ABSTRACT

THE EVALUATION OF PLANT GROWTH REGULATORS ON SCARLET ROYAL TABLE GRAPES TO DETERMINE POST HARVEST QUALITY

The incidence of Botrytis cinerea and other diseases as well as berry quality parameters were evaluated on Scarlet Royal after post-harvest storage using different combinations of plant growth regulators (PGR) during the growing season. Three treatments including a Control (no PGR applied), 5 ppm

Gibberellic Acid (GA3) and a combination of 5 ppm GA3 + 6 ppm Forchlorfenuron (CPPU) were applied. After commercial cold storage was completed, berries were evaluated for Botrytis bunch rot, berry crack, berry shatter, other diseases and other damage. Normal berry quality parameters were also evaluated including pH, titratable acidity, total soluble solids, berry firmness, berry diameter, berry length and skin color.

Results showed no significant differences for Botrytis bunch rot incidence, berry crack, other diseases or other damage. However, berry shatter was significantly higher for the 5 ppm GA3 treatment. The three treatments showed no effect on berry firmness, berry diameter, berry length, juice pH, titratable acidity and total soluble solids. Furthermore, berry color presented no differences in lightness, chroma or hue.

Victoria Towers May 2015

THE EVALUATION OF PLANT GROWTH REGULATORS ON SCARLET ROYAL TABLE GRAPES TO DETERMINE POST HARVEST QUALITY

by Victoria Towers

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Viticulture and Enology in the Jordan College of Agricultural Sciences and Technology California State University, Fresno

May 2015 APPROVED

For the Department of Viticulture and Enology:

We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.

Victoria Towers Thesis Author

Sonet Van Zyl (Chair) Viticulture and Enology

Kaan Kurtural Viticulture and Enology

Anil Shrestha Plant Science

For the University Graduate Committee:

Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION

OF MASTER’S THESIS

X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.

Permission to reproduce this thesis in part or in its entirety must be obtained from me.

Signature of thesis author: ACKNOWLEDGMENTS

I would like to express my most sincere gratitude to the members of my committee Dr. Kaan Kurtural and Dr. Anil Shrestha for their help and guidance and in particular to my adviser Dr. Sonet Van Zyl for giving me the opportunity to work with her, for her guidance and her friendship. I would also like to extend my gratitude to The California State University Agricultural Research Institute and The California Table Grape Commission for their financial support for this project and to Scatagglia Growers and Shippers for providing the vineyard as well as their cold storage facilities and staff. I would particularly want to thank Craig Calandra, Vincent Silva and Darryl Alchian for their help, expertise and patience in the field. Others who are appreciated for their hard work and sincere friendship are my coworkers and lab team Amanda Burke, Laura Richaud, Erin Palumbo, Thomas Duvall, Humberto Topete, Alexander Pineda and Jaqueline

Chenoweth. Finally, I would like to express my gratitude to my family and friends for their support and encouragement throughout my time at Fresno State without whom I would not have received this degree.

Thank you. TABLE OF CONTENTS Page

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: LITERATURE REVIEW ...... 3 Table Grapes: Importance and Production in the World and the United States ...... 3

Fresno County Climate ...... 5

Scarlet Royal Table Gapes ...... 8

Production Problems and Special Considerations for Scarlet Royal ...... 10

Pre and Post-Harvest Pathological Problems in Table Grapes ...... 10

Plant Growth Regulators: Gibberellins and Cytokinins ...... 25

Cluster and Berry Thinning ...... 33

CHAPTER 3: MATERIALS AND METHODS ...... 36

Site Selection ...... 36

Experimental Design ...... 36

Treatment Applications ...... 36

Post-Harvest Decay Forecasting ...... 37

Parameters Measured and Instruments Used ...... 38

Statistical Analysis ...... 40

CHAPTER 4: RESULTS AND DISCUSSION ...... 41

Post-Harvest Decay Forecasting ...... 41

Experimental Results ...... 43

Discussion ...... 45

CHAPTER 5: CONCLUSION ...... 48 vi vi Page

REFERENCES ...... 49

APPENDIX: TABLE GRAPE TYPES: SURFACE AREA IN HECTARES BY VARIETY AND YEAR PLANTED IN CALIFORNIA ...... 57 LIST OF TABLES

Page

Table 1: Acreage Standing by County and Year Planted in California for Table Grapes (CDFA, 2014)...... 4

Table 2: Cultural Practices for Scarlet Royal Table Grapes...... 9 Table 3: Fungicide Efficacy for Botrytis and Summer Bunch Rot Control (Adaskaveg et al., 2013)...... 18

Table 4: Treatments, Concentration Rates and Commercial Product ...... 37 Table 5: Percentage of Botrytis cinerea and Other Diseases Affecting Control, GA3 Treatment and GA3+CPPU Treatment in 2013 and 2014...... 41

Table 6: Effect of GA3 and GA3+CPPU on Percentage of Botrytis cinerea Incidence, Berry Shatter, Berry Crack, Other Damage and Other Disease Incidence at Post-Harvest on Scarlet Royal Table Grapes ...... 43

Table 7: Effect of GA3 and GA3+CPPU on Berry Length, Berry Width, Berry Firmness and Berry Color (Lightness, Chroma and Hue) at Post- Harvest on Scarlet Royal Table Grapes...... 44

Table 8: Effect of GA3 and GA3+CPPU on Berry Juice pH, Titratable Acidity and Total Soluble Solids at Post-Harvest on Scarlet Royal Table Grapes...... 45

LIST OF FIGURES

Page

Figure 1: Average Temperature from 1971-2007. Adapted from Annual Climatology: Fresno, CA (Fresno County, 2014) and The Weather Channel: Monthly Weather for Fresno (The Weather Channel, 2014). Accessed 02/2014...... 6 Figure 2: Average Precipitation from 1971-2007. Adapted from Annual Climatology: Fresno, CA (Fresno County, 2014) and The Weather Channel: Monthly Weather for Fresno (The Weather Channel, 2014). Accessed 02/2014...... 7 Figure 3: Calendar of Weather Events for the Southern San Joaquin Valley (Vasquez et al., 2013)...... 8

Figure 4: Botrytis cinerea life cycle (Marois et al., 1992)...... 13 Figure 5: Grapes Arranged for Post-Harvest Decay Forecasting Showing Infected Berries...... 42 Figure 6: From Left to Right: Botrytis Infected Berry, Penicillium Infected Berry, Cladosporium Infected Berry and Aspergillus Infected Berry...... 42

CHAPTER 1: INTRODUCTION

The total table grape production in the world currently exceeds 18.1 million tons per year. China is the leading table grape producer followed by Turkey and the European Union. According to the United States Department of

Agriculture (USDA) the United States is the 6th largest producer with a total of 1,017,000 tons for the 2013/14 season (USDA, 2014a). The total world trade for fresh table grapes is around 2.5 million tons a year. Chile and the United States are the main table grape exporters with 755,000 and 416,000 tons per year, respectively (USDA, 2014a). The California Table Grape Commission (CTGC) reported that table grapes are one of the preferred fresh fruits consumed in the United States along with bananas and apples with a mean consumption of 3.52 kg per capita per year (USDA, 2014b). Of the commercial fresh grapes grown in the United States, California produces 99% (CTGC, 2013) with a total of 36,912 ha. California exports 41% of the total production as fresh fruit to over 50 overseas markets, and according to the California Department of Food and Agriculture this represents a total of 812.3 million USD per year (CDFA, 2013a; USDA, 2014a). Botrytis cinerea causes the fungal disease known as gray mold. It is considered the most damaging disease in table grapes since it can infect the berries in the field and continue its development during cold storage (Gubler et al., 2006). This fungus can affect the entire vine including succulent tissue and stressed or dead tissue. The most susceptible cultivars generally present a vigorous canopy, tightly arranged clusters and thin-skinned berries. The susceptibility increases when growing conditions are humid (Bettiga and Gubler, 2013). 2 2

Gray mold can be controlled in the vineyard and during post-harvest by applying an integrated approach. This approach considers the combination of different practices such as chemical control, vineyard sanitation, canopy management, irrigation management, berry damage reduction practices and plant resistance (Bettiga and Gubler, 2013).

Plant growth regulators (PGR) such as Gibberellic Acid (GA3) and Forchlorfenuron (CPPU) are usually applied to table grapes at several growth stages at different concentrations with the main purpose of reducing fruit set and increasing berry size (Christodoulou et al., 1968). These applications also have an effect on cluster tightness, berry skin thickness and pedicel rigidity which in combination affects susceptibility to disease infection and storage potential of the harvested fruit (Ben-Arie et al., 1998) The main objective of this research project was to evaluate the effect of PGR applications on Scarlet Royal table grapes to determine their incidence on gray mold and quality parameters during post-harvest storage.

CHAPTER 2: LITERATURE REVIEW

Table Grapes: Importance and Production in the World and the United States Table grapes are one of the main three fresh fruits consumed in the US along with bananas and apples. From the total consumed tonnage 53.5% is grown in California, while the remaining 46.5% corresponds to imported produce (USDA, 2014a; USDA, 2014b). The total area planted with table grapes in California for 2013 was 36,912 hectares with 34,047 bearing hectares and 2,865 non-bearing hectares (see Appendix). There are currently more than 70 different table grape varieties grown in California, but the majority of the commercialized volume is accounted only by a dozen of them. These main 12 varieties include Autumn King (1,580 ha); Autumn Royal (1,845 ha); Crimson Seedless (5,021 ha); Flame Seedless (7,394 ha); Perlette (414 ha); Princess (1399 ha); Red Globe (4,456 ha); Ruby Seedless (621 ha); Scarlet Royal (1,831 ha); Sugraone (2,369 ha); Summer Royal (391 ha); Vintage Red (501 ha) and Thompson Seedless (71,008 ha not considered in the total acreage for table grapes due to its variety of uses) (CDFA, 2014). The majority of the table grape acreage in California is located in District 13 and 14 (Table 1). In district 13 the production is concentrated in the counties of Tulare (10574 ha); Fresno (5020 ha) and Madera (880 ha) while in district 14 the counties of Kern (16364 ha) and Kings (404 ha) are the primary table grape producers. The remainder of the total acreage is distributed throughout California in numerous counties that include Alameda (4 ha); Imperial (72 ha); Merced (54 ha); San Bernardino (216 ha); San Joaquin (88 ha); San Luis Obispo

(40 ha); Solano (4 ha) and Stanislaus (4 ha) (CDFA, 2013b; CDFA, 2014).

