glycerol Fallahi et al., 1992 B-1956 alkyd resin alkylphenyl polyethylene glycol Triton X-100 Kopcke et al., 2002 ether alkylphenyl polyethylene glycol Triton X-114 Kopcke et al., 2002 ether

Tween 20 C58H114O26 Bound, 2006 and 2010 McFerson, 2005; Lombardini et al., vegetable oil emulsion canola, corn, or soybean 2003; Schmidt et al., 2011 YI-1066 2-hydroxyethyl-n-octyl-sulfide Byers, 1997 Essential Oils and Extracts Basak, 2006; Rom and McFerson, Biochicol 020 PC chitosan products 2006 Bioczos and garlic unspecified formulation Basak, 2006 soap Pfeiffer and Reuss, 2002; Rom and bioneem; Rimulgan neem oil product McFerson, 2004 black pepper oil unspecified formulation Rom and McFerson, 2006 camphor oil unspecified formulation Rom and McFerson, 2006 (2R,3S)-2-(3,4- catechin dihydroxyphenyl)-3,4-dihydro- Rom and McFerson, 2006 2H-chromene-3,5,7-trio cedarwood oil unspecified formulation Rom and McFerson, 2006 cinnamon oil unspecified formulation Rom and McFerson, 2006 clove oil unspecified formulation Rom and McFerson, 2006 coumarin 2H-chromen-2-one Rom and McFerson, 2006 cypress oil unspecified formulation Rom and McFerson, 2006 eucalyptus oil unspecified formulation Rom and McFerson, 2006 a.i. 2-methoxy-4-(2- eugenol Miller and Tworkoski, 2010 propenyl)phenol fir needle oil unspecified formulation Rom and McFerson, 2006 ginger oil unspecified formulation Rom and McFerson, 2006 Goemar algae substrate Weibel et al., 2012 grapefuit oil unspecified formulation Rom and McFerson, 2006 lavender oil unspecified formulation Rom and McFerson, 2006 lemon oil unspecified formulation Rom and McFerson, 2006 linseed oil unspecified formulation Rom and McFerson, 2006 Miller and Tworkoski, 2010; Rom Matran EC clove oil and McFerson, 2006

Common and/or Chemical formula or description Author(s) chemical name Essential Oils and Extracts (cont.) (1R,2S,5R)-2-Isopropyl-5- menthol Pfeiffer and Reuss, 2002 methylcyclohexano molasses unspecified formulation Byers and Wolf, 2003 pine needle oil unspecified formulation Rom and McFerson, 2006 Proflo oil cotton seed oil extract Byers and Wolf, 2003 seaweed extract unspecified formulation Byers and Wolf, 2003 spruce oil unspecified formulation Rom and McFerson, 2006 tangerine oil unspecified formulation Rom and McFerson, 2006 tea tree oil unspecified formulation Rom and McFerson, 2006 thistle oil unspecified formulation Pfeiffer and Reuss, 2002 thymol 2-isopropyl-5-methylphenol Rom and McFerson, 2006 vinasse sugar beet byproduct Weibel et al., 2012 walnut leaves - tea unspecified formulation Pfeiffer and Reuss, 2002 witch hazel unspecified formulation Byers and Wolf, 2003 ylang ylang III oil unspecified formulation Rom and McFerson, 2006

Fertilizers and salts

ammonium sulfate (NH4)2(SO4) Rom and McFerson, 2006 ammonium H N O S Numerous, see text thiosulfate (ATS) 8 2 3 2 Azolon methylene urea molecules Handschack and Alexander, 2002 Looney et al., 2002; Rom and calcium chloride CaCl 2 McFerson, 2004 and 2006

calcium nitrate Ca(NO3)2 Handschack and Alexander, 2002 cor-clear 34.5% Ca Looney et al., 2002

ferric sulfate Fe2(SO4)3 Rom and McFerson, 2006 Kasil-6 (potassium K OSiO H O Embree and Foster, 1999 silicate) 2 2 2 mono-ammonium NH H PO Embree and Foster, 1999 phosphate (MAP) 4 2 4 magnesium/calcium chloride Byers and Wolf, 2003; Looney et NC99 brine al., 2002; Schmidt et al., 2011 potassium bisulfate K2SO4 Rom and McFerson, 2006 potassium iodide KI Rom and McFerson, 2006 potassium meta- K S O Rom and McFerson, 2004 and 2006 bisulfate 2 2 5 potassium sulfate K2SO4 Rom and McFerson, 2004 and 2006

Common and/or Chemical formula or description Author(s) chemical name

Fertilizers and salts (cont.) potassium thiosulfate Bound and Wilson, 2004; Milic et K O S (KTS) 2 3 2 al., 2011 Alegre and Alins, 2007; Byers and Wolf, 2003; Embree and Foster, sodium chloride NaCl 1999; Myra et al., 2006; Rom and McFerson, 2006; Stopar, 2008 sodium hydroxide NaOH Rom and McFerson, 2004 and 2006 sodium meta- Na2S2O5 Rom and McFerson, 2006 bisulfate sodium nitrate NaNO3 Auchter and Roberts, 1933 Stopit 12% Ca + surfactant Looney et al., 2002 Embree and Foster, 1999; urea CO(NH ) 2 2 Handschack and Alexander, 2002

Films and coatings Anti-Stress acrylic polymers Embree and Foster, 1999 Pine oil (Nuflim) + dust of active black oil Weibel et al., 2012 carbon BrilliantSchwarz black food colorant Hegele et al., 2010 (E151) dextrin unspecified formulation Stopar, 2008 Masbrane (Gao-Zhi- dodecyl alchol Embree and Foster, 1999 Mo) milk unspecified formulation Pfeiffer and Reuss, 2002 natural lecithin unspecified formulation Rom and McFerson, 2006 polymeric film Nufilm unspecified formulation Pfeiffer and Reuss, 2002 Nurti-Save N,O-carboxymenthylchitosan Embree and Foster, 1999 Olejan 85 EC di-1-P-menthene Basak, 2006

PEG-1000 HO(-CH2CH2O-)NH Embree and Foster, 1999 potato starch unspecified formulation Pfeiffer and Reuss, 2002 Safer-Soap potassium salts of fatty acids Embree and Foster, 1999 Myra et al., 2006; Alegre and Alins, Surround kaolin clay 2007

wheaten flour unspecified formulation Pfeiffer and Reuss, 2002

Common and/or Chemical formula or description Author(s) chemical name

Fungicides Agri-mycin 17 streptomycin Yi et al., 2003 Hegele et al., 2010; Weibel et al., Armicarb potassium-bicarbonate; KHCO 3 2012 Methyl [1- Church and Williams, 1977; Fell et benomyl [(butylamino)carbonyl]-1H- al., 1983 benzimidazol-2-yl]carbamate (RS)-(2-Butan-2-yl-4,6- binapacryl dinitrophenyl) 3-methylbut-2- Church and Williams, 1977 enoate 5-Butyl-2-ethylamino-6- bupirimate methylpyrimidin-4- Church and Williams, 1977 yldimethylsulphamate Church and Williams, 1977; Eaton, N-Trichlorormethylthio-4- captan 1963; Fell et al., 1983; Yi et al., cyclohexene-1,2-dicarboximide 2003 Auchter and Roberts, 1933; Rom copper sulfate CuSO4 and McFerson, 2006 Dikar 76 mancozeb + dinocap Fell et al., 1983 (2,6-Dinitro-4- octylphenyl)crotonat und (2,4- dinocap Church and Williams, 1977 Dinitro-6-octylphenyl)crotonat, Isomerengemisch 2,3-Dicyano-9,10-dioxo-1,4- dithianon Church and Williams, 1977 dithiaanthracen

Ecocarb potassium-bicarbonate; KHCO3 Bound, 2010 ethylene diamine tetraacetic EDTA Kopcke et al., 2002 acid Elgetol dinitro-ortho-cresol; DNOC Numerous, see text Church and Williams, 1977; Fell et Funginex Triforine al., 1983

Kaligreen potassium-bicarbonate; KHCO3 Byers and Wolf, 2003 (E)-2-methoxyimino-2-[2- Kresoxim-methyl (otolyloxymethyl)phenyl] Kopcke et al., 2002 acetate lime sulfur calcium polysulfide Numerous, see text

Common and/or Chemical formula or description Author(s) chemical name

Fungicides(cont.)

manganese mancozeb ethylenebis(dithiocarbamate) Church and Williams, 1977 (polymeric) Nova 40W myclobutanil Yi et al., 2003 Polyram 80W metiram Fell et al., 1983 Reynoutria sachalinensis (F. Regalia Delong, 2016; Peck et al., 2016 Schmidt) extract

Remedy potassium-bicarbonate; KHCO3 Byers and Wolf, 2003 sodium bicarbonate NaHCO3 Pfeiffer and Reuss, 2002 Church and Williams, 1977; Kopcke sulfur unspecified formulation et al., 2002 Church and Williams, 1977; Fell et Topsin-M 70W thiophanate-methyl al., 1983

Herbicides and Acids Bound, 2010; Byers and Wolf, 2003; Rom and McFerson, 2006; acetic acid CH COOH 3 Myra et al., 2006; Stopar, 2008; Weibel et al., 2012 cinnamic acid (E)-3-phenylprop-2-enoic acid Rom and McFerson, 2006 2-hydroxypropane-1,2,3- citric acid Rom and McFerson, 2006 tricarboxylic acid 7,oxybicylo(2,2,2)heptane-2-3 endothall Numerous, see text dicarboxylic acid glutamic acid 2-Aminopentanedioic acid Rom and McFerson, 2006 hydrogen peroxide H2O2 Byers, 2003 Pfeiffer and Reuss, 2002; Rom and oxalic acid ethanedioic acid McFerson, 2006 salicylic acid 2-Hydroxybenzoic acid Rom and McFerson, 2006 sodium hypochlorite NaOCl Rom and McFerson, 2006 Thinex (MYX4801) pelargonic acid Byers, 1997; Fallahi, 1997 sulfacarbimide (D-88); 1- Byers, 1997; Fallahi, 1997; Webster Wilthin aminomethanamide and Spencer, 1999; Williams, 1992 dihydrogen tetraoxosulfate

Common and/or Chemical formula or description Author(s) chemical name

Plant Growth Regulators (+)-8'-acetylene ABA analouge McArtney et al., 2014 abscisic acid 4-CPA 4-chlorophenoxy acetic acid Yokota et al., 1995 N-(2-chloro-4-pyridyl)-N´- CPPU Greene, 1995 phenylurea (2, 4-dichlorophenoxy) dichlorprop propionic acid triethanolamine Yokota et al., 1995 salt Byers, 1997; Fallahi et al., 1992; Dormex hydrogen cyanimide Fallahi, 1997; Fallahi et al., 1998; Kacal and Koyncu, 2012 Irving et al., 1989; Jones et al., ethephon (2-chloroethyl) phosphonic acid 1993; Koen et al., 1988 ethyl 5-chloro-1H-3-imidazol-3- ethychlozate Yokota et al., 1995 yl acetate ethyl 4-(4-chloro-2- Looney et al., 1997; Guak et al., MCPB-ethyl methylphenoxy)butanoate 2002; Yokota et al., 1995 Methyl (1R,2R)-3-Oxo-2-(2Z)-2- methyl jasmonate Rom and McFerson, 2006 pentenyl-cyclopentaneacetate Irving et al., 1989; Koen et al., NAA 1-naphthaleneacetic acid 1988; Looney et al., 1997; Guak et al., 2002; Yokota et al., 1995 NAD naphthaleneacetamide Greene et al., 2015 N-(1,3-dimethyl- 1H-pyrazol-5- NSK-905 yl)-2-[(3, 5, 6-trichloro-2- Yokota et al., 1995 pyridinyl oxy] acetamide N-(phenylmethyl)-1H-purine-6- promalin Greene, 1995 amine plus gibberellins A4+7 (2Z,4E)-5-[(1S)-1-hydroxy-2,6,6- trimethyl-4-oxocyclohex-2-en- s-abscisic acid (ABA) McArtney et al., 2014 1-yl]-3-methylpenta-2,4-dienoic acid 1-phenyl-3-(1,2,3-thidiazol-5-yl) thidiazuron Greene, 1995 urea

Timing of chemical blossom thinning applications

While bloom thinner sensitivity to temperature is not nearly as important as with post bloom thinning, one inherent challenge with bloom thinning is the short period of time that growers have to apply treatments over large acreage (Moran and Southwick,

2000). Additionally, the number of blossoms that are open can vary widely within a block or tree (Byers, 2003). One typical protocol is to apply blossom thinning sprays after the grower estimates that a sufficient number of king blossoms were fertilized. King blossoms are thought to have a competitive advantage when compared to side bloom, due to a superior vascular connection (Westwood et al, 1967) and/or correlative inhibition

(Jackopic et al., 2015). While this relationship is true for some cultivars, it has been broadly extrapolated despite evidence that king blossom dominance is not inherent in all cultivars (Ferree et al., 2001). Another established protocol is to time, application of blossom thinners based on a visual estimate of open blossoms. Common timings include:

20%, 60%, 80% and/or 100% bloom, and single or multiple applications can be carried out at various bloom stages. In general, multiple applications of blossom thinners resulted in increased thinning (Byers, 2003). Blossom thinners applied at full bloom or later often increased fruit marking and russeting, which are unacceptable for fresh market fruit

(Byers, 2003).

Models were developed to estimate the rate of pollen tube growth in apple styles.

Child (1966) made one of the first attempts to evaluate apple pollen tube growth rates in vivo in the cider cultivar ‘Michelin’. Detached blossoms were subjected to constant temperatures (5 to 24 ˚C), and pollen tube growth rates were estimated using fluorescence microscopy (Child, 1966). Using similar techniques, Williams (1970) developed an index

that estimated pollen tube growth based on temperature was developed for growers; however, only low temperatures were evaluated (7 to 15 ˚C) using detached blossoms.

Jefferies and Brain (1984) measured pollen tube growth rate in detached flowers at a range of incubation temperatures over 24 d. This effort was initiated to aid research in controlled laboratory conditions. A relatively complex model was produced, and the authors indicated a few shortcomings, such as the overestimation of pollen tube growth at low temperatures, and that pollen tube growth under varying temperature regimes was not evaluated.

The effort to model pollen tube growth rate was revisited in 2003 using attached blossoms. Mature trees on ‘M.27’ were grown in root bags and were placed in growth chambers at a range of night and day temperatures (Yoder et al., 2009), and ‘Snowdrift’ crabapple pollen was applied to emasculated king blossoms. Using this system, pollen tube growth rates of several apple cultivars were determined and modeled (Yoder et al.,

2013). Maternal cultivar, temperature, and style length are inputs in the model. Influence of the paternal pollen source on pollen tube growth rate is being evaluated (DeLong,

2016). While pollen genotype can influence pollen tube growth rates in vivo, these relationships are complex and are dependent on maternal cultivar and temperature

(DeLong, 2016). Pollen source is an unlikely input in pollen tube growth models.

Pollen tube growth models were tested extensively to aid timing of LS + FO applications in Washington. These models were made available to the public, and data suggest that the model is an effective timing aid for blossom thinning programs (Yoder et al., 2013). The model works on the predicted mortality of growing pollen tubes, but the observed photosynthetic inhibition of LLS + FO treatments is not accounted for in the

model. The duration of Pn reduction in Washington is typically shorter than that of eastern fruit producing regions (Schmidt and Elfving, 2007).

Mechanical blossom thinning

In theory, use of non-chemical means of crop load management would overcome concerns that inhibit product development of chemical thinners, such as the risks of phtyotoxicity and, product liability, as well as the high costs of product registration.

Environmental conditions proximal to application date may be less restrictive than when using chemical thinners, and such practices would more readily conform to organic production standards. Blossom thinning with mechanical devices has been evaluated with several thinning machines and tree fruit crops, and the results on apple have been contradictory (Table 3). The first machine was a rotating string thinner (Darwin 300,

Fruit-Tec, Deggenhausertal, Germany) designed by Hermann Gessler, a German grower, to remove apple blossoms in organic orchards. A description of the string thinner and details of its application can be found in Berschinger et al. (1998). A similar device with three rotors was also developed for use on apple (Damerow et al., 2007). Both of these thinning machines are only compatible with tree canopies that are narrow, planar, and lack permanent scaffold branches. Alternatively, hand-held mechanical thinners could be used on any production system, albeit more labor intensive (Martin-Gorriz et al., 2011).

In general, apple mechanical thinning results from European trials have been positive, while results from North American trials suggest some limitations. In the US, adoption of mechanical thinning technology has occurred with peach (Schupp et al.,

2008), but has been limited with apple. A significant barrier for adoption is the possible transfer of Erwinia amylovora, the bacterial pathogen that causes fire blight, with

mechanical thinners (Ngugi and Schupp, 2009). Current mechanical blossom thinners are non-selective, and significant damage to spur leaf tissue can occur (Kon et al., 2013;

Sauertieg et al, 2015). Vision systems and end effectors, used to detect and remove blossoms with selective or semi-selective mechanical thinners, are under development

(Lyons et al., 2015; Wouters et al., 2015).

