rHt"‘“' ‘flhfll‘f :- .41... -' " ' ‘ \=.;- -{ F LIBRARY ~ MW ; limuuligmmlwillinglxiwii f pmvcmty {4: This is to certify that the thesis entitled CUTICLE DEVELOPMENT AND INCIDENCE 0F RUSSET ON 'GOLDEN DELICIOUS'APPLE AS INFLUENCED BY SUBCLONE SUSCEPTIBILITY AND SHELTERS presented by Stephen Michael Long ; has been accepted towards fulfillment of the requirements for 0- 71cm, Major professor Date MO 0-7639 OVERDUE FINES: 25¢ per day per item ggumm LIBRARY mgams: Place in book return to remove charge from circulation records CUTICLE DEVELOPMENT AND INCIDENCE OF RUSSET ON 'GOLDEN DELICIOUS' APPLE AS INFLUENCED BY SUBCLONE SUSCEPTIBILITY AND SHELTERS BY Stephen Michael Long A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture w" f9 ‘3' -//6 (/5, .9. '. C 2.... 9’ ABSTRACT CUTICLE DEVELOPMENT AND INCIDENCE OF RUSSET ON 'GOLDEN DELICIOUS' APPLE AS INFLUENCED BY SUBCLONE SUSCEPTIBILITY AND SHELTERS BY Stephen Michael Long Fruit cuticle characteristics of 'Golden Delicious'(Malus pumila Mill.), Frazier Spur, Smoothee and 'Red Delicious' were examined. Scanning electron micrographs (SEM) indicated that 'Red Delicious' fruit were covered with more projecting wax structures than fruit of the 'Golden Delicious' strains. Total membrane, epicuticular wax, cuticular wax, total wax and cutin matrix (cutin acid plus carbonate soluble material) weights were not different among the 'Golden Delicious' strains 30 days after petal fall, and thin-layer chromatography re- vealed no differences in wax composition. There were no significant correlations between cuticular component weights 30 days after petal fall and russet severity at harvest. Cuticle from russeted and non-russeted areas of 'Golden Delicious' and Frazier Spur fruit was examined. SEM revealed massive cuticle disruption in russeted areas, while some epi- cuticular wax was visible. Russeted areas from both 'Golden Delicious' strains contained less epicuticular and cuticular wax than non-russeted areas. Cold storage reduced the rate of water loss from russeted fruit, but even under these conditions the rate of water loss from 'Golden Delicious' and Frazier Spur fruit was double the rate from 'Red Delicious' fruit. Shelters were used to eliminate rain, alter light quality, and decrease light quantity on 'Golden Delicious' trees. Black polyethylene shade (8.1% full sun) resulted in 27.3% fruit suitable for the fresh market, while full sun controls resulted in 1.6%. Different colored cellophane reduced russet formation, however no relation between light wavelengths and russet formation was established. Clear plastic rain shelters resulted in 82.7% fruit suitable for the fresh market, while unsheltered controls resulted in 0.3%. Light quality and quantity were found to affect the formation of russet, and to affect the quantities of cuti- cular components present in fruit. However, no correlation was found between these components and russet severity. DEDICATION To Susie, whose love and understanding made it possible. ii ACKNOWLEDGMENTS I would like to express my thanks to Dr. James A. Flore for guidance throughout my graduate experience. I wish to thank Drs. M. J. Bukovac, R. Rotz and D. Linville for their assistance and participation on my graduate committee. I wish to thank my fellow graduate students for their support and friendship. I'll miss them down the line. iii TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O O O O O O O O O 0 LIST OF FIGURES . . . . . . . . . . . . . . . . . . . INTRODUCTION 0 O O O O O O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . The Development of Russet Tissues . . . . . . . . Factors Influencing Russet Development . . . . . . Internal Factors . . . . . . . . . . . . . . . . External Factors . . . . . . . . . . . . . . . . Remedies . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . SECTION I RUSSET VARIATION AND FRUIT CUTICLE CHARACTERISTICS OF SEVERAL VARIETIES OF DELICIOUS APPLES . . . . . . . . Abstract . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . Russet Evaluation . . . . . . . . . . . . . . . Cuticular Components . . . . . . . . . . . . . . Thin Layer Chromatography . . . . . . . . . . . Surface Fine Structure . . . . . . . . . . . . . Statistical . . . . . . . . . . . . . . . . . . Results. . . . . . . . . . . . . . . . . . . . . . Russet Evaluation . . . . . . . . . . . . . . . Surface Fine Structure . . . . . . . . . . . . . Cuticular Components . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . Literature Cited . . . . . . . . . . . . . . . . . SECTION II CHARACTERIZATION OF CUTICLE FROM RUSSET AND NON-RUSSET AREAS OF 'GOLDEN DELICIOUS' APPLE . . . . . . . . . . Abstract . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . Plant Materials . . . . . . . . . . . . . . . . Storage Behavior . . . . . . . . . . . . . . . . Surface Fine Structure . . . . . . . . . . . . . Cuticular Components . . . . . . . . . . . . . . Statistical . . . . . . . . . . . . . . . . . . iv Page vi viii H P‘Hld qcnham~dwto 19 20 21 22 22 23 24 25 25 25 25 26 32 36 38 Results Storage Behavior Surface Fine Structure Cuticular Components . Discussion Literature Cited THE EFFECT OF SHELTERS ON DEVELOPMENT OF Abstract Introduction . Materials and Methods General Polyethylene Shade Material SECTION III Colored Cellophane . Plastic Shelter Statistical Results Polyethylene Shade M Colored Cellophane . Plastic Shelter Discussion Literature Cited APPENDICES RUSSET FORMATION 'GOLDEN DELICIOUS' aterial APPLE. FRUIT EPICUTICULAR WAX CHANGES DURING STORAGE CUTICLE GEOMETRIC APPROXIMATIONS OF FRUIT SURFACE AREAS. ETHYLENE PRODUCTION AS AN INDICATOR OF RUSSET FORMATION . FRUIT GROWTH RATES BIBLIOGRAPHY Page 44 47 47 51 53 54 55 56 57 57 57 58 58 60 63 63 69 73 77 79 82 85 89 92 LIST OF TABLES Table 1. 2. 10. 11. 12. Degree of russet among different strains of 'Golden Delicious' apple fruit at harvest. . . . Cuticular components of different strains of 'Golden Delicious' and 'Red Delicious' apple 30 days after petal fall . . . . . . . . . . . . . Correlation between cuticular components and russet severity of 'Golden Delicious' apple 30 days after petal fall and at harvest . . . Cuticular components of different strains of 'Golden Delicious' and 'Red Delicious' apple at harvest 0 O O O O O O O C O O O O O O O O O O O Cuticular components from russeted and non- russeted areas of 'Golden Delicious' and Frazier Spur apple fruit . . . . . . . . . . . . The effect of shelter materials on transmittance of sunlight under full sun conditions. . . . . . The effect of black polyethylene shade material on the degree of russet on 'Golden Delicious' apple fruit at harvest . . . . . . . . . . . . The effect of black polyethylene shade material on cuticular components of 'Golden Delicious' apple 30 days after petal fall and at harvest. . The effect of black polyethylene shade material on the correlation between cuticular components and russet severity of 'Golden Delicious' fruit at harvest. . . . . . . . . . . . . . . . . . . The effect of different colored cellOphane material on the degree of russet on 'Golden Delicious' apple fruit at harvest . . . . . . . The effect of different colored cellophane material on cuticular components of 'Golden Delicious' apple at harvest . . . . . . . . . . The effect of cellOphane material on the correla- tion between cuticular components and russet severity of 'Golden Delicious' fruit at harvest. vi Page 27 33 33 35 49 60 64 64 65 65 67 70 The effect of clear polyethylene shelter on degree of russet on 'Golden Delicious' apple 14. 15. fruit at harvest . . . . . . . . . . . . The effect of clear polyethylene shelter on cuticular components of 'Golden Delicious' 30 days after petal fall and at harvest. The effect of clear polyethylene shelter on the apple 70 71 correlation between cuticular components and russet severity of 'Golden Delicious‘ fruit at harvest . . . . . . . . . . . . . . . . . The effect of black polyethylene shade, clear polyethylene shelter, and colored cellophane on the correlation between cuticular components and russet severity of 'Golden Delicious' fruit at harvest . . . . . . . . . . . . . . . . . . . . Changes in epicuticular wax for 'Golden Deli- cious', Frazier Spur, and 'Red Delicious' fruit B1. B2. C1. C2. C3. during storage at 1°C. . . . . . . . . . Large fruit surface areas (cmz) : S.E. as estimated by five geometric approximation formulas . . . . . . . . . . . . . . . . Small fruit surface areas (cmz) :S.E. as estimated by five geometric approximation formulas. . . . . . . . . . . . . . . . Average ethylene production at petal fall + 11 days (ul/kg/hr). . . . . . . . . . Average ethylene production at petal fall + 21 days (ul/kg/hr) . . . . . . . . . . . Mature fruit russet evaluations from branches earlier sampled for ethylene production. vii 81 84 84 86 88 88 LIST OF FIGURES Figure Page 1. Scanning electron micrographs illustrating epicuticular wax fine structure of different strains of 'Golden Delicious' and 'Red Deli- cious' apple. Columns, left to right: Smoothee, 'Golden Delicious', Frazier Spur, 'Red Delicious'. Row A: fruit harvested at petal fall (PF), 54X. Row B: fruit har- vested at PF, 1000X. Row C: fruit harvested at PF + 9 days, 54X. Row D: fruit harvested at PF + 9 days, 1000X . . . . . . . . . . . . 29 Scanning electron micrographs illustrating epicuticular wax fine structure of different strains of 'Golden Delicious' and 'Red Deli- cious' apple. Columns, left to right: Smoothee, 'Golden Delicious', Frazier Spur, 'Red Delicious'. Row A: fruit harvested at PF + 17 days, 1000X. Row B: fruit harvested at PF + 30 days, 1000X. Row C: fruit har- vested at PF + 129 days, 1000X . . . . . . . . 31 Thin-layer chromatograms of epicuticular wax (deve10ped in chlorogorm/ethyl acetate 7:3). Position (a) cabbage wax for reference, (b) 'Golden Delicious', (c) Frazier Spur, (d) Smoothee, (3) 'Red Delicious' . . . . . . . . 34 The effect of russet severity on relative water loss of 'Golden Delicious', Frazier Spur and 'Red Delicious' fruit stored at 1°C. . . 45 The effect of russet severity on relative water loss of 'Golden Delicious' and Frazier Spur fruit stored at 20°C. . . . . . . . . . . . . 46 Scanning electron micrographs illustrating epicuticular wax fine structure of russeted and non-russeted areas of 'Golden Delicious' fruit. (A) non-russeted area, lOOX. (B) non-russeted area, 1000X. (C) russeted area, 100x. (D) russeted area, 1000X.. . . . . . . . 48 Think layer chromatograms of wax extracted from russeted and non-russeted areas of 'Golden Delicious' fruit (developed in chloro- form/ethyl acetate 7:3). Position (a) cabbage wax for reference, (b) russeted area, (c) non- russeted area . . . . . . . . . . . . . . . . 50 viii Figure 8. 10. 11. 12. D1. Shelters: (A) black polyethylene shade, (B) and (C) colored cellophanes, (D) clear polyethylene rain shelter. . . . . . . . The effect of black and clear polyethylene materials on light transmittance under full sun. 0 O O O O O O I O O O O O I O O O O O O O The effect of colored cellophant materials on light transmittance under full sun conditions. Thin-layer chromatograms of epicuticular wax extracted from fruit grown under control and black polyethylene shade conditions (developed in chloroform/ethyl acetate 7:3). Position (a) cabbage wax for reference, (b) control, (c) 32.0% FS, (d) 8.1% FS. . . . . . . . . . . Thin-layer chromatograms of epicuticular wax extracted at harvest from fruit grown under control, cellophane, and plastic shelter con- ditions (deve10ped in chlorogorm/ethyl acetate 7:3). Position (a) cabbage wax for reference, (b) control, (c) clear, (d) orange, (e) green, (f) red, (9) blue, (h) purple, (1) yellow, (j) unsheltered control, (k) plastic shelter re- moved, (1) plastic shelter kept on . . . . . Fruit growth rates of 4 apple strains as measured by the change in fruit diameter . ix Page 59 61 62 66 68 9O INTRODUCTION 'Golden Delicious' is the second most important apple cultivar in the U.S. (18). However, fruit russeting in eastern and midwestern States is a long-standing problem for apple growers (8,9,16,18,65). Consumer preference is for smooth-skinned apples, in spite of the fact that the eating quality of russeted fruit is just as high as that of smooth- skinned (29). Another major problem for russeted apples is their poor storage quality. Russeted fruit lose more water during storage than smooth fruit (2,20,57), and as a result often have shriveled skins, reducing consumer acceptance. However, such water loss is reduced by storage in high humid- ity with low temperature (2,29,51). The serious problem of consumer preference for smooth fruit remains unsolved (9,10, 12,13,16,25,26,32,50). Methods to eliminate russeting of 'Golden Delicious' apples are desired. Russet may cover entire fruit, or only small areas of fruit (7). It may occur on fruit throughout the entire tree or be limited to some branches of the tree (46,47,48). Rus- seting may be influenced by many factors, including: genetic make-up, cultural practices, water relations, pesticide sprays, nutrition, environmental effects, frost and mechanical injury (2,5,16,26,27,39,43,44,65). The present study was initiated to investigate the influence of several of these factors on 2 russet formation. 