4 4

Table 1: Acreage Standing by County and Year Planted in California for Table Grapes (CDFA, 2014).

2004 & 2004

Earlier County 2005 2006 2007 2008 2009 2010 2011 2012 Bearing Non- Total

Bearing Alameda 4 0 0 0 0 0 0 0 4 0 4 Amador 0.4 0 0 0 0 0 0 0 0.4 0 0.4 Butte 4 0 0 0 0 0 0 0 4 0 4 Calavera 0.4 0 0 0 0 0 0 0 0.4 0 0.4 Contra Costa 1 0.4 0 0 0 0 0 0 1.4 0 1.4 El Dorado 2 0.4 0 0 0 0 0 0 2.4 0 2.4 Fresno 3681 173 169 213 116 155 373 75 4507 513 5020 Humboldt 0.4 0 0 0 0 0 0 0 0.4 0 0.4 Imperial 72 0 0 0 0 0 0 0 72 0 72 Kern 10452 919 594 788 684 546 1207 662 13984 2381 16364 Kings 218 67 0 17 73 14 0 0 390 14 404 Lake 0 0 0 0 0 0.4 0 0 0.4 0 0.4 Madera 633 28 30 11 36 0 82 61 739 143 882 Mendocino 1 0 0 0 0 0 0 0 1 0 1 Merced 5 0 0 0 0 0 0 0 5 48 54 Monterey 0.4 0 0 0 0 0 0 0 0 0 0.4 Napa 0.4 0 0 0 0 0 0 0 0 0 0.4 Placer 1 0 0 0 0 0 0 0 1 0 1 Riverside 2412 70 80 95 18 52 46 7 2779 19 2797 San Bernardino 215 0 0 0 0 0 0 0 216 0 216 San Diego 0.4 0 0 0 0.4 0 0 0 0.4 0 0.4 San Joaquin 87 0 0 1 0 0 0 0 88 0 88 San Luis Obispo 0 0 40 0 0 0 0 0 40 0 40 Santa Cruz 2 0 0 0 0 0 0 0 2 0 2 Shasta 3 0 0 0.4 0 0 0 0 3.4 0 3.4 Siskiyou 0 0 0 0 0 0 0 0 0 0 0 Solano 1 0 0 0 3 0 0 0 4 0 4 Sonoma 1 0 0 0 0 0 0 0 1 0 1 Stanislaus 3 0 0 1 0 0 0 0 4 0 4 Tehama 3 0 0 0 0 0 0 0 3 0 3 Tulare 6992 482 512 458 619 366 511 305 9430 1145 10574 Yolo 7 1 0 0 0 0 0 0 8 0 8 State Total 24804 1740 1425 1585 1550 1132 2218 1110 32288 4263 36551 5 5

As with every crop, table grape production is influenced by the weather characteristics of a specific growing region. The research presented in this project is based on a table grape production site in the Fresno area, thus the climate characteristics for this county will be described in this chapter. Fresno County is located in the San Joaquin Valley (SJV) and its climate is greatly influenced by the surrounding mountain ranges. The Pacific moisture flow is blocked by the Diablo Range that generates a dry climate in Fresno area. Summers are characterized by hot temperatures and sunshine while rainfall events, though infrequent, do occur but generally in small amounts. During the fall season, temperatures will slightly cool down and precipitation can become more frequent. By mid-October and November frontal passages become more common bringing the first widespread rains of the season (Stachelski and Sanger, 2008).

Fresno County Climate Fresno County is located in the California Climate Zone 13, which has Fresno city as main reference. The characterization of this region includes data collected mainly from Fresno, Bakersfield, Visalia and Porterville. It is located at Latitude of 36.46 N and a Longitude of 119.43 W (Paciffic Energy Center, 2008) The mean elevation is 99.97 in Fresno with a variation across the SJV between 30.48 and 182.88 meters above sea level (Stachelski and Sanger, 2008).

Fresno County Temperatures The summers in Fresno are characterized by almost constant sunshine and high temperatures. The mean daily temperatures during the three months of summer are 24.5°C for June, 27.27°C for July and 26.55°C for August. Maximum 6 6 temperatures are usually reached it in the month of July with an average of 35.9°C. Temperatures tend to slightly decrease during the fall (Figure 1).

Figure 1: Average Temperature from 1971-2007. Adapted from Annual Climatology: Fresno, CA (Fresno County, 2014) and The Weather Channel: Monthly Weather for Fresno (The Weather Channel, 2014). Accessed 02/2014.

During the winter temperatures are usually mild but occasionally temperatures can drop to or below freezing. The mean daily temperatures during winter months are 7.33°C for December, 7.78°C for January and 10.77°C for February. The lowest temperatures are registered generally in December with a mean of 2.77°C. During spring, the weather transitions from the winter storms season to the hot and dry summer (Stachelski and Sanger, 2008).

Fresno County Precipitation Fresno county is subject to a Mediterranean climate which means that the summers are hot and dry while winters have mild temperatures and relatively light precipitation (Fresno County, 2014). The normal annual precipitation for Fresno is 294.05 mm of which 90% is received from November through April (Figure 2). The remaining 10% is spread throughout the remaining 6 months and 7 7 can be described as early or late rain events. Data collected in the last 30 years (1977-2007) showed that 20% of the time early rain events may occur in August while chances increase to 43% in September and 80% in October (Stachelski and Sanger, 2008). These early season rains often represent a hazard in fruit production areas by providing humidity which in combination with warm temperatures provides adequate conditions for disease proliferation (Bettiga and Gubler, 2013).

Figure 2: Average Precipitation from 1971-2007. Adapted from Annual Climatology: Fresno, CA (Fresno County, 2014) and The Weather Channel: Monthly Weather for Fresno (The Weather Channel, 2014). Accessed 02/2014.

Calendar of Weather Events: Southern San Joaquin Valley The rain hazard on fruit for the southern SJV is present from mid-August through the beginning of December since this period coincides with the harvest time of numerous table grape varieties (Figure 3) (Vasquez et al., 2013).

8 8

Figure 3: Calendar of Weather Events for the Southern San Joaquin Valley (Vasquez et al., 2013).

Scarlet Royal Table Gapes

Origins and Description of Scarlet Royal Table Grapes Scarlet Royal is red seedless grape cultivar that was developed by Dr. David Ramming and Ronald Tarailo from the USDA in Parlier, CA. This variety was evaluated as B1 that resulted from the highly complex cross C33-30 X C51- 63, and was released in 2006.

Scarlet Royal is medium in vigor and has an average production of 27 kg per vine when trained to quadrilateral system, pruned to 2 bud spurs and grown on a Y or open gable trellis system. The clusters are conically shaped and are large in both size and length. The berries are oval shaped, with a medium to thick skin and a firm, meaty textured flesh. Each berry has 3 to 4 aborted seeds, which are imperceptible when consumed. Its taste has been described to be sweet and neutral. This variety has a dark red color when fully ripened and can achieve full coloring even when grown under full canopy conditions. At harvest, berries present 22.0% soluble solids and a Titratable acidity (TA) of 0.55 g/100 mL of juice (Ramming and Tarailo, 2006). 9 9

This variety can be planted on its own roots or grafted on to different rootstocks depending on site-specific soil pest and/or soil physical and chemical conditions. Popular rootstocks used for Scarlet Royal include Freedom and 1103- P. Differences in yield, fruit quality and vine performance for each rootstock are yet to be determined (Hashim-Buckey and Ramming, 2008) . Scarlet Royal is a mid-season variety, ripening uniformly from mid to late August in the California SJV and can be kept in cold storage for 2 months without compromising berry firmness. The commercialization of Scarlet Royal covers the market window between Flame Seedless and Crimson seedless (Ramming and Jones, 2005). Considering the total grape volume by variety in California in the past three years, Scarlet Royal occupied 4th place with a total of 8,672,565 tons in 2012 (CTGC, 2013).

Cultural Practices for Scarlet Royal The main cultural practices for Scarlet Royal include spur pruning to 2 buds during dormant season, shoot thinning and cluster thinning (see Table 2).

Usually, 2 GA3 applications are performed, the first one at bloom and the second one for sizing. For Scarlet Royal girdling is not recommended and ethephon applications are not necessary (Andris et al., 1985; Hashim-Buckey and Ramming, 2008).

Table 2: Cultural Practices for Scarlet Royal Table Grapes.

Variety Type of Number Number Shoot GA3 GA3 Girlding Ethephon Cluster pruning of spurs of buds thinning Bloom Sizing applicati thinning per vine per spur sprays spray on

Scarlet Spur 30-40 2 Yes 2-2.5 20 No No Yes

Royal ppm ppm 10 10 Production Problems and Special Considerations for Scarlet Royal Scarlet Royal can develop bitter flavors or skin astringency if the time of harvest is delayed or the total soluble solids (TSS) are over 23%. Harvest should begin when the fruit is sweet, well balanced and soluble solids are equal or over 17%. To ensure a palatable, high quality fruit, harvest should be continued until the TSS reach 22% (Hashim-Buckey and Ramming, 2008). Form the information collected, Scarlet Royal shows a high susceptibility to Botrytis bunch rot both in the vineyard and during cold storage. This problem is enhanced by the fact that this is a late variety that might be subject to rain, which increases berry cracking due to high humidity. Furthermore, Scarlet Royal tends to have tight clusters and a lack of a thick epicuticular wax that contributes to its susceptibility to bunch rot.