Table 1-3. A summary of several recent mechanical thinning studies. Machine Reduced Increased Reduced Increased Author Spindle and tractor speeds Typez Fruit Set Fruit Size Yield Return Bloom Damerow et al., 2007 Bonner 220-320 rpm at 2.5 km/h Yesy Yes No No 300 rpm at 9 km/h and 320 rpm Dorigoni et al., 2008 Darwin Yes Yes No No at 8 km/h Dorigoni et al., 2010 Darwin 210 and 230 rpm at 6 km/h Yes Yes Yes Yes Hehnen et al., 2012 Bonner 220 and 360 rpm at 2.5 km/h Yes Yes Yes No 2 of 3 Kon et al., 2013 Darwin 180-300 rpm at 4.8 km/h Yes Yes No studies 300- 420 rpm at 5 km/h; 300-480 Kong et al., 2009 Bonner Yes Yes Yes NAx rpm at 7.5 km/h McClure and Cline, 1 of 2 1 of 2 Darwin 180-240 rpm at 3.2 km/h Yes NA 2015 years years Schupp and Kon, Darwin 210 rpm at 4.8 km/h No No Yes Yes 2014 Schupp et al. 2008 Darwin 245 rpm at 4 km/h Yes Yes Yes NA 360 and 420 rpm and 5.0 and 7.5 Seehuber et al., 2014 Bonner Yes Yes Yes NA km/h Sinatsch et al., 2010 Darwin 200 and 220 rpm at 8 km/h Yes Yes No NA Solomakhin and 300-420 rpm at 5 km/h; 360-480 Bonner Yes Yes Yes NA Blanke, 2010 rpm at 7.5 km/h Stadler et al., 1996 Darwin 4km/hw Yes NA NA NA Strimmer and Darwin 200 rpm at 5, 7, and 8 km/h Yes Yes No Yes Kelderer, 1997 Veal et al., 2011 Bonner 240-360 rpm at 2.5, 5 and 7 km/h NA Yesv Nov Yesv Weibel et al., 2008 Darwin 300 rpm at 10-12 km/h Yes NA NA Yes zSee Schupp et al., 2008 (Darwin) and Damerow et al., 2007 (Bonner) for descriptions of the machines. y“Yes” indicates a significant (P>.05) difference reported between the control and a mechanical thinning treatment or significant relationship. “No” indicates that response variable was tested, but there was no difference between the control and any of the mechanical thinning treatments listed or no relationship. xNA indicates that the study did not investigate the listed response variable. wSpindle speed not presented. vStatistical analysis not presented.

Post bloom thinners: status and new chemistries

In general, three classes of chemistries are used in post bloom chemical thinning programs: 1) plant growth regulators, 2) insecticidal carbamates, and 3) photosynthetic inhibitors. Plant growth regulators make up the majority of the thinning options, and include synthetic auxins, NAA and NAD, a cytokinin [6-benzyladenine (6-BA)], and the ethylene producing compound ethephon. Two carbamate insecticides, carbaryl (1- naphthyl methylcarbamate), and oxamyl (oximino oxamyl) have mild thinning activity and can be used in conjunction with other products or alone when less thinning is

required. There are currently no registered thinning products classified as photosynthetic inhibitors in the US. While each of these compounds can result in a reduction in fruit set, the mode of action of many of these products is poorly understood (Dennis, 2002; Byers,

2003).

The number and diversity of chemical compounds that can legally be applied in a conventional thinning program is expected to be reduced in the European Union (EU).

Carbaryl can harm pollinators and beneficial insects, and has been banned in the EU since 2008. The legal status of NAA, NAAm, 6-BA, and ethephon as thinning compounds varies widely across the EU, and the European directive EU 91/414 seeks to standardize legal thinning compounds (Costa et al., 2013). The future legal status of these compounds is unclear.

Two recently developed post-bloom thinning products, ACC (1- aminocyclopropane carboxylic acid; McArtnety and Obermiller, 2012; Schupp et al.,

2012) and metamitron (Dorogoni and Lezzer, 2007; McArtney et al., 2012; Stern 2014), have shown great potential but are not yet labeled for use in the US. ACC, the precursor to ethylene, was an effective thinner at 20 mm fruit diameter in multiple trials, and also showed some activity at 10 mm. Metamitron is a Photosystem II inhibitor (McArtney et al., 2012), received a label as a thinning product in Serbia, and is anticipated to be registered in other countries.

ABA has been evaluated as a post-bloom thinner, with some promising results, although leaf yellowing and abscission may be detrimental to commercialization (Greene et al., 2011; McArtney et al., 2014). ABA may have utility when used in combination with other thinning products at lower rates (McArtney et al., 2014), but more work is

needed to elucidate product interactions (Greene et al., 2011). Developing effective and sustainable post-bloom thinners remains a research priority for apple producing regions worldwide.

Post bloom thinners: advances in application timing

It is somewhat unusual in agronomic and/or horticultural crops to intentionally impose stress during the production cycle, since this action has potential to reduce yield

(Schmidt, 2006). In the instance of applying a post-bloom thinner, the goal is to create a transient stress, long enough to stimulate a portion of the fruits to abscise, but not so long as to cause over-thinning, or reduce the growth potential of the persisting fruits.

Application timing of post-bloom apple thinners has evolved over time. Fruitlet diameter and days after full bloom have been used as a timing aid (Donoho, 1965;

Marini, 2003), along with the temperature and light conditions on days in close relation to the date of thinning application (Byers, 2003). Temperature and light conditions are important factors to consider during application of any thinning product, since these factors contribute to the production and utilization of carbon. Spring temperatures in many fruit growing regions are unpredictable and erratic, which further compounds thinning decisions. Since both environmental factors and cultural practices play important roles in efficacy of post-bloom chemical thinners, variable results occur from year-to- year (Stover and Greene, 2005).

More recently, the carbon balance during the thinning window has been estimated using predictive models (Lakso et al., 1999, 2001, 2006, 2007, 2015). The Cornell carbon balance model has been utilized as timing aid for post bloom thinners in the eastern US and Midwest, and daily temperature and light data are inputs in the model. While carbon

balance accounts for the effect of the environment on the susceptibility of the tree, environment also affects chemical uptake and activity. In order for a plant growth regulator to be effective, it must be absorbed by the leaf, transported to the site of activity, where it regulates some metabolic activity of the tree or the fruit. Figure 1 illustrates the interactions between the chemical thinner, the environment, and the tree to obtain successful fruit thinning (Schupp and Crassweller, 2016).

Figure 1-1. The chemical thinning triangle. The Cornell carbon balance model is useful to estimate environmental effects on thinning susceptibility, but the other interactions must also be understood for successful post-bloom chemical thinning.

After application of a given post bloom thinner(s) or stress, affected fruitlets will not abscise for approximately 7-14 days, depending upon temperature. A predictive model has been developed to estimate if a fruit will persist or abscise, based on fruit growth rate (Greene et al., 2013). An estimate of the percentage of fruit that will abscise

can be made 7-8 days after thinner application, providing the opportunity for re-thinning if needed.

Integrated crop load management strategies

Another significant change in crop load management is the concept of deploying multiple strategies in an integrated practice. Return bloom promoting programs are applied in the previous season to assure adequate blossom cluster density (McArtney et al, 2007). Some growers sample spur buds during dormancy to determine the number of reproductive buds using light microscopy to influence management decisions. Thinning practices start with pruning shears and ends after June drop, with some reports that apple growers delay hand thinning to achieve optimum fruit size with large fruited cultivars.

Crop load can be manipulated at several timings throughout the growing season. Typical timings include: dormant pruning, bloom, petal fall (5-7 mm fruit diameter), post-bloom

(9-16 mm), rescue (18-25 mm), and green fruit (> 30 mm). This wide window of intervention is a noteworthy departure from chemical thinning programs of old.

The combined use of bud, blossom and fruitlet counts, predictive models, and multiple chemical thinning applications have been structured and promoted as a crop load management program for Eastern and Midwestern apple growers (Robinson et al., 2013,

2014). This program is currently being tested and implemented by some apple growers, and consists of up to 6 periods of intervention (pruning, bloom, petal fall, post-bloom, rescue thinning, and hand thinning) and requires frequent assessments of fruit number and fruit diameter. However, unpredictable environmental conditions and the lack of precision in weather prediction are problematic. Little research has been published to determine the efficacy of these multi-step programs, partly due to the difficulty in

replicating such trials from year-to-year. When using the Cornell carbon balance model, chemistry, concentration, and timing will be partly dictated by environmental conditions and a range of other factors (Stover and Greene 2005). In an assessment of a multi-step thinning program (Stover et al., 2002) observed that the addition of a petal fall thinning application was of benefit in a two-year trial, but the addition of a bloom thinning spray with endothal was not. Long term evaluations of multi-step thinning programs should be considered in future research, with focus on economic and physiological implications.

Conclusions

Since the loss of Elgetol, numerous blossom thinning products have been evaluated in the lab and field. The mode of action of most of these blossom thinners is unclear. The majority of blossom thinning products are destructive by nature, and there is often a risk of crop damage. Finding a registrant willing to accept such risk is difficult.

Due to concerns regarding phytotoxicity, inconsistency, fruit finish, and a lack of proprietary exclusivity adoption of blossom thinners has been limited to only a few apple producing regions or states. Apple blossom thinning in the east has been relatively limited due to the erratic nature of environmental conditions in the spring. Additionally, the general efficacy of post-bloom thinner applications in the east has been a major reason for the limited evaluation of blossom thinners.

Renowned pomologist Art Thompson described several significant changes in pomology through the majority of the 20th century (Thompson, 1979), and briefly summarized the changes in chemical thinning in stone fruit. “Extensive investigations over the past 38 years with at least 9 compounds have been conducted for chemical thinning of the peach, apricot, and plum. All have failed to due to various reasons, but

largely due to inconsistency of response and/or plant injury. Today the cost realities of new chemical development in the face of total government regulation have brought this effort to a standstill in the US”

The statement above was a candid assessment of the progress (or lack thereof) made with thinning compounds for use on stone fruit. This review was conducted to assess the current status of apple blossom thinning. Over the past 35 years, at least 150 blossom thinning compounds have been screened, 5 have been extensively tested, and up to three are registered for use in one state. LS and endothal are registered in Washington.

The only registered bloom thinner in the majority of apple producing states is NAD, a hormonal thinner developed in the 1930’s. Inconsistent thinning response, plant injury, and a lack of registered products prompted growers to rely on other crop load management strategies in the US. The cost realities of new chemical development in the face of total government regulation have brought this effort to a standstill in the US.

While the author attempted to create a comprehensive account of all materials screened for use as a blossom thinning products since 1989, I acknowledge that this list is likely incomplete. Blossom thinning data may be presented at various meetings or appear in proprietary technical reports to prospective registrants, but never appear in the literature. In some cases, perhaps due to negative outcomes, this information is not published at all. While the prospect of publishing negative data is not an attractive option for researchers, this information is useful to advancing our understanding of blossom thinning of tree fruit.

A number of blossom thinning studies utilized experimental designs that prove difficult to interpret. Multiple factors such as, thinning chemistry, timing of application,

number of applications, concentration, cultivar, addition of adjuvant, addition of post bloom thinners, and/or supplemental hand-thinning after June drop were combined in a solitary experiment. Frequently, these studies were analyzed via mean separation. While practical information can be obtained from these data, single degree of freedom contrasts could be utilized to isolate treatment effects of blossom thinners from other factors

(Marini, 2003). Additional research on blossom thinners that utilize simple experimental designs with clear objectives and structure would provide specific information, which would be useful to develop a clearer understanding of the potential role of blossom thinners in an integrated crop load management program.

Additional study of the effect of blossom thinner chemistries on leaf function is also needed. Phytotoxicity from blossom thinners is sometimes referenced vaguely, or described as being ephemeral. Phytotoxicity of leaf tissue is not a reversible phenomenon; suggesting that damaged leaves either abscised or was obscured by developing shoot leaves. In some cases, spur leaf injury is not documented at all. Given the importance of spur leaves in fruit development (Ferree and Palmer, 1982), documenting and/or quantifying this injury should be conducted in all blossom thinning trials. Determining sub-lethal effects of blossom thinners on photosynthesis is also warranted.

Given the lack of bloom thinning compounds that are registered for use, further evaluation of blossom thinners should occur. Such studies would be particularly valuable for commercially important cultivars that are subject to small fruit size, (‘Gala’ and ‘Pink

Lady’), or that are prone to biennial bearing (‘Golden Delicious’, ‘Fuji’, ‘Honeycrisp’,

‘Delicious’, etc.). Early thinning is an effective measure to increase fruit size and reduce

the incidence of biennial bearing (Batjer, 1965). Much of our understanding of blossom thinning and its place in crop load management stems from the evaluation of Elgetol over

50 years ago. There are little data with modern chemistries to support this paradigm. Due to the lack of efficacy of post-bloom thinners and return bloom sprays in the western

United States, blossom thinning research is likely to continue. While long-term west coast blossom thinner efficacy trials have been conducted since the 1990’s, the results of these trials remain largely unpublished. Coordinated, long term evaluation of blossom thinning products has not occurred in the eastern United States.

The development of decision-making aids to improve the timing of thinner applications has been a significant contribution in crop load management research. Continued evaluation and refinement of these models should occur. Developing and/or refining technologies to consistently reduce bud, flower, or fruit number are certainly areas for future research, especially development of selective thinning technologies.

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Chapter 2

Evaluation of chemical blossom thinners using ‘Golden Delicious’ and ‘Gala’ pollen tube growth models as timing aids

Introduction

The first opportunity to visually assess apple tree crop potential is at bloom. The fragile and ephemeral nature of apple blossoms presents an early opportunity for adjusting crop load. Several chemicals have been evaluated as potential blossom thinners, with variable efficacy. One potential source of variability is the lack of precision in timing blossom thinner applications.

Application timing of apple blossom thinners is often based on arbitrary visual estimates of the percentage of open blossoms. Common timings include: 20%, 60%, 80% and/or 100% full bloom, and single or multiple applications can be applied. Inconsistent blossom thinning responses may be attributed to application timing, since pollen tubes can reach the base of the style within 48 h (Yoder et al, 2009). Blossom thinner applications that occur at full bloom or later were attributed to increased fruit marking and russeting, which is unacceptable for fresh market fruit (Byers, 2003). The number of blossoms that are open can vary widely within a block or tree (Byers, 2003). An inherent challenge with blossom thinning is the short period of time that growers have to apply treatments over large acreage (Moran and Southwick, 2000).

Models were developed to estimate the rate of pollen tube growth in apple styles.

Child (1966) made one of the first attempts to evaluate apple pollen tube growth rates in

vivo in the cider cultivar ‘Michelin’. Detached blossoms were subjected to constant temperatures (5 to 24 ˚C), and estimates of pollen tube growth rates were determined using fluorescence microscopy (Child, 1966). Using similar techniques, Williams (1970) developed an index that estimated pollen tube growth based on temperature; however, only low temperatures were evaluated (7 to 15 ˚C) and estimates were based on detached blossoms. Jefferies and Brain (1984) measured pollen tube growth rates in detached blossoms at a range of incubation temperatures over 24 d. This effort was initiated to aid in research in controlled laboratory conditions. A relatively complex model was produced; however, the authors indicated that the model overestimated pollen tube growth at low temperatures, and noted that pollen tube growth under varying temperature regimes was not evaluated.

The effort to model apple pollen tube growth rates was revisited in 2003. Mature trees grafted on ‘M.27’ were grown in rootbags, placed in growth chambers at a range of specific night and day temperatures (Yoder et al., 2009) and ‘Snowdrift’ crabapple pollen was applied to emasculated king blossoms. While Williams (1965) found 5 to 7 d was required for pollen tubes to reach the style base in detached blossoms, Yoder et al. (2009) demonstrated that pollen tubes could reach the style base in less than 48 h in attached blossoms. Pollen tube growth rates of several apple cultivars were determined and modeled (Yoder et al., 2013). Pollen tube growth models were tested extensively in

Washington as a timing aid for blossom thinning applications of lime sulfur (LS; calcium polysulfide) and fish oil (FO) (Yoder et al, 2013). These models proved to be robust, were made available to the public, and were adopted as timing aids for blossom thinning programs. Maternal cultivar, temperature, and style length are inputs in the model.

Evaluation of the influence of the paternal pollen source on pollen tube growth rate is underway (DeLong, 2016). While pollen genotype can influence pollen tube growth rates in vivo, these relationships are complex and depend on maternal cultivar and temperature

(DeLong, 2016).

The fungicide LS was one of the first chemical constituents with recognized activity to inhibit fruit set of apple (Bagenal et al,.1925), but has only recently been utilized for the purpose of crop load management. LS has multiple sites of action – the inhibition of pollen tube growth and reduced net photosynthesis (Pn) (McArtney et al.,

2006; Yoder et al., 2009). A 2% LS + 2% fish oil (FO) application prevented pollen tubes from reaching the base of the style when applied within 24 h of the pollination event, but later applications (48 h) did not influence the number of pollen tubes that reached the base of the style (Yoder et al., 2009). Since LS inhibited growth of pollen tubes that have partly traversed the style, Schmidt and Elfving (2007) suggested that LS thinning programs may have a longer application window when compared to other products. Photosynthetic inhibition following LS + FO treatments is not considered in the pollen tube growth model(s).