'Golden Delicious' cultivars of different russet susceptibilities were compared, as were russet and non-russet tissues. The quantity and quality of light in- cident on fruit was altered, and fruit were grown with protection from rainfall. LITERATURE REVIEW Literature Review I. The deve10pment of russet tissues Russet is initiated within about 30 days after full bloOm (5,6,15,33,45,46,47,48). Cork (periderm) formation has been observed as early as 20 days after fruit set at 10X magnification (47). The greatest increment of tangential growth in apples has also been observed within this early time period (30). The greatest susceptibility to russet for- mation due to spray injury occurs between pink and about 14 days or more after petal fall (39). Several microscopic studies have followed the earliest histological development of russet tissue. In 1937 Bell (4,5,6) found epidermal development of 'Golden Russet' to be the same as in other varieties up until full bloom. During and shortly after full bloom, epidermal cells of 'Golden Russet' underwent distinctive tangential divisions which did not occur in other varieties. The cuticle was seen to develop rapidly during early stages of growth, forming a continuous layer over epidermal cells. Most epidermal cells divided to form a 2-4 cell thick layer of epidermis. However, some epidermal cells did not divide, leaving areas of epidermis only 1 cell thick. In the fol- lowing weeks cuticle was observed to extend down between and under many epidermal cells. An active cork cambium was ini- tiated in lower epidermal regions which underwent rapid cell divisions. Subsequent cork cell develOpment was towards the outermost epidermal cells. Upon maturity the cambial cells were observed to form rows of cork cells. 4 The original epidermal cells were crushed by the con- tinued outward expansion of cork cambium, with subsequent dispersal of cellular contents. Eventually the cuticle itself ruptured, forming a network of fine cracks considered typical of russeted apples. After the initial formation of a russet layer, cork cells continued to push outward. Slough- ing-off of cuticle, dead epidermal cells, and cork cells was observed throughout the growing season. Though some areas of intact cuticle remained, cork or periderm eventually be- came the dominant protective layer of the russeted apples. Meyer (33) confirmed Bell's observation that early devel- opment of the epidermis in russeted 'Golden Delicious' apples proceeded in the same manner as in non-russeted fruit of other varieties. That is, as fruit began to expand, epi- dermal cells divided radially while increasing tangentially. However, 30 days after full bloom epidermal cells of 'Golden Delicious' fruit underwent considerable periclinal division not observed in non-russeted fruit. This critical difference resulted in an irregular epidermal layer with frequent under- lying air spaces. Cracks were observed in the cuticle, and these widened and extended into epidermal and hypodermal cell layers as the fruit continued to enlarge. Cells adjacent to these cracks were often left unprotected by cuticle or cork, thus allowing excessive water loss and shrinkage of stored fruit. More recent studies by Simons gt a1. (44,45,46,47,50,51) indicate a different development of russeted 'Golden Delicious' fruit. Simons noted a lack of periclinal and anticlinal 6 divisions in the epidermal cell layer at about 35 days after full bloom. The hypodermal layer of russeted fruit had 25% less growth than in non-russeted fruit by 14 days after fruit set. This lack of cell divisions resulted in fruit tissues unable to increase in size without disruption of the outermost protective layers. As the fruit increased in size during normal growth, extensive disruption of the epidermal and hypodermal cell layers occurred. Cork cambium cells began to divide and push outward, eventually rupturing the cuticle. Simons also observed the abnormal appearance of many air Spaces in cortical tissues of russeted fruit” (Russet development was thereafter observed to continue just as des- cribed by Bell and Meyer. Studies by Skene in 1965 (52) with 'Cox's Orange Pippin' fruit, indicated a close relationship between russet initia- tion and the occurrence of cracks and dead cells. Skene's count in young fruit showed that 5% of epidermal cells were dead in russeted apples. Cell divisions common to russeting were frequently found directly beneath these dead epidermal cells. And epidermal cracks were often found associated with dead cells. Skene hypothesized that these dead cells could be instrumental in the initiation of russet by their influ- ence on surrounding cells. However, Skene also noted that some russet varieties examined had virtually no dead cells present. DeVries in 1968 (61) found a "second" cuticle formed beneath outer necrotic russeted tissues late in their deve10p- ment. He also found many invaginations of cuticular material 7 between disrupted epidermal cells. In 1972 Pratt (40) studied the anatomical origin of periderm in 'Stark' apples and the russet-forming sport 'Stark 287'. Pratt noted periclinal divisions of epidermal cells and the subsequent death of many epidermal cells. However, epidermal cells in russeted fruit were observed to cease division sooner than in non-russeted fruit. Normal growth of inner tissues then disrupted the epidermis and phellogen layer (active cork cambium). The phellogen layer was observed to originate in hypodermal cells, with some involvement of derivatives of epidermal cells. Pratt's work was in agreement with that of Simons (47) which indicated that russet was initiated by the loss of functioning outer cells, that is, death of epidermal cells triggered the de- velopment of periderm. Electron microsc0py studies in 1972 by Gough and Shutak (23) and by Faust and Shear (17) indicated very fine surface cracks throughout the epicuticular wax of 'Golden Delicious' apples. Of several varieties examined, only 'Golden Deli- cious' had this extensive wax cracking. Faust and Shear took these cracks to be evidence of failure of the wax to expand fast enough to keep up with the growth of internal tissues. II. Factors influencing russet develOpment The factors which influence the initiation and develop- ment of russet may be divided into two basic groups (16,48, 65). These are: 8 a) internal factors, including genetic make-up, which affect the susceptibility of fruit to russet formation, and b) external factors, especially environmental condi- tions, which promote the initiation and/or development of russet. Internal factors. Among apple cultivars, 'Golden Delicious' is one of the most susceptible to russet formation. However, this sus- ceptibility is known to vary between individual 'Golden Deli- cious' subclones (8,10,18,46,47,49,52,57). Individual trees have been observed to contain branches which produce severely russeted fruit, while other branches on the same tree pro- duce normal-appearing fruit (19,46). After gamma irradiation, some 'Golden Delicious' buds produced fruit with russet-free sectors. Un—irradiated con- trols produced no such clean sectors (11). Irradiation of russeted sports 'Stark 287' by Pratt (40) also produced russet-free sectors on fruit. Pratt also reported reversions to non-russeted fruit among controls. Similar work on both irradiated and control 'Sargeant Golden Delicious' showed no reversions to russet-free fruit. It was suggested that the russeted sports may be periclinal chimeras, with a mutation for abnormal development of fruit epidermis in the first layer or layers of the apical meristem. No subclone of 'Golden Delicious' has yet been estab- lished to be completely russet-free. Evaluations of Spur strains (Schell, Thompson, Elliot, Thornton, Goldspur, Morrison, Columbia River, Templin, Frazier, Yellowspur and 9 Starkspur) have shown them to consistently produce lower quality fruit with more russet than regular 'Golden Deli- cious' (16,18). Russet-resistant sports have been reported, including: Smoothee, Kelly, and Magnolia Gold (49). Of these, only Smoothee has been shown to have a high quality approaching that of regular 'Golden Delicious', while pro- ducing fruit with less russet (10,18,49). Some evidence exists that rootstocks can affect russet formation. Walter (65) mentioned studies in Belgium and Denmark where less russeting was found on more vigorous rootstocks such as: MM 106, MM 104, and M 16. Work by Chandler and Mason (8) indicated that some favorable effects could be gained by grafting unto "low russet" rootstocks. However, no significant data was obtained. Cuticle thickness and structure are important factors related to the occurence of russet. Apple cultivars with thin cuticles (4), or areas of fruit with thin cuticles (such as the stem-end of Yellow Newtown apples) are more likely to russet (7). Simons (45) found cuticle thickness on normal 'Golden Delicious' apples significantly greater than on russeted beginning at 37 days after full bloom. However, Gough and Shutak (23) found 'Golden Delicious' to have thicker cuticle than non-russeting 'Cortland' and 'McIntosh' varieties. Epicuticular wax structure of 'Golden Delicious' fruit as well as that of other varieties was examined with scanning electron microsc0py by Faust and Shear (l7), and by Gough and Shutak (23). 'Golden Delicious' had an amorphous 10 cuticle with wax platelets embedded in a structureless matrix, while non-russeting varieties had epicuticular wax arranged in free platelets. No qualitative and only slight quantitative differences were found between cutin acids (fatty acids not removed by boiling in MeOH and CHC13) from russeted and non-russeted fruit (62,63). The cutin acids from both types of tissue were predominantly fatty acids with chain lengths of 16 and 18 carbon atoms. Deposition of wax above the epidermal cells was retarded in russeted fruit as compared to non—russeted (61). Cuticle elasticity may also be an important factor in russeting. A direct relationship has been found in tomatoes between the ability of the skin to stretch and the resistance to cracking (3,60). Microsc0pic examinations by Meyer (33) and Skene (52), as well as electron microsc0pe work by Faust and Shear (l7) and by Gough and Shutak (23) all suggested that 'Golden Delicious' cuticle is unable to stretch suffi- ciently to accomodate the expansion of the growing fruit. The growth rate of fruit will also affect the formation of russet. Many interactions between internal and external factors are involved with these growth rates, including: tree vigor, age, nutritional status, water status, and temperature (16,24,26,65). When conditions are favorable for rapid fruit growth, cultivars with thin, inelastic cuti- cles and unfavorable wax structure may be induced to russet (17). It is also known that cool climates increase epi- cuticular wax and humid conditions decrease epicuticular 11 wax (l). Cuticle cracks (4,5,14,20,21,23,38,52,57) and dead epidermal cells (52) have been associated with russet areas and are apparently due to high tension during growth. In addition to the over-all fruit growth rate, another factor may be the diurnal fluctuations in fruit size. Apples have been found to decrease in size during the day, and in- crease in size during the night (14,24,56,58). Eggert (12) found russet to be more severe when trees were given treat- ments causing a greater diurnal fluctuation in fruit size. It is possible that fruit less subject to this fluctuation would be less subject to cracking of surface waxes and russet formation (17). External factors Temperature was first linked to russet formation in relation to spray injury. In 1930 MacDaniels and Heinicke (31) reported that low temperature (about freezing) before or during bloom caused russet that was often mistaken for spray injury. Other studies (27) supported this View of harmful effects of low temperatures. In 1942 work by Chandler and Mason (8) indicated no relationship between temperature and russeting. Faust and Shear (16) in 1972 suggested that russet is less likely to develop in areas with low night temperatures. This view was supported by Taylor in 1975 (55). Slower fruit growth rates are partially a result of lower temperatures, and result in less russet formation. In 1977 Creasy published results (9) which showed that temperature is a critical factor in russet formation from full bloom 12 until 10 days after full bloom. Higher temperatures during this period resulted in more russet formation. Chandler (8) found no significant relationship between rainfall and russet formation. However, Palmiter in 1944 mentioned a prevailing theory that wet or humid weather during spraying increased russet (39). Also Montgomery in 1959 (38) observed that high rainfall during June and August coincided with the formation of heavy fruit russet. Work by Hatch in 1975 involved the covering of apple trees with plastic canopies (25). The prevention of rainfall from striking the apples resulted in less russet than that formed on uncovered trees. Studies by Edgerton gt 31. (12, 13) found high rainfall increased russet also. In 1977 Creasy (9) found precipitation to be a critical factor in russet formation during the period between 10 and 20 days after full bloom. During this period, higher rainfall gave higher russet. The study also found that rain