Pre and Post-Harvest Pathological Problems in Table Grapes Pre- and post-harvest decay on table grapes is one of the causes of financial loss in numerous counties. During pre-harvest, decay is mainly caused by fungi infections such as Botrytis, Rhizopus, Aspergillus and Penicillium as well as bacteria and yeasts. Post-harvest decay on the other hand is mostly caused by Botrytis cinerea. However, the abovementioned pathogens, as well as Alternaria, can damage the stored fruit depending on the storage conditions. Three factors directly influence the onset and evolution of fruit decay during the pre-harvest period: a) the presence of pathogenic material, b) favorable environmental conditions and c) host susceptibility (Fourie, 2008). During storage, the development of decay causing pathogens is reduced, but not completely inhibited, by the use of low temperatures. If the storage 11 11 temperature is above 0°C or fluctuates with time, many fungi can mature and cause damage (Fourie, 2008).

Botrytis Bunch Rot in Table Grapes Botrytis cinerea and other types of Botrytis are major pathogens for a large number of field and orchard crops as well as stored and transported products, which includes table grapes. Epidemics caused by Botrytis cinerea can be severe and economically damaging in conditions conductive to infection (Elmer and Michailides, 2007). Botrytis cinerea causes the disease commonly known as gray mold or bunch rot. It is of great importance in table grapes since it can infect the berries in the field and then continue to grow inside them during cold storage. The level of infection in the field determines the degree of susceptibility of the grapes to the disease before cold storage as well as the variety, the condition of the fruit at harvest and the effectiveness of control measures (Gubler et al., 2006).

This fungus can grow on any succulent grape tissue such as shoots, young leaves and flower parts or even on stressed or dead tissue. The most susceptible cultivars are the heavy canopy cultivars with tight clusters and thin-skinned berries, especially when growing conditions are humid (Bettiga and Gubler, 2013).

Symptoms of Botrytis Bunch Rot In the vineyard, Botrytis bunch rot symptoms can be seen on foliage, fruit and canes. On the leaves, soft brown tissue forms in sections that are followed by the death of the infected part. Yield can often be reduced by the death of smaller infected shoots and their inflorescences and older shoots can wilt or break at the infection site. In the majority of the cases, as a result of water accumulation, 12 12 infections take place on the joints of the leaves and inflorescences axis with the main shoot (Bettiga and Gubler, 2013; Emmet et al., 2007).

Bunch rot usually starts when blossoms are infected with spores during rainfall. These spores become dormant after infecting the flowers until veráison, when the pulp gets infected (Bettiga and Gubler, 2013). This initial stage of Botrytis bunch rot is known as the “slip-skin” stage. This distinctive characteristic is a result of the loosening of the overlaying skin of the grape that can be easily separated from the pulp. After the first stage, a brown discoloration develops which results in copious production of brown spores (Snowdon, 1990). Weather conditions like moderate temperatures, high moisture and low wind speed can favor the cracking of the berries’ epidermis in which mycelium and spores are generated giving the characteristic gray velvety appearance of the Botrytis bunch rot. The disease can spread from berry to berry and give the appearance of a nested infected cluster. Furthermore, the percentage of infected berries can increase if favorable conditions are maintained and if the fungus reaches the rachis, the affected berries can raisin (Bettiga and Gubler, 2013). The shoots, spots of soft brown rot develop on shoot stems. The infected shoots can break at the nodes where an internal brown discoloration can be seen (Emmet et al., 2007). In storage, the appearance of symptoms can be delayed due to low storage temperatures. The first symptoms are water soaked lesions on the berry skins which can progress to slip-skin in 1-4 days at 0°C. Following the slip-skin phase, gray-brown lesions develop on the surface of the grape (this symptom may not be evident in red or black grapes) and subsequently, mycelium starts growing out of the berry. Within 1 week, the mycelium can infect adjacent berries and results in the development of infection nests with a gray-white mycelium. In the 13 13 final stages of decay, the berries become dark and lose their juice. The rachis can also become infected and turn brown with subsequent mycelium formation. This mycelium can move through the stems and infect other berries and berries attached to infected stems can dry out (Bettiga and Gubler, 2013).

Botrytis cinerea Life Cycle Overwintering: Botrytis can survive the winter either on the surface or inside colonized tissue as sclerotinia (dormant structure), inside the vine canopy or on the ground. The main sources of these dormant structures are mummy clusters form the previous year and same year infected canes. During spring, the sclerotinia germinate after rain or irrigation events and produce conidia (spores) that are disseminated mainly by wind (Figure 4) (Bettiga and Gubler, 2013; Emmet et al., 2007).

Figure 4: Botrytis cinerea life cycle (Marois et al., 1992). 14 14

Germination: With continuous free water and nutrients, spores of Botrytis cinerea can germinate on the surface of healthy or damaged berries (Bettiga and

Gubler, 2013). The length of the moisture period necessary to produce infection varies with the ambient temperature (Nelson, 1950), where hot temperatures accelerate the drying of the fruit which reduces germination. Periods of no available water of 15 minutes or less are sufficient to halt germination. Germination and infection takes place at an optimum temperature of 22°C. At 32°C and above, spores cease to grow but they can do it at a slow pace during storage with temperatures as low as 1°C (Bettiga and Gubler, 2013). Infection: Under humid conditions the spores can infect flowers, succulent young stems and leaves in the early spring. Later in the season, the berries become more prone to infection as the sugar content increases and the skin softens. The spores can penetrate the grape through wounds or directly through intact skin. The main protection against Botrytis infections comes from the berry skin and epicuticular wax, therefore any factor (for example: cultural practices and chemical applications) that can alter the physical and chemical characteristics of these, will affect the susceptibility of the berries to infection (Bettiga and Gubler, 2013). Dead and infected flower parts that remain within the cluster can become a source of infection (Emmet et al., 2007) Infected berries show cracks where more spores are developed, and these spores can rapidly spread to uninfected grapes especially after a rain event late in the season. If high temperatures and low humidity conditions prevail, infected berries may dry up but the fungus will remain alive and continue to grow once favorable conditions are restored (Bettiga and Gubler, 2013). 15 15

In stored grapes the initial infection starts in the vineyard and spreads within the storage container. At this stage, the spores produced asexually

(conidia) and are not an important source of infection (Snowdon, 1990).

Effect of Cluster Tightness on Botrytis Bunch Rot and Other Infections Cluster architecture has a dramatic influence on the development of Botrytis bunch rot epidemics since it directly impacts the berry’s surface microclimate. The compactness of the cluster determines the length of time that a cluster retains water and consequently tight clusters take longer to dry than lose clusters (Vail and Marois, 1991). Having a wet surface provides the needed conditions of free water and humidity for conidia to germinate and infect the berries (Carre, 1985). Tight clusters provide a high ratio of interior to exterior berries which results in high surface contact area between them (Vail and Marois, 1991). The direct contact between grapes interferes with the normal development of epicuticular wax, making the berries more susceptible to Botrytis infections (Marois et al., 1986). This is important for disease prevention infections since the epicuticular wax serves as a protective layer against Botrytis infections and other threats such as dissecation, insect attack, physical abrasion, frost and radiation, bacterial infections and wind injuries (Martin and Juniper, 1970). Also, grape varieties that present a tight arrangement of the berries in the cluster are more susceptible to berry splitting and cracking which allows colonization of the berry by numerous disease spores consequently causing berry breakdown (Barbetti, 1980). Generally, the more berries per centimeter a cluster has, the higher the rot incidence and severity it presents. Compactness may affect the number of retained floral debris to the time of berry ripening and can 16 16 increase contact between berries and debris facilitating retained debris to have an important role in rot development (Hed et al., 2009).

Furthermore, cluster compactness can affect the efficacy of fungicide sprays applied to control diseases. During ripening, when berries are highly susceptible to bunch rot, the pesticides may not penetrate to the inside surface of compact bunches which reduces the effectiveness of disease control (Hed et al., 2009).

Botrytis Bunch Rot Pre- and Post- Harvest Decay Control Botrytis bunch rot control in the vineyard can best be achieved by applying an integrated approach that considers the combination of different practices. The main target of the integrated program is to adequately manage the fruit zone to reduce humidity and facilitate the drying of bunches after a pre- harvest rain event. Canopy density and phenological stages of the clusters are key factors in determining chemical application efficacy. By applying both cultural and chemical control methods the disease can be properly managed. Cultural practices: These include canopy management, sanitation practices, irrigation management, berry damage reduction practices and plant resistance (Bettiga and Gubler, 2013; Pearson and Goheen, 1998). Chemical control: During the dormant period lime sulfur is commonly used as a clean-up product to reduce the overwintering sclerotia. Applications are usually performed at a rate of 93.61 l/ha in a high volume of water (Bettiga and Gubler, 2013). During the growing season several fungicides can be used to control diseases such as Cyprodinil, Fenhexamid, Iprodione, Pyraclostrobin,

Captan, Dichloran and Mancozeb (Table 3) (Gubler et al., 2014). To optimize the fungicide coverage, adjuvants are generally added to the tank mix. To prevent 17 17 the development of resistance, fungicides with different modes of actions should be rotated. Spray programs are planned according to the vineyard’s Botrytis history, cultivar susceptibility and weather conditions that can favor the development of the disease. Usually vines are sprayed at bloom, cluster pre- close, veráison and pre-harvest. During bloom spray applications multiple applications may be necessary depending on the abovementioned conditions. The best time for fungicide application is when the favorable environmental conditions for rot development have been forecasted and before rain events (Bettiga and Gubler, 2013; Emmet et al., 2007; Pearson and Goheen, 1998). Sanitation: Since the fungus can survive on mummy clusters, it is recommended that during pruning all clusters are removed from the vines and incorporated into the soil. By maintaining a clean vineyard with no fruit left on the vines, the source of inoculum for the following year can be reduced (Bettiga and Gubler, 2013).