The fertilizer ammonium thiosulfate (ATS) was shown to desiccate floral tissues of peach (Byers and Lyons, 1985) and apple (Byers, 1997). Pollen tube growth was inhibited in vitro and in vivo following ATS application (Embree and Foster, 1999; Myra et al., 2006). Schroder (2001, as cited in Schroder and Bangerth (2006)) suggested the mode of action of ATS is a combination of damaged floral tissue and reduced photosynthesis due to leaf injury. In some experiments, ATS caused unacceptable leaf phytotoxicity (Embree and Foster, 1999; Byers, 1997), which resulted in reduced fruit

growth (Wertheim, 2000). Conversely, Schmidt and Elfving (2007) suggested that ATS primarily influences blossoms that have recently opened and have not received pollen, and applications early in the flowering period (~20% bloom) increased efficacy (Bound and Wilson, 2007). However, ATS was a potent inhibitor of pollen tube growth in vivo, and reduced pollen tube growth when applied 12 h before (Myra et al., 2006) or 24 h after pollination (Embree and Foster, 1999). Multiple applications at low rates of ATS were more effective than a single application (Bound and Wilson, 2007). Given the assumptions of the pollen tube growth model, model-based application timing may improve consistency of ATS applications.

An aquatic herbicide, endothal [7, oxybicylo(2,2,2)heptane-2-3 dicarboxylic acid;

ThinRite; TR], has been evaluated as an apple blossom thinner since 1993, and was registered for use in Washington in 2014. The mode of action of endothal was assumed to be as a desiccant (Williams et al., 1995) and reduced the number of pollen tubes that reached the style base when applied 24 h after pollination (Embree and Foster, 1999).

Two applications of endothal during bloom reduced fruit set and improved fruit size when compared to a single application (Bound and Wilson, 2007; Greene, 2004;

Williams et al., 1995), though this relationship was not consistent (Byers, 1997). Since the proposed mode of action of endothal is similar to ATS, model-based application timing may be of benefit.

Naphthaleneacetamide (NAD) is a hormonal thinner developed in the 1930’s.

NAD was developed as a milder auxin-based thinner for use on summer ripening cultivars and has efficacy at bloom (Greene, 2002). While NAD use has been limited in recent years, there is renewed interest in the use of NAD as a bloom thinner in the eastern

US (Greene et al., 2015). Greene et al. (2015) observed thinning activity at bloom and petal fall, with no observed dose response. Since activity of hormonal thinners is influenced by temperatures proximal to application (Greene, 2002), pollen tube growth models may not be suitable.

Using predictive models developed to determine application timing, the efficacy of several blossom thinning chemistries on ‘Golden Delicious’ and ‘Gala’ was compared.

Blossom thinning treatments were evaluated as the sole method of ‘Golden Delicious’ crop load management, and as part of a ‘Gala’ crop load management program in a commercial orchard.

Materials and Methods

‘Golden Delicious’

Experiments were conducted in 2014 and 2015 at Pennsylvania State University’s

Fruit Research and Extension Center in Biglerville, PA. Uniform ‘Golden Delicious’/

‘Budagovski 9’ trees were selected at pink bud stage. Trees were planted in 2004 at 1.5 x

4.6 m spacing and trained to vertical axis. Treatments were randomly assigned to single tree plots, with buffer trees between each treatment. Two to three uniform limbs were selected and flagged on each tree. Circumference was measured, and blossom clusters were counted on uniform limbs.

At the late balloon stage, 50 king blooms from adjacent trees were collected, and the length of the longest style was measured with digital calipers. The average length of the longest style was calculated and used as an input in the model. Flagged limbs were used to determine the timing of model initiation. Limbs were fitted with a hand-held gauge to provide a target crop density (Equilifruit; INRA, Montpellier, France, described

in Kon and Schupp, 2013). Once the number of blossoms that were open matched the F value (~ 6 fruit per cm2 branch cross-sectional area; BCSA), the ‘Golden Delicious’ pollen tube growth model was initiated. In the spring of 2014, freeze injury was observed to flower buds and was documented. Bud mortality of all flowers on 10 randomly selected limbs was evaluated. Approximately 30% of flower buds were visibly injured, but sufficient viable blossoms remained to conduct the experiment. To account for this injury, the initiation of the model was delayed until approximately 12 blossoms per cm2

BCSA were open on selected limbs in 2014. Since cold injury was not observed in 2015,

6 fruit per cm2 BCSA was set as the target crop density.

Hourly temperature data from an onsite weather station was used as an input in the model. Two blossom thinner applications were applied in accordance with current recommendations for use of the pollen tube growth model (Yoder et al., 2013). After estimated pollen tube length was equivalent to the mean style length, the 1st application of the thinning treatments occurred (Fig. 2-1). After the initial application, the model clock was reset to track the growth of pollen tubes in side blossoms. The second application occurred at or before the model estimate of pollen tube length was 70% of the mean style length.

The following treatments were applied in both years: 1) unthinned control

(Control), 2) hand-thinned control (HT), 3) 2% (v:v) LS (Miller Chemical and Fertilizer,

LLC, Hanover PA) and 2% (v:v) stylet oil (SO; JMS Flower Farms, Inc., Vero Beach,

FL) 4), 2% (v:v) ATS, and 5) 1.5 mL∙L-1 TR (Thin Rite; United Phosphorus, Inc., King of Prussia, PA). In 2015, 50 mg∙L-1 NAD; (Amid-Thin, AMVAC, Los Angeles, CA) was also evaluated.

The experiment was a completely randomized design with five replications. All chemical thinning treatments were applied with a CO2 sprayer at 276 KPa (Bellspray,

Inc., Opelousas, LA) to single tree plots. Caustic products were applied until the canopy was thoroughly wetted and NAD was applied until runoff. HT was conducted during bloom, and crop load was manually adjusted using scissors. Using the aforementioned hand-thinning gauge, crop load was manually adjusted to ~12 or 6 blossoms per cm2

BCSA to reflect the targeted crop load in 2014 and 2015, respectively. Open king blossoms were retained preferentially.

30 Cumulative pollen tube growth (mm) Temperature (˚C) Style length (mm) 10

8 20

15 (mm)

10 4 Temperature (˚C) Temperature 5 2

0 0 Cumulative pollentube Cumulativegrowth

A. Time 30 10

25 8 20 6 15 4

10 (mm)

Temperature (˚C) Temperature 5 2

0 0 Cumulative pollentube Cumulativegrowth

B. Fig. 2-1. Graphical representation of ‘Golden Delicious’ pollen tube growth model application timings (indicated by arrows), estimates of pollen tube growth rates, and temperature in 2014 and 2015 (A and B, respectively). The model was initiated at 0900 HR on 4 May 2014 and 1400 HR on 29 Apr 2015.

One week after treatment, leaf phytotoxicity was visually rated (1 to 5 scale: 1=

No visible damage; 2 = trace to 10% damage; 3 = 11% to 24% damage; 4 = 25% to 49% damage; 5 = >50% damage). Fruit were counted on selected limbs at ~ 9 mm fruit diameter and after June drop. Crop density (fruit no. per cm2 BCSA) and fruit set was calculated at both timings.

Fruit was harvested from entire trees at a commercially acceptable level of maturity, and fruit number, yield, and average fruit weight was determined using an electronic fruit sizer (Durand Wayland, LaGrange, GA). A 20 fruit sample was collected and russet was evaluated using digital image analysis adapted from Winzeler and Schupp

(2013). Seeds were extracted from the 20 fruit sample and were counted and recorded.

During dormancy, trunks were measured 30 cm above the graft union and number of fruit per cm2 trunk cross-sectional area (TCSA) was calculated. The following spring, return bloom density was determined by counting the number of blossom clusters on two or three representative limbs on each tree and expressing the number of blossom clusters per cm2 BCSA.

In 2015, king blossoms and lateral blossoms on five spurs were selected and tagged on each tree immediately after the model was initiated. The spurs were selected with king bloom at anthesis, king blossoms were labeled, and the furthest developed side blossom (generally at balloon stage) was labeled. Similar methods to those of Embree and Foster (1999) were used to visualize pollen tube growth. Blossoms were harvested 48 h after the first application timing, placed in labeled vials containing 5% sodium sulfite, and stored at 4˚C. Samples were autoclaved at 121 ˚C for 10 min to soften tissues for

slide preparation. Prior to microscopic examination, blossoms were rinsed with distilled- deionized water and styles were removed with a scalpel at the junction with the hypanthinum. Remaining floral tissues were discarded. Styles were rinsed, separated, and soaked in a water-soluble fluorescence solution of 0.01% Aniline Blue stain in 0.067 M

K2HPO4 on a microscope slide. Styles were squashed between two microscope slides.

Samples were incubated overnight and observed via fluorescence microscopy at 100X

(Olympus BX51; Tokyo, Japan), equipped with a UV/DAPI long pass filter cube (19000-

AT-UV/DAPI; Chroma Technology Corporation, Bellows Falls, VT). Pollen density on the stigmatic surface was visually rated using a 0 to 10 scale, described in Yoder et al.

(2009). Pollen tubes that entered the style and those that reached the style base were counted. Longest pollen tube length and style length were measured with an ocular micrometer. Pollen tube length was standardized by style length to estimate pollen tube growth (pollen tube growth = (pollen tube length/style length) *100).

‘Gala’

In 2015, a trial was conducted in a commercial orchard of mature ‘Buckeye Gala’/

‘Nic 29’. Trees were trained to tall spindle at 1.5 m x 4.6 m spacing. Twenty-four uniform ‘Gala’ trees were selected and flagged. Treatments were randomly assigned to single tree plots, with buffer trees between each treatment. Model initiation and application timing of the ‘Gala’ pollen tube growth model was conducted as previously described. The target crop load was ~ 6 fruit per cm2 BCSA.

Using the ‘Gala’ pollen tube growth model (Fig. 2-2), the following blossom thinning treatments were applied to whole trees with a CO2 sprayer at 276 kPa until

thoroughly wetted: 1) control, 2) LS + SO, 3) ATS, and 4) TR. Rates used were identical to that of the previous experiment. All trees received the following post bloom thinning application via airblast sprayer: 100 mg∙ L-1 6-benzyladenine (6-BA, Excelis Plus, Fine

Americas, Inc., Walnut Creek, CA) + 1 pint 1-naphthyl methylcarbamate (carbaryl)+ 1- pint adjuvant (Level 7; Winfield Solutions, LLC., St. Paul, Mn). Post bloom thinner was applied via airblast sprayer calibrated to apply 843 L water per hectare on 14 May 2015.

The experiment was a completely randomized design and was replicated six times.

30 12

25 10

20 8

15 6

Temperature (˚C) Temperature 10 4

5 2 pollentube Cumulativegrowth (mm)

0 0

Time

Cumulative pollen tube growth (mm) Temperature (˚C) Style length (mm)

Fig. 2-2. Graphical representation of ‘Gala’ pollen tube growth model application timings (indicated by arrows), estimates of pollen tube growth rates, and temperature in 2015. The model was initiated at 1500 HR on 30 Apr 2015.

With a few exceptions, all response variables described in the previous experiment were documented and evaluated. A 20 fruit sample was collected and the percentage of apple peel exhibiting russet was quantified by visual estimations, rather than digital image analysis. Additionally, fruit shape appeared to differ among treatments.

The length and diameter of the 20 fruit sample was measured and recorded. Fruit length:diameter ratio was calculated to characterize fruit shape. In the spring of 2016, a series of cold weather events resulted in some flower bud mortality. To determine if blossom thinning treatments influenced frost tolerance, flower bud mortality on three limbs per tree was evaluated. The number of flowers was counted, then each individual flower was dissected and the pistil was rated as dead or alive. The percentage of viable flowers was calculated.

Statistical analysis

Data was analyzed using the personal computer version of SAS (SAS 9.3; SAS

Institute, Cary, NC). Analysis of variance was performed using PROC MIXED. Tukey’s honest significance test was utilized to compare treatment means at P = 0.05.

Results and Discussion

‘Golden Delicious’

Several promising blossom thinning chemistries were evaluated using the pollen tube growth model as a timing aid. The number of blossom thinning applications was restricted to two, since the majority of blossoms were open at the time the 2nd blossom thinning spray was applied. In some years or geographical areas, the bloom period may

be protracted due to environmental conditions, requiring additional blossom thinner applications (Moran and Southwick, 2000).

Pollen tube growth metrics for king blossoms were not influenced by any treatment, suggesting that selected open king blossoms were not susceptible to blossom thinners (Table 2-1). LS + FO was a potent inhibitor of pollen tube growth when applied at 4 and 24 h after pollination, but was ineffective 48 h after pollination (Yoder, et al.,

2009). Similarly, ATS and TR reduced the number of pollen tubes that reached the style base when applied 24 h after pollination (Embree and Foster, 1999). Based on the assumptions of the pollen tube growth models, open king blossoms received pollen at the initiation of the model. Model estimates of pollen tube growth rates are based on hourly temperature data. In theory, pollen tubes in king blossoms should reach the hypanthinum before the first application of thinner. Pollen tube growth occurs at the tip, and cytoplasm is physically separated from the rest of the pollen tube by callose plugs. After reaching the hypanthinum, it is assumed that pollen tubes are unlikely to be influenced by chemical thinning treatments. Therefore, limited influence on pollen tube growth of selected king blossoms was a desirable outcome and our data suggests that the model was successful in preventing premature application of blossom thinning chemicals.

Differences among treatments were observed in measured responses of pollen tube growth in side blossoms. LS + SO and ATS reduced pollen tube density on the stigma, the number of pollen tubes that entered the style, and pollen tube growth. When compared to the control, LS + SO and ATS reduced the number of pollen tubes that entered the style of side blossoms by 75% and 63%, respectively. LS + SO reduced the

number of pollen tubes that reached the style base, but ATS did not. Several experiments demonstrated that LS + oil and ATS were effective in reducing pollen tube growth in vivo

(Embree and Foster, 1999; McArtney et al, 2006; Myra et al. 2006; Yoder et al., 2009).

Pollen tube growth was not affected by TR or NAD. The lack of effects observed on pollen tube growth with TR was unexpected; since TR had previously been shown to reduce the number of pollen tubes that reach the style base when applied 24 h after pollination (Embree and Foster, 1999). Williams et al. (1995) suggested TR acts as a desiccant of floral tissue, thereby preventing fertilization. However, our data shows that inhibition of pollen tube growth is an improbable mechanism for TR and NAD, since responses did not differ from the control.

Table 2-1. Comparison of blossom thinning treatments on pollen density rating, number of pollen tubes that entered the style, number of pollen tubes that reached the style base, and pollen tube growth in king and side bloom of 'Golden Delicious'/'Budagovski 9' apple trees in 2015.z Pollen Pollen Pollen tubes Pollen tubes at density on tube Thinning entering style style base stigma growth treatmentyx (0-10 (no. (no. v w (%) rating) visible/flower) visible/flower) King blossom Control 3.2 au 124 a 8.7 a 86 a HT 2.4 a 100 a 7.4 a 79 a LS + SO 3.0 a 130 a 6.0 a 75 a ATS 3.3 a 119 a 5.3 a 73 a TR 3.3 a 131 a 9.0 a 87 a NAD 2.8 a 123 a 7.7 a 82 a

Side blossom Control 2.8 ab 141 b 7.8 a 78 a HT 3.5 a 188 a 6.1 ab 76 a LS + SO 2.1 b 35 c 0.5 b 24 b ATS 2.0 b 52 c 2.8 ab 37 b TR 3.5 a 154 ab 9.2 a 80 a NAD 2.8 ab 135 b 4.4 ab 65 a zMeans of four observations. yTiming of chemical treatments were determined with the ‘Golden Delicious' pollen tube growth model. In 2015, the model was initiated at 1400 HR on 29 Apr. Chemical treatments were applied at 1000 HR on 3 May 2015 and 1600 HR 4 May 2015. The hand-thinned control treatment was applied on 3 May 2015. xControl = unthinned control; HT = hand-thinned at bloom; LS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA); NAD = 50 mg∙L- 1 Amid-Thin (AMVAC, Los Angeles, CA). wA visual rating scale was used to quantify pollen density on the stigma: 0 = no pollen tubes visible on the surface; 1 = 1% to 10 % area covered; 2 = 11% to 20%; 3 = 21% to 30%; 4 = 31% to 40%; 5 = 41% to 50%; 6 = 51% to 60%; 7 = 61% to 70%; 8 = 71% to 80%; 9 = 81% to 90%; 10 = 91% to 100% of surface covered by pollen tubes. vPollen tube growth = (the length of the longest pollen tube / style length)*100 uMean separation within columns by Tukey’s honest significance test at P = 0.05.

While TR did not influence pollen tube growth in 2015, TR increased damage to spur leaf tissue in both years of this trial (Table 2-2). Symptoms included marginal leaf injury, goose-necking, and round necrotic lesions (Fig. 2-3). Spur leaf injury was rated 7 d after treatment. Shortly after evaluation, some of the spur leaves exhibiting injury abscised. Any reduction in spur leaf area or leaf function was not formally quantified.