coverings prevented russet. It is widely accepted that russet of 'Golden Delicious' is more severe in growing areas with high humidity than in areas with low humidity (9,10,16,18,65). Verner's study of fruit cracking (59) indicated that high humidity around fruit would result in increased hydration, which could in- crease cracking if the cuticle was unable to stretch ade- quately. It has also been found that when water is allowed to pool atop fruit, a localized, severe russet occurs (57,66). Other work by Tukey (57) and Watanabe (66) indicated that covering apples with moisture-proof materials (beginning 3 13 weeks and 10 days after petal fall, respectively) resulted in high humidity and high russet. Mink (34) and Creasy (7) discussed the commerical use of water-permeable newspaper coverings in Japan, which results in russet-free apples. In contrast, Hatch (25) recently indicated that apples grown under plastic canopies had less russet than controls, in spite of higher humidity under the canOpies (70% vs.40%). Hatch also grew 'Golden Delicious' trees in a greenhouse which produced smooth, almost russet-free fruit. The over-all water status of the apple tree can also in- fluence russet formation. Both water stress, and an over- abundance of water (from excessive rain or irrigation) can lead to extensive skin cracking and russet formation on fruit (14,21,22). Some evidence has been obtained concerning the role of sunlight and fruit exposure in russet formation. Verner sug— gested that shaded fruit may have more elastic cells than those exposed to sunlight. Exposed fruit had rigid cells which were unable to expand during great increases in turgor, and therefore were more likely to burst (59). On the other hand, Tukey (57) observed that russet occurred over entire fruit surfaces, not just those exposed to sunlight. From this he concluded that sunlight was not an important factor. Mont- gomery (28) believed that a poor foliage cover (due to disease or improper nutrition) exposed fruit to excessive climatic influences, and resulted in greater russeting. Various paper and plastic materials have been used to cover apples. In addition to restricting gas and water 14 movements, these coverings also produced a partial shade over the apples (9,34,41,66). Proctor and Lougheed (41) found that covered 'Golden Delicious' fruit were signifi- cantly firmer than controls, but gaverurdata on russet. Tukey (57) reported that apples covered in paper bags and in clear, fairly permeable plastic bags had no russet, and had a good oily and waxy finish. Mink reported that apples grown inside paper bags in Japan had a good finish with no russet, but also had reduced color and storage quality. The effect of sunlight on russet formation was studied by Watanabe (66) by covering apples with various paper, plas- tic, and cellophane materials. In addition to reducing russet formation, several types of coverings also signifi- cantly altered the cuticles of the apples. Coverings made of newspaper, cellophane, and glass all increased the amount of waxy substance present compared to uncovered apples. Cover- ings of black vinyl and polyethylene showed no change in the amount of waxy substance present. All of the covering materials resulted in apples with a slightly greater weight of cuticlar membrance weight versus uncovered fruit. Watanabe also exposed 'Golden Delicious' apples to U.V. light at various times throughout the growing season. Apples exposed very early in the season developed localized heavy russet, while those exposed later in the season showed no effects from the U.V. exposure. DeVries (64) indicated that sunlight may exchange the polymerization of cutin acids into an amor- phous matrix which is more susceptible to cracking. 15 Frost damage incurred on immature apples is known to persist as russet bands upon the fruit when they mature (2, 26,27,29,38,43,38,51,65). Similar russet forms as a result of accidental mechanical injury. The injury may be caused by hail striking young fruit, abrasion from wind-blown dust, abrasion from fruit and branches colliding via wind, machin- ery bumping fruit, and by abrasion from the force of pesti- cide sprayer (65). Several studies have been done conerning apples injured by frost and by deliberate mechanical means (2,26,29,43,44,61). These studies indicated that such russet formation proceeded in a manner very similar to that of "normal" russet. Outer cells were greatly disrupted, and reversion to meristematic activity occurred to produce the periderm characteristic of russet. However, russet due to frost and mechanical injury generally remains localized and develops rapidly, unlike the slow and dispersed formation of "normal" russet. Several studies investigating the effects of nutrition on russet indicate that high nitrogen applications led to greater russeting (15,54), presumably because high nitrogen induced cell enlargement (28,61). However, others have shown no relationship between nitrogen and russet (15,25), and a positive relationship between smooth fruit and potassimm(15). Similarly, low phosphorous applications resulted in more russet (15), apparently because high phosphorous levels produce correlation exists between magnesium levels and russet formation (15,25,38), while various levels of boron applications did not affect the amount of russeting (8,25). 16 Levels of calcium, manganese, iron, copper, zinc and sodium were also found to not affect russet formation (25). Many studies indicated that certain spray materials in- creased russet formation when applied near the full bloom period. The damage was most severe when adverse weather was involved, namely temperature near or below freezing (27), or excessive moisture (39). Bordeaux mixture, c0pper, sulfur, lime, lead, and arsenate were the pesticide chemicals found to be harmful to 'Golden Delicious' apples (6,27,35,36, 37,38,39,42,53). Later studies indicated that oil—containing sprays (9) and benzyladenine sprays (55) increased russet when applied during the critical period from petal fall until about 15-20 days after petal fall. Hatch (25) indi- cated that certain fungicides plus rain produced greater russeting than the same fungicides without rain. III. Remedies Russet formation can be reduced to some extent by proper cultural practices, namely: prOper nitrogen and phosphorous nutrition (15,38), minimization of water imbalances (21, 22, 65), proper pesticide usage (35,36,37,39,42,53), and selec- tion of russet-free subclones of 'Golden Delicious' (10,18,49 66). While covering the apple fruit with paper bags is known to eliminate russet formation (34,66), this practice is not economically feasible in the United States. Several studies have indicated that the use of Captan reduces russet on 'Golden Delicious' apples (27,38,53). However, as Faust and Shear pointed out (16), no method for effective 17 reduction or elimination of russet on 'Golden Delicious‘ apples was available. Recent work suggests that some reduction in russet formation is associated with certain chemical sprays. Taylor (55) found that applications of GA (4+7) at petal fall and petal fall +7 days reduced russet significantly. As the concentration of GA was increased from 25 ppm to 200 ppm, the reduction of russet was increased. However, Taylor also reported unfavorable side-effects as a result of the GA appli- cation. These included a decrease in flower initiation, a decrease in seed number, and an increase in the formation of spindly shoots. Work by Edgerton et a1. (12) and by Meador (32) showed that Apasil (a commercial preparation containing silicon dioxide) applied at 2.5% at petal fall and petal fall +7 days, significantly reduced russet formation. No harmful side-effects were observed with the use of Apasil. Addi- tional work by Edgerton and Veinbrants in 1979 (13) showed that a combination of Apasil and GA (4+7) provided better russet control than either compound used alone. The most successful mixtures involved the use of Apasil at 2.5%, and GA (4+7) at 25 and 50 ppm (to avoid harmful side-effects). Exogenous GA apparently affects cell division or elongation, while Apasil probably affects the moisture conditions on fruit surfaces (12). IV. Conclusions In spite of significant reductions of russet by the use of Apasil and GA (4+7), the complete control of russeting 18 on 'Golden Delicious' apples has not yet been accom— plished (13). A combination of many of the several factors mentioned may contribute to the occurrence and severity of russet each year in any given orchard (13,16,32). Further research is needed in order to better understand the phy- siological basis for russet formation, and to improve methods for controlling it. SECTION I RUSSET VARIATION AND FRUIT CUTICLE CHARACTERISTICS OF SEVERAL VARIETIES OF DELICIOUS APPLE l9 20 RUSSET VARIATION AND FRUIT CUTICLE CHARACTERISTICS OF SEVERAL VARIETIES OF DELICIOUS APPLE Abstract Fruit cuticle characteristics of 'Golden Delicious' (Malus pumila Mill.), Frazier Spur, Smoothee and 'Red De- licious' fruit were examined. Scanning electron micrographs indicated that 'Red Delicious' fruit were covered with a greater number of projecting wax structures than fruit of the 'Golden Delicious' strains throughout the growing season. Total membrane, epicuticular wax, cuticular wax, total wax and cutin matrix (cutin acid plus carbonate soluble material) weights were not different among the 'Golden Delicious' strains 30 days after petal fall. However, at harvest Smoothee fruit had greater total membrance, cuticular wax and total wax than 'Golden Delicious' and Frazier Spur fruit, and 'Red Delicious' fruit had more epicuticular wax than those 'Golden Delicious' strains. At harvest correlations between all cuticular components (except epicuticular wax) and russet severity were very significant, however corre- lations were not significant 30 days after petal fall. The data presented indicates that russet formation is not re- lated to the quantity of cuticular components present in the fruit of the 'Golden Delicious' strains, nor to the composition of the waxes present. 21 Introduction Among apple cultivars, 'Golden Delicious' is one of the most susceptible to fruit russet formation. Russet appears as an uneven, rough surface composed of cork-like periderm cells. Susceptibility to russet formation is known to vary among individual 'Golden Delicious' trees, and among 'Golden Delicious' subclones (6,7,11,19,20,21,22,24). Indi- vidual trees have been observed to contain branches which produce severely russeted fruit, while other branches on the same trees produce russet-free fruit (14,19). Individual trees and branches also vary from year to year in russet susceptibility (21). Evaluations of spur strains have shown them to consis- tently produce lower quality fruit with more russet than regular 'Golden Delicious' (9,11). Russet-resistent sports have been reported, including 'Smoothee', 'Kelly' and 'Mag- nolia Gold' (21). Of these sports only Smoothee has been shown to have a high quality similar to that of regular 'Golden Delicious', and to produce less russet (7,11,21). However, no sport or subclone of 'Golden Delicious' is com- pletely russet-free. Fruit susceptibility to russet formation is greatest between petal fall and 30 days after petal fall (4,16,18). Two hypotheses currently exist: firstly, susceptible varieties have thin cuticles (3,24) which are easily damaged by factors 22 such as frost, rainfall, pesticides and mechanical injury, which cause russet to form as a protective response; secondly, abnormal growth of underlying epidermal cells causes internal pressures to deveIOp which cause cracking of cuticular membranes unable to sufficiently expand. Cell death has been observed underneath cuticle cracks (22). Active cork cambium initiated in nearby cells as a repair mechanism, thus giving rise to cork cells and the appearance of russet (9). The present study made us of four apple strains with known differences in russet susceptibility: Frazier Spur (subclone of 'Golden Delicious' , severe russet), 'Golden Delicious' (moderate russet), Smoothee (mutation of 'Golden Delicious', light russet) and 'Red Delicious' (no russet). The purpose of the study was to examine fruit cuticle com- ponents in relation to russet formation for each of these types of apple. Materials and Methods Russet evaluation. Mature fruit (30-100/tree) were har- vested at random (9/24/79) from 4 trees each of 'Golden Deli— cious' (14-year-old, seedling rootstock), Frazier Spur (13 year-old Malling-Merton 111 rootstock), and 'Red Delicious' (14-year-old, seedling rootstock) and from 8 trees of Smoothee (ll-year-old, Malling 9 rootstock) grown on the Horticultural Research Center, East Lansing, Michigan. Fruit were evalu— ated for russet severity using a 1-5 subjective scale by comparing individual fruit with photographs of representative 23 fruit in each class (12). A russet rating of "1" indicates fruit free of russet, while higher ratings indicate the presence of progressively more russet. Fruit with ratings of "1" and "2" are considered suitable for fresh sales, and fall within the "U.S. Fancy" grade (1). Russet indexes were also calculated for each group of fruit by multiplying the number of fruit in each russet class by the class value (1-5), and