Canopy management: The objective of canopy management is to create a non-favorable microclimate for fungus development by exposing the clusters to light and increased wind speed to reduce drying time after wetting. Canopy management starts with the vineyard design. The selection of rootstocks and scion, trellis system, pruning method and plant spacing are important since they will have an effect on canopy density (Pearson and Goheen, 1998). The orientation of the rows can also influence canopy microclimate and it should be selected according to the site’s climate conditions. Other practices, which can be annually manipulated such as irrigation and fertilization, can affect the canopy density by influencing main and lateral shoot growth. For short-term canopy manipulation several practices are recommended. Shoot thinning, shoot positioning, leaf removal and hedging practices reduce canopy density and 18 18

Table 3: Fungicide Efficacy for Botrytis and Summer Bunch Rot Control (Adaskaveg et al., 2013).

Trade Name Active Ingredient Botrytis Summer Bunch Rot

Abound Azoxystribin + -- Flint Trifloxystrobin ++ ++ Elite/Orius/Tebuzol Tebuconazole ++ ++ Quadris Top Azoxystrobin/difenoconazole ++ ++ Inspire Super Difenoconazole ++++ ++ Luna Experience Fluopyram/tebuconazole ++++ ++ Luna Tranquility Fluopyram/pyrimethanil ++++ ++ Mettle Tetraconazole --- + Pristine Pyraclostrobin/boscalid ++++ +++ Sovran Kresomix-methyl ++ ++ Topsin-M/T Thiophanate-methyl ++ ++ Copper Copper ++ +++ Elevate Fenhexamid ++++ ++ Ph-D Polixin-D +++ +++ Rovral + Oil Iprodione ++++ --- Scala Pyrimethanil ++++ ++ Switch Cyprodinil/fludioxonil ++++ ++ Vangard Cyprodinil ++++ ++ Captan Captan +++ +++ Dithan/Manzate/Pen Mancozeb ++ --- ncozeb Rovral/Iprodione/Ne Iprodione +++ --- vado Ziram Ziram + + Rating: ++++ = Excellent and consistent, +++ = Good and reliable, ++ = Moderate and variable, + = Minimal and often ineffective and --- = ineffective. 19 19 increase light penetration and air movement. Timing is a key factor in the use of these practices since the effect is only temporary and canopy regrowth may occur

(Bettiga and Gubler, 2013). Moreover, these practices will vary in different climate regions. In warm weather production areas leaf removal should not be excessive since fruit sunburn may occur and if removed later in the season the damage can be severe. If leaves are removed early in the season at cluster set, the berries can develop a thick cuticle that helps prevent both sunburn and Botrytis infection (Gubler et al., 2014). Irrigation: Selecting the correct type and managing the level and timing of irrigation can be a tool to help control the disease. The use of overhead sprinklers should be avoided specially close to harvest to reduce Botrytis levels. Irrigation timing should be adjusted to the prevalent climate since it is recommended that clusters should not remain wet for more than 15 hours. Drip and furrow irrigation should also be utilized thoughtfully because high volumes of water can result in dense canopies that provide favorable conditions for rot development (Bettiga and Gubler, 2013). Berry damage reduction practices: Controlling insects (such as leafroller caterpillars) and birds that feed on berries reduces the wounds that serve as entry points for fungal infections. Other diseases such as powdery mildew can cause berry cracking, therefore controlling this disease also helps prevent Botrytis bunch rot infections. Furthermore, all injuries related to canopy and cluster management should be minimized as they all contribute to the development of the disease (Bettiga and Gubler, 2013; Emmet et al., 2007). Plant resistance: The combination of numerous factors determines a variety susceptibility or resistance to Botrytis bunch rot. Generally, white grape varieties with thin-skinned berries are more susceptible to Botrytis cinerea. On the 20 20 other hand, research has shown that cluster architecture and tightness have a greater impact on susceptibility rather than the characteristic of individual berries (Bettiga and Gubler, 2013; Pearson and Goheen, 1998). Research has shown that some of the characteristics of the more resistant varieties were a thick epidermis and external hypodermis with numerous cell layers, low number of pores and a thick cuticle (Gabler et al., 2003). Post-harvest control begins with correct management of the disease in the vineyard. Harvest during rainy periods should be avoided especially when clusters are wet and only be resumed once they are completely dry. During harvest, the damaged and decayed berries should be trimmed from the clusters. Trimmed grape clusters should be placed in an adequately chosen box without over packing it (Gubler et al., 2013). After harvest, the fruit should be rapidly cooled and handled with care.

Sulfur dioxide (SO2) is generally used for disease control in cold storage. The SO2 gas is an effective fungistat that prevents new infections by killing spores and inhibiting mold growth on the berries surface although it cannot stop established infections. Sulfur dioxide can be applied by using fumigation technology or in- package SO2 generators. These rely on potassium or sodium metabisulphite and can be found as chemical impregnated sheets, plastic sachets that contain either the solution or powder formulation (Snowdon, 1990). Traditional fumigation practices include initially gassing fruit with 5000 ppm SO2, followed by supplementary fumigations at 7 to 10-day intervals with 2500 to 5000 ppm. Currently, a more developed system is applied to decrease residue and environmental pollution while increasing worker safety. This modern system consists of an initial application combined with forced-air cooling and additional 21 21 fumigations follow at weekly intervals. The applied quantity depends on the number of boxes in storage and the packaging material (Gubler et al., 2013).

Problems associated with the use of SO2 for post-harvest Botrytis bunch rot control includes the presence of residues that exceed the 10mg/kg tolerance for most countries and the impossibility of its use on organically certified grapes. Furthermore, repeated or high dosage fumigations may produce bleaching injuries on the surface of the berries which affects the commercializing potential.(Gabler and Smilanik, 2001). Different control methods are currently being researched to escape the problems associated with SO2 use. Sub-lethal levels of ethanol in combination with potassium sorbate proved to be effective in controlling post-harvest Botrytis disease (Karabulut et al., 2005). With brief immersions of detached berries in ammonium bicarbonate, sodium bicarbonate and ethanol, gray mold postharvest infections could be controlled with similar effectiveness as SO2 fumigations. Furthermore, these treatments have minimal environmental and worker safety issues and because of their low toxicity they pose a minimal ingestion hazard (Gabler and Smilanik, 2001).

Blue Mold Rot and Rhizopus Rot in Table Grapes Blue mold rot: Following Botrytis bunch rot, blue mold rot is considered the second most damaging disease for stored grapes. It is caused by a number of Penicillium species such as Penicillium canescens Sopp and Penicillium citrimun Thom (India), Penicillium cyclopium Westling (Israel) and Penicillium expansum (USA, Chile and Germany). Along with other fungi (Aspergillus spp, Alternaria tenuis, Cladosporuim spp and Rhizopus arrizhus) Penicillium species are usually involved in summer bunch rot complexes (Bettiga and Gubler, 2013). 22 22

The symptoms for blue mold include abundant presence of white mold that subsequently produces green-blue powdery spores on stems and berries.

The infected grapes become watery and soft and release a distinctive moldy odor (Snowdon, 1990). Penicillium spp. life cycle: Conidia from Penicillium species survive on decaying plant material and can be dispersed by wind, water and insects. Injured berries are predisposed to infection throughout the season and mold can even spread through the bunches during refrigerated storage (Snowdon, 1990). Rhizopus rot: This disease is caused by different species of Rhizopus, such as Rhizopus oryzae Went & Prinsen Geerligs, Rhizopus stolonifer Lind and Rhizopus arrizhus. Rhizopus rot can be recognized by the presence of spherical spore-heads that will change from white to black, covering the surface of berries in the cluster (Snowdon, 1990). This fungus can also be found in summer bunch rot complexes. The presence of this pathogen in stored grapes indicates that storage conditions were mismanaged since the fungus growth is inhibited at temperatures below 4°C (Bettiga and Gubler, 2013). Rhizopus spp life cycle: The Rhizopus spores (sporangiospores) are present in the soil and on plant debris and disseminated by wind. The berries are subject to infection during the entire grape growing season. Injured berries are the entry points for primary infections but Rhizopus oryzae can also penetrate healthy berry skins when exuded grape juice is present. Once the disease is established, adjacent sound berries can be rapidly infected especially in high temperature conditions (Snowdon, 1990). 23 23 Aspergillus Rot and Cladosporium Rot in Table Grapes Aspergillus rot: This fungus commonly prevails in production areas with prolonged hot temperatures and is also often associated with bunch-rot complexes. The presence of this fungus in harvested fruit can be a result of prolonged high temperatures in storage thus, in normal storage conditions Aspergillus rot is not usually a problem (Nelson, 1979). This disease is caused by two Aspergillus species named Aspergillus niger and Aspergillus carbonarius. These are usually the first fungus species to colonize wounded berries in the SJV and are generally an important contributor to summer bunch rot complexes (Bettiga and Gubler, 2013). The spores can easily be seen and are usually black and but in some cases another form with brown spores can infect the berries. The fungus destroys the berry pulp, creating a pale and watery spot below the mycelium while releasing a sour odor (Snowdon, 1990).