Williams et al. (1995) suggested that some cultivars, particularly ‘Golden Delicious’, were sensitive to TR. He observed bud and leaf damage at 1.0 and 1.5 ml·L-1 on ‘Golden

Delicious’, however, fruit size and quality were not affected (Williams et al., 1995). In this study, similar injury to ‘Golden Delicious’ was observed at 1.5 ml·L-1. The legal rate of TR can range between 1.25 and 2.50 mL ∙ L-1. The label for TR indicates that multiple applications should not be made within a 24 h period, and applications should not be made if temperature is greater than 29.4 ˚C within 24 h of application. The observed environmental conditions permitted compliance with these restrictions. In 2014, but not in 2015, ATS slightly increased leaf phytotoxicity when compared to the control (trace to

10% leaf damage). ATS injured apple foliage at lower concentrations than were utilized in this study (Balkoven-Baart and Wertheim,1997). Since environmental effects, such as temperature and relative humidity, can influence drying conditions, incidents of ATS phytotoxicity can be unpredictable (Wertheim, 2000). When compared to the control,

HT, LS + SO, and NAD did not increase visible leaf phytotoxicity in either year.

Table 2-2. Comparison of blossom thinning treatments on phytotoxicity, fruit set, and crop density of 'Golden Delicious'/'Budagovski 9' apple trees in 2014 and 2015.z Initial Final

Crop Crop Leaf Fruit set Fruit set Phytotoxicity density density Thinning yxw (no. (no. treatment v 2 t 2 (1-5) fruit/cm (%) fruit/cm (%) u BCSA) BCSA) 2014 Control 1.0 cs 99.3 a 287 a 18.5 a 55 a HT 1.0 c 15.8 c 49 c 14.1 ab 44 a LS + SO 1.2 c 65.8 ab 178 b 17.6 a 54 a ATS 2.0 b 39.9 bc 124 b 15.5 ab 49 a TR 2.6 a 49.1 bc 181 b 9.7 b 37 a 2015 Control 1.0 b 122.7 a 389 a 19.1 a 59 a HT 1.0 b 8.3 c 24 c 7.0 b 20 c LS + SO 1.6 b 60.4 b 186 b 12.6 ab 39 abc ATS 1.6 b 79.8 b 235 b 13.4 ab 40 abc TR 2.6 a 129.6 a 344 a 16.5 a 43 ab NAD 1.0 b 136.3 a 352 a 14.9 a 38 bc zMeans of five observations. yTiming of chemical treatments were determined with the 'Golden Delicious' pollen tube growth model. Model was initiated at 0900 HR on 4 May 2014. Treatments were applied at 1800 HR on 8 May 2014 and 1100 HR on 10 May 2014. The hand-thinned control was applied on 8 May 2014. xTiming of chemical treatments were determined with the ‘Golden Delicious' pollen tube growth model. Model was initiated at 1400 HR on 29 Apr. Treatments were applied at 1000 HR on 3 May 2015 and 1600 HR 4 May 2015. The hand-thinned control treatment was applied on 3 May 2015. wControl = unthinned control; HT = hand-thinned at bloom; LS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA); NAD = 50 mg∙L-1 Amid-Thin (AMVAC, Los Angeles, CA). vThe following visual rating scale was used to quantify leaf phytotoxicity: 1= No visible damage; 2 = trace to 10 % damage; 3 = 11 - 24 % damage; 4 = 25-49 % damage; 5 = 50 % damage or greater. uBCSA = branch cross-sectional area. tFruit set = (no. fruit / no. flower clusters)*100. sMean separation within columns by Tukey’s honest significance test at P = 0.05.

Fig. 2-3. Leaf phytotoxicity observed on ‘Golden Delicious’ treated with 1.5 mL∙L-1 TR (Thin Rite; United Phosphorus, Inc., King of Prussia, PA) in 2014.

Source limitations for carbohydrates are not thought to occur until fruitlet diameter is greater than 9 mm, and unfertilized blossoms abscise shortly after bloom.

Therefore, initial fruit set is an indirect indicator of blossom thinning treatment efficacy.

When compared to the control, LS + SO, ATS, and TR reduced initial fruit set by 37% to

57% in 2014. In 2015, LS + SO and ATS reduced initial fruit set (51% and 35%, respectively), but TR did not differ from the control. The observed reduction in pollen tube growth metrics corresponded with initial fruit set. As expected, HT resulted in the lowest initial fruit set in both years, since blossom number was reduced manually to 49% or 24% fruit set (2014 and 2015, respectively). In HT treatments, very few fruit abscised

between measurements of initial and final fruit set, suggesting that HT reduced competition among fruitlets and limited June drop.

On the other hand, final crop density is an estimate of whole tree crop density cm2

TCSA. There were some differences in final crop density among treatments, but our discussion will focus on whole-tree crop density. While whole-tree crop density was reduced by HT and TR in 2014, it was not affected by LS + SO or ATS (Table 2-3). This may be partly explained by the delay in initiation of the model to a target crop load of 12 fruit per cm2 BCSA. A spring frost resulted in 30% flower bud mortality in 2014. To account for the loss of viable flower buds, the model was delayed until ~ 12 blossoms per cm2 BCSA were open. Despite a reduction in crop density by HT and TR in 2014, yield was not influenced by either treatment. This is likely explained by the observed increase in fruit weight as a result of HT and TR. In 2015, LS + SO and TR reduced crop density by 39% and HT by 68% when compared to the control. Both LS + SO and TR resulted in near optimal crop loads for ‘Golden Delicious’ (6 to 8 fruit per cm2 TCSA), while HT over-thinned. Pollen tube growth models were developed for LS blossom thinning programs, and Pn is not a factor in the model. Reduced Pn as a result of a LS program has been confirmed in partial leaf (Noordijk and Schupp, 2003; McArtney et al., 2006), leaf

(Hoffman, 1935), and whole tree experiments (Lombardini et al., 2003; Whiting, 2007).

In the Pacific Northwest, this stress was reported to last between 4 to 10 d (Schmidt and

Elfving, 2007), but spur leaf Pn was reduced for more than 57 d in New Zealand

(McArtney et al., 2006). The reduction in crop density observed with TR was surprising, since it did not appear to reduce pollen tube growth or initial fruit set in 2015. However,

the observed damage and subsequent abscission of leaf tissue may explain the reduction in crop density.

Table 2-3. Comparison of blossom thinning treatments on fruit number, crop density, yield, and fruit weight of 'Golden Delicious'/'Budagovski 9' apple trees in 2014 and 2015.z

Fruit Crop density Yield Fruit wt Thinning yxw 2 treatment (no. fruit/cm (no./tree) v (kg/tree) (g) TCSA) 2014 Control 545 au 14.7 a 53.3 a 98 c HT 318 b 9.5 b 46.3 a 146 a LS + SO 438 ab 11.4 ab 49.1 a 114 bc ATS 393 b 11.6 ab 44.7 a 113 bc TR 371 b 7.8 b 48.2 a 132 ab

2015 Control 500 a 12.2 a 55.9 a 113 c HT 166 c 3.9 c 37.7 b 228 a LS + SO 333 abc 7.5 b 53.2 ab 160 b ATS 390 ab 9.1 ab 55.3 a 141 bc TR 318 bc 7.5 b 47.4 ab 156 b NAD 454 ab 10.5 ab 61.2 a 135 bc zMeans of four observations. yAll chemical treatments were applied in accordance with the ‘Golden Delicious' pollen tube growth model. Model was initiated at 0900 HR on 4 May 2014. Treatments were applied at 1800 HR on 8 May 2014 and 1100 HR on 10 May 2014. The hand-thinned control was applied on 8 May 2014. xTiming of chemical treatments were determined with the ‘Golden Delicious' pollen tube growth model. Model was initiated at 1400 HR on 29 Apr. Treatments were applied at 1000 HR on 3 May 2015 and 1600 HR 4 May 2015. The hand-thinned control treatment was applied on 3 May 2015. wControl = unthinned control; HT = hand-thinned at bloom; LS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA); NAD = 50 mg∙L-1 Amid-Thin (AMVAC, Los Angeles, CA). vTCSA = trunk cross-sectional area. uMean separation within columns by Tukey’s honest significance test at P = 0.05.

Despite reducing initial fruit set in both years, ATS did not reduce crop density, yield, or increase fruit weight. In sweet cherry, several physiological measurements demonstrated that ATS had a minor impact on leaf function (Lenean and Whiting, 2006) and ethylene induced abscission was not likely a causal factor (Jandoui and Flore, 2005).

ATS reduced pollen tube growth when applied 12 h before (Myra et al., 2006) or 24 h after pollination (Embree and Foster, 1999). These studies suggest that the primary mode of action of ATS can be attributed to the desiccation of floral tissues and subsequent inhibition of pollen tube growth in vivo. While the scorching of leaf tissue may affect leaf function, apple leaves can tolerate a minor reduction in functional leaf area without reducing Pn. Physical removal of up to 10% of spur leaves did not reduce Pn (Hall and

Ferree, 1976). In this experiment, visual estimates of leaf injury by ATS did not exceed a rating of 2 (trace to 10% damage).

The hormonal thinner, NAD reduced final limb crop density by 35% but did not reduce whole tree crop density. NAD was an effective thinner while using the pollen tube growth model as a timing aid on ‘Honeycrisp’ (Peck et al., unpublished). While the maximum legal rate of NAD was utilized on ‘Golden Delicious’ in this experiment, thinning activity was limited. Lack of efficacy may be partly explained by cultivar, as

‘Golden Delicious’ is considered a difficult-to-thin variety. The model estimates the rate of pollen tube growth based on hourly temperature, but temperatures proximal to application timing are not an important factor. Conversely, the efficacy of hormonal thinning products, particularly products that induce generation of ethylene, are influenced by temperatures proximal to application timing (Greene, 2002). Evaluation of NAD

bloom application in combination with adjuvants or other thinners on hard-to-thin cultivars should be considered in future work.

Table 2-4. Comparison of blossom thinning treatments on fruit russet, seed number, and return bloom of 'Golden Delicious'/'Budagovski 9' apple trees in 2014 and 2015.z Return Fruit russet Seeds Bloom

Thinning (no. yxw treatment blossom (%) (no./fruit) clusters/cm2 BCSA)v 2014 Control 1.8 bu 8.3 a 0.0 b HT 6.2 a 7.3 a 4.9 a LS + SO 4.2 ab 7.9 a 3.0 ab ATS 1.8 ab 8.2 a 0.8 ab TR 2.8 ab 7.5 a 0.4 b

2015 Control 6.2 a 7.0 a 0.8 b HT 5.7 a 5.9 a 26.2 a LS + SO 7.1 a 6.4 a 5.0 b ATS 5.3 a 5.5 a 5.3 b TR 6.1 a 7.2 a 3.6 b NAD 3.2 a 6.8 a 2.3 b zMeans of three observations. yTiming of chemical treatments were determined with the ‘Golden Delicious' pollen tube growth model. Model was initiated at 0900 HR on 4 May 2014. Treatments were applied at 1800 HR on 8 May 2014 and 1100 HR on 10 May 2014. The hand-thinned control treatment was applied on 8 May 2014. xTiming of chemical treatments were determined with the ‘Golden Delicious' pollen tube growth model. Model was initiated at 1400 HR on 29 Apr. Treatments were applied at 1000 HR on 3 May 2015 and 1600 HR 4 May 2015. The hand-thinned control treatment was applied on 3 May 2015. wControl = unthinned control; HT = hand-thinned at bloom; LS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA); NAD = 50 mg∙L-1 Amid-Thin (AMVAC, Los Angeles, CA). vBCSA = branch cross-sectional area. uMean separation within columns by Tukey’s honest significance test at P = 0.05.

Fruit russet was greater on fruit from HT trees in 2014, but this increase was inconsequential and would not reduce the grade of the fruit (Table 2-4). USDA grading standards indicate that greater than 10% of the fruit surface must be russeted to be considered damaged. There were no differences in fruit russet in 2015 and none of the treatment means exceeded 10% russet. Fast drying conditions occurred during both years of the experiment at the time of application, which was suggested to reduce russet incidents (Byers, 2003). While seed number can be reduced by caustic products (Bound,

1997; Bound and Wilson, 2007), seed number was not influenced by any treatment in either year.

When compared to the control, HT increased return bloom in both years. Despite the early reduction in initial fruit set for LS + SO, ATS, and TR, return bloom did not differ from the control for any of these treatments. In 2014, HT reduced crop density to ~

14 fruit per cm2 BCSA. The resulting return bloom in the spring of 2015 was 4.9 blossom clusters per cm2 BCSA. This blossom density would be adequate to set a commercial crop. Schmidt et al. (2009) suggested that blossom densities between 2 and 6 blossom clusters per cm2 TCSA were acceptable. Reducing crop load early in the season has potential to increase return bloom; however, in some strongly biennial cultivars, summer bloom promoting sprays are required to ensure consistent cropping potential (Ferree and

Schmid, 2000; McArtney et al., 2007). While not evaluated in this trial, research is needed to understand the interaction between blossom thinners and products that promote return bloom.

‘Gala’

Table 2-5. Comparison of four blossom thinning treatments on pollen density rating, number of pollen tubes that entered the style, number of pollen tubes that reached the style base, and pollen tube growth in king and side bloom of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015.z Pollen Pollen density Pollen tubes Pollen tubes tube Thinning on stigma entering style at style base growth treatmentyxw (no. (no.visible/ (0-10 rating)v (%)u visible/flower) flower) King blossom Control 2.1 at 71 ab 1.7 a 58 a LS + SO 2.1 a 44 b 1.4 a 45 a ATS 2.7 a 63 ab 3.4 a 51 a TR 2.9 a 79 a 2.2 a 59 a

Side blossom Control 4.0 a 180 a 2.8 a 58 a LS + SO 1.9 bc 15 c 0.0 b 5 b ATS 1.2 c 19 c 0.0 b 6 b TR 2.8 ab 120 b 1.4 ab 47 a zMeans of four observations. yTiming of chemical treatments were determined with the 'Gala' pollen tube growth model. Model was initiated at 1500 HR on 30 Apr 2015 .Treatments were applied at 0600 HR on 3 May 2015 and 1100 HR on 4 May 2015. xLS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA) wOn 14 May 2015, all trees received a post bloom thinning application: 100 mg∙ L-1 6- benzyladenine (6-BA, Excelis Plus, Fine Americas, Inc., Walnut Creek, CA) + 1 pint 1- naphthyl methylcarbamate (carbaryl)+ 1 pint adjuvant (Level 7; Winfield Solutions, LLC., St. Paul, Mn). vThe following visual rating scale was used to quantify pollen density on the stigma: 0 = no pollen tubes visible on the surface; 1 = 1% to 10 % area covered; 2 = 11% to 20%; 3 = 21% to 30%; 4 = 31% to 40%; 5 = 41% to 50%; 6 = 51% to 60%; 7 = 61% to 70%; 8 = 71% to 80%; 9 = 81% to 90%; 10 = 91% to 100% of surface covered by pollen tubes. uPollen tube growth = (length of the longest pollen tube / style length)*100. tMean separation within columns by Tukey’s honest significance test at P = 0.05.

Effects on pollen tube growth were similar to observed responses on ‘Golden

Delicious’, indicating that LS + SO and ATS were potent inhibitors of pollen tube growth in side blossoms (Table 2-5). In general, pollen tube growth responses in king blossoms were not influenced by any treatment. LS + SO reduced the number of visible pollen tubes that entered the style, but did not affect the number of tubes that reached the style base. In side blossoms, LS + SO and ATS were effective inhibitors of pollen germination and growth in the style. TR application resulted in a minor reduction in the number of pollen tubes that entered the style (33%), but did not reduce the number of pollen tubes that reached the style base or pollen tube growth percentage.

ATS and TR caused leaf phytotoxicity (Table 2-6) and ATS injury primarily occurred at the leaf margin (Fig. 2-4). Leaf phytotoxicity may be reduced by using multiple applications of low ATS rates (0.8% to 1.5%), with similar thinning outcomes to a single application at higher rates (Bound and Wilson, 2007). Leaf phytotoxicity symptoms of TR were similar to the damage observed in the trial on ‘Golden Delicious’.

Bound and Jones (1997) observed increased phytotoxicity with increasing concentration of TR on ‘Delicious’, and rates of 1.25 to 1.5 ml·L-1 were suggested to reduce crop load without excessive damage. Our treatments were within this range, and it is unclear why phytotoxicity was increased with TR. The author is unaware of evidence suggesting

‘Gala’ is sensitive to TR, and drying conditions after treatments were ideal.

Initial and final crop densities were not affected by treatment. Since initial fruit set and whole tree crop load were not different, these estimates may not have been sensitive enough to detect differences. Initial fruit set was reduced by ATS and TR (42%

and 35%, respectively). Since pollen tube growth was not reduced with TR, the reduction in initial fruit set may be due to other factors, such as carbohydrate limitations. Loss of photosynthetic spur leaf area during bloom negatively affected fruit set (Ferree and

Palmer, 1982).