then dividing the total by the number of fruit in the group. Both the % of fruit suitable for the fresh market and the russet index give a concise measure of the severity of russet present within a group of fruit. Cuticular components. Fruit (40—50) were randomly col- lected from each of the 4 cultivars 30 days after petal fall (6/20/79) and cuticular components were determined using a method similar to that described by Flore and Bukovac (13). Briefly, discs (8 mm diam, approx 1 mm thick) were punched from fruit and randomly assigned to 3 groups of 50 each. Epicuticular waxes were extracted by dipping the discs into 2 successive 50 ml portions of redistilled chloroform for 20 seconds. The extracts were pooled and the solvent was removed under reduced pressure on a rotary evaporator (40°C). The waxes were transferred to tared test tubes and epicuti- cular wax weight was determined by subtraction. This tech- nique gave similar results to those obtained when wax extraction was performed prior to the punching of cuticle discs (12). Cuticular membranes were then isolated in a solution of 5.0% (w/v) pectinase (Nutritional Biochem. Corp., Cleveland, 24 Ohio) plus 0.2% (w/v) cellulase (Nutritional Biochem. Corp., Cleveland, Ohio) buffered at pH 3.2 (dibasic sodium phosphate/ citric acid) under mechanical agitation for approx 72 hours. Fresh enzyme solution was substituted after the first 24-36 hours of digestion. After cuticle isolation the discs were agitated in distilled water for approx 24 hours, and then air-dried to a uniform weight. Cuticular waxes were Soxhlent extracted (chloroform/ methanol 9:1 vol/vol) for 2 hours at 45°C. Solvent was removed under reduced pressure on a rotary evaporator and waxes were transferred to tared test tubes for weight deter- mination by subtraction. Discs were again air-dried to determine the weight of the remaining cutin matrix, which is composed of cutin acids and carbonate soluble materia1(13). Cutin matrix was added to total wax to give total membrane weights. Cuticular components for mature fruit were deter- mined using the same procedure with sample discs 12 mm in diameter, approx 1 mm thick. Data are expressed on a weight/ unit area basis (Aq/cmz), and represent the average of 3 samples from each cultivar. Thin layer chromatography. Epicuticular and cuticular wax components were separated by TLC using precoated/silica gel thin-layer plates (Uniplate, 250 um, Analtech, Inc., Newark, Delaware), which were prewashed in benzene and dried at 110°C for 30 minutes. The waxes were dissolved in 1:1 (vol/vol) chloroform/ethyl acetate (5 mg/ml 30 days after petal fall, 10 mg/ml at harvest) and spotted (2 ul) onto the plates along with cabbage wax for reference. The plates 25 were developed in chloroform/ethyl acetate (7:3 vol/vol), and the components were located by charring (160°C) after spraying with 5% KZCrO4in 50% H2804. Surface fine structure. Freeze dried fruit cuticle sections were attached to aluminum studs, coated with gold (approx 20 nm), and observed with a JEOL Model JSM 35C scan- ning electron microscope Operated at 15 kV. Sections from 3 fruit/cultivar were examined from several collection dates, and representative sections were photographed and used for detailed observation. Statistical. Data were subjected to analysis of vari- ance and significance between treatment means was determined by Duncan's multiple range test (23). Regression analysis was performed using cuticular component weights (uglcmz) as the independent variable and the degree of russet at harvest as the dependent variable. A Control Data Corp. 6500 com- puter and the Statistical Package for the Social Sciences (17) were used to analyze the data. Results Russet evaluation. Significant differences in russet severity between strains was evident at harvest with Smoothee being the least susceptible, 'Golden Delicious' intermediate, and Frazier Spur most susceptible (Table 1). No russet was observed on 'Red Delicious'. The relative rankings of these strains is similar to that found from 1975-1978, although the magnitude changed from year to year (12). 26 Surface fine structure. Fruits of all cultivars were covered with trichomes (similar in size and shape) from petal fall to 9 days after petal fall (Fig. 1A,C). .An amorphous wax with occassional granular masses was evident early in the season, while flat droplets and free standing platelets became visible at later sample dates (Figs. 1,2). 'Golden Delicious' surfaces exhibited few projecting wax platelets 9 days after petal fall (Fig. ID), but none were visible on later sample dates. The wax appeared generally amorphous 17, 30 and 129 days after petal fall while exhibit- ing small wax drOplets (Fig. 2A,B,C). Smoothee surfaces remained amorphous until 17 days after petal fall when projecting platelets became visible (Fig. 2A). These platelets were more pronounced 30 days after petal fall but an amorphous surface was present 129 days after petal fall (Fig. 2B,C). Frazier Spur surfaces also remained amor- phous until 17 days after petal fall when flattened wax platelets were visible (Fig. 2A). Later samples appeared to have the same surface structure as 'Golden Delicious' (Fig. ZB,C). Projecting wax platelets were observed on 'Red Delicious' cuticles 9 days after petal fall (Fig. 1D), and covered the surface extensively 17 and 30 days after petal fall (Fig. 2A,B). 'Red Delicious' surfaces were similar to those of 'Golden Delicious' and Frazier Spur 129 days after petal fall (Fig. 2C). 27 Table 1. Degree of russet among different strains of 'Golden Delicious' apple fruit at harvest. Russet evaluation Fruit in each class(%)2 Suitable for Indexw 1 2 3 4 5 fresh market(%)y value Golden x Delicious 2.8 21.4 52.2 20.0 3.6 24.2b 2.98 Frazier Spur 0.5 4.9 37.5 46.1 11.0 5.4c 3.63 Smoothee 13.1 45.7 34.8 5.0 1.4 58.8a 2.27 21= no russet; 5= severe russet y % in classes 1 and 2 X Mean separation by Duncan's multiple range test, 5% level w Summation of the number of fruit in each class times the class number, divided by the total number of fruit Fig. l 28 Scanning electron micrographs illustrating epicuti- cular wax fine structure of different strains of 'Golden Delicious' and 'Red Delicious' apple. Columns from left to right: Smoothee, 'Golden Delicious', Frazier Spur, 'Red Delicious'. Row A: fruit har- vested at petal fall (PF), 54x. Row B: fruit harvested at PF, 1000X. Row C: fruit harvested at PF +9 days, 54X. Row D: fruit harvested at PF + 9 days, 1000X. 29 r/ I \/~\/. \ 331’s» 1 / if : , i .6 4\ as: . ‘NTJ-fl Fig. 2. 30 Scanning electron micrographs illustrating epicu- ticular wax fine structure of different strains of 'Golden Delicious' and 'Red Delicious'. Columns left to right: Smoothee, 'Golden Delicious', Frazier Spur, 'Red Delicious'. Row A: fruit harvested at PF + 17 days, 1000X. Row B: fruit harvested at PF + 30 days, 1000X. Row C: fruit harvested at PF + 129 days, 1000X. 31 32 Cuticular components. There were no significant quan- titative or qualitative differences in the cuticular components between the different strains of 'Golden Deli— cious' 30 days after petal fall (Table 2, Fig. 3). 'Red Delicious' had significantly less epicuticular wax, total wax and total membrance weight than the 'Golden Delicious' strains. Regression analysis revealed no significant correla- tion between cuticular components and russet 30 days after petal fall (Table 3). At harvest 'Red Delicious' had significantly more epi- cuticular wax than the other samples, and 'Golden Delicious' had significantly more than Frazier Spur, but no Smoothee (Table 4). Smoothee had significantly more cuticular wax than the other sample, and cutin matrix weights greater than those of Frazier Spur. Total membrane weights for Smoothee were significantly greater than those of Frazier Spur and 'Red Delicious', while 'Golden Delicious' had more total membrane than Frazier Spur. Smoothee had significantly more total wax than the other samples, while 'Red Delicious' had more than Frazier Spur. TLC again revealed no differ- ences between the wax compositions of the 'Golden Delicious' strains, but 'Red Delicious' had an additional epicuticular wax component with an R value of .84-.88 which co-chroma- f tographed with cabbage wax ketones (Fig. 3). Very significant positive correlations were found be- tween cuticular wax, total membrane and total wax weights vs. 33 russet (Table 3). A significant positive correlation was also present between cuticular matrix weights and russet. Table 2. Cuticular components of different strains of 'Golden Delicious' and 'Red Delicious' Cuticular cgmponent (fig/cm ) Total Epicuticular Cuticular Total Cutin membrane wax wax wax matrix Golden Delicious 1973.7az 415.0a 197.8ab 612.8a 1266.02 Frazier Spur 1913.8a 415.0a 233.6a 648.7a 1265.3ab Smoothee 2069.0a 416.6a 212.1ab 628.7a 1440.4a Red Delicious 1594.3b 366.1b 127.3b 493.4b 1100.9b zMean separation within columns by Duncan's multiple range test, 5% level. Table 3. Correlation between cuticular components and russet severity of 'Golden Delicious' apple 30 days after petal fall and at harvest. Date of Cuticular analysis 30 days after petal fall At harvest Cuticular r F signi- r F signi- Component value ficance(%) value ficance(%) Total membrane .20602 59.5 .51572 15.5 Epitucular wax .27928 46.7 .82976 0.6 Cuticular wax .51288 15.8 .72238 2.8 Total wax .38420 30.7 .90419 0.1 Cutin matrix .22236 56.6 .91171 0.1 34 30 days after petal fall at harvest esters Ketones primary alcohols _ triterpenoid acids . . . . . . . . origin a b c d e a b c d e Fig. 3. Thin-layer chromatograms of epicuticular wax (de— veloped in chloroform/ethyl acetate 7:3) Position (a) cabbage wax for reference, (b) 'Golden Delicious' (c) Frazier Spur, (d) Smoothee, (e) 'Red Delicious'. 35 Table 4. Cuticular components of different strains of 'Golden Delicious' and 'Red Delicious' apple at harvest Cuticular component (Ag/cm ) Total Epicuticular Cuticular Total Cutin membrane wax wax wax matrix GoldenDelicious 3379.9abz 630.316 485.616 116.4bc 2263.5ab Frazier Spur 2875.8c 564.5c 479.2b 1043.7c 1832.1b Smoothee 3723.0a 601.8bc 781.6a 1383.4a 2339.6a Red Delicious 3115.9bc 745.0a 452.2b 1197.2b 1918.7ab zMean separation within columns by Duncan's multiple range test, 5% level. 36 Discussion Russet evaluations confirmed the differing russet sus- ceptibilities of 'Golden Delicious', Frazier Spur, and Smoothee (Table 1). No significant correlations were found between cuticular components 30 days after petal fall and the amount of russet present at harvest (Table 3). TLC re- vealed no differences in the wax compositions of the 'Golden Delicious' strains (Fig. 3). Since it is generally accepted that russet initiation occurs between petal fall and 30 days after petal fall, it appears that russet formation is not related to the amounts of cuticular components present in the strains, nor to the composition of the waxes present. Examination of fruit surface fine structure revealed that 'Red Delicious' fruit were covered with projecting wax platelets at 9, and eSpecially 17 and 30 days after petal fall (Figs. 1,2). In contrast, fruit of the 'Golden Delicious' strains exhibited such projecting platelets in smaller quan- tities and only briefly during this crucial time period. Water repellancy is known to be greatest when wax has a rough surface in the form of projecting rods or other struc- tures (15). The relative shortage of projections on fruit of the 'Golden Delicious' strains may result in more water diffusing into epidermal cells through cracks, causing increased turgor and possible cell rupture and possibly more russet (9). It is possible that an upright arrangement of wax platelets allows the wax of russet-resistent varieties to better accomodate the expansion of underlying fruit 37 tissues (10). The generally amorphous wax on fruit of all 3 'Golden Delicious' strains may be less able to expand during normal fruit growth. The appearance of the surface fine structure of fruit of the 'Golden Delicious' strains did not indicate possible causes for the differing russet susceptibilities of these strains. The differences in cuticle component weights at harvest (Table 4) may be due to normal cuticle development throughout the growing season, primarily after russet formation has occurred. This would explain the close correlations between cuticular component weights and russet found at harvest, while no significant correlations were found 30 days after petal fall. Russet formation could be related to the growth rate of fruit tissue, which may result in cracking of amorphous waxes such as those observed on fruit of the 'Golden Delicious' strains (Figs. 1,2). Russet formation could also be related to the composition of the cutin matrix (not investigated in the present study), or to the cuticular elasticity of dif- ferent apple varieties. Studies with tomato have shown a relationship bewteen the ability of cuticles to stretch and the occurrence of fruit cracking (2,25). An experiement is presently underway to investigate this relationship in apples. 10. ll. 12. 13. 14. 38 Literature Cited Anon, Apple grader's manual. Cooperative Extension Ser- vice, Michigan State University, Extension Bulletin E-747. Batal, K.M., J.L. Weigle, and D.C. Foley. 