Aspergillus niger life cycle: The fungus survives on plant debris in the soil and its development is favored by warm temperatures between 25°C and 35°C. Air currents disseminate the conidia which infect the berries through surface wounds (Snowdon, 1990). Aspergillus only infects the berries after the phonological stage of veráison. Once the fungus invades the tissue, the pulp goes through rapid decay which enables colonization of other fungi and yeasts as well as insect visitation (Bettiga and Gubler, 2013). High temperature conditions accelerate the spreading of the fungus through the cluster. The infections can continue through post-harvest storage (Snowdon, 1990). Cladosporium rot: This fungus is commonly found in stored grapes due to its ability to grow at very low temperatures (0°C). It is caused by Cladosporium herbarum and the symptoms include circular black spots under the skin that 24 24 subsequently forms velvety olive-green mold (Snowdon, 1990). Cladosporium cladosporioides can also be a problem in stored grapes and both species are usually found in summer bunch rot complexes. If the berries are subject to sunburn in the vineyard, this fungus can colonize the injured tissue. During cold storage the affected tissue begins to break down and brown spots appear on the surface of the berry. When these berries are removed from storage, a green sporulation is produced on the rotten tissue (Bettiga and Gubler, 2013). Cladosporium spp. life cycle: This fungus survives on dead plant material in the soil. Spores (conidia) are disseminated in the air and usually infect berries through open wounds that were caused by rain or rough handling. The fungus is also capable of direct penetration through the intact skin. Primary infection occurs before harvest and high incidence of disease is frequently associated with wet conditions (Snowdon, 1990).

Summer Bunch Rot Complex Pre- and Post-Harvest Control The most suitable control for summer bunch rot complex (Aspergillus spp, Penicillium spp, Cladosporium spp and Rhizopus spp) is minimizing berry injuries before, during and after harvest. Controlling damage caused by canopy and cluster management, insects, birds and early-season powdery mildew infections can significantly reduce the incidence of bunch rot complex in the vineyard. Berry cracking can be prevented by thinning the grape clusters that will reduce tightness and overcrowding of berries. There are numerous approved fungicides currently available and used (Table 3, p. 18) to control summer bunch rot problems (Adaskaveg et al., 2013). Nonetheless, chemical treatments have proved to be somewhat ineffective since most of them only target one or two pathogens rather than the entire bunch rot complex (Muthuswamy et al., 1971). 25 25

During cold storage the fruit must be handled with care to avoid injury and SO2 applications should be performed to kill existing spores on the berry surface. The fruit must be stored at an adequate and constant low temperature (Snowdon, 1990).

Plant Growth Regulators: Gibberellins and Cytokinins Plant growth regulators are a set of organic substances that occur naturally and have a direct impact on the physiological processes of the plant at low concentrations. Cell growth, differentiation and development are directly influenced by hormones but other processes can also be affected by hormonal activity like stomatal movement (Davies, 2004).

Gibberellins Gibberellins or GAs are a group of substances based on the ent- gibberellane structure and include more than 125 members in this group of hormones. The main gibberellin in plants is GA1, but the most abundant one is a fungal product, GA3. This hormone regulates many stages of the higher plant’s development. The effects of gibberellins include stem growth (GA1 stimulates cell division and elongation), bolting in long day plants, induction of seed germination, enzyme production during germination, fruit setting and growth (in grapes, exogenous applications can induce fruit set and growth) and induction of maleness in dioecious flowers (Davies, 2004).

Effects of Gibberellic Acid (GA3) on Grapes Gibberellic acid is a metabolic product derived from the fungus Gibberella fujikuroi first found in rice in Japan. This hormone can be obtained by fermenting large quantities of the Gibberella fujikuroi (Brian, 1959). 26 26

Gibberellic acid has been used in table grape production for many years with a wide variety of applications and purposes. Application timing and concentrations have different effects on different grape varieties. Pre-bloom applications: Can have numerous effects such as seedlessness, flowering acceleration, extension or shortening of flowering period, increasing or decreasing berry size, high or low presence of shot berries. The course of these effects is highly dependent on application timing, concentration and grape variety. The varieties ‘Delaware’ and ‘Early Campbell’ showed optimum outcomes if applications are conducted between 23 and 10 days before full bloom. This means that full bloom can be accelerated, flowering period can be extended and seedlessness can be achieved with proper application timing. Other varieties like Vitis labrusca Baile, Kyoho, Muscat Bayley A have responded to GA3 application by reduction seedlessness. This effect may be a result of the sensitivity of the ovules to gibberellins that induces parthenocarpic berry development or by inhibition of pollen germination and pollen tube growth. (Fukunaga and Kurooka, 1987; Jeong et al., 1998; Kimura et al., 1996; Motomura and Ito, 1972). Bloom applications: Often used when fruit set is excessive and results in very tight clusters when other practices such as trunk girdling are not enough to solve the problem. As in pre-bloom applications, the effect that bloom applications may have is also dependent on timing, concentration and variety.

Nevertheless, some generalizations can be made on the effects of GA3 sprays during bloom regarding fruit set, berry size and fruit ripening. Applications at 25-75% capfall in Thomson Seedless usually results in loose clusters and enlarged berries where later applications may not have the same effect since it is believed that the apical berry tissue decreases its response to GA3. These late bloom 27 27 treatments also favor berry elongation but have little effect on berry width which tends to stay constant regardless of application time (Christodoulou et al., 1968).

Other varieties may have later optimum application time such as Crimson Seedless. A single spray between 80-100% bloom significantly reduces berry set, increases berry size and length without reducing cluster weight or cluster number per vine. This single treatment is enough to cause cluster thinning and reduce the compactness but if successive applications follow, thinning can be excessive and a high number of shot berries may appear (Dokoozlian and Peacock, 2001). Data supports that the increase in berry weight is a result of the hormone induction to movement of assimilates into the berries shortly after application (Weaver et al., 1968). Repeated bloom applications can also have an effect on fruit maturation on Thomson Seedless. Growers, as a common practice, spray the clusters with

GA3 up to eight times between bloom and fruit set to increase berry size. It has been demonstrated that the effect on size decreases after the fourth application, but maturation can be significantly affected. Repeated sprays can result in reduced TSS and high acidity level which signifies a delay in ripening (Ben-Tal,

1990). The concentration at which GA3 is used can vary depending on the desired results. Low concentrations (2.5 to 5 ppm) can be sufficient to cause cluster loosening without significantly increasing berry size. On the other hand, concentrations from 5 to 40 ppm increase berry weight and elongation. These different concentrations tend to change berry size by affecting its length while keeping berry diameter constant (Christodoulou et al., 1968; Weaver et al., 1968).

GA3 levels between 20-50 ppm can affect sugar concentration by increasing fructose and glucose quantities at harvest in varieties like Cardinal, Michele Palieri and Black Corinth (Rusjan, 2010; Weaver et al., 1968). Conversely, 28 28 organic acid and amino acid concentrations usually decrease with high GA3 levels (Weaver et al., 1968). High dosages or over application of GA3 can have negative effects during post-harvest storage, increasing hairline cracking incidences and shatter after 30 to 60 days in cold storage at 0°C (Rusjan, 2010; Zoffoli et al., 2009). Fruit-set applications: Used for berry sizing usually after one bloom application of 15 ppm. In some varieties such as Thompson Seedless, fruit-set sprays can be repeated up to three times to increase berry length (Singh et al., 1978). In Thompson Seedless, Ruby Seedless and Black Corinth fresh and dry berry weights increase with GA3 applications (Harrell and Williams, 1987; Weaver et al., 1968). At this stage, clusters become a stronger sink for assimilates and therefore the berries have a stronger response to the hormone application by significantly increasing size and weight (Weaver et al., 1968). Gibberellic acid induces cell enlargement, which increases berry size but decreases cell density so treated berries present a thinner skin than untreated berries. This reduction in cell density and thinned berry skin can increase susceptibility to decay during post-harvest storage (Ben-Arie et al., 1998).

High GA3 concentrations at this stage can negatively affect berry adherence which results in an increase of berry shatter after 25-30 days in cold storage. Moreover, although pedicel diameter increases, its flexibility along with the rachis flexibility is reduced with increasing GA3 concentrations (Retamales and Cooper, 1993; Singh et al., 1978). The increase in pedicle rigidity restricts the accommodation of the berries during harvest and post-harvest manipulation which results in high percentages of berry detachment (Retamales and Cooper,

1993). 29 29

Veráison applications: GA3 sprays can have an effect on berry texture, increasing both flesh and skin firmness (Singh et al., 1978).

Pre-harvest applications: Late applications of GA3, especially at high dosages, can result in delayed fruit ripening and increased berry blemishes for Sultanina and Waltham Cross grapes (Wolf and Loubser, 1992).

Cytokinins Cytokinins or CKs are derived from adenine. The most common CK in plants is Zeatine and its biosynthesis takes place in roots and seeds. The effects of this hormone include cell division, growth and development. Exogenous applications induce cell division when auxin is present and it is endogenously found in gall tumors. It also promotes morphogenesis as it initiates shoot elongation. Furthermore, CK play a role in lateral bud growth, leaf expansion, chloroplast development (which increases chlorophyll accumulation) and it delays leaf senescence (Davies, 2004). CPPU is a synthetic CK also known as

Forchlorfenuron [N-(2-chloro-4pyridyl)-N’-phenylurea] commonly used as a plant growth regulator in numerous crops (Pubchem, 2014).

Effects of Cytokinin (CPPU) on Grapes The effects of CPPU applications on table grapes are numerous and the response level is related to the stage of berry development at the time of treatment. Mostly, at low concentrations, this hormone promotes berry set and development (Nickell, 1985). Furthermore, synthetic CK can cause different reactions depending on the cultivar and amount applied (Strydom, 2013).

Pre-bloom and bloom applications: berry set percentage increases when CPPU is applied at pre-bloom or bloom. Conversely, berry size is hardly affected 30 30 with pre-bloom applications and slightly affected with bloom applications (Nickell, 1986a).