Table 2-6. Comparison of four blossom thinning treatments on phytotoxicity, crop density, and fruit set of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015.z Initial Final

Leaf Crop Crop Fruit set Fruit set phytotoxicity density density Thinning yxw (no. (no. treatment v 2 t 2 (1-5) fruit/cm (%) fruit/cm (%) u BCSA) BCSA) Control 1.0 cs 76.2 a 306 a 4.2 a 15 a LS + SO 1.5 bc 61.5 a 242 ab 2.2 a 9.5 a ATS 2.3 a 53.9 a 176 b 2.3 a 9.2 a TR 2.2 ab 54.7 a 200 b 2.3 a 7.8 a zMeans of five observations. yTiming of chemical treatments were determined with the 'Gala' pollen tube growth model. Model was initiated at 1500 HR on 30 Apr 2015 .Treatments were applied at 0600 HR on 3 May 2015 and 1100 HR on 4 May 2015. xLS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA) wOn 14 May 2015, all trees received a post bloom thinning application: 100 mg∙ L-1 6- benzyladenine (6-BA, Excelis Plus, Fine Americas, Inc., Walnut Creek, CA) + 1 pint 1-naphthyl methylcarbamate (carbaryl)+ 1 pint adjuvant (Level 7; Winfield Solutions, LLC., St. Paul, Mn) v1 = No visible damage; 2 = trace to 10 % damage; 3 = 11 - 24 % damage; 4 = 25-49 % damage; 5 = 50 % damage or greater. uBCSA = branch cross-sectional area. tFruit set = (no. fruit / no. flower clusters)*100. sMean separation within columns by Tukey’s honest significance test at P = 0.05.

Fig. 2-4. Leaf phytotoxicity observed on ‘Buckeye Gala’ treated with 2% (v:v) ammonium thiosulfate in 2015.

All treatments reduced crop density below the optimal for ‘Gala’ (6 to 8 fruit per cm2 TCSA; Table 2-7). All treatments, including the control, received a post bloom thinner application. This treatment resulted in over-thinning. LS + SO and ATS did not reduce crop density when compared to the control, suggesting that chronic effects of the blossom thinner application did not cause additional over-thinning. However, TR reduced the number of fruit per tree, crop density, and yield. Research was conducted on interactions between TR and post bloom compounds in multi-step thinning programs

(Bound and Wilson, 2007; Stover et al., 2002). While assessing a multi-step thinning program, Stover et al. (2002) observed that the addition of a petal fall thinning

application was beneficial in a two-year trial, but the addition of a blossom thinning spray with TR was not. Despite a significant reduction in crop load, TR reduced fruit weight when compared to the control. At high rates of TR (3.0 ml·L-1), Bound and Jones (1997) suggested that spur leaves damage had negative consequences on fruit weight.

Table 2-7.Comparison of four blossom thinning treatments on fruit number, crop density, yield, and fruit weight of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015.z

Fruit Crop density Yield Fruit wt Thinning yxw 2 treatment (no. fruit/cm (no./tree) v (kg/tree) (g) TCSA) Control 110 au 4.5 a 18.6 a 170 ab LS + SO 70 ab 2.7 ab 12.7 ab 180 a ATS 80 ab 3.0 ab 14.3 ab 181 a TR 44 b 2.1 b 7.1 b 161 b zMeans of four observations. yTiming of chemical treatments were determined with the 'Gala' pollen tube growth model. Model was initiated at 1500 HR on 30 Apr 2015 .Treatments were applied at 0600 HR on 3 May 2015 and 1100 HR on 4 May 2015. xLS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA) wOn 14 May 2015, all trees received a post bloom thinning application: 100 mg∙ L-1 6- benzyladenine (6-BA, Excelis Plus, Fine Americas, Inc., Walnut Creek, CA) + 1 pint 1- naphthyl methylcarbamate (carbaryl)+ 1 pint adjuvant (Level 7; Winfield Solutions, LLC., St. Paul, Mn). vTCSA = trunk cross-sectional area. uMean separation within columns by Tukey’s honest significance test at P = 0.05.

Fruit russet increased slightly with ATS and TR, but treatments resulted in less than 1% of the fruit surface covered with russet (Table 2-8). Seed number was not influenced by any treatment. Some reports document that TR can result in the flattening of apple fruit (Bound and Jones 1997), while others found no effects on fruit shape

(Greene, 2004). In this trial, TR reduced the L:D ratio when compared to LS + SO and

ATS, but was not different than the control. Therefore, while statistically significant, the minor effect on fruit flattening may not be of practical significance. In general, higher TR concentrations increased the effect of fruit flattening (Bound and Jones 1997).

Table 2-8. Comparison of four blossom thinning treatments on fruit russet, seed number, fruit LD ratio, and return bloom of 7th leaf ‘Buckeye Gala’/‘Nic 29’ apple trees in 2015 .z

Fruit russet Seeds LD ratio Return Bloom Thinning yxw treatment v Fruit length : (no. blossom (%) (no./fruit) 2 u fruit diameter clusters/cm BCSA) Control 0.21 bt 4.3 a 0.95 ab 22.9 a LS + SO 0.33 ab 4.7 a 0.96 a 20.4 a ATS 0.62 a 4.5 a 0.96 a 20.4 a TR 0.64 a 3.2 a 0.93 b 21.4 a zMeans of four observations. yTiming of chemical treatments were determined with the 'Gala' pollen tube growth model. Model was initiated at 1500 HR on 30 Apr 2015 .Treatments were applied at 0600 HR on 3 May 2015 and 1100 HR on 4 May 2015. x LS + SO = 2% (v:v) LS (Miller Chemical and Fertilizer, LLC, Hanover PA) and 2% (v:v) stylet oil (JMS Flower Farms, Inc., Vero Beach, FL); ATS = 2% (v:v) ammonium thiosulfate; TR = 1.5 mL∙L-1 Thin Rite (United Phosphorus, Inc., King of Prussia, PA) wOn 14 May 2015, all trees received a post bloom thinning application: 100 mg∙ L-1 6- benzyladenine (6-BA, Excelis Plus, Fine Americas, Inc., Walnut Creek, CA) + 1 pint 1-naphthyl methylcarbamate (carbaryl)+ 1 pint adjuvant (Level 7; Winfield Solutions, LLC., St. Paul, Mn). vFruit russet = visual estimate of apple peel that exhibited russet. A 20 fruit sample was evaluated from each tree. uBCSA = branch cross-sectional area. tMean separation within columns by Tukey’s honest significance test at P = 0.05.

Return bloom was not influenced by any treatment. ‘Gala’ is an annual bearing cultivar and all treatments had sub-optimal crop loads in 2014. Blossom thinning treatments did not influence flower bud mortality in the year after treatment (data not presented). Blossom thinning treatments improved freeze tolerance of peach flower buds

in the following year (Byers and Marini, 1994; Edgerton, 1948), but no such evidence exists for apple.

Conclusions

While a decision-making aid that estimates pollen tube growth rates to inform timing of blossom thinning applications was utilized, two products that had apparent or potential impacts on Pn were the most effective thinners in this trial. The documented leaf injury and subsequent leaf abscission observed with TR likely had a negative effect on Pn. LS reduced Pn, and multiple applications can prolong the duration of Pn reduction

(McArtney et al., 2006). Pn was not measured in this experiment, but the limited influence of TR on pollen tube growth and initial fruit set suggest that an additional stress was imposed. Perhaps the partial reduction in primary spur leaf area reduced fruit set

(Ferree and Palmer, 1982).

TR reduced crop load in all experiments, and negatively impacted fruit size of

‘Gala’, but not ‘Golden Delicious’. In one of two years, LS + SO resulted in a near ideal crop load and increased fruit weight. ATS was effective in reducing pollen tube growth and initial fruit set in ‘Golden Delicious’ and ‘Gala’, but did not reduce whole tree crop density. NAD had limited efficacy on ‘Golden Delicious’ at the concentrations and application timings used in this trial.

In our opinion, if blossom thinning is to be adopted in the eastern US, partial crop load reduction is the desired outcome. Unpredictable weather conditions during apple bloom, particularly, the risk of spring frosts, are prevalent in the east. While best results were observed with HT, this practice would carry great risk and expense. While using the

pollen tube growth model as a timing aid, none of the chemistries evaluated over-thinned or increased fruit injury to commercially unacceptable levels. Also, initial fruit set was reduced by compounds that were proven to inhibit pollen tube growth in vivo, which is a desirable outcome. Limited fruit injury was attributed to favorable drying conditions, which minimized the amount of time that spray solutions rested on the fruit surface.

Initiation of the model is a critical and time sensitive step. Current methods to estimate the number of open blossoms require frequent counts on limbs or trees and the number of blossoms that are open can vary widely within a block or tree (Byers, 2003).

Development of improved sampling methods to estimate the number open blossoms should be considered in future research with the pollen tube growth model(s).

Pollen tube growth models were developed using king blossoms as the principal experimental unit. Differences in pollen tube growth rates existed when comparing king and side blossoms in ‘Golden Delicious’ (Losada and Herrero, 2013). Additional research is needed to determine if this relationship is true across commercially important cultivars.

Additionally, there were significant differences in the average style length between king and side blossoms. The current heuristic for the timing of secondary or tertiary applications using the pollen tube growth model is to wait until the estimated cumulative pollen tube growth is 60% to 70% of the average style length. If positional differences in pollen tube growth rates exist, perhaps modeling of pollen tube growth in side and/or lateral blossoms would increase the accuracy and precision of the model.

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Whiting, M. 2007. Impact of cultural practices on apple canopy physiology. Final project report #AH-04-412 to Washington Tree Fruit Res. Commission.

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214.

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Chapter 3

Thermal shock temperature and timing effects on apple stigmatic receptivity, pollen tube growth, and leaf injury

Introduction

2009).

There has been a significant effort to develop new blossom thinners for apple over the past 35 years. Approximately 150 chemistries and multiple iterations of tractor driven and hand-held mechanical thinners were evaluated (Bertschinger et al., 1998; Damerow et al., 2007; Embree and Foster, 1999; Martin-Gorriz et al., 2011; Rom and McFerson,

2006). Despite these efforts, apple growers in the majority of the United States do not have consistent, registered blossom thinning options. Though existing blossom thinners may reduce fruit set, there are negative consequences associated with chemical and mechanical blossom thinners, such as erratic responses from year-to-year (Byers, 1997;

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Webster and Spencer, 1999), a chronic reduction in spur leaf photosynthesis (McArtney et al, 2006), and chemical or mechanical injury to vegetative structures (Byers, 1997;

Kon et al., 2013). Spur leaves are important in promoting fruit growth, thus injury to these tissues had negative impacts on fruit size, fruit set, and fruit mineral content (Ferree and Palmer, 1982). A key to developing effective blossom thinners for apple is to limit injury to these important vegetative tissues.

The application of short duration thermal treatments to plant canopies is not a novel concept. Thermal treatments were applied to weeds via hot water, steam (Leon and

Ferreira, 2008), flaming, infrared (Ascard, 1998), and microwave (Brodie et al., 2012).

Many thermal weed control methods were effective in weed injury/mortality and inhibition of weed seed germination. However, thermal weed control has not been a cost effective measure as compared to conventional herbicides, and weed species differ in their thermal tolerance (Leon and Ferreira, 2008).

The possibility of using short duration applications of thermal energy (thermal shock; TS) as a blossom thinning method has not been investigated. Apple blossoms are sensitive to temperature stress. Apple styles and ovules are more sensitive to freezing temperatures when compared to surrounding floral tissues (Palmer et al., 2003). Pistils of apple blossoms at pink to full bloom can be killed at -3.9 to -1.7 ˚C with only 1 hour of exposure (Ballard et al., 1981). Temperature stress in plants is a complex interaction of the intensity, duration, and rate of temperature change (Wahid et al., 2007). As demonstrated with an ornamental lily, pollen tube growth in vivo was temporarily arrested with 10 s exposure to 45 ˚C (Pierson et al., 1993). Unlike leaves and pistils,

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mature pollen does not have heat tolerance mechanisms and is very sensitive to heat stress (Snider and Oosterhuis, 2011).

Heat stress effects on plant sexual reproduction has received considerable attention in the literature (Snider and Oosterhuis, 2011; Wahid et al., 2007; Zinn et al.,

2010). While increased ambient temperatures decreased the length of the effective pollination period (EPP; a concept devised to estimate the duration of flower receptivity;

(Williams, 1965), it is unknown if TS influences EPP of fruit trees. High temperatures increased the rate of apple pollen tube growth, but reduced the longevity of ovule and stigma lifespan/receptivity. Stigmatic receptivity can be a limiting factor in the length of the EPP (Sanozol, 2001). Since the purpose of the stigma is to adhere pollen grains and support pollen grain hydration and germination, damaging the stigma may result in reduced fruit set. Elevated air temperatures can reduce carbohydrate availability in pistils, resulting in reduced pollen tube growth and poor fruit set (Snider and Oosterhuis, 2011).

The onset of thermal injury to leaf tissue occurred over a very narrow temperature range. For example, there was no observable injury to soybean leaves that were subjected to a hot water bath of 53 ˚C for 1 min, but exposure to 54 ˚C caused chlorosis of leaf tissue and 55 ˚C caused necrosis (Daniell et al., 1966). Lethal temperatures caused disintegration of cellular membranes. TS could present a new method of blossom thinning apple, if application(s) prevents fertilization of later blossoms and spur leaf injury is minimal.

The purpose of this work is to determine: 1) effects of TS treatments on the duration of stigmatic receptivity, 2) effects of TS temperature and timing on pollen tube

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growth in vivo and visible spur leaf injury, and 3) if TS has potential as a crop load management strategy.

Materials and Methods

Trials were conducted in 2014 and 2015 at the Pennsylvania State University’s

Fruit Research and Extension Center in Biglerville, PA. ‘Crimson Gala’/’M.9’ trees planted in 2011 at 0.9 m x 3.7 m spacing were used for all experiments.

Expt.1: TS effects on stigmatic receptivity of ‘Crimson Gala’ apple.

Ninety spurs on 2- to 3-year-old-wood were selected when the king blossom was at late balloon stage. All side bloom was removed. Selected king bloom was emasculated and the spur was excluded from pollinators with a bag made of spunbonded row cover material to prevent unregulated pollination. Treatments were randomly assigned to solitary blossoms and flagged. On the following day, a range of TS treatments were applied to apple blossoms. A variable temperature heat gun set to an air flow rate of 0.50 m3 · min-1 was used to apply all treatments (Milwaukee 8988-20, Brookfield, WI). A gas-powered generator supplied electricity to the heat gun in the field. A data logging thermocouple (EL-GFX-TC; Lascar Electronics Inc., Erie, PA) was used to monitor the output temperature of the heat gun. The thermocouple probe was attached to the heat gun

2 cm away from the heat gun outlet. For a given treatment, a nominal heat value was set on the heat gun, and actual thermal output was recorded and is reported. After the heat gun temperature stabilized, all replicates of a given heat treatment were applied in ascending order. The start and stop time was recorded. The heat gun was positioned perpendicular to the calyx when heat treatments were applied to the pistil. Distance of the heat gun aperture from the pistil and duration of application were held constant (2 cm and

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2 s, respectively). All treatments were applied to nine leaves within one hour. Mean, standard deviation, and variance was determined for each level of temperature.

Descriptive statistics of TS treatments is provided in Table 3-1. In all subsequent tables and figures, mean output temperatures are presented as the explanatory variable.

Table 3-1. Expt. 1: Descriptive statistics for thermal shock (TS) output temperatures applied to ‘Crimson Gala’ in 2014 and 2015.z Mean Treatment ID (˚C) Standard Deviation 2014 1 15y − 2 48z 0.5 3 66 0.4 4 76 0.4 5 86 0.8 2015 1 19 − 2 48 3.9 3 63 4.2 4 78 3.4 5 87 3.6 zTreatments were applied on 3 May 2014 and 29 Apr 2015. In both years, all treatments were applied within 1 h. yControl treatment = ambient air temperature at timing of treatment. xAll heat treatments were applied with a variable temperature heat gun for 2 s. Distance from the heat gun aperture and the pistil was 2 cm.

Fifteen blossoms were hand pollinated with ‘Rome’ pollen using a painters brush on 0, 2, 4, 6, 8, and 10 d after treatment. Blossoms were collected 24 h after pollination, placed in labeled vials containing 5% sodium sulfite, and stored at 4 ˚C until analysis. A

106 modified version of the method of Embree and Foster (1999) was used to visualize pollen tube growth. Prior to microscopic examination, samples were autoclaved at 121 ˚C for 10 min to soften tissues. Blossoms were rinsed with distilled deionized water and the style was removed with a scalpel at the junction with the hypanthinum. Remaining floral tissues were discarded. Styles were rinsed, separated, and soaked in a water-soluble fluorescence solution of 0.01% Aniline Blue stain in 0.067 M K2HPO4 on microscope slide. Styles were squashed between two microscope slides and were incubated overnight at room temperature. Samples were observed using fluorescence microscopy at 100X

(BX51; Olympus Optical Co., Tokyo, Japan). A high pressure mercury vapor light source and UV/DAPI long pass filter cube was used (part 19000; Chroma Technology Corp,

Bellows Falls, VT). Style damage was visually rated (1-6 scale; 1 = no visible injury; 2 = trace to 10% style damaged; 3 = 11 to 25% style damaged; 4 = 26 to 50 % style damaged;

5 = 51 to 75% style damaged; 6 = 76 to 100% style damaged). Pollen tubes that germinated on the stigma, and entered the style were counted. The percentage of stigmas and stylets that supported pollen tube growth was calculated.