1970. Relation of stress-strain properties of tomato skin to cracking of tomato fruit. HortScience 5:223-224. Bell, H.P. 1937. The protective layers of the apple. Can. J. Res. 15:391-402. .1937. The origin of russeting in the 'Golden Russet' apple. Can. J. Res. 15:560-566. .1941. The origin and histology of bordeaux spray russeting of the apple. Can. J. Res.19:493-499. Chandler, F.B. and J.C. Mason. 1942. Russeting of 'Golden Delicious' apples. Proc. Amer. Soc. Hort. Sci. 40:120-122. Cummins, J.N., P.L. Forsline, and R.D. Way. 1977. A com- parison of russeting among 'Golden Delicious' subclones. HortScience 12:241-242. Eggert, D.A. and A.E. Mitchell. 1966. Russeting of 'Golden Delicious' apples as related to soil applications of sodium nitrate. Proc. Amer. Soc. Hort. Sci. 99:1-8. Faust, M. and C.B. Shear. 1972. Russeting of apples, an interpretive review. HortScience 7:233-235. and .1972. Fine structure of the fruit surface of three apple cultivars. J. Amer. Soc. Hort. Sci. 97:351-355. Ferree, D.C. and R. Lich. 1978. The search for a russet- free strain of 'Golden Delicious'. Ohio Report 63(4): 51—53. Flore, J.A. 1975. Unpublished data. Michigan State Uni- versity, East Lansing, Michigan. and M.J. Bukovac. 1976. Pesticide effects on the plant cuticle: II. EPTC effects on leaf cuticle morpho- logy and composition in Brassica oleracea L. J. Amer. Soc. Hort. Sci. 101:586-590. Gardner, V.R., W. Toenjes, M. Giefel, and J.C. Kremer. 1948. Variability and segregation in the 'Golden Russet' apple. J. Agric. Res. 76:231-240. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. ' 39 Martin, J.T. and B.E. Juniper. 1970. The cuticles of plants. Edward Arnold Pub1., Edinburg. Meyer, A. 1944. A study of the skin structure of 'Golden Delicious' apples Amer. Soc. Hort. Sci. 45:105-110. Nie, H.C., H. Hull, J.G. Jenkins, K. Steinbrenner, and D.H. Bent. 1975. Statistical package for the social sciences. McGraw-Hill, New York. Simons, R.K. 1960. Developmental changes in russet sports of 'Golden Delicious' apples - morphological and anatomi- cal comparison with normal fruit. Proc. Amer. Soc. Hort. Sci. 76:41-51. .1962. Spontaneous russet sports of 'Golden Deli- cious' apples - morphological and anatomical comparisons with normal fruit. Proc. Amer. Soc. Hort. Sci. 80:79-89. . 1965. The origin of russeting in russet sports of the 'Golden Delicious' apple. Hort. Res. 5:101-106. . 1974. Russeting of 'Golden Delicious' and re- lated strains of apples. Trans. Ill. State Hort. Soc. 107:69-74. Skene, D.S.1965. Cracking and russeting in apple fruits. Ann. Rpt. E. Malling Res. Sta. for 1964: 99-101. Steel, R.G. and J.H. Torrie. 1960. Principles and pro- cedures of statistics, McGraw-Hill, New York. Tukey, L.D. 1959. Observations on the russeting of apples growing in plastic bags. Proc. Amer. Soc. Hort. Sci. 74:30-39. Voisey, P.W., L.H. Lyall, and M.Klock. 1970. Tomato skin strength - its measurement and relation to cracking. J. Amer. Soc. Hort. Sci. 95:485-488. SECTION II CHARACTERIZATION OF CUTICLE FROM RUSSET AND NON-RUSSETED AREAS OF 'GOLDEN DELICIOUS' APPLE 40 41 CHARACTERIZATION OF CUTICLE FROM RUSSET AND NON-RUSSET AREAS OF 'GOLDEN DELICIOUS' APPLE Abstract Cuticle from russeted and non-russeted areas of 'Golden Delicious' (Malus pumila Mill.) and Frazier Spur was examined. Scanning electron microscopy revealed massive cuticle disrup- tion in russeted areas, however epicuticular wax fine structure was still evident. Frazier Spur fruit cuticle was generally composed of less epicuticular wax, cuticular wax, cutin matrix (cutin acid plus carbonate soluble material), and total membrane weight than cuticle from 'Golden Delicious' fruit. Russeted cuticle from fruit of both 'Golden Delicious' and Frazier Spur contained less epicuticular and cuticular wax than non-russeted cuticl. At room temperature (20°C) russeted fruit lost water at a greater rate than non-russeted fruit. In cold storage (1°C) russeted fruit initially lost water at a greater rate than non-russeted, but the rates were essentially the same during later storage. However, even under cold storage conditions 'Golden Delicious' and Frazier Spur fruit lost water twice as fast as 'Red Delicious' fruit. 42 Introduction Histological studies indicate that apple russet results from irregular cell divisions which lead to the formation of an uneven, disrupted layer of epidermal cells (1,2,7,8). Active cork cambium is initiated in lower epidermal layers which divides toward the outermost epidermal cells and even- tually ruptures the fruit surface. Russet is observed throughout the remaining growing season as a sloughing of cuticle, dead epidermal cells and cork cells. Russeted fruit are generally not acceptable to consumers, and they are more susceptible to water loss and shriveling in storage than are non-russeted fruit (6,11). The present study was initiated in order to characterize differences between russet and non-russet cuticles in relation to water loss during storage. Since little quantitative in- formation is available concerning russet and non-russet cuti- cular components, these were determined for both types of cuticles. 'Golden Delicious' (moderate russet) and its sub- clone Frazier Spur (severe russet) were used to determine possible differences in russet tissue characteristics when different russet susceptibilities are known to exist. Materials and Methods Plant Materials. Mature fruit were harvested from 14- year-old 'Golden Delicious' trees (seedling rootstock) and from 13-year-old Frazier Spur trees (MM 111 rootstock) grown on the Horticultural Research Center, East Lansing, Michigan. 43 Storage behavior. 'Golden Delicious' and Frazier Spur fruit were evaluated for russet severity as previously des- cribed (3,5) and separated into "moderate russet" and "severe russet" groups. The moderate russet group contained fruit rated 1-3, and the severe russet group contained fruit rated 3-5. Samples of 10 fruit each were randomly selected from these groups and the russet index was calculated for each as previously described (5). Fruit samples were placed into cold storage (1°C) and kept at room temperature (20°C). Moderate and severe russet samples of 'Golden Delicious' and Frazier Spur were replicated 5 times in each storage tempera- ture. Five replicate samples of non-russet 'Red Delicious' fruit were also put into cold storage for comparison. Initial sample weights were recorded and each sample was weighed periodically thereafter. Fruit kept at room tempera- ture were discarded after 5 weeks because of severe deteriora- tion. Fruit kept in cold storage were sampled for 12 weeks. The weight loss during each interval between weighings was divided by the previous sample weight, and then by the number of days between weighings to give the relative weight loss per day for each storage interval. Surface fine structure. Fruit tissue samples were col- lected and prepared for S.E.M. examination as previously described (5). Representative examples of russet and non- russet fruit surfaces were photographed for detailed observation. Cuticular components. Fruit tissues were selected as russeted and non-russeted, and cuticular components were 44 isolated and analyzed as previously described (5). Three replicate samples of 50 discs (12 mm diam) were analy- zed for each type of tissue. Statistical. Standard deviations were calculated from the relative water loss/day values for each treatment. Cor- relation coefficients (r) were calculated using relative water loss/day as the dependent variable and the russet index as the independent variable. Cuticular component data were subjected to analysis of variance and significance between treatment means was determined by Duncan's multiple range test (10). Results Storage behavior. Cold storage greatly reduced water loss for both moderate and severe russet fruit of the 'Golden Delicious' strains (Figs. 4,5). The rate of water loss for all treatments except 'Red Delicious' were generally highest during the first week of storage and gradually decreased thereafter. In cold storage all 'Golden Delicious' and Frazier Spur fruit lost water at similar rates, however the rate for severe russet fruit was much higher for both from 10/8/-10/12 (7 days of storage) and for Frazier Spur from 10/23-10/30 (27 days of storage) than that observed for moderate russet fruit. The relative water loss rate of 'Red Delicious' in cold storage was much lower than that of all 'Golden Delicious' treatments on all sample dates. Severe russet caused significantly greater rates of water loss at room temperature storage than those which 45 N0 .UOH um emooum uwoum .maoHowHoo com. com Hzaw uoflumum .mzowowamo cocaoo. mo mmoH nouns o>wumHoH co huwuo>om ummmsu mo uoommo one v .mflm Hw>¢ou mam» wacmOFw um _ «v. t, «n, . «N. t Nwr i, N_. )8 .. m u “U .. zHu 0A w 3 .. 6 M 115 u. .1 lb M E .C.wu II xw its. IOLS .39 8a .9. my $333 5 .E .1 __ .. 3 E4382 .3 E... w mamim do good. a A 35:50: .30 400.6. 46 «v .Uoom um omnoum Denna Noam uoflnmum cam .msOHUAHmo cooaoo. no mood Houm3 o>wuwaou co >uwuo>om oommsu mo poommo one .m .mflm 530. m2: mammog mm Nut Na 1&- 4L ‘) p E [P I 5 Jl 1 HERE .3 E .1 fiiwooz A .E .6. $325 do good. 93.23002 :50 .._00 .0. L ) H 55 (ii-01X l-OJM 1"} 6w OZH 5w AUG 83c! 980-) 8315M HAIlU—IB 47 occurred in moderate russet samples (Fig. 5). Rates for severe russet samples of 'Golden Delicious' and Frazier Spur were not significantly different in most cases, and the same was true for moderate russet samples of the two strains stored at room temperature. A very significant positive correlation (r=0.59) was present between the russet index and relative water loss per day in room temperature storage. Surface fine structure. Extensive cuticle disruption was observed on russeted tissue sample from both 'Golden Delicious' and Frazier Spur fruit (Fig. 6). While non- russet surfaces were seen to be continuous coverings of epicuticular waxes, the russet surfaces were discontinuous and broken. Underlying epidermal cell outlines were also clearly visible in russet areas. It was evident that in— ternal tissues were still somewhat protected in russet areas since wax deposits were visible. The fruit surfaces of non- russet areas adjacent to russet areas appeared to be iden- tical to non-russet areas present anywhere on the fruit. Cuticular components. More cuticular matter of every type was present in non-russet 'Golden Delicious' than in non-russet Frazier Spur, and more was present in russet Gol- den Delicious' than in russet Frazier Spur (Table 5). Al- though the differences were not all significant, the trend suggests that Frazier Spur fruit normally have less cuticular matter than 'Golden Delicious' fruit. Russet areas contained significantly less epicuticular and cuticular waxes than 48 .xOOOH .moum Umuomwsu An. .xooH .moum wouommsu AU. .xoooa .monm poummmsnico: Amy .xOOH .monm pouommsu Icoc Aaom Hmoao on .oomnm wcoawacuohaom xomHn Adv ”muouamnm .m .meh 60 Table 6. The effect of shelter materials on transmittance2 of sunlight under full sun conditions. Material Transmittance (% of full sun) Clear cellOphane 100.0 Purple 80.6 Yellow 75.1 Blue 62.1 Orange 56.9 Green 42.4 Red 37.5 92% black polyethyleney 8. 1 55% black polyethylene 32. 0 Clear polyethylene 93.2 2Calculated from data determined by spectroradiometry. yManufacturer's shade designations, referred to as 8.1% FS and 32.0% FS respectively. leaving the lower 2-3 feet Open for air movement (Fig. 8). Six trees selected for uniform size and flowering were covered while 6 adjacent trees were selected as controls. The plastic shelters were removed from 3 of the trees 40 days after petal fall ("shelter removed") and from the remaining trees at harvest ("shelter kept on"). Spectroradiometer measurements showed that the plastic did not affect light quality, while it decreased FS by 6.8% (Fig. 9, Table 6). Statistical. Data were subjected to analysis of variance and significance between treatment means was determined by Duncan's multiple range test (14). Regression analysis was performed using cuticular components from the various treat- ments as the independent variables and the degree of russet at harvest as the dependent variable. A Control Data Corp. 61 A55 .mcowuapcoo com Hasm Hops: mocmuqumcmnu unmaa co mamfiuoume ocoahnuoaaom Hmoao pom xowan mo uoomuo one .m .oflm Ebzmnm>¢z car can amm— mu— mafiwniu w”. Xmm‘u U333; m + RED + BLUE 1’ -l- YELLOW -.- PURPLE 35" *CLEAR :6» _ - 770 I 610 I 1660 ' :1'00 330 1170 ' 1610 HRVELENGTH (nm) Fig. 10. The effect of colored cellophane Intericls on light transmittance under full sun conditions. 