Fruit-set applications: The application of CPPU at this stage has different effects on the berries and the magnitude of these effects is highly concentration dependent with the best results obtained between 5 ppm and 15 ppm. In numerous varieties such as Flame Seedless, Red Globe, Crimson Seedless, Thompson Seedless, Sultanina, Sovereign Coronation, Simone and Summerland selections low CPPU concentrations increase berry size and fruit set (Navarro et al., 2001; Retamales et al., 1995; Reynolds et al., 1992; Strydom, 2013). Treated berries of Thompson seedless and Kyoho were heavier and rounder since length is reduced due to a proportionate increase in berry diameter. (Dokoozlian et al., 1994; Han and Lee, 2004). Also, Flame Seedless, Himrod and Perlette treated berries are generally firmer than non-treated berries and the firmness increases in a linear fashion with increasing CPPU concentrations (Ben-Arie et al., 1998; Peppi and Fidelibus, 2008; Zabadal and Bukovac, 2006). This change in firmness may be a result of skin thickening due to increased cell division (and cell density) caused by CPPU applications. Furthermore, susceptibility to disease was reduced in treated berries due to the thickened skin, that provides mechanical resistance to pathogen invasion and therefore storage potential is increased (Ben-Arie et al., 1998). For all the tested varieties the general appearance of the clusters was also affected. Cluster length, diameter and weight increased (Han and Lee, 2004) as well compactness (Zabadal and Bukovac, 2006). In Sultanina, Perlette and Superior table grapes the rachis and pedicels thicken and increase in weight, which strengthens the attachment of the berry and reduces post-harvest shatter (Ben-Arie et al., 1998; Navarro et al., 2001; Retamales et al., 1995). 31 31

Treatments with CPPU can also delay berry maturity, which is to be expected since cytokinin-like compounds are known to slow senescence. At harvest, this effect results in reduced TSS, and pH while TA is increased. This may become a viticultural concern for late-season varieties, or those where an early market is critical (Navarro et al., 2001; Peppi and Fidelibus, 2008; Reynolds et al., 1992). The reduction in TSS is inversely related to the CPPU concentration, and it can be reduced by 4-10% (Zabadal and Bukovac, 2006). Moreover, both skin anthocyanin concentration and color were found to significantly decrease in Flame Seedless; Sultanina; Kyoho and Sovereign Coronation grapes with CPPU applications (Han and Lee, 2004; Navarro et al., 2001; Reynolds et al., 1992). Flame seedless berries become uniformly light colored with less red and more green which translates into increased Lightness (L*) and Hue (hº) values (Peppi and Fidelibus, 2008). Many varieties show improved post storage quality with CPPU applications since rachis necrosis and berry abscission are significantly reduced after 30 days in cold storage at 1°C (Dokoozlian et al., 2000; Zabadal and Bukovac, 2006). Pre-veráison and veráison applications: When CPPU is applied past 9 mm of berry diameter in Himond table grapes, the response for berry size and mass are similar than at fruit set and only the magnitude of the response decreases as berry diameter increases (Zabadal and Bukovac, 2006). In Red Globe grapes, parameters such as TSS, TA and pH are not affected by CPPU applications at this stage but berry firmness can be increased with concentrations from 6-9 ppm (Avenant and Avenant, 2006). 32 32 Effects of the Combination of GA3 and CPPU on Grapes The combination effects of plant growth regulators on table grapes has not been extensively studied and the results vary greatly depending on numerous factors like grape variety, GA3 concentration, number of GA3 applications, CPPU concentration and time of application. In varieties such as Flame Seedless, Red Globe, Crimson Seedless and

Sultanina the combination of GA3 and CPPU results in larger berries and heavier clusters than non-treated clusters (Strydom, 2013). Other berry parameters are affected differently depend mostly on the grape variety and CPPU concentration applied. When GA3 and CPPU are combined, berry firmness in Flame Seedless is reduced (Strydom, 2013) while firmness increase in Red Globe (Avenant and Avenant, 2006). In Thompson Seedless there is no apparent effect (Ben-Arie et al., 1998). Red Globe, Flame seedless and Crimson Seedless sugar accumulation and maturity is delayed. The effect on TA differs greatly depending on the variety, while in Red Globe TA levels decrease in Flame Seedless. Titratable acidity is increased with the combination of both hormones (Avenant and Avenant, 2006; Strydom, 2013). Berry quality in cold storage is influenced by the management of plant growth regulators during the grapevine growing season (Zoffoli et al., 2009).

Pedicel thickness increases in clusters treated with both GA3 and CCPU in varieties such as Thompson Seedless, Red Globe, Ruby Seedless and Sultanina (Navarro et al., 2001; Zoffoli et al., 2009) which can result in high percentages of loose berries (shatter) (Strydom, 2013; Zoffoli et al., 2009). Furthermore, cell density and skin thickness of berries treated with both plant growth regulators are equal to those only treated with GA3 which can affect susceptibility to disease infection (Ben-Arie et al., 1998). Thompson Seedless and Ruby Seedless develop 33 33 hairline cracks during storage when treated with GA3 and CPPU and can present a high incidence of gray mold (Zoffoli et al., 2009).

In general, all the CPPU effects are depressed with GA3 (Ben-Arie et al.,

1998) and all GA3 effects are reduced with the addition of CPPU but general tendencies remain (Navarro et al., 2001).

Cluster and Berry Thinning

Effects of Cluster and Berry Thinning on Grapes Cluster architecture has an effect on disease incidence. Wine grape varieties such as Carignane, Chenin Blanc, Zinfandel, Barbera; Semillon and French Colombard present tightly arranged clusters, and are more affected by Botrytis bunch rot than those varieties with loosely arranged clusters like Muscat of Alexandria and Cabernet Sauvignon (Vail and Marois, 1991). Even different clones of the same variety like Chardonnay wine grapes can present differences in susceptibility to bunch rot depending on the tightness of the clusters (Vail et al., 1998). Cluster thinning consists of eliminating whole clusters after bloom once the berries have set while berry thinning consists on removing only certain portions of the cluster by eliminating branches of the cluster and the tip of the rachis (Winkler, 1931). The process of removing clusters has numerous effects on the remaining fruit. By eliminating small and misshapen and overly large clusters, the vine’s energy is utilized by the remaining clusters that will present higher marketable quality (Winkler, 1931). 34 34

Cluster thinning can be performed at various stages of the vines reproductive cycle, from pre-bloom through pre-harvest. Pre-bloom thinning entails removing the inflorescences at an early stage by pinching. If the number of leaves is kept constant then the retained clusters will benefit from a higher nutrient supply and consequently present better fruit set and a high percentage of well-developed berries. Also, the cost of thinning can be reduced if performed at this stage. However, since cluster shape and berry set are unknown at this stage, pre-bloom thinning can be risky. Post-berry set is the optimal and most common time to perform bunch thinning since cluster shape and number of berries per cluster is easily determined. Total yield can be decreased when clusters are removed but the packable yield increases because the remaining fruit generally presents high quality attributes. The remaining clusters have an increased berry weight and uniformity along with increased TSS concentration and color development

(Dokoozlian and Hirschfelt, 1995; Winkler, 1931). Cluster thinning after berry softening reduces berry growth and packable yield in early and mid-season ripening grapes (Dokoozlian and Hirschfelt, 1995). Berry thinning is normally used in varieties with compact clusters. It improves fruit quality by increasing berry weight and improving skin color. Berry thinning can be performed several times but the best results are achieved as soon as normal berry drop occurs after bloom. Generally, the earlier thinning is performed, the greater the berry weight gain will be. Thinning can also advance berry maturity and can result in more uniform and earlier development of color (Winkler, 1931). In Red Globe table grapes berry thinning affects organic acids by increasing malic and tartaric acid concentrations. In this variety TSS and TA are higher in hand thinned clusters than non-hand thinned ones (Keskin et 35 35 al., 2013). In Rhine Riesling wine grapes, non-thinned clusters present a higher percentage of infected berries than thinned clusters and usually there is a positive correlation between cluster weight and the number of rotted berries. The majority of infected berries frequently have either concentric splitting or cracking while healthy berries hardly show any open wounds. This shows that by thinning berries from tightly arranged clusters can reduce berry damage and subsequently minimize bunch rot incidence (Barbetti, 1980). Finally, reduction of cluster compactness by berry thinning can increase spray penetration and efficacy of chemical control programs by allowing the surface of inside berries to be exposed to the fungicides (Hed et al., 2009).

CHAPTER 3: MATERIALS AND METHODS

Site Selection The research project was conducted in a commercial Scarlet Royal table grape vineyard managed by Scattaglia Growers and Shippers, LLC (SGS), located in Kingsburg, California. All cultural practices performed for the commercial blocks were executed in the research plot except for no application of

GA3 for sizing. The total surface area utilized for the study was of 0.556 ha that comprised of four rows with 208 vines each. The plant spacing was 3.65 m x 1.82 m. The vineyard was established in 2007 and all vines are own-rooted, meaning no rootstocks were used for grafting.

Experimental Design The three different treatments were arranged in a Complete Randomized

Design (CRD). Each treatment was replicated six times and three vines per replicate were used. One vine between each set of three vines was left as a buffer. The two adjacent rows to the experimental vines were left untreated to create a buffer zone from regular PGR applications. The first four vines at both ends of the rows were excluded in the design as to maintain uniformity of the conditions of the treated vines.

Treatment Applications The different treatments (Table 4) were applied on 30 May 2013 and 29 May 2014 at fruit set (E-L Stage 31). Application sprays were performed with

11.35 L hand sprayers and directed to the fruiting zone. Experimental solutions 37 37 were prepared in a 400 L tank with Tripleline Foam-Away and Latron*B 1956 Spreader-Sticker as application adjuvants.