Expt. 2: TS effects on pollen tube growth in vivo.

Eighty ‘Crimson Gala’ spurs were selected using the same criteria described previously. Selected king blossoms were emasculated and excluded from pollinators. On the following day, all blossoms were hand pollinated with ‘Rome’ pollen using a painters’ brush. Using the methods described in Expt. 1, TS treatments were applied to apple blossoms at two distinct timings: 1) just prior to pollination, and 2) 24 h after pollination. Descriptive statistics of TS treatments for 2014 and 2015 are provided in

Table 3-2 and 3-3, respectively. In all subsequent tables and figures, mean output

107 temperature datum is presented as the explanatory variable. Blossoms were harvested 120 h after pollination and placed in a labeled vial containing 5% sodium sulfite and stored at

4 ˚C. Samples were prepared using the previously described methods.

Pollen density on the stigmatic surface was visually rated using a 0 to 10 scale, described in Yoder et al., 2009. Pollen tubes that entered the style were counted. Longest pollen tube length and style length were measured with an ocular micrometer.

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Table 3-2. Expt.2:Descriptive Table 3-3. Expt. 2:Descriptive statistics for thermal shock statistics for thermal shock treatments applied to ‘Crimson treatments applied to ‘Crimson zyx zyx Gala’ in 2014. Gala’ in 2015. Timing Mean Standard Timing Mean Standard w w (HAP) (˚C) Deviation (HAP) (˚C) deviation 0 15 − 0 12 − 36 0.4 35 1.4

43 0.6 43 2.0 51 0.3 55 2.8 54 0.5 61 2.7 60 0.4 64 2.4 63 1.5 66 3.0

64 4.6 72 2.1 66 3.4 79 1.9 68 4.5 84 1.4 24 14 − 24 18 − 27 0.3 39 1.5

37 0.9 52 2.4 47 0.4 62 1.9 49 1.0 67 2.8 55 1.7 73 3.0 58 1.8 77 2.4

63 2.3 84 1.9 67 1.1 89 1.8 70 1.9 95 1.4 zControl treatment was the zControl treatment was the ambient air temperature at ambient air temperature at timing of treatment. timing of treatment. yWith the exception of the yWith the exception of the controls, all heat treatments controls, all heat treatments were applied with a variable were applied with a variable temperature heat gun for 2 s. temperature heat gun for 2 s. Distance from the heat gun Distance from the heat gun aperture and the pistil was 2 cm. aperture and the pistil was 2 cm. At each timing, all treatments At each timing, all treatments were applied within 1 h. were applied within 1 h. xTiming 0 HAP was applied on 3 xTiming 0 HAP was applied on 1 May 2014 and 24HAP was May 2015 and 24 HAP was applied on 4 May 14. applied on 2 May 2015. w HAP = h after pollination. wHAP = h after pollination.

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Expt. 3: TS effects on visible spur leaf injury.

In 2015, 30 flowering spurs were randomly selected. Spurs were manipulated to permit unobstructed airflow to test leaves. At each spur, all blossoms were removed and three fully expanded spur leaves were selected and the rest were removed. Treatments were randomly assigned to spurs and were flagged. Using the methods described in Expt.

1, TS treatments were applied to persisting spur leaves at each spur. Spur leaf blades were held in a fixed position perpendicular to the heat gun aperture using forceps. The adaxial surface of the leaf was treated. Descriptive statistics of TS treatments are provided in Table 3-4. Visual injury to spur leaves was visually rated 7 d after treatment and the percentage of leaf tissue that exhibited injury was estimated.

Table 3-4. Expt. 3: Descriptive statistics for thermal shock output temperature applied to ‘Crimson Gala’ spur leaf tissue in 2015.zyx Tre M Standard atment ID ean (˚C) deviation 1 28 − 2 60 3.4 3 64 3.6 4 67 3.9 5 73 4.0 6 79 4.3 7 80 4.1 8 86 2.7 9 92 2.9 10 95 3.0 zControl treatment was the ambient air temperature at timing of treatment. yWith the exception of the control, all heat treatments were applied with a variable temperature heat gun for 2 s. xDistance from the heat gun aperture and the leaf was 2 cm.

110 Statistical analysis

All experiments had a completely randomized design. Two factors were evaluated in Expt.1, temperature and pollination day, and were structured in a factorial arrangement. Five levels of temperature (quantitative variable) and six pollination days

(qualitative variable) were tested – a total of 30 treatments. The experiment was replicated three times.

Two factors were evaluated in Expt. 2 - temperature and timing - and were structured in a factorial arrangement. Ten levels of temperature and two levels of timing were tested; a total of 20 treatments. The experiment was replicated four times. In Expt.

3, ten levels of temperature were evaluated and the experiment was replicated three times.

The PC version of SAS (Version 9.3; SAS Institute, Cary, NC) was used for all statistical analysis. Where appropriate, analysis of variance was used to test main effects and interactions and regression analysis was conducted via PROC GLM.

Results and Discussion

Expt.1: TS effects on stigmatic receptivity of ‘Crimson Gala’ apple.

In both years, TS temperature influenced stylar injury and pollen tube growth responses (Table 3-5). However, the effect of pollination day and the interaction between temperature and pollination day were not significant. We recognize that time (pollination day) is an influential factor in the duration of floral longevity. Maximal stigmatic receptivity of apple blossoms occurred at anthesis, and coincided with a loss in turgidity of stigmatic papillae (Losada and Herrero, 2012; Sheffield, et al., 2005). Secretions from the stigma support pollen adhesion, germination, and growth. Floral tissues are

111 ephemeral and receptivity of apple stigmas diminished with later pollination days

(Losada and Herrero, 2013). In this experiment, a total loss of stigmatic receptivity was observed on pollination day 8 or 6 (2014 and 2015, respectively), across all treatments.

However; parametric tests assume equal variance among samples. In 2014, responses on pollination days 8 and 10 violated assumptions of equal variance, and were not included in tests of main effects and interactions. Similarly, in 2015, blossoms pollinated on days 8 and 10 abscised prior to collection, and blossoms collected on pollination day 6 violated assumptions of equal variance and were not included in tests of main effects and interactions. There was no interaction between temperature and pollination day, indicating that slopes for each pollination day did not differ. This suggests that thermal injury to floral tissue and the subsequent reduction in pollen germination, and growth was acute. Therefore, linear and quadratic models were evaluated for each response variable using temperature as a predictor variable.

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Table 3-5. Main effects and interactions of thermal shock temperature and pollination date on stylar browning rating, pollen tube growth on the stigma, pollen tube growth in the style, percentage of receptive stigmas, and the percentage of stylets supporting pollen tube growth of 'Crimson Gala' in 2014 and 2015.z Significance (P>F) Response variable Temperature Pollination day Interaction 2014y Stylar browning < 0.0001 0.8869 0.4139 No. pollen tubes on stigmatic surface < 0.0001 0.4277 0.6256 No. pollen tubes in style < 0.0001 0.0707 0.2201 Receptive stigmas < 0.0001 0.8569 0.2515 Stylets supporting pollen tubes < 0.0001 0.2913 0.5049 2015x Stylar browning < 0.0001 0.2579 0.7730 No. pollen tubes on stigmatic surface 0.0004 0.3845 0.7243 No. pollen tubes in style 0.0018 0.3110 0.5306 Receptive stigmas 0.0021 0.9978 0.9367 Stylets supporting pollen tubes < 0.0001 0.6717 0.9500 zExperiment was a completely randomized design with a factorial treatment structure (5 quantitative temperatures x 6 quantitative pollination days) yTo avoid violating assumptions of equal variance, data from pollination days 8 and 10 were omitted from the data set (2014). xTo avoid violating the assumptions of equal variance, data from pollination days 6, 8, 10 data was omitted from the data set (2015).

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Table 3-6. Effects of thermal shock temperature on stylar browning rating, pollen tube growth on the stigma, pollen tube growth in the style, percentage of receptive stigmas, and percentage of stylets supporting pollen tube growth of 'Crimson Gala' in 2014 and 2015.z Temperature Stylar Browning No. pollen tubes No. pollen tubes in Receptive stigmas Stylets supporting (˚C) Rating (1-6)y on stigma style (%)x pollen tubes (%)w 2014 15 1.3 26.1 15.6 95 80 48 1.6 21.3 10.8 95 87 66 1.6 20.5 9.1 93 75 76 2.5 8.8 4.1 55 41 86 3.7 2.6 0.6 28 13 Significance Qv Q L Q Q P-value <0.0001u <0.0001 <0.0001 <0.0001 <0.0001 r2 0.51 0.44 0.37 0.59 0.55 2015 19 1.5 29.2 18.9 98 93 48 1.5 32.5 22.6 100 91 63 1.6 26.2 15.9 98 89 78 2.1 21.4 12.6 98 71 87 3.2 3.4 1.5 50 18 Significance Q Q Q Q Q P-value <0.0001 0.0002 0.0016 <0.0001 <0.0001 r2 0.60 0.34 0.26 0.43 0.58 zMeans of 5 observations. yStyle damage was visually rated (1-6 scale; 1=no visible injury; 2=trace; 3=25% style damaged; 4=50% style damage; 5=75% style damage; 6=100% style damaged). xReceptive stigmas = (no. of stigmas supporting pollen germination/ no. stigmas)*100. wStyles supporting pollen tubes = (no. of stylets supporting pollen germination/ no. stylets)*100. vL = linear model; Q = quadratic model. uP-value is for the model: n = 60 and 45 in 2014 and 2015, respectively.

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Chemicals can have detrimental effects on the morphology of the stigma, potentially accelerating papillae collapse (Yi et al., 2003). As TS temperature increased, a curvilinear increase in visible injury to stylar tissue was observed in both years (Table 3-

6). The onset of this injury corresponded with reduced pollen germination on the stigmatic surface and pollen tube growth in the stylar tissue. Similarly, chemical blossom thinners can cause oxidative injury to apple styles, and stylar injury ratings correlated well with reduced pollen tube growth and/or fruit set (Jandoui and Flore, 2005; Rom and

McFerson, 2006).

The highest level of TS temperature tested in each year (86 and 87 ˚C) had a strong inhibitory effect on pollen tube growth on the stigmatic surface and in the style.

When compared to ambient temperatures (control), these temperatures reduced the number of germinated pollen tubes on the stigmatic surface by 90% and 88% (2014 and

2015, respectively). Similarly, the highest level of temperature reduced numbers of pollen tubes that entered the style to 0.6 or 1.5 (2014 and 2015, respectively). In the style, pollen tube growth occurs in the transmitting tissue, a longitudinal sector of specialized cells, which provides proteins and polysaccharides to support pollen tube growth (Cresti et al.,

1980; Pratt, 1988). Damage to stigmatic papillae or the transmitting tissue may be detrimental to pollen tube growth and subsequent fruit set. In theory, fruit set may result if only one pollen tube fertilized one ovule. However, reduced pollen load resulted in poor fruit and seed set (Janse and Verhaegh, 1993). Most apple flowers have 5 carpels with 2 to 4 ovules per carpel (Pratt, 1988; 10-20 ovules per blossom). For potential of a full complement of seeds, however; at least 10 pollen tubes must enter the style and

115 fertilize the ovules (McArtney et al., 2006). Blossoms with limiting pollen tube number was observed at TS temperatures >66˚C and 86 ˚C in 2014 and 2015, respectively.

As TS temperature increased, a quadratic reduction in the proportion of stigmas and stylets that supported pollen tube growth was observed. Low TS temperatures did not reduce the percentage of receptive stigmas below 93%. Even at high temperatures, the percentage of receptive stigmas was 28% or 50% in 2014 and 2015, respectively.

Quantifying stigmatic receptivity as the percentage of stigmas that were capable of supporting pollen germination may be somewhat misleading. The germination of one pollen grain resulted in classifying a stigma as being receptive. When screening chemical blossom thinners, pollen tube growth in the upper style was significantly reduced, but not totally eliminated with effective chemicals (Embree and Foster, 1999). The highest level of temperature tested in each year (86 and 87 ˚C) reduced the percentage of styles that contained pollen tubes to less than 20%. Assuming a five carpel pistil, less than one stylet supported pollen tube growth per flower at these temperatures.

Expt. 2: TS effects on pollen tube growth in vivo.

When using the variable temperature heat gun to apply TS, consistent nominal output temperatures were set in each year, and actual thermal output was recorded with a data-logging thermocouple. Since the air source was not supplied, ambient air temperature and/or relative humidity appeared to affect the actual output temperature of the heat gun despite using the same nominal heat settings in each year (refer to Tables 3-2 and 3-3). Therefore, the range of TS temperatures tested differed in each year.

116 In both years, TS temperature influenced pollen tube growth responses (Table 3-

7). In 2014, TS temperature had a weak linear relationship with pollen density

(P=0.0039; r2=0.10) and the number of pollen tubes that entered the style (P=0.0347; r2=0.06) (Fig. 3-1). The weakness and trend of these relationships is likely explained by the limited high range of TS temperatures evaluated. In 2014, the highest temperature evaluated was 70 ˚C. In Expt. 1, pollen tube germination and growth was not consistently reduced/inhibited at < 86 ˚C. This suggests the range of TS temperatures evaluated in

2014 were too low to have an influence of practical significance on pollen germination and growth in vivo.

Table 3-7. Main effects and interactions of thermal shock temperature and pollination date on pollen density on the stigma, no. of pollen tubes that entered the style, and length of the longest pollen tube of 'Crimson Gala' in 2014 and 2015. Significance (P>F) Response variable Temperature Timing Interaction 2014 Pollen density 0.0022 0.1345 0.2181 No. pollen tubes entering style 0.0201 0.4866 0.9234 Length of longest pollen tube 0.0058 0.7816 0.8709 2015 Pollen density < 0.0001 0.0026 0.0006 No. pollen tubes entering style < 0.0001 0.0501 0.0371 Length of longest pollen tube < 0.0001 0.2785 0.2733 zAll heat treatments were applied with a variable temperature heat gun for 2 s. Distance from the heat gun aperture and the pistil was 2 cm. yExperiment was a completely randomized design with a factorial treatment structure (10 temperatures (quantitative) x 2 timings (qualitative).

117 In 2015, higher temperatures were applied resulting in a significant interaction term with pollen density (P = 0.0006) and the number of pollen tubes entering style (P =

0.0371). TS treatments applied 24 HAP provided ample time for pollen to germinate, and begin to grow down the style. Pollen grains germinated by 2 h after pollination and penetrated the style by 10 h (Losada and Herrero, 2014). A curvilinear reduction in pollen density and no. pollen tubes entering style was observed when TS was applied at 0 HAP.

Less than 10% of the stigma was covered in germinated pollen (pollen density rating = 1) and < 10 pollen tubes entered the style at temperatures > 79 ˚C.

118

24 HAP Y=2.485 - 0.016x 0 HAP 2 2015 y = -0.0131x + 3.3861 2014 r = 0.10 y = -0.0026x2 + 0.2531x - 2.7263 R² = 0.1505 P = 0.0039 r² = 0.631 5 5 P=0.0194 P=<0.0001 4.5 4.5

4 4

3.5 3.5 3 3 2.5 0 HAP 2.5

2 24 HAP 2 Pollen density Pollen Pollen density Pollen 1.5 1.5 1 1 0.5 0.5 0 0 A A0 20 40 60 80 100 B 0 B 20 40 60 80 100

45 y = 17.05 - 0.109x 45 0 HAP 24 HAP

r2 = 0.06 y = -0.0223x2 + 2.1711x - 22.644 y = -0.0093x2 + 0.7992x + 12.667 40 P = 0.0347 40 R² = 0.72 R² = 0.615 35 35 P=<0.0001 P=<0.0001 30 30 25 25 20 20 15 15 10 10

5 5

No . . . tubestubespollenpollen NoNoenteringentering style style 0 0 0 20 40 60 80 100 0 20 40 60 80 100 Temperature (˚C) Temperature (˚C) C C D D Fig. 3-1. Effects of thermal shock temperature and timing on pollen density (A and B) and the number of pollen tubes that enter the style (C and D) in 2014 and 2015. All heat treatments were applied with a variable temperature heat gun for 2 s. Distance from the heat gun aperture and the pistil was 2 cm. A visual rating scale was used to quantify pollen density on the stigma: 0 = no pollen tubes visible on the surface; 1 = 1% to 10 % area covered; 2 = 11% to 20%; 3 = 21% to 30%; 4 = 31% to 40%; 5 = 41% to 50%; 6 = 51% to 60%; 7 = 61% to 70%; 8 = 71% to 80%; 9 = 81% to 90%; 10 = 91% to 100% of surface covered by pollen tubes.