63 6500 computer and the Statistical Package for the Social Sciences (8) were used to analyze the data. Results Polyethylene shade material. The 8.1% FS treatment significantly increased the % of fruit suitable for the fresh market as compared to untreated fruit (Table 7). The 32.0% FS treatment also improved fruit quality as evidence by 61.1% in russet class 3 and no fruit in russet class 5, compared to 29.5% in class 3 and 17.1% in class 5 for control fruit. Cuticular component weights of both shade treatments and controls were not significantly different 30 days after petal fall, nor at harvest (Table 8). No significant corre- lations were detected between cuticular component weights and russet (Table 9). TLC of both epicuticular and cuti- cular waxes from both sample dates showed no differences between any of the treatments (Fig. 11). Colored cellophane. Russet formation was significantly reduced by red, blue, purple and clear cellophanes (Table 10). The % of fruit suitable for the fresh market from the blue cellOphane was the highest of all the cellophanes, and this treatment also produced no fruit rated in the most severe russet classes (4 and 5). Clear and blue treated fruit had significantly more epi- cuticular wax at harvest than those of the other treatments and controls (Table II). Epicuticular wax weights of purple and red treated fruit were not significantly different than controls. Fruit from red, green and yellow treatments had 64 Table 7. The effect of black polyethylene shade material on the degree of russet on 'Golden Delicious' apple fruit at harvest. Russet evaluation Fruit in each class (%)z Suitable for the Index Treat- y w ment 1 . 2 3 4 5 fresh market value Full sun 0.0 1.6 29.5 51.9 17.1 1.616x 3.87 32.0%FS 0.0 2.8 61.1 36.1 0.0 2.8b 32.7 8.1%FS 6.1 21.2 54.5 18.2 0.0 27.3a 2.87 z l=no russet; 5=severe russet. y% in classes 1 and 2. xMean separation by Duncan's multiple range test, 5% level wSummation of the number of fruit in each class times the class number, divided by the total number of fruit. Table 8. The effect of black polyethylene shade material on cuticular components of 'Golden Delicious' apple 30 days after petal fall and at harvest. Cuticularzcomponent Gig/cm ) Sample Total Epicuticular Cuticular Total Cutin Date Treatment membrane — wax wax wax matrix 30 days Full sun 1712.12 383.2 187.0 570.2 1141.9 after 32.0% FS 1752.7 386.0 175.1 561.0 1191.7 petal 8.1% FS 1661.2 397.9 195.0 592.9 1068.3 fall At Full sun 3504.6 802.5 401.4 1203.9 2300.7 harvest 32.0% FS 3390.5 786.9 455.0 1241.9 2148.6 8.1% FS 3444.3 800.6 422.6 1223.2 2221.1 2No significant differences were detected within columns for each sample date by Duncan's multiple range test, 5% level. 65 Table 9. The effect of black polyethylene shade material on the correlation between cuticular components and russet severity of 'Golden Delicious' fruit at at harvest. Cuticle component r value F significance (%) Epicuticular wax .03775 92.3 Cuticular wax .26534 49.0 Total wax .18780 62.8 Cutin matrix .02187 95.5 Total membrane .12536 74.8 Table 10. The effect of different colored cellophane material on the degree of russet on 'Golden Delicious' apple fruit at harvest. Russet evaluation Fruit in each class (%)z Suitable for the Index Treatment 1 2 3 4 5 fresh markety value Control 0.0 0.8 10.9 35.2 53.1 0.8cx 4.40 Clear 8.3 37.5 45.8 8.3 0.0 45.8ab 2.63 Orange 12.5 20.8 50.0 16.7 0.0 33.3bc 2.80 Green 11.1 22.2 63.0 3.7 0.0 33.3bc 2.55 Red 23.1 30.8 38.5 7.7 0.0 53.9ab 2.20 Blue 27.8 61.1 11.1 0.0 0.0 88.9a 2.20 Purple 18.2 40.9 31.8 9.1 0.0 59.1ab 2.20 Yellow 11.1 33.3 27.8 27.8 0.0 44.4bc 2.58 z 1=no russet; 5=severe russet y% in classes 1 and 2 XMean separation by Duncan's multiple range test, 5% level. wSummation of the number of fruit in each class times the class number of fruit. 66 30 days after petal at harvest fall e ters aIkanes Ketones primary alcohols triter- penoid acids origin Fig. 11.Thin-layer chromatograms of epicuticular wax extracted from fruit grown under control and black polyethylene shade conditions (developed in chloroform/ethyl acetate 7:3). Position (a) cabbage wax for reference, (b) control (c) 32.0% FS, (d) 8.1% FS. 67 Table 11. The effect of different colored cellophane on cuticular components of 'Golden Delicious' apple at harvest. Cuticular component (Hg/cm ) Total Epicuticular Cuticular Total Cutin Treatment membrane wax wax wax matrix Control 3218.3bz 608.7cd 542.4bcd 1151.0ab 2067.3 Purple 3115.7bc 595.2cd 429.9cd 1020.2b 2095.5 Red 3387.5a 561.1d 766.2a 1327.4a 2060.2 Green 3143.5bc 592.9cd 651.3abc 1244.2ab 1899.3 Orange 3097.5c 639.5bc 569.4abdc 1208.9ab 1888.6 Blue 3227.8b 666.0ab 501.7bcd 1167.7ab 2060.2 Clear 3174.8bc 669.6a 365.5d 1065.1b 2109.7 Yellow 3152.0bc 609.6cd 701.5ab 1311.1a 1840.9 zMean separation within columns by Duncan's multiple range test, 5% level. significantly more cuticular wax than controls, and the red treatment resulted in significantly more cuticular wax than did the blue, purple and clear treatments. No total wax weights from the treatments were significantly from the controls, while those from red and yellow treatments were significantly higher than those from purple and clear treatments. No Significant differences were present between cutin matrix weights from treatments and controls. Only the red treatment produced significantly higher total membrane weights than the controls, while the orange treatment pro- duced weights significantly lower than the controls. TLC revealed no differences in epicuticular wax composi- tions from any treatments and controls (Fig. 12). Although 68 esters, alkanes Ketones primary alcohols triterpenoid acids origin Fig. 12. Thin-layer chromatograms of epicuticular wax ex- tracted at harvest from fruit grown under control, cellophane, and plastic shelter conditions (developed chloroform/ethyl acetate 7:3). Position (a) cabbage wax for reference, (b) control, (c) clear, (d) orange, (e) green, (f) red, (g) blue, (h) purple, (i) yellow, (j) unsheltered control, (k) plastic shelter kept on. (i) shelter removed. O 69 many significant differences in russet formation and cuti- cular formation and cuticular component weights resulted from the cellophane treatments, no significant correlations were found between these data (Table 12). Plastic shelter. Both plastic shelter treatments caused a significant increase in the % of fruit suitable for the fresh market as compared to unsheltered controls (Table 13). At harvest a small decrease in fruit quality was observed on the trees from which the plastic shelters were removed 40 days after petal fall. This treatment resulted in fewer fruit suitable for the fresh market than was present when the shel- ters were left on (68.9% vs. 82.7%, respectively). This difference appears to be due to the larger % of fruit in russet class 3 when the shelters were removed (26.2%) as com- pared to when the shelters were left on (11.9%). Fruit grown under plastic shelters had significantly more epicuticular wax than controls 30 days after petal fall (Table 14). Control fruit had significantly higher cutin matrix weights than the treated fruit at this time. At har- vest control fruit had significantly higher epicuticular wax and total membrane weights than fruit with the shelters left on. Both control fruit and fruit with the shelters removed had significantly greater total wax weights than fruit with the shelters left on. TLC revealed no differences in the compositions of epi- ticular and cuticular waxes on both sampling dates from the shelter treatments and controls (Fig. 12). Correlations 70 Table 12. The effect of cellophane material on the cor- relation between cuticular components and russet severity of 'Golden Delicious' fruit at harvest. Cuticle component r value F significance (%) Epicuticular wax .16867 35.6 Cuticular wax .05570 76.2 Total wax .01094 95.3 Cutin matrix .06426 72.7 Total membrane .09317 61.2 Table 13. The effect of clear polyethylene shelter on degree of russet on 'Golden Delicious' apple fruit at harvest. Russet evaluation Fruit in each class (%)z Suitable for Index Treatment 1 2 3 4 5 fresh market(%)yvalue Control 0.0 0.3 9.8 40.8 49.0 0.3bx 4.30 Shelter removed 17.2 51.7 26.2 4.8 0.0 68.9a 2.20 Shelter kept on 20.2 62.5 11.9 4.2 1.2 82.7a 2.03 z1=no russet; 5=severe russet. y% in classes 1 and 2. xMean separation by Duncan's multiple range test, 5% level. wSummation of the number of fruit in each class times the class number, divided by the total number of fruit. 71 Table 14. The effect of clear polyethylene shelter on cuticular components of 'Golden Delicious' apple 30 days after petal fall and at harvest. Cuticularzcomponent (us/cm ) Sample Total Epicuti- Cuticular Total Cutin date Treatment membrane cular wax wax wax matrix 30 days Control 1819.5 380.8bz 124.5 505.3 1314.2a after petal fall Shelter 1802.4 407.0a 179.1 586.1 1216.3b At Control 3050.7a 526.6a 483.3 1009.9a 2040.7 harvest Shelter removed 2995.6ab 522.9ab 492.1 1015.1a 1980.6 Shelter kept on 2831.2b 502.2b 422.6 924.9b 1906.3 zMean separation within columns for each sample date by Duncan's multiple range test, 5% level. between cuticular component weights and russet severity were not significant. Previous results (10) showed that russeted cuticles have significantly less epicuticular wax than non- russeted. Thus it is not surprising that a significant positive correlation between epicuticular wax weight and russet reduction was detected when data from all treatments were combined. 72 Table 15. The effect of clear polyethylene shelter on the correlation between cuticular components and russet severity of 'Golden Delicious' fruit at harvest. Cuticle component r value F significance (%) Epicuticular wax .56847 11.0 Cuticular wax .42928 24.9 Total wax .53521 13.8 Cutin matrix .49727 17.3 Total membrane .58116 10.1 Table 16. The effect of black polyethylene shade, clear polyethylene shelter, and colored cellophane on the correlation between cuticular components and russet severity of 'Golden Delicious' fruit at harvest. Cuticle component r value F significance (%) Epicuticular wax .23354 7.5 Cuticular wax .07516 57.2 Total wax .06600 61.9 Cutin matrix .01342 92.0 Total membrane .03034 82.0 73 Discussion Very low incident light (8.1% FS) was favorable for less russet formation, while somewhat higher incident light (32.0% FS) caused a lesser reduction in russet formation (Table 7). While no differences in cuticular components or waxes were found as a result of the shade treatments, it is possible that these treatments produced more elastic fruit cuticles as postulated by Verner (16), and thus were able to resist cuticle cracking and russet formation to some degree. It is also possible that in spite of the structural design used, temperature was increased, or rainfall was somewhat excluded underneath the black shade materials, thus confound- ing the shade effects. Russet initiation is believed to occur during the first 30 days after petal fall (3). At the end of this period, fruit grown under clear plastic shelters had significantly more epicuticular wax than unsheltered control fruit (TabLe14). However, cuticular wax and total wax from sheltered fruit and all waxes from fruit grown under black polyethylene shade material were not significantly different from waxes of control fruit at this time (Tables 8, 14). At harvest, sheltered fruit had less total wax than control fruit and fruit from which the shelters had been removed. This de- crease in wax quantity may have been caused by the humid environment present underneath the plastic shelters through- out the growing season (15). The increase in russet on fruit from which the shelters had been removed (Table 13) 74 suggests that the severity of russet present at harvest can be affected by the environmental conditions present after the primary russet initiation period has passed. Factors other than cuticular components may have been responsible for the differences in russet formation which occurred as a result of the various treatments. Rainfall or high humidity may cause pooling of water at0p fruit, which presumably causes epidermal cells to absorb water by diffusion, and can lead to over-expansion and bursting of these cells, and thus to russet formation (15, 20). Rain- fall striking very young fruit may cause sufficient physical abrasion to damage epidermal cells and induce russet for- mation as is known to occur by frost or deliberate mechanical injury (1,7,11,13,17). The data presented here also indicate the importance of rain for russet formation. Rain shelters resulted in very little fruit russet when kept over trees throughout the growing season, while removal of the shelters 40 days after petal fall resulted in more heavily russeted fruit (Table 17). It seems doubtful that the russet reduction obtained under the cellOphane treatments was due to the different light wavelengths applied. Clear cellophane produced significantly less russeted fruit (Table 10), yet this cellophane was shown to have no effect on the incident sunlight (Table 6, Fig. 10). The other most successful cellOphane colors had very different spectra (Fig. 10), and very different light transmittance (Table 6). Red 75 cellOphane admitted almost no light below 551mn,and admitted almost all above 650rmu Both blue and purple admitted at least half of the light below 550 nm, and while purple admitted almost all of the light above 650 nm, blue admitted less than half between and 550 and 675 nm. Clear cellophane transmitted 100% of the incident sunlight, purple transmitted 80.6%, blue transmitted 62.1%, and red transmitted only 37.5% of the sunlight. The quantity and quality of light transmitted through the various cellOphane colors had variable success in russet reduction, and various effects on cuticular component weights. However, no relationship between light quality and quantity and russet formation was established (Table 12). Since all the cellophane treatments did reduce fruit russet (Table 10), it is possible that the success was due to rain sheltering as was observed under the clear plastic shelters. The cello- phane treatments were applied in such a manner as to allow as much rainfall penetration as possible, while the plastic shelters were applied so as to totally exclude rainfall. The markedly lower russet reduction which resulted from the cellophane treatments as compared to fruit grown under plastic shelters may have been a result of the celloPhanes only partially blocking rain from the fruit. Artificial shelters did alter the amount of cuticular components present 30 days after petal fall and at harvest (Tables 11,14). However, from the data presented it seems unlikely that either epicuticular wax, cuticular wax, or 76 membrane weight are associated with russet formation. No significant differences in cuticle component weights were present 30 days after petal fall as a result of black poly- ethylene shade treatments (Table 8), yet the shade treat- ments resulted in fruit with less russet (Table 7). Cuticle component weights of fruit grown under cellophanes and plastic shelters were not correlated with russet severity (Tables 12, 15). Only epicuticular wax weight was related (7.5% level) to russet severity when component weights from all treatments were combined (Table 16), and this relation- ship was shown to be due to the presence of less epicuti- cular wax on russeted areas of fruit at harvest (10). Rainfall, or an interaction between rainfall and light may be responsible for the reduction of russet observed. The composition of the cutin matrix may be affected by en- vironmental conditions and may be involved in russet formation (18). Cuticle elasticity may also be an important factor in russet formation in relation to cell growth and expansion, and it is being investigated at the present time. 10. 