Table 4: Treatments, Concentration Rates and Commercial Product Treatment Concentration Rate Commercial Product

Control No PGR ------

GA3 5 ppm GibGro® 4LS. (4% GA)

GA3+CPPU 5 ppm GA + 6 ppm CPPU GibGro® 4LS (4% GA) +

KimzallTM (0.8% CPPU)

All clusters were manually thinned pre-veráison (E-L Stage 33-35). Third shoulders and small individual berries from tightly set clusters were removed as well as weak and poorly positioned clusters.

Post-Harvest Decay Forecasting Prior to harvest a sample of 100 berries per treatment was taken for decay forecasting as a method to estimate the type and amount of decay that would develop on the fruit during storage. An adaptation of the protocol for Forecasting Decay in Table Grapes for Storage was used (Usda, 1984). For each treatment the total number of collected berries was divided into two groups and placed in individual plastic boxes on a mesh rack with a moisturized paper towel placed underneath. The boxes were left uncovered over night at the pre-cooler facilities at SGS, LLC for SO2 surface sterilization of the berries. After SO2 fumigation under sterile conditions, berries were rearranged in the box to avoid direct contact between the berries. Paper towels were re-moisturized with deionized water and the boxes were kept for three weeks at 15°C for rot induction. Berry decay was quantified by counting the number of infected berries 38 38 and calculating the percentage of each type of disease using the total number of berries per box. The observation of natural incidence of diseases provided information as to what type of mold could potentially affect the grapes during storage.

Treatment Harvest All the treatment plots were handpicked during commercial harvest on 18 September 2013 and 29 August 2014. Each treatment replication was placed in an individual 9 kg cardboard box and clusters were packed in breathable plastic bags. After harvest, the fruit was stored at the SGS, LLC commercial cold storage facility for four weeks at a temperature of 0°C. The boxes were moved to the Viticulture and Enology Research Center at California State University, Fresno and kept in cold storage for one week at 7.5°C to simulate the conditions of the commercial shelf life for table grapes. To prevent further fruit decay each box contained an individual UVASYS slow release SO2 pad.

Parameters Measured and Instruments Used

Post-Harvest Decay Assessment For every individual box the total weight of the berries was recorded in grams. Subsequently, every berry in the box was inspected and those presenting any abnormalities were placed into a corresponding group. There were a total of five groups: 1 = Botrytis cinerea infected berries; 2 = Shattered berries; 3 = Cracked berries (longitudinal or neck cracks); 4 = Berries infected by disease other than Botrytis cinerea; 5 = Physically damaged berries or insect damaged berries.

39 39

Botrytis infected berries were identified by the presence of gray/brown velvety mycelia, faded or pale berry skin color and skins that easily slipped off while leaving the pulp intact (Bettiga and Gubler, 2013) Shattered berries were those that easily detached from the rachis when the cluster was gently shaken and berries that remained loose in the box after all clusters were examined (Singh et al., 1978) Cracked berries presented small skin fractures, either around the neck circling the pedicel or running along the berry length. Berries with other damage presented insect or bird injuries as well as trimming shear damage. Berries with diseases other than Botrytis were affected by numerous molds such as Aspergillus, Cladosporium, Rhizopus or Penicillium. After the inspection and classification of the fruit, each receptacle containing the abnormal berries was weighed and the incidence of each group was calculated as a percentage of the total weight of the box.

Post-Harvest Berry Quality Assessment Two sub-samples of 50 unblemished berries were collected from each box to obtain further quality parameters. The first 50 berries were used to record berry length, berry width (diameter), berry firmness and berry skin color. Berry length (mm) was obtained with a hand held digital caliper (General® Ultratech, USA). Firmness (g/mm) was determined by the amount of force required in grams to cause a one mm deflection in the berry (FirmTech2, BioWorks, Wamego, KS). Berry width (mm) was also recorded with the FirmTech2 equipment. Skin color was recorded with a handheld spectrophotometer (Konica Minolta CM-700d, Japan). The CIELab L*a*b* color space was used where L* 40 40 specifies lightness (values go from 0 for black to 100 for white), a* specifies chroma (low values indicate green color while high values indicate red color) and b* specifies hue (low values indicate blue color while high values indicate yellow color). The second set of 50 berries was macerated and filtered through a strainer. From the collected juice TA, pH and TSS was recorded. Titratable acidity (g/L) and pH were measured with an automatic titrator (Metler Toledo DL15 Titrator, Switzerland) while TSS (°Brix) was measured with an automatic refractometer (ATAGO model PAL-1, Japan).

Statistical Analysis Statistical Package for the Social Sciences (SPSS)TM 10.0 Software was used to perform multiple comparisons using Analysis of Variance (ANOVA). For those variables that showed significant differences at a 0.05 level of significance between treatments, a post-hoc test was performed using Tukey’s Honest

Significant Difference Test (HSD) for treatment mean separation. Year and replication were considered as random effects and the treatments as fixed effects.

CHAPTER 4: RESULTS AND DISCUSSION

Post-Harvest Decay Forecasting The rot development trials for the different treatments in 2013 and 2014 provided a guide of the mold types that could potentially develop during cold storage (Figure 5). The results showed that for both years the grapes were affected predominantly by four different mold species: Botrytis cinerea, Aspergillus spp., Cladosporium spp. and Penicillium spp (Figure 6). In 2013, the control treatment showed 9% of the berries were affected by

Botrytis while other types of mold affected 39%. The GA3 and the GA3+CPPU treatments were affected by 2% and 6% Botrytis respectively and by other types of mold 19% and 39% respectively (Table 5). In 2014, the percentage of Botrytis affected grapes for the control, GA and

GA3+CPPU treatments were 11%, 23% and 23% respectively. The total percentage of berries affected by other types of mold was 22% for the control, 16% for the

GA3 treatment and 11% for the GA3+CPPU treatment (Table 5).

Table 5: Percentage of Botrytis cinerea and Other Diseases Affecting Control, GA3 Treatment and GA3+CPPU Treatment in 2013 and 2014.

Treatment 2013 2014

Botrytis cinerea (%) Other Disease (%) Botrytis Cinerea (%) Other Disease (%)

Control 9 39 11 22

GA3 2 19 23 16

GA3+CPPU 6 39 23 11 Statistical analysis was not performed for the trial, as the objective was only to determine the type of mold that could potentially develop during post-harvest cold storage.

42 42

Figure 5: Grapes Arranged for Post-Harvest Decay Forecasting Showing Infected Berries.

Figure 6: From Left to Right: Botrytis Infected Berry, Penicillium Infected Berry, Cladosporium Infected Berry and Aspergillus Infected Berry.

43 43 Experimental Results

Post-Harvest Decay Assessment The percentage of Botrytis cinerea incidence after 4 weeks of cold storage at 0°C and 1 week at 7.5°C on Scarlet Royal showed no significant differences for any of the three applied treatments (P = 0.866) (Table7). However, the percentage of shattered berries after post-harvest storage was significantly affected by the application of GA3 (P = 0.000, see Table 7). The data showed that the application of GA3 at a rate of 5 ppm at fruit set resulted in the highest percentage of shatter at 2.0348% compared to the control treatment and the GA3+CPPU treatment that showed 0.6494% and 0.3701% shatter respectively.

The application of GA3 and the combination of GA3+CPPU had no significant effect after post-harvest storage on the remaining variables, percentage of cracked berries (P = 0.842), percentage of other damage (P = 0.374) and percentage of other disease (P = 0.969, see Table 6).

Table 6: Effect of GA3 and GA3+CPPU on Percentage of Botrytis cinerea Incidence, Berry Shatter, Berry Crack, Other Damage and Other Disease Incidence at Post- Harvest on Scarlet Royal Table Grapes Treatment Botrytis Berry Shatter Berry Other Damage Other Disease cinerea (%) (%) Crack (%) (%) (%)

Control 1.5292 a 0.6494 a 1.8305 a 3.4252 a 4.0815 a

GA3 2.0579 a 2.0348 b 1.5059 a 5.0025 a 4.1693 a

GA3+CPPU 1.7713 a 0.3701 a 2.0822 a 5.1977 a 3.6950 a

P 0.866 0.000 0.842 0.374 0.969 Mean values within a column followed by the same letter are not significantly different according to Tukey’s HSD test (P ≤ 0.05). 44 44 Post-Harvest Berry Quality Assessment The application of GA3 and the combination of GA3+CPPU at fruit set had

no significant effect on normal berry quality parameters after post-harvest cold

storage (Table 7).

Berry length was not affected by the application of either GA3 or

GA3+CPPU when compared to the control treatment (P = 0.203, see Table 7).

Furthermore, the differences in berry width and in berry firmness were small

and not significant (P = 0.289 and P = 0.883, see Table 7). Berry skin color was also

not affected regarding any of the three CIELab parameters Lightness, chroma

and Hue (P = 0.988, P = 0.782 and P = 0.996 see Table 7).

Table 7: Effect of GA3 and GA3+CPPU on Berry Length, Berry Width, Berry Firmness and Berry Color (Lightness, Chroma and Hue) at Post-Harvest on Scarlet Royal Table Grapes.

Treatment Berry Length Berry Width Berry Firmness Lightness (L*) Chroma Hue (b*)

(mm) (mm) (g/mm) (a*)

Control 27.9142 a 20.8813 a 290.3766 a 45.4816 a 4.5317 a 6.2290 a

GA3 28.4288 a 22.2118 a 287.4603 a 45.3333 a 4.4494 a 6.2067 a

GA3+CPPU 28.9319 a 23.5598 a 283.4788 a 45.5447 a 4.3037 a 6.1295 a

P 0.203 0.289 0.883 0.988 0.782 0.996 Mean values within a column followed by the same letter are not significantly different according to Tukey’s HSD test (P ≤ 0.05).