119

Blossoms in this experiment remained in situ on the tree for 5d prior to sampling; however, pollen tubes did not reach the style base in 2014 (Fig. 3-2). While pollen tubes can grow to the style base in less than 48 h, cool temperatures reduced pollen tube growth rates (Yoder, 2009). Cool spring temperatures were observed in 2014 (mean daily temp =

14.6 ˚C), and pollen tubes did not have sufficient time to grow to the style base before collection. The weakness of the relationship between TS temperature and pollen length

(r2 = 0.09) in 2014, may be explained in part by inhibition of all pollen tube growth caused by cool spring temperatures. Additionally, the narrow range of actual TS temperatures applied in 2014 may have been a function of the cool ambient temperatures.

Influential data points were observed at the higher temperatures (>63 ˚C) evaluated in

2014.

Warmer ambient temperatures occurred during bloom in 2015, and we observed pollen tubes at the style base after the 5 d incubation period (mean daily temp = 19.4 ˚C).

A curvilinear reduction in pollen tube length was observed with increased temperature in

2015. Temperatures greater than 79˚ C reduced pollen tube length to less than the average style length. Application timing and the interaction of temperature and timing did not affect pollen tube length in either year. The absence of an interaction between timing and temperature is important and may have practical implications. One of the most consistent apple blossom thinners lime sulfur (calcium polysulfide) was effective in reducing pollen tube growth up to 24 h after treatment (Yoder et al., 2009). The ability of a blossom thinner to reduce or inhibit pollen tube growth over a wide period of time is very desirable, since blossom thinning is a time sensitive operation and the number of

120

applications may be reduced (Schmidt and Elfving, 2007). Future research should consider the efficacy of TS at later timings and/or in relation to thermal time (i.e. growing degree days). Timing TS treatments with GDD may increase the repeatability of results and increase the precision of thinning outcomes.

14 y = 6.381 - 0.0396x r2 = 0.09 12 P = 0.0079

4 Pollen tube Pollen length(mm) 2

0 A 0 10 20 30 40 50 60 70 80 90 100

10 0 HAP 8 24 HAP 6

y = 10.52 - 0.0603x + 0.004693x2 - 0.000056x3 Pollen tube Pollen length(mm) 2 r2 = 0.69 P = <0.0001 0 0 10 20 30 40 50 60 70 80 90 100 Temperature (˚C) B

Fig. 3-2. Effects of thermal shock temperature on the average length of the longest pollen tube in 2014 and 2015 (A and B, respectively). All heat treatments were applied with a variable temperature heat gun for 2 s. Distance from the heat gun aperture and the pistil was 2 cm.

121

Expt. 3: TS effects on visible spur leaf injury.

Visible injury to leaf tissue was not observed with TS treatments below 67 ˚C, and was not included in analysis. As TS temperature increased, a curvilinear increase in leaf tissue that exhibited visible injury was observed (Fig.3-3). In general, injury to spur leaf tissue is undesirable. Spur leaf injury thresholds have been determined, by excision or mechanical wounding of spur leaf area. Hall and Ferree (1976) found that Pn was unaffected by a 10% removal of individual apple leaves, but higher rates of leaf removal reduced Pn. Ferree and Palmer (1982) demonstrated that removal of >33% of spur leaf area had negative consequences on fruit set, fruit size, and mineral nutrition. We observed >10% visible spur leaf injury at 86˚C, and >33% injury at 95˚C. The onset of visible leaf injury occurred over a relatively narrow temperature range, which accords with results of Daniell et al. (1969). In this experiment, minor leaf injury (<10%) occurred at temperatures that reduced or inhibited pollen tube growth in vivo.

50 y = 0.058x2 - 8.1456x + 285.79 r² = 0.7355 40 P= <0.0001

20 injury 10 0

0 10 20 30 40 50 60 70 80 90 100 % of leaf tissue with visible tissue of leaf % visible with Temperature (˚C)

Fig. 3-3. Effect of thermal shock temperature on visible spur leaf injury in 2015. The percentage of spur leaf tissue that exhibited injury was estimated visually. Visible injury to leaf tissue was not observed with TS treatments below 67 ˚C, and was not included in analysis. All heat treatments were applied with a variable temperature heat gun for 2 s. Distance from the heat gun aperture and the pistil was 2 cm.

122

Conclusions

This was exploratory work to determine the potential of a previously untested crop load management strategy. A series of small-scale trials using a variable temperature heat gun to apply various TS output temperatures to solitary blossoms and spur leaves was conducted. Specifically, a range of TS temperatures that reduced apple pollen tube growth in vivo was identified. TS temperatures >86 ˚C had a strong and consistent inhibitory effect on pollen tube growth on the stigmatic surface and in the style. At effective temperatures, TS effects were acute and did not influence the duration of the stigmatic receptivity. Additionally, pollen tube growth was reduced up to 24 h after the pollination event, TS temperatures > 79˚ C reduced pollen tube length to less than the average style length. While minimal spur leaf injury (<10%) occurred at 86˚C, unacceptable leaf injury (>33%) was observed at 95 ˚C. While our data show that TS was effective in reducing pollen tube growth in vivo, the onset of visible injury to leaf tissue occurred at similar temperatures. Additional work is required to validate and further develop this concept.

Some challenges in using forced heated air as a TS delivery system were identified. Much like chemical thinning, environmental effects appeared to influence the efficacy of the treatment. The likely influence of the environment on heat gun output temperatures is problematic, and resulted in application of lower output temperatures than expected. While not considered in this study, several factors may influence heat transfer by convention the initial temperature of tissues, relative humidity, wind speed, and physical structure of the tissues may influence the amount of heat energy transferred by

123

convection (Hamer, 1985; Tsilingiris, 2008). Additional application considerations, such as duration of treatment and canopy distance from the heat source, would influence thermal transfer to reproductive or vegetative tissues of apple. Use of forced heated air is a relatively inefficient method of heat transfer. More efficient methods of heat transfer, such as infrared or steam should be considered in future research. However, given the relatively narrow difference in thermal sensitivity between reproductive and vegetative tissues, use of inefficient methods of heat transfer may be advantageous to limit injury to non-target tissues.

124

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Chapter 4

Apple pollen tube growth and spur leaf injury in response to thermal shock temperature and duration

Introduction

Blossom thinning can confer significant benefits to apple growers, including increased fruit size and annual bearing (Batjer, 1965). Early thinning reduces competition among fruit, which increases fruit cell division and size potential (Lakso et al., 1996). However, apple growers in the majority of the United States do not have consistent, registered blossom thinning options. While existing chemical and mechanical blossom thinners may reduce apple fruit set, blossom thinners are destructive by nature and can cause injury to non-target tissues.

As described previously (Chapter 3), the use of short duration applications of thermal energy (thermal shock; TS) as an apple blossom thinning method was not previously investigated. Apple blossoms are sensitive to temperature stress. Apple styles and ovules are more sensitive to freezing temperatures when compared to surrounding floral tissues (Palmer et al., 2003). Temperature stress in plants is a complex interaction of the intensity, duration, and rate of temperature change (Wahid et al., 2007). While reproductive tissues are more sensitive to chronic heat stress when compared to vegetative tissues (Snider and Oosterhuis, 2011), information comparing tissue sensitivity to short duration TS is limited. The individual and combined effects TS temperature and treatment duration on pollen tube growth in vivo and visible spur leaf injury was evaluated.

130 Materials and Methods

Trials were conducted in 2014 and 2015 at the Pennsylvania State University’s

Fruit Research and Extension Center in Biglerville, PA. Experiments were carried out on

‘York Imperial’/Bud. 9’ planted in 2004 at 1.5 m x 4.6 m spacing.

Expt.1: Effects of TS temperature and treatment duration on stigmatic receptivity of ‘York’ apple.

Eighty spurs on 2- to 3-year-old-wood were selected when the king blossoms were at late balloon stage. All side blooms were removed, and selected king blossoms were emasculated. The spur was excluded from pollinators with a bag made of spunbonded row cover material to prevent unregulated pollination. Treatments were randomly assigned to solitary blossoms and flagged. On the following day, blossoms were hand pollinated with ‘Rome’ pollen. A range of TS treatments were applied to solitary blossoms 24 h after pollination, using a variable temperature heat gun set to an air flow rate of 0.50 m3 · min-1 (Milwaukee 8988-20, Brookfield, WI). Effects of output temperature (five levels) and treatment duration (four levels; 0.5, 1, 2, or 4 s) were evaluated in a completely randomized design with a factorial treatment structure and was replicated four times. A gas-powered generator supplied electricity to the heat gun in the field. A data logging thermocouple (EL-GFX-TC; Lascar Electronics Inc., Erie, PA) was used to monitor the output temperature of the heat gun. The thermocouple probe was attached to the heat gun 2 cm from the heat gun outlet. For a given treatment, a nominal heat value was set on the heat gun, and actual thermal output was recorded. After the heat gun temperature stabilized, all replicates of a given heat treatment were applied in ascending order. The start and stop time was recorded. The heat gun was positioned

131 perpendicular to the calyx when heat treatments were applied to the pistil. Distance of the heat gun aperture from the pistil was held constant at 2 cm. All treatments were applied within one hour. Mean and standard deviation was determined for each level of the factor temperature. Descriptive statistics of TS treatments is provided in Table 4-1. In all subsequent tables and figures, mean output temperatures are presented as the explanatory variable.

Table 4-1. Expt. 1: Descriptive statistics for thermal shock (TS) output temperatures applied to ‘York’ blossoms in 2014 and 2015.z Mean Standard Nominal treatment (˚C) Deviation 2014 Control 18y − 49 41x 0.4 66 56 0.5 82 73 1.1 93 83 0.8 2015 Control 23 − 49 51 3.6 66 66 4.4 82 81 3.5 93 89 3.8 zIn both years, all treatments were applied within 1 h. yControl treatment = ambient air temperature at timing of treatment. xAll heat treatments were applied with a variable temperature. Distance from the heat gun aperture and the pistil was 2 cm.

Blossoms were collected 24 h after pollination, placed in a labeled vial containing

5% sodium sulfite, and stored at 4 ˚C until analysis. A modified version of the method of

132 Embree and Foster (1999) was used to visualize pollen tube growth. Prior to microscopic examination, samples were autoclaved at 121 ˚C for 10 min to soften tissues. Blossoms were rinsed with distilled deionized water and the style was removed with a scalpel at the junction with the hypanthinum. Remaining floral tissues were discarded. Styles were rinsed, separated, and soaked in a water-soluble fluorescence solution of 0.01% Aniline

Blue stain in 0.067 M K2HPO4 on a microscope slide. Styles were squashed between two microscope slides and were incubated overnight at room temperature. Samples were observed using fluorescence microscopy at 100X (BX51; Olympus Optical Co., Tokyo,

Japan). A high pressure mercury vapor light source and UV/DAPI long pass filter cube was used (part 19000; Chroma Technology Corp, Bellows Falls, VT). Style damage was visually rated (1-6 scale; 1 = no visible injury; 2 = trace to 10% style damage; 3 = 11 to

25% style damage; 4 = 26 to 50 % style damage; 5 = 51 to 75% style damage; 6 = 76 to

100% style damage). Pollen density on the stigmatic surface was visually rated using a 0 to 10 scale, described in Yoder et al., 2009. Pollen tubes that entered the style and the number that reached the style base were counted. Longest pollen tube length and style length were measured with an ocular micrometer.

Expt. 2. Effects of TS temperature and treatment duration on visible spur leaf injury of ‘York’ apple.

Sixty flowering spurs on 2- to 3-year-old-wood were selected. Spurs were manipulated to permit unobstructed airflow to test leaves. At each spur, all blossoms were removed, three fully expanded spur leaves were selected, and the rest were removed. Treatments were randomly assigned to spurs and flagged. Using the methods

133 described in Expt. 1, TS treatments were applied to persisting spur leaves at each spur.

Spur leaf blades were held in a fixed position perpendicular to the heat gun aperture using forceps. The adaxial surface of the leaf was treated. Descriptive statistics of TS treatments is provided in Table 4-2. One week after treatment, leaf injury was visually rated (1-6 scale; 1 = no visible injury; 2 = trace to 10% leaf damage; 3 = 11 to 25% leaf damage; 4 = 26 to 50 % leaf damage; 5 = 51 to 75% leaf damage; 6 = 76 to 100% leaf damage).

Table 4-2. Expt. 2: Descriptive statistics for thermal shock (TS) output temperatures applied to ‘York’ spur leaves in 2014 and 2015.z Nominal Mean Standard Treatment (˚C) Deviation 2014 Control 28y − 49 56x 0.9 66 70 1.2 82 81 2.2 93 92 1.9 2015 Control 28 − 49 57 3.1 66 71 3.6 82 84 2.8 93 94 3.3 zIn both years, all treatments were applied within 1 h. yControl treatment = ambient air temperature at timing of treatment. xAll heat treatments were applied with a variable temperature heat gun. Distance from the heat gun aperture and the pistil was 2 cm.

134 Effects of output temperature (five levels) and treatment duration (four levels; 0.5,

1, 2, or 4 s) were evaluated in a completely randomized design with a factorial treatment structure. The experiment was replicated three times.

Statistical analysis

The PC version of SAS (Version 9.3; SAS Institute, Cary, NC) was used for all statistical analysis. Main effects and interactions were determined using analysis of variance. In several cases, the interaction between temperature and duration was significant. In these instances, regression analysis was conducted via PROC GLM at each level of duration.

Results and Discussion

Expt.1: Effects of TS temperature and treatment duration on stigmatic receptivity of ‘York’ apple.

Visible injury to stylets was influenced by the interaction between temperature and duration in both years (Table 4-3). As TS temperature increased, greater visible injury to styles was observed; however, the magnitude of the observed increase in injury was greater with longer treatment durations (Table 4-4). Specialized glandular cells in the style (transmitting tissue) were shown to provide resources to support pollen tube growth

(Losada and Herrero, 2014). In chemical thinning trials, stylar browning corresponded with blossom thinner efficacy (Rom and McFerson, 2006). Regardless of treatment, it was rare to observe stylets with greater than 75% of the style exhibiting injury (injury rating = 6). Since apple styles are fused at the base, basal portions of the style presumably have a greater thermal tolerance when compared to the upper style.

135

Significance (P>F) Response variable Temperature Duration Interaction

2014 Stylar browning < 0.0001 0.5275 0.0129 Pollen Density 0.3026 0.2887 0.4379 No. pollen tubes in style 0.4671 0.3417 0.2499 Length of longest pollen tube < 0.0001 0.6896 0.0038 No. pollen tubes at style base < 0.0001 0.4519 0.0191 2015 Stylar browning < 0.0001 0.0048 <0.0001 Pollen Density 0.2414 0.8484 0.4648

No. pollen tubes in style 0.0010 0.1675 0.0038 Length of longest pollen tube <0.0001 0.0195 <0.0001

No. pollen tubes at style base 0.4055 0.1436 0.0088

The interaction between temperature and duration was significant for all response variables evaluated, with two exceptions: pollen density rating (means not presented,

2014 and 2015) and the number of pollen tubes entering the style (2014 only). Pollen was applied to all blossoms 24 h before TS application. This provided ample time for pollen grains to germinate, as Losada and Herrero, (2014) showed that apple pollen germination occurred within 2 h after deposition on the stigmatic surface (. Pollen tube cell walls contain callose (Currier, 1957), a polysaccharide and plant cell constituent. Stigmatic and stylar transmitting tissues are devoid of callose (Losada and Herrero, 2014). When viewed via fluorescence microscopy and stained with Aniline Blue, callose present in germinated pollen glows vividly. In this experiment, callose tissue from germinated pollen grains was still visible despite visible damage to the stylar tissue. When chemical blossom thinners were applied to blossoms that were hand pollinated 24 h prior to

136 treatment, pollen tubes were still visible, but were brown and faded (Embree and Foster,

1999). Similar injury to germinated pollen tubes described by Embree and Foster (1999) with TS treatments was observed.

The number of pollen tubes that penetrated the style was not influenced by TS temperature, duration, or the interaction between temperature and duration in 2014. This corresponds with the limited effect of TS treatments on pollen germination, and is a function of the delayed application timing (24 HAP) evaluated in this experiment. In

2015, there was no relationship between TS temperature and the pollen tubes entering the style at 0.5 s duration. However, at 2.0 and 4.0 s durations, the number of visible pollen tubes entering the style was reduced linearly as temperature increased. At the highest temperature evaluated (89 ˚C) the number of pollen tubes entering the style was reduced

43% and 83% at 2 and 4 s treatment durations, respectively.