11. 12. 13. 77 Literature Cited Baker, C.B. 1930. A study of the skin structure of the Grimes apple as affected by different types of injury. Proc. Amer. Hort. Soc. 27:75-81. Creasy, L.L. 1977. Can russet of 'Golden Delicious' be controlled? Proc. Ann. Meetinng.Y. State Hort. Soc. 122:160-162. Faust, M. and C.B. Shear, 1972, Russeting of apples, an interpretive review, HortScience 7:233-235. Ferree, D.C. and R. Lich. 1978. The search for a russet- free strain of 'Golden Delicious'. Ohio Report 63(4):51-53. Flore, J.A. 1975. Unpublished data, Michigan State Uni- veristy, East Lansing, Michigan. Hatch, A.H. 1975. The influence of mineral nutrition and fungicides on russeting of Goldspur apple fruit. J. Amer. Soc. Hort. Sci. 100:52-55. Havis, L. and J.H. Gourley. 1934. The russeting of apples. Ohio Agric Exp. Sta. Bimonthly Bul. 19:147-155. Nie, H.C., H. Hull, J.G. Jenkins, K. Steinbrenner, and D. H. Bent. 1975. Statistical package for the social sciences. McGraw-Hill, New York. Long, S.M. 1980. Russet variation and fruit cuticle char- acteristics of several varieties of Delicious apple. MS Thesis, Michigan State University, E. Lansing, Michigan. Long, S.M. 1980. Characterization of cuticle from russet and non-russet areas of 'Golden Delicious' apple. MS Thesis, Michigan State University, E. Lansing, Michigan. Lott, R.V. 1957. The quality and keepability of 'Golden Delicious' apples having russet bands caused by a frost. Proc. Amer. Soc. Hort. Sci. 69:56-64. Simons, R.K. 1957. Frost injury on 'Golden Delicious' apples - morphological and antomical characteristics of russeted and normal tissue. Proc. Amer. Soc. Hort. Sci. 69:48-55. and M.P. Aubertin. 1959. Development of epidermal, hypodermal and cortical tissues in the 'Golden Delicious' apple as influenced by induced mechanical injury. Proc. Amer. Soc. Hort. Sci. 74:1-9. 14. 15. 16. 17. 18. 19. 20. 78 Steel, R.G. and J.H. Torrie. 1960. Principles and pro- cedures of statistics. McGraw-Hill, New York. Tukey, L.D. 1959. Observations on the russeting of apples growing in plastic bags. Proc. Amer. Soc. Hort. Sci. 74:30-39. Verner, L. 1938. Histology of fruit tissue in relation to cracking. J. Agric. Res. 57:813-824. Vries, H.A.M. de. 1968. DeveloPment of the structure of the russeted apple skin. Acta. Bot. Neerl. 17:405-415. . 1970. Polymerization of the cutin acids of the apples skin. Acta. Bot. Neerl. 19:41-48. Walter, T.E. 1967. Russeting and cracking in apples: a review of world literature. Ann. Rpt. E. Malling Res. Sta. for 1966:83-95. Watanabe, S. 1969. Histological studies on the cause of russet in apples. Bul. of the Yamagata Univ. 5(4):882-890. APPENDICES 79 APPENDIX A Fruit epicuticular wax changes during storage. Equipment and time limitations cause cuticular compo- nent analysis for many samples to be conducted over a period of several weeks. If changes in epicuticular wax weights occur during cold storage of sample fruit, wax weights determined on one date could not be correctly compared to those determined on other dates. Epicuticular waxes were extracted from mature 'Golden Delicious', Frazier Spur and 'Red Delicious' fruit after several periods of cold storage to test for any change . Whole fruit of the three cultivars were measured for length and width with a caliper, and then epicuticular waxes were extracted. The fruit were immersed two separate times for 20 sec. in fresh redistilled chloroform. Five fruit selected randomly were used for each sample. Samples for each variety and length of storage were replicated 3 times. Extracts for each sample were combined and the solvent was removed under reduced pressure on a rotary evaporator. Epi- cuticular waxes were transferred to tared test tubes and then were allowed to air-dry to a constant weight. The combined wax weights for each 5 fruit samples were divided by the combined surface area of these fruit (see Appendix II) to give ug/cm2 of fruit surfact. The initial extraction was carried out on fruit kept in cold storage (1°C) and on fruit kept at room temperature 80 (20°C) for 1 month. The following extractions were per- formed on fruit kept in cold storage for 2 and 3 months. No consistent change in epicuticular wax weight was observed over the 3 month period of cold storage (Table A1). Fruit stored for 1 month at room temperature had somewhat higher epicuticular wax weight than those kept at cold storage temperatures. Similar results have been observed on 'Cox' (E. A. Baker, 23 21. 1963. Bristol U. Agr. and Hort. Res. Sta. Ann. Rpt. for 1962:69-76) and 'Sturmer' apples (I.M. Morice and F.B. Shorland. 1973. J. Sci. Fd. Agr. 24: 1331-1339), while increases in epicuticular wax during storage have been observed on 'Bramley', 'Granny Smith' and 'Dougherty' apples. 81 .HHOHM wusumfi m mo oomomfioo mos onEMm comma mm.mom HH.mHh m hm.mmm MH.Nmb mm.mmm N Hm.h¢n on.H¢m NH.Mhm mm.mmn m~.Hmm mm.owo H .Hoo com mH.o~m 0H.mov mv.mHm m Hm.mHo om.Hom mm.~mv mh.Hmw m mm.vmm mm.mmm mm.mm¢ m¢.va mm.¢Hm mm.mmm no.mmv vH.Mhm H .mm MOHNcum nm.wmm om.mmm mv.th m mv.mmo em.Hmm mm.m>m mm.omm N ov.mmm mm.¢mm Hm.omm mm.mmm h>.mmm hH.hmm om.vmm mm.mom H .Hoo copHoo m\H m\~H m\HH oH\HH m\H m\~H meHH oH\HH moom\sooHun> mommno>¢ MHOHHm>\oumo omcuoum CHOU Eoum :oxme mama mews HH< I .833 xm3 HmHSOHuOOHmm .OoH um ommnoum mcHuOo uHsHm .msoHOHHoo com. can spam HwHumHm ..mOOHOHHoo cooHoo. How xm3 HmHsOHusOHmm CH momccno .Hm oHnt 82 APPENDIX B Geometric approximations of fruit surface areas. The study of Galbreath (1975. New Zealand J. of Agr. Res. 19:543-544) concluded that simple geometric approxi- mations of fruit surface areas were little different than estimates obtained by the use of more complex and tedious methods such as measuring the area of peelings. The present study was initiated in order to determine differences be- tween surface area estimations obtained using two geometric approximations for both large and small apple fruits. The length and width of fruit harvested at petal fall + 30 days and at maturity were measured with a caliper. Since apple fruit are neither perfect spheres nor perfect ellipses, variations were made in the geometric approxima- tions used. Both the length and width measurements were used as the diameter component within the calculations. Thus five different formulas were used to estimate fruit surface areas as listed below. Based on the surfact area of an ellipse: 1) 2 x diameter x profile length; width used as diameter. 2) 2 x diameter x profile length; length used as diameter. Based on the surface area of a sphere: 3) Pi x (diameter)2: width used a diameter. 4) Pi x (diameter)2; length used as diameter. 5) Pi x (diameter)2; average of length and width used as diameter. 83 Profile lengths were calculated by: 2 2 where a and b are semi-axes of the ellipse, here 1/2 length and 1/2 width. The results showed little difference between the 5 geometric approximation formulas (Table B1 and B2). The use of lengths as diameters gave slightly larger surface area estimates than did the use of widths (formulas 2 and 4 vs. formulas 1 and 3). The use of length and width averages as diameters gave estimated surface areas of intermediate size (formula 5 vs. formulas 3 and 4). An examination of the Standard Errors (S.E.) obtained with the different formulas showed that smaller values were obtained when formula 1 was used to estimate the surface area of large fruit. For this reason formula 1 may be preferred when geometric approximations of fruit surface areas are used. 84 Table B1. Large fruit surface areas (cmz)z 1 S.E. as estimated by five geometric approximation formulas. Formula Used Variety 1 2 3 4 5 Golden Delicious 141.04 141.68 140.84 142.12 141.23 13.54 15.01 14.39 16.60 13.86 Frazier Spur 131.45 134.40 130.02 135.91 132.93 15.27 15.31 15.33 15.44 15.26 Red Delicious 155.46 146.18 160.27 141.78 150.61 17.37 16.84 18.70 17.69 16.67 zEach variety sample was composed of 9 fruit, values given are the average for each sample. Table BZ. Small fruit surface areas (cmz)z 1 S.E. as estimated by five geometric approximation formulas. Formula Used Variety 1 2 3 4 5 Golden Delicious 17.28 19.26 16.32 20.27 18.23 10.62 10.79 10.58 10.90 10.70 Frazier Spur 19.95 22.32 18.80 23.53 21.08 10.60 10.59 10.69 10.70 10.58 zEach variety sample was composed of 12 fruit, values given are the average for each sample. 85 APPENDIX C Ethylene production as an indicator of russet formation. Russet formation occurs as a major disruption of the epidermal cells followed by periderm cell proliferation. Since russet develOpment resembles a wound protection res- ponse, ethylene production may be higher within fruit which are develOping severe russet than within fruit develOping little or no russet. Ethylene production rates by fruit during the critical period of russet initiation (from petal fall to petal fall + 30 days) may be an indication of the severity which will be present at harvest. In order to test for this relationship, ethylene pro- duction of fruit from cultivars known to have differing russet susceptibilities was measured and remaining fruit from the same branches were evaluated for russet severity at bar- vest. Additional measurements were made on fruit which had been deliberately wounded by the use of COpper sulfate sprays and by U.V. light exposure. A preliminary trial showed that ethylne was being pro- duced by small fruit in sufficient quantities for detection by gas chromatography (Varian Aerograph Series 1400) using standard methods (activated 60/80 mesh alumina column, 80°C). Branches were selected on 3 trees each of 'Golden Delicious', Frazier Spur, Smoothee and 'Red Delicious'. For each ehtylene sample 3 fruit were placed into sealed 10 cc plastic syringes in the field. Once in the lab syringes were kept at a constant temperature (259C) using a water bath. 1.cc gas 86 samples were extracted while 1 cc of the sample volume was pushed out. Three selected 'Golden Delicious' branches were Sprayed with a COpper sulfate solution at petal fall + 10 days. Fruit on 3 selected Frazier Spur branches were exposed to U.V. light for 5 mintes, and on 3 branches for 10 minutes at petal fall + 20 days. Ethylene production rates over a 24-hour period were measured at petal fall + 11 days. (Table C1). The highest rates were produced by Smoothee and copper-treated fruit, while Frazier Spur and 'Red Delicious' fruit had the lowest 'Golden Delicious' ethylene production rates at all times. fruit had intermediate rates. Table C1. Average ehtylene production at petal fall + 11 days (HQ/kg/hr). Sample Time Sample 11 A.M. 1 P.M. 4 P.M. 7 P.M. 9A.M. Golden Del. 2.898 2.016 1.147 0.925 0.741 COpper 3.508 3.008 2.173 1.501 1.414 Smoothee 3.664 2.205 1.216 0.916 0.556 Red Del. 1.263 0.768 0.469 _0.398 0.407 Frazier Spur 1.077 1.115 0.750 0.585 0.577 Ethylene production rates 3 hours after sample collection were measured at petal fall + 21 days (Table C2). The highest rates were present in U.V. treated fruit (10 min. exposure), 'Golden Delicious' and cOpper-treated fruit. Smoothee and 'Red Delicious' fruit had the lowest ethylene production rates, while those of Frazier Spur and U.V. treated (5 min. exposure) had intermediate rates. 