— ,‘, _ > _ — —_ — — — —_ ‘— — — — —— — — lilillllllillllllllllill'lllllllllllllllillI 31293 00908 5717 This is to certify that the thesis entitled Adjuvants to Enhance Calcium Penetration Into Cauliflower Leaves presented by Shuangling Guo has been accepted towards fulfillment of the requirements for M. S. degree in Horticulture Major fessor Date Novelnber, 4; 1991 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRAFW _ Michigan Slate L University vv‘ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE l fifl MSU Is An Affirmative ActiorVEqual Opportunity Institution cmmh.‘ ADJUV ANTS TO ENHANCE CALCIUM PENETRATION INTO CAULIFLOWER LEAVES BY Shuangling Guo A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1991 - M3 ’7 A. I IA 5:" ABSTRACT ADJDVANTS TO ENHANCE CALCIUN PENETRATION INTO CAULIFLOWER LEAVES BY Shuangling Guo Nine adjuvants and two salt sources of Ca were applied to detached ’White Rock’ cauliflower leaves and leaf discs to increase Ca penetration and uptake under laboratory conditions. An ethoxylated alkyl phenol adjuvant (Flo Mo 845) was the most effective adjuvant to enhance Ca penetration and uptake. L-77, x2-5309, 02-5152 and Flo Mo 6T were also effective. Ca absorption into leaves increased as Ca concentration of the applied solution and duration of exposure to Ca solution increased, and leaf age decreased. In field experiments, CaCl2 and C:a(NO3)2 and two adjuvants (L-77 and x2-5309) were applied in foliar spray to cauliflower plants. Foliar application of Ca with adjuvants significantly increased the Ca concentration of leaf tissues and reduced leaf tipburn. There was no significant difference between CaCl2 and Ca (N03)2 as Ca sources or L-77 and X2-5309 as adjuvants in increasing Ca penetration and reducing tipburn. As the number of applications increased, tipburn decreased. At least three applications were required to significantly reduce tipburn. ACENCNLEDGENENTS I would like to express my gratitude to my major professor Dr. Bernard H. Zandstra for the guidance, encouragement, and support during my graduate studies and research. I would also like to thank the other members of my committee, Dr. Irvin Widders and Dr. Donald Penner for their advice and helpful suggestions. I owe thanks to Joseph G. Hasabni and Ronald V. Gruesbeck for their help in my field work. Finally, I would like to thank my husband, Shifeng Pan, for his help, encouragement, and understanding. iii TABLE OF CONTENTS Page LIST OF TABLES...OOOOOOOOOOOOOOOOOOOOOOO0.0...OOOOOOOOOOOOVi LIST OF FIGURESOOOOOOOOOOOOOOOOOOOO0........OOOOOOOOOOOOViii CRAFT“ 1. REVIEW OF LITERATUREOOOOOOOOOOOOO..OOOOOOOOOOOOOI I. II. III. Calcium Deficiency in Cauliflower...................1 A.Introduction......................................1 B. Calcium Uptake and Transportation................2 C. Factors Affecting Ca Deficiency and Leaf Tipburn Development......... ....... .........3 Adjuvants and Foliar Sprays.........................9 A. Introduction.....................................9 B. Factors Affecting the Efficiency of