137

Table 4-4. Effects of thermal shock temperature and duration on pollen density and the number of pollen tubes penetrating the style of hand-pollinated 'York Imperial' blossoms in 2014 and 2015.z Mean Temperature Duration (s) (C˚) 0.5 1.0 2.0 4.0 0.5 1.0 2.0 4.0 Stylar browning (1-6)y No. pollen tubes penetrating style 2014 18 1.1 1.1 1.3 1.9 15.9 16.8 13.9 9.9 41 1.0 1.0 1.1 1.0 14.4 19.6 16.0 19.1 56 1.2 1.0 2.8 4.8 12.8 16.8 12.3 13.0 73 1.0 2.6 4.8 5.0 16.8 14.9 7.4 23.0 83 3.0 5.0 5.0 5.0 14.8 14.4 23.0 18.0 Significance Qx Q L L − − − − P-value 0.0038w <0.0001 <0.0001 <0.0001 − − − − r2 0.50 0.87 0.69 0.62 − − − − 2015 23 1.6 1.6 1.5 1.4 20.1 16.8 25.0 21.5 51 1.5 1.2 1.6 1.3 17.4 23.1 14.9 21.2 66 1.2 1.7 2.3 3.2 24.3 24.6 22.5 10.4 81 1.5 2.4 3.7 4.3 15.9 20.0 8.7 8.8 89 1.2 2.8 4.3 4.9 21.5 17.1 14.3 3.6 Significance NS L Q Q NS Q L L P-value − 0.0383 <0.0001 <0.0001 − 0.0442 0.0290 <0.0001 r2 − 0.22 0.76 0.89 − 0.31 0.24 0.56 zAll thermal treatments were applied with a variable temperature heat gun at 2 cm distance from the pistil. yStyle damage was visually rated (1-6 scale; 1 = no visible injury; 2 = trace to 10% style damaged; 3 = 11 to 25% style damaged; 4 = 26 to 50 % style damaged; 5 = 51 to 75% style damaged; 6 = 76 to 100% style damaged). x In cases where a significant interaction was observed, each level of duration was analyzed separately. L = linear model; Q = quadratic model; NS = not significant. wP-value is for the model.

138

In 2014, a linear reduction in the pollen tube length with 2 and 4 s treatment durations was observed (Table 4-5). At 2 and 4 s durations, temperatures > 56 ˚C resulted in pollen tube length being less than 50% of the average style length (9.2 mm) and prevented pollen tubes from reaching the style base. While a minor reduction in pollen tube length was observed with increased temperature at 0.5 s treatment duration, these short duration treatments did not affect the number of pollen tubes that reached the style base.

In 2015, there was no relationship between TS temperature and pollen tube length with shorter duration treatments (0.5 and 1.0 s). However, at 2 and 4 s durations, curvilinear reductions were observed in pollen tube length with increasing TS temperature. The average style length in 2015 was 8.8 mm (data not presented), and high temperatures (> 81 ˚C) at 4.0 s duration limited pollen tube length to 50% of the average style length. TS temperature did not influence the number of pollen tubes that reached the style base with 0.5, 1.0, and 2.0 s treatment durations. At 4.0 s duration, a curvilinear reduction in the number of pollen tubes that reached the style base was observed.

TS temperature effects on the number of pollen tubes that reached the style base were not consistent among years. In 2014, TS temperatures > 56 ˚C prevented pollen tubes from reaching the style base at 2 and 4 s durations. However, much higher temperatures were required to prevent pollen tubes from reaching the style base in 2015.

The reasons for this inconsistency are unclear. While TS treatments were applied at the same timing in each year (24 h after pollination), the rate of pollen tube growth in vivo may have been more rapid in 2015 due to warm temperatures during the 24 h period

139 following pollination (mean temperature = 19.8 ˚C). Perhaps pollen tubes penetrated far enough into the style to avoid injury caused by TS treatments. At constant temperatures >

24 ˚C, pollen tubes reached the style base of ‘Golden Delicious’ within 24 h (Yoder et al.,

2009). Maternal cultivar can influence pollen tube growth rates (Yoder et al., 2013).

Pollen tube growth rates in ‘York Imperial’ have not been evaluated, making a direct comparison difficult. During pollen tube growth, the cytoplasm and sperm cells are positioned near the apex of the pollen tube. Deposits of callose (i.e. callose plugs) isolate the actively growing portion of the pollen tube (Losada and Herrero, 2014). To arrest the growth of pollen tubes with a blossom thinner, the actively growing pollen tube tip or adjacent transmitting tissue must be disrupted or damaged. It is possible that our treatments were applied too late to have an impact in 2015 because the pollen tube growing point had progressed beyond the more distal regions of the stylar tissue that were affected by treatment.

140

Table 4-5. Effects thermal shock temperature and duration on the length of the longest pollen tube and number of pollen tubes at style base of hand-pollinated 'York Imperial' blossoms in 2014 and 2015.zx Mean Temperature Duration (s) (C˚) 0.5 1.0 2.0 4.0 0.5 1.0 2.0 4.0 Length of longest pollen tube (mm) No. pollen tubes at style base 2014 18 8.7 8.7 8.2 8.9 3.2 3.1 4.5 4.1 41 10.0 9.1 8.6 8.6 5.0 6.2 5.1 2.5 56 9.1 8.1 6.2 4.3 4.4 3.7 0.0 0.1 73 8.9 8.4 3.3 3.9 3.9 2.4 0.2 0.0 83 7.5 7.5 5.3 3.8 3.3 1.2 0.1 0.0 Significanceu Q NS L L NS Q L Q P-valuet 0.0337 − 0.0048 0.0006 − 0.0097 0.0003 <0.0001 r2 0.35 − 0.40 0.51 − 0.44 0.55 0.78 2015 23 8.7 9 8.9 8.9 4.8 2.7 5.5 4.1 51 8.92 9.2 9 8.7 3.9 8.5 3.7 9.5 66 9.1 9.1 8.1 7.6 5.8 4.1 6.4 2.7 81 8.8 8.8 7.4 4.4 6 7.5 2.65 1.5 89 8.4 8.3 5.5 1.3 8.8 4.1 1.8 0.05 Significance NS NS Q Q NS NS NS Q P-value − − 0.0025 <0.0001 − − − 0.0018 r2 − − 0.51 0.77 − − − 0.52 zBlossoms were hand pollinated 24 h prior to treatment. xThermal treatments were applied with a variable temperature heat gun at 2 cm distance.

141

Table 4-6. Effects thermal shock temperature and duration on visible injury ratings of 'York Imperial' spur leaves in 2014 and 2015.zy Mean Temperature Duration (s) (C˚) 0.5 1.0 2.0 4.0 Visible leaf injury (1-6) 2014 28 1 1 1 1 56 1 1 1 1.3 70 1 1 2.7 4.3 81 1.3 2 3.3 5.3 92 1.3 2.3 5 6 Significance NS Ly Q Q P-value − 0.0004x <0.0001 <0.0001 r2 − 0.73 0.89 0.95 2015 28 1 1 1 1 57 1 1 1.1 1.2 71 1 1 2 4.2 84 1 1.2 4.7 5.1 94 1 1.9 5 6 Significance − L L Q P-value − 0.0127 <0.0001 <0.0001 r2 − 0.48 0.92 0.95 zOne week after treatment, leaf injury was visually rated (1 to 5 scale: 1= No visible damage; 2 = trace to 10% damage; 3 = 11% to 25% damage; 4 = 26% to 50% damage; 5 =51% to 75% damage; 6 = 76% damage to 100% damage). yL = linear model; Q = quadratic model; NS = not significant. xP-value is for the model

Expt. 2. Effects of TS temperature and treatment duration on visible spur leaf injury of ‘York’ apple.

Relationships between visible injury and TS treatments to spur leaves were very consistent in both years of this trial. At 0.5 s duration, TS temperatures did not influence visible spur leaf injury. While, spur leaf injury increased when temperatures increased at

142 1.0 s duration, none of the temperatures evaluated resulted in injury to spur leaves >33 %.

Ferree and Palmer (1982) demonstrated that removal of >33% of spur leaf area removal had negative consequences on fruit set, fruit size, and mineral nutrition. At 2.0 and 4.0 s, visible injury to spur leaf tissue exceeded a rating of 4 (4 = 26% to 50% damage) at temperatures greater than 84 ˚C and 70 ˚C, respectively.

Conclusions

At the range of TS temperatures tested, short duration forced air treatments (0.5 and 1.0 s) were ineffective in arresting pollen tube growth in vivo and had negligible effects on spur leaf injury. A minimum of 2.0 s TS treatment duration was required to elicit a meaningful reduction in pollen tube growth in vivo. Leaf injury responses to TS were consistent in both years of this trial, and suggest that TS strategies based on forced air have an upper threshold of 70 ˚C for 2 s to avoid visible leaf injury.

TS was inconsistent across years in reducing pollen tube growth in the style, and we speculate that the lower efficacy in 2015 was primarily a timing issue. Based upon the

2014 outcomes, 56 ˚C for 2 s would prevent fertilization, at a temperature well below that required to damage leaves. Use of a pollen tube growth model, and the ability to deploy

TS treatments rapidly would be essential to its successful deployment as a thinner. The strong interaction between temperature and duration in arresting pollen tube growth suggests that longer durations (4 s) may present a strategy to extend the efficacy of TS in seasons when ambient temperature favors rapid pollen tube growth.

143

Literature Cited

Batjer, L.P. 1965. Fruit thinning with chemicals. Agri. Res. Bull. No. 289, Ag. Res.

Service, USDA, Washington DC, 27 pp.

Currier, H.B. 1957. Callose substance in plant cells. Amer. J. Bot. 44(6):478-488.

Embree, C.G. and A. Foster. 1999. Effects of coatings and pollenicides on pollen tube growth through the stigma and style of ‘McIntosh’ apple. J. Tree Fruit Prod. 2(2):19-32.

Ferree, D.C. and J.W. Palmer. 1982. Effect of spur defoliation and ringing during bloom on fruiting, mineral level, and net photosynthesis of ‘Golden Delicious’ apple. J. Amer.

Soc. Hort. Sci. 107:1182-1186.

Lakso, A.N., T.L. Robinson, and M.C. Goffinet. 1996. Influence of fruit competition on size, and the importance of early thinning. New York Fruit Qrtrly.4(1):7-9.

Losada, J.M. and M. Herrero. 2014. Glycoprotien composition along the pistil of Malus x domestica and the modulation of pollen tube growth. Plant Biol. 14:1-14.

Palmer, J.W., J.P. Privé and D.S. Tustin. 2003. Temperature, p. 217-236. In: D.C. Ferree and I.J. Warrington. (eds.). Apples: botany, production and uses. CAB International,

Oxfordshire, UK.

Rom, C. R. and J. McFerson. 2006. Alternative apple crop thinning strategies. Final project report #AH-04-415 to Washington Tree Fruit Res. Commission.

144 Snider, J.L. and D.M. Oosterhuis. 2011. How does timing, duration and severity of heat stress influence pollen-pistil interactions in angiosperms? Pl. Signaling Behavior.

6(7):930-933.

Wahid A, Gelani S, Ashraf M, Foolad MR. 2007. Heat tolerance in plants: an overview.

Environ. Expt. Bot. 61:199–223.

Yoder, K.S., G.M. Peck, L.D. Combs and R.E. Byers. 2013. Using a pollen tube growth model to improve apple bloom thinning for organic production. Acta Hort. 1001:207-214.

145 Chapter 5

Summary and Conclusions

Early crop load management practices for apple was reviewed and blossom thinning products that were tested on apple since 1989 was catalogued (Chapter 1).

Approximately 150 blossom thinning chemistries were screened within this time frame.

While promising results were observed with several screened blossom thinners, many of these products lack proprietary exclusivity. Blossom thinner product development has been limited due to the risk of crop damage and high costs of product registration. Only four blossom thinning chemicals are currently used in commercial apple production in the

United States, and use of these products is restricted to specific locations. Therefore, improving the consistency of existing blossom thinners and/or developing non-chemical crop load management strategies is a research priority. The focus of this research was to compare the efficacy of currently available blossom thinning products while using a predictive model, and to evaluate the potential of short duration heat treatments (thermal shock; TS) as an apple crop load management strategy.

A recently developed predictive model was used to determine application timing in a comparison of several blossom thinning products (Chapter 2). While thinning responses were observed with several of the products evaluated, products with apparent or potential impacts on Pn were the most effective. While endothal (TR) application did not influence measured responses of pollen tube growth, TR was effective in reducing crop load in all experiments. TR resulted in excessive foliar injury on two cultivars, and caused excessive thinning in an experiment conducted in a commercial orchard.

Commonly anticipated problems associated with blossom thinner applications (increased

146 russet, excessive phytotoxicity, over-thinning, etc.), with calcium polysulfide (LS), ammonium thiosulfate (ATS), and naphthaleneacetamide (NAD) was not observed.

Partial crop load reduction was achieved with LS, ATS, and TR. The hormonal thinner

NAD had limited efficacy on ‘Golden Delicious’, which is a difficult-to-thin cultivar. The inclusion of blossom thinners in multi-step crop load management should be evaluated in future work, and physiological and economical effects of these treatments should be quantified. While the pollen tube growth model was a useful tool, developing efficient sampling procedures may further improve consistency.

Effects of TS temperature and timing were evaluated in multiple experiments

(Chapter 3). TS effects on stigmatic receptivity were consistent in a two-year study. TS temperatures > 86 ˚C resulted in reduced pollen germination and growth in stylar tissue in

‘Crimson Gala’. Effects on stigmatic receptivity were acute, and did not affect the duration of stigmatic receptivity. The data indicated that a prophylactic application of TS may be effective in limiting pollen tube growth in vivo, reducing the probability of fertilization and subsequent fruit set. Timing (0 or 24 h after pollination) was not an influential factor, indicating that effective TS temperatures reduced pollen tube growth up to 24 h after the pollination event. The onset of thermal injury to vegetative tissues occurred at similar TS temperatures that inhibited pollen tube growth in vivo, and unacceptable leaf injury (>33%) was observed at 95 ˚C.

Effects of TS temperature and treatment duration on pollen tube growth in vivo and leaf injury was evaluated (Chapter 4). Short duration treatments (0.5 and 1.0 s) were ineffective in arresting pollen tube growth in vivo. In 2014, TS temperatures > 56 ˚C inhibited pollen tubes from reaching the style base at 2.0 and 4.0 s durations. In 2015, TS

147 temperatures > 81 ˚C at 4.0 s prevented pollen tubes from reaching the style base.

Excessive injury to spur leaf tissue was observed at temperatures greater than 84 ˚C and

70 ˚C (2.0 and 4.0 s, respectively),

Inconsistent TS temperatures and/or responses were observed in these experiments. Environmental effects may have influenced the results. Ambient air temperature influenced heat gun output temperatures and resulted in application of lower output temperatures than expected (Chapter 3). Inconsistent effects of TS on pollen tube growth metrics was observed in 2015, and was attributed to treatments being applied too late, due to optimal conditions for pollen tube growth in the intervening 24 hr period after the pollination event. Bloom thinning using chemical and thermal treatments is a time sensitive operation, and efficacy is influenced by environmental conditions. TS is perhaps even more time sensitive, since the effects of TS last only seconds.

While not evaluated in this work, differences in floral structure among cultivars could play a role in the efficacy of TS treatments. To the best of our knowledge, limited research has been conducted on floral structure differences among new cultivars.

Identifying and exploiting structural differences between apple blossoms and vegetative spur leaves may provide insight to future development of TS, or other attempts at developing selective thinning technologies. While our evaluation was focused on king blossoms, comparing the thermal sensitivity of side/lateral blossoms would be of value.

Since king blossoms are larger than lateral blossoms, heat transfer to king blossoms may differ.

For purposes of consistency, solitary blossoms and spur leaf blades were held in a fixed position perpendicular to the heat gun aperture. Also, test blossoms were

148 emasculated to control the timing of the pollination. That this method would not mimic air flow in a plant canopy using convective heat transfer. If application of TS is evaluated on whole plant canopies, more efficient methods of thermal transfer to plant canopies should be considered.

Regardless of the method of heat transfer, canopy structure and distance of the heat source from the target are important considerations. The relatively recent adoption of high density orchards with narrow “tree wall” canopies could facilitate the application of

TS to whole plant canopies.

VITA Thomas M. Kon

Education

Ph.D. 2012 – Present. The Pennsylvania State University. Degree in Horticulture

M.S. 2012. The Pennsylvania State University. Degree in Horticulture

B.S. 2009. University of Nebraska. Degree in Horticulture

Manuscripts published in refereed journals

Schupp, J.R. and T.M. Kon. 2014. Mechanical blossom thinning of ‘GoldRush’ / M.9 apple trees with two string types and two timings. J. Amer. Pomol. Soc. 68:24-32.

Kon, T.M. and J.R. Schupp. 2013. Thinning tall spindle apple based on estimations based made with a hand-thinning gauge. HortTechnology 23:830-835.

Schupp, J.R., T.M. Kon, and H.E. Winzeler. 2012. 1-Aminocyclopropane carboxylic acid shows promise as a chemical thinner for apple. HortScience 47:1308-1311. Review article

Kon, T.M. and J.R. Schupp. 2015. Pollen tube growth in apple: a review. J. Amer. Pomol. Soc. 69(3):158-163. Awards

2015. J. Franklin and Agnes T. Styer Scholarship in Horticulture. College of Agricultural Sciences – Penn State University. 2015. U.P. Hedrick Award; 1st Place Paper by a Student. Pollen tube growth in apple: a review. American Pomological Society. 2014. 1st Place Oral Presentation by a Student: Mechanical blossom thinning ‘GoldRush’ apple trees with two string types and two timings. Northeastern Region of the American Society for Horticultural Science. 2012. Frederick H. Brown Scholarship in Floriculture; Penn State Horticulture Department. 2011. 1st Place Poster Presentation in Health and Life Sciences: Mechanical blossom thinner effectively removes apple blossoms, but may injure trees. The Graduate Exhibition; The Graduate School of Penn State.