87 Smoothee fruit had very little russet at maturity, while Frazier Spur produced few clean fruit and 'Golden Delicious' had fruit of intermediate quality (Table C3). No fruit from the COpper treatment were suitable for the fresh market due to extensive russet formation. While the U.V. light expo- sure was seen to produce severe localized russet, nearby control fruit were also severely russeted (naturally). For this reason no difference in russet ratings was apparent between the U.V. treatments and controls. The COpper treatment gave severe russet, yet no real difference in ethylene production rates was seen between these fruit and untreated 'Golden Delicious' fruit. A comparison of the ethylene production rates of Smoothee, 'Golden Delicious' and Frazier Spur on both dates gave no indication of the large differences in russet severity which existed between the cultivars at harvest. This evidence suggests that no predictive ability exists from early season ethylene production rates for the severity of russet at harvest. However, it must be realized that no russet evalu- ation was possible of the fruit actually sampled for ethylene production. So it is not known if the measured fruit actually differed in russet severity. Since branches from which ethylene samples were taken did develOp fruit with differ- ences in russet severity, it seems reasonable to expect that sampled fruit would have differed in both russet severity and ethylene production if a relationship did exist between the two. 88 Table C2. Average ethylene production at petal fall + 21 days (ul/kg/hr). Sample Golden Delicious 1.133 COpper 1.078 Smoothee 0.639 Red Delicious 0.605 Frazier Spur 0.842 U.V. - 5 min. 0.846 U.V. - 10 min. 1.183 Table C3. Mature fruit russet evaluations from branches earlier sampled for ethylene production. % of Fruit in Each Russet Classz 3:?2231e Treatment # of Fruit 1 2 3 4 5 for fresh Market Copper 46 0.0 0.0 0.0 8.7 91.3 0.0 Golden Del. 60 15.0 46.7 38.3 . 0.0 61.7 (also COpper cont.) Smoothee 59 16.9 57.6 22.0 1.7 1.7 74.5 Frazier Spur 229 1.7 17.0 51.5 26.7 3.1 18.7 U.V. - 5 min. 6 0.0 0.0 0.0 1.7 83.3 0.0 U.V. - 5 min. 8 0.0 0.0 0.0 50.0 50.0 0.0 (cont.) U.V. 10 min. 9 . . 0.0 33.3 66.7 0.0 U.V. 10 min. 16 . 0.0 37.5 37.5 25.0 0.0 z 1= no russet; 5= severe russet. 89 APPENDIX D Fruit Growth Rates. Ten clusters of 3 fruit each were selected over 3 trees of 'Golden Delicious', Frazier Spur, Smoothee and 'Red De- licious'. The diameters of these fruit were measured through- out the first month after petal fall at 7 a.m. and 2 p.m. on selected days, and at 2 p.m. only on other days. Fruit drop reduced the number of fruit being measured to about 10/ variety by 30 days after petal fall. The fruit diameters were measured using a photographic method. A piece of white cardboard with a ruler attached was held directly behind each cluster, which was then photographed (35 mm, Tri-X film). The film negatives were develOped using standard procedures. The negatives were projected using a film enlarger. A ruler was used to measure the mag- nified (projected) size of the photographed ruler increments, and to measure the magnified diameter of the photographed fruit. The proportion of the magnified ruler increments to the actual ruler increments was used to calculate the actual fruit diameter from the magnified fruit diameter measured. Results. The fruit growth rates observed were very similar for the different apple varieties (Fig. 01). Diurnal fluctuations in fruit size were detected by the photographic method on all days when 2 measurements were taken: 10, 15, 24 and 29 days after petal fall. However, no significant differences were found between varieties in the % change in diameter on any given day and for all dates combined. 90 .Houmemwo uHsum cH omcoso man an consumes mm mCHmuum OHmmm e no nouns suzoum anum .Ho .mHm .....c... ...:wm «mph—c w>¢o 68.8 . 80MB , 838 . 8am: . 838 1 8m; . o 2...? can? 8.7m. 686 1: PT? M a I I; 68 S 0 I Nu nu a: w... 68 m: 8 33.0. H .l\ m ..\w m \ 68.8 .. 8.3 91 Correlations between the average % change in diameter vs. the average % of fruit suitable for the fresh market were not significant. Neither the rate of fruit growth, nor the amount of diurnal size fluctuation appeared to be related to the dif- ferent russet susceptibilities of the 4 apple varieties. However, since only 9-11 fruit/variety were measured through petal fall + 30 days, it is possible that not enough fruit were measured to detect possible differences. Since indi- vidual trees and branches differ in russet susceptibility, it is also possible that the fruit measured were not representative of russeting and non-russeting fruit. BIBLIOGRAPHY 10. 11. 12. 13. BIBLIOGRAPHY Baker, E.A. 1974. The influence of environment on leaf wax development in Brassica oleracea var. Gemmifera. New Phytol. 73:955-966. Baker, C.E. 1930. A study of the skin structure of the Grimes apple as affected by different types of injury. Proc. Amer. Soc. Hort. Sci. 27:75-81. Batal, K.M., J.L. Weigle, and D.C. Foley. 1970. Relation of stress-strain prOperties of tomato skin to cracking of tomato fruit. HortScience 5:223-224. Bell, H.P. 1937. The origin of russetin in the "Golden Russet' apples. Can. J. Res. 15:560566. .1937. The protective layers of the apple. Can. J. Res. 15:391-402. . 1941. The origin and histology of bordeaux Spray russeting of the apple. Can. J. Res. 19:493-499. Brown, D.S. and E.C. Koch. 1962. Stem-end russet of Yellow Newtown apples. Proc. Amer. Soc. Hort. Sci. 40:120-122. Chandler, F.B. and J.C. Mason. 1942. Russeting of 'Golden Delicious' apples. Proc. Amer. Soc. Hort. Sci. 40:120-122. Creasy, L.L. 1977. Can russet of 'Golden Delicious' be controlled? Proc Ann. Meeting N.Y. State Hort Soc. 122:160-162. Cummins, J.N., P.L. Forsline, and R.D. Way. 1977. A com- parison of russeting among 'Golden Delicious' subclones. HortScience 12:241-242. Decourtye, L. 1967. Russet-free sectors on fruits of 'Golden Delicious' apple trees after gamma irradiation. Proc. Amer. Soc. Hort. Sci. 91:73-77. Edgerton, L.J., N. Veinbrants, and J.F. Hutchinson. 1976. Foliar sprays of silicon dioxide-containing compound reduce russeting in 'Golden Delicious' apple fruits. HortScience 11:508-509. , and N. Veinbrants. 1979. Reduction in russeting of 'Golden Delicious' apples with Silicon dioxide formulations and gibberellins A 4+7. HortScience 14:40-41. 92 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 93 Eggert, D.A. 1964. Russeting of the apple (Malus domes- tica Bork.), cultivar Golden Delicious, as reIated to nutritional factors influencing the physiology and his- tology of develOping fruits. Ph.D. Thesis, Michigan State University, East Lansing, Michigan. , and A.E. Mitchell. 1966. Russeting of 'Golden Delicious' apples as related to soil applications of sodium nitrate. Proc. Amer. Soc. Hort. Sci.90:1-8. Faust, M. and C.B. Shear. 1972. Russeting of apples, an interpretive review. HortScience 7:233-235. and .1972. Fine structure of the fruit surface of three apple cultivars. Proc. Amer. Soc. Hort. Sci. 97:351-355. Ferree, D.C. and R. Lich. 1978. The search for a russet- free strain of 'Golden Delicious'. Ohio Report63(4):51-S3. Gardner, V.R., W. Toenjes, M. Giefel, and J.C. Kremer. 1948. Variability and segregation in the 'Golden Russet' apple. J. Agric. Res. 76:231-240. and E.G. Christ. 1953. Studies on cracking in the Stayman apple. Hort. News N.J. Hort. Soc. 34(7): 2701, 2710-2712. Goode, J.E., Fuller, M. Morwenna, and K.H. Hyrycy. 1975. Skin cracking of Cox's Orange Pippin apples in relation to water stress. J. Hort Sci. 50:265-269. , K.H. Higgs, and K.J. Hyrycy. 1978. Abscissic acid applied to archard trees of 'Golden Deli- cious' apple to control water stress. J. Hort. Sci. 53:99-103. Gough, R.E. and V.G. Shutak. 1972. Fruit structure of the apple cuticle and storage scald. HortScience7:561-562. Harlay, C.P. and M.P. Masure. 1938. Relation of atmos- pheric conditions to enlargement rate and periodicity of Winesap apples. J. Agric. Res. 57:2 109-124. Hatch, A.H. 1975. The influence of mineral nutrition and fungicides on russeting of Goldspur apple fruit. J. Amer. Soc. Hort. Sci. 100:52-55. Havis, L. and J.H. Gourley. 1934. The russeting of apples. Ohio Agric Exp. Sta. Bimonthly Bul. 19:147-155. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 94 Kretchman, D.W. and A.E. Mitchell. 1958. The interrela- tionship of temperature and pesticide chemicals as affecting the finish of 'Golden Delicious' apples. Mich. State Exp. Sta.1guart. Bul. 40:620-627. Letham, D.S. 1961. Influence of fertilizer treatment on apple fruit composition and physiology I. Austral. J. Agric Res. 12:600-611. Lott, R.V. 1957. The quality and keepability of 'Golden Delicious' apples having russet bands caused by a frost. Proc. Amer. Soc. Hort. Sci. 69:56-64. Lotter, J. de V. 1965. Study of the development of the fruit of Malus domestica, cultivar Golden Delicious, MS Thesis, Univ. of Stellenbosh, South Africa. MacDaniels, L.H. and A.J. Heinicke. 1930. To what extent is "spray burn" of apple fruit caused by freezing of the flowers? Phytopath. 20:903-906. Meador, D.B. 1977. Reducing russet on 'Golden Delicious' apples with silicon dioxide formulation foliage Sprays. HortScience 12:504-505. Meyer, A. 1944. A study of the skin structure of 'Golden Delicious' apples. Amer. Soc. Hort. Sci. 45:105-110. Mink, 6.1. 1973. The apple industry in Japan. HortScience 8:81-86. Mitchell, A.E. 1951. The use of organic protective fungi- cides. Ann. Rpt. State Hort. Soc. of Mich. 81:22-24. . 1954. Apple spraying problems and the selection of spray materials. Ann. Rpt. State Hort. Soc. Mich. 84:14-18, 85-87. . 1955. Performance of Spray chemicals in 1955. Ann. Rpt. State Hort. Soc. of Mich. 85:111-114. Montgomery, H.B.S. 1959. Russeting and cracking of Cox's Orange Pippin apples. Ann Rpt. E. Malling Res. Sta. for 1958:163-164. Palmiter, D.H. 1944. Relation of spray materials to rus- seting of Delicious and 'Golden Delicious' apples. Proc. Amer. Soc. Hort. Sci. 45:113-118. Pratt, C. 1972. Periderm develOpment and radiation sta- bility of russet-fruited Sports of apple. Hort. Res. 12:5-12. ‘ 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 95 Proctor, J.T.A. and E.C. Lougheed. 1976. The effect of covering apples during development. HortScience11:108-109. Rasmussen, E.J. 1943. Effect of Spray materials on finish and keeping quality of apples. Mich. Agric. Exp. Sta. Quart.Bul. 25:263-271. Simons, R.K. 1957. Frost injury on 'Golden Delicious' apples - morphological and anatomical characteristics of russeted and normal fruit. Proc. Amer. Soc. Hort. Sci. 69:48-55. , and M.P. Aubertin. 1959. DevelOpment of epidermal, hypodermal and cortical tissues in the 'Golden Delicious' apple as influenced by induced mechanical injury. Proc. Amer. Soc. Hort. Sci. 74:1-9. . 1960. DevelOpmental changes in russet sports of 'Golden Delicious' apples - morphological and anatomi- cal comparison with normal fruit. Proc. Amer. Soc. Hort. S91. 76:41-51. . 1962. Spontaneous russet sports of 'Golden Delicious' apples - morphological and anatomical compari- sons with normal fruit. Proc. Amer. Soc. Hort. Sci. ' 80:79-89. . 1965. The origin of russeting in russet sports of the 'Golden Delicious' apple. Hort Res.5:101-106. . 1954. Apple Spraying problems and the selec- tion of spray materials. Ann. Rpt. State Hort. Soc. Mich. 84:14-18, 85-87. .1974. Russeting of 'Golden Delicious' and related strains of apples. Trans Ill. State Hort. Soc. 107:69-74. .1976. Russeting of the 'Golden Delicious' apple: morphological and anatomical development. Trans. Ill. State Hort. Soc. 109:25-30. and M.C. Chu. 1978. Periderm morphology of mature 'Golden Delicious' apples with special reference to russeting. Scientia Horticulturae. 8:333-340. Skene, D.S. 1965. Cracking and russeting in apple fruits. Ann Rpt. E. Malling Res. Sta. for 1964:99-101. Stiles, W.C., N.F. Childers, M.J. Prusik, and T.N. Kom, 1959. Effects of urea sprays and pesticides on russeting and cracking of the Stayman apple. Proc. Amer. Soc. Hort. Sgi. 74:25-29. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 96 Stubbins, W.A.K. and D.K. Strydom, 1965. Russeting of 'Golden Delicious' apples in the Elgin area. Deciduous Fruit Grower 15:149-151. Taylor, B.K. 1975. Reduction of apple skin russeting by gibberellin A 4+7. J. Hort. Sci. 50:169-172. Tukey, L.D. 1959. Periodicity in the the growth of fruits of apples, peaches, and sour cherries with some factors influencing this development. Penn. Exp. Bul. 661. .1959. Observations on the russeting of apples growing in plastic bags. Proc. Amer. Soc. Hort. Sci. 74:30-39. Verner. L. 1837. A study of growth rates in Stayman Winesap apples. Amer. Soc. Hort. Sci. Proc. 35:128-131. . 1938. Histology of apple fruit tissue in relation to cracking. J. Agric. Res. 57:813-824. Voisey, P.W., L.H. Lyall, and M. Klock. 1970. Tomato skin strength - its measurement and relation to cracking. J. Amer. Soc. Hort. Sci. 95:485-488. Vries, H.A.M. de. 1968. Development of the structure of the russeted apple skin. Acta. Bot. Neerl. 17:405-415. .1969. The cutin acids of smooth and russeted 'Golden Delicious' apples. Acta. Bot. Neerl. 18:589-596. . 1970. Rapid examination of cutin acids by gas-liquid chromatography. Acta. Bot. Neerl. 19:36-40. . 1970. Polymerization of the cutin acids of the apple skin. Acta. Bot. Neerl. 19:41-48. Walter, T.E. 1967. Russeting and cracking in apples: a review of world literature. Ann. Rpt. E. Malling Res. Sta. for 1966:83-95. Watanabe, S. 1969. Histological studies on the cause of russet in apples. Bul. of the Yamagata Univ. 5(4): 822-890.