Post-Harvest Juice Quality Assessment

As shown in Table 8, there were no differences between GA3 and

GA3+CPPU applications for the juice quality parameters measured after storage (Table 8). The pH (P = 0.543), TA (P = 0.918) and TSS were not significantly affected by the application of either plant growth regulator (P = 0.945). 45 45

Table 8: Effect of GA3 and GA3+CPPU on Berry Juice pH, Titratable Acidity and Total Soluble Solids at Post-Harvest on Scarlet Royal Table Grapes.

Treatment pH TA (g/L) TSS%

Control 4.0020 a 7.5922 a 21.4500 a

GA3 4.0110 a 7.4044 a 21.7500 a

GA3+CPPU 4.0720 a 7.4769 a 21.7900 a

P 0.543 0.918 0.945 Mean values within a column followed by the same letter are not significantly different according to Tukey’s HSD test (P ≤ 0.05).

Discussion Table grape varieties with tightly arranged clusters, such as Scarlet Royal, have a high susceptibility to disease infection. These clusters retain humidity for long periods of time and they are more prone to berry splitting and cracking. Moreover, the high surface contact area between berries disrupts the development of the epicuticular wax, which serves as protection. All these conditions provide favorable conditions for mold spores to germinate and infect the berries (Barbetti, 1980; Carre, 1985; Marois et al., 1986; Martin and Juniper, 1970; Vail and Marois, 1991). Furthermore, fungicide efficacy for disease control can be reduced in compact clusters (Hed et al., 2009). Germination conditions for Botrytis cinerea include temperatures around 22°C and continuous free water on the berry surface. High temperatures, usually above 35°C, can slow growth and readily dry clusters which reduces overall spore germination (Gubler et al., 2013; Nelson, 1950). In this experiment, the lack of differences between treatments and the low percentages of Botrytis infection shown in the rot development trials can be a result of unfavorable conditions for spore germination. With temperatures over 35.5°C experienced during the ripening period in 2013 and 2014 and the lack of rain to provide free water may 46 46 have reduced overall spore germination in the field which consequently reduced mold growth during cold storage (Gubler et al., 2006). Moreover, due to USDA spacing problems and government shut down during September of 2013, the information from the rot development trials was not sufficient to determine Botrytis incidence for the different treatments and was only used as a guide for type of mold present.

The application of 5 ppm of GA3 at fruit set resulted in higher berry shatter than the control or the combination of GA3+CPPU. The use of GA3 has been proven to cause poor berry adherence by decreasing pedicel flexibility that restricts the movement of berries and therefore they can easily detach (Retamales and Cooper, 1993; Singh et al., 1978; Zoffoli et al., 2009). For the GA3+CPPU treatment the detaching effect of the GA3 was presumably cancelled by the capacity of the CPPU to thicken the rachis and pedicels and strengthen berry attachment therefore reducing post-harvest shatter (Ben-Arie et al., 1998;

Navarro et al., 2001; Retamales et al., 1995). In 2014, the second year of the experiment, the management of the commercial block at Scattaglia Growers and Shippers LLC changed and viticulture practices differed from 2013. Girdling was performed on all vines before fruit set during 2014, despite this practice not being recommended for Scarlet Royal (Hashim-Buckey and Ramming, 2008). Numerous authors proved that girdling vines results in larger berries compared to ungirdled vines (Dokoozlian et al., 1999; Reynolds and De Savigny, 2004; Winkler, 1962). The lack of significant differences between the application of PGR and the control might be a result of the effect of girdling masking the effect of both GA3 and CCPU regarding berry enlargement. 47 47

Finally, the results show that the mean TA for the control and the PGR treatments was higher than the 5.5 g/L value described for Scarlet Royal by

Hashim-Buckey and Ramming (2008). Several management and viticultural practices can cause elevated TA values such as excessive soil moisture during growth stage III, reduced cluster exposure to sunlight at veráison and high crop load (Jackson and Lombard, 1993).

CHAPTER 5: CONCLUSION

Gibberellic acid and CPPU were applied to Scarlet Royal table grapes to determine the effect on post- harvest quality parameters as well as disease incidence. Gibberellic acid was applied at a concentration of 5 ppm and in combination with CPPU at a concentration of 5 ppm of GA3 and 6 ppm of CPPU. The different treatments had no effect on the incidence of Botrytis cinerea or any other fungal disease or quality parameter tested. Moreover, PGR applications had no effect on berry length, weight, firmness, berry skin color, titratable acidity, total soluble solids or pH. However, berry shatter was significantly increased by the application of GA3. The weather conditions for the 2013/14 production years were abnormal for the Fresno area, receiving no rain in the months of August and September. Consequently, field conditions for mold development were not optimal and further research is necessary to understand the effects of PGR applications on

Botrytis cinerea during post-storage.

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APPENDIX: TABLE GRAPE TYPES: SURFACE AREA IN HECTARES BY VARIETY AND YEAR PLANTED IN CALIFORNIA

Table Grape Types: Surface Area in Hectares by Variety and Year Planted in California, (CDFA, 2014).

Earlier

2005 & 2005 2013

Variety 2006 2007 2008 2009 2010 2011 2012 2013 Bearing Non- Total

Bearing

Arra 127 0 0 6 0 0 0 4 4 133 8 141 Autumn King 26 32 204 267 214 409 213 187 29 1152 428 1580 Autumn Royal 1542 60 33 57 25 25 24 47 28 1745 99 1845 Autumn Seedless 27 8 8 0 15 0 0 0 0 58 0 58 Beauty Seedless 80 0 0 0 0 0 0 0 1 58 1 81 Black Monukka 31 0 0 0 0 0 0 0 0 81 0 31 Black Morocco 117 0 0 0 0 0 0 0 0 31 0 117 Blanc Seedless 123 0 29 0 0 0 0 0 0 117 0 153 Calmeria 220 0 3 0 0 0 0 4 0 153 4 228 Castlerock Red 13 0 61 0 0 0 0 0 0 224 0 74 Christmas Rose 120 0 0 0 0 0 0 0 0 74 0 121 Concord 41 0 0 3 0 4 0 0 1 121 2 50 Crimson Seedless 4553 85 89 96 142 17 0 16 0 48 16 5021 Early Muscat 32 0 0 0 0 0 0 0 0 5005 0 32 Early Sweet 34 0 0 0 0 0 0 0 0 32 0 34 Emerald Seedless 153 12 6 22 13 10 0 0 0 34 0 217 Emperatriz 32 0 0 0 0 0 0 0 0 217 0 32 Emperor 201 0 0 0 0 0 0 0 0 32 0 201 Fantasy Seedless 265 0 0 9 6 2 0 28 2 201 31 315 Flame Seedless 6824 93 88 60 132 63 85 35 3 7272 122 7394 Flame Tokay 62 0 27 0 0 0 0 0 0 89 0 89 Flaming Red 45 0 0 0 0 0 0 0 0 45 0 45

58

Golden Globe 132 0 0 0 0 0 0 17 0 132 17 149 Jade Seedless 31 0 0 0 0 0 0 0 0 32 0 31 Kyoho 31 1 0 0 0 2 0 0 0 34 0 34 Luisco 13 0 15 15 8 51 20 59 15 104 95 198 Malaga 23 0 0 0 0 0 0 0 0 23 0 23 Marroo Seedless 40 0 0 0 0 0 0 0 1 40 1 41 Muscat Flame 17 0 0 0 15 212 0 0 0 244 0 244 Niabell 19 20 0 0 1 0 0 0 0 39 0 39 Nicolo 17 0 0 0 8 0 0 0 8 24 8 32 Olivette Blanche 4 0 0 0 0 6 0 15 0 10 15 24 Perlette 400 14 0 0 0 0 0 0 0 414 0 414 Prime Black 31 0 0 0 0 0 0 0 0 31 0 31 Seedless Princess 862 323 56 47 27 41 8 13 19 1358 41 1399 Red Globe 3975 51 67 56 78 93 76 15 39 4327 129 4456 Ribier 85 0 0 0 0 0 0 0 0 85 0 85 Rouge 186 0 0 0 0 0 0 0 0 187 0 187 Royal Black 22 0 0 0 0 12 0 0 0 23 0 23 Seedless Ruby Seedless 936 31 0 0 0 1 57 0 0 969 57 621 Scarlet 18 58 189 17 0 1 0 0 27 283 27 310 Scarlet Royal 65 199 217 491 166 362 166 132 34 1524 307 1831 Sugranineteen 114 66 119 0 0 0 0 0 0 299 0 299 Sugraone 1754 125 151 54 70 131 27 19 34 2301 68 2369 Sugrasixteen 27 0 0 0 0 7 0 0 0 34 0 34 Sugrathirteen 137 0 39 0 0 0 0 0 0 176 0 176 Summer Royal 198 71 67 13 9 7 5 17 4 365 26 391

59

Sweet 0 0 0 0 1 134 21 85 0 136 106 242 Celebration Sweet Scarlet 43 27 0 0 66 30 0 0 0 166 0 166 Sweet Sunshine 0 0 0 0 40 0 0 0 0 40 0 40 Thomcord 0 0 4 0 0 6 5 2 9 10 16 26 Timco 8 0 0 0 8 0 22 43 31 16 95 111 Tudor 4 0 0 0 0 0 0 0 16 4 16 21 Vintage Red 93 8 8 94 55 48 94 19 55 333 168 501 90-3618 74 0 0 0 29 0 0 0 0 103 0 103 Other Varieties 171 200 182 215 141 582 380 474 106 3038 960 3998 Total Varieties 25740 1492 1681 1538 1270 2244 1205 1231 466 34047 2865 36912

The underlined varieties represent the 12 major varieties in California.

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Victoria Towers

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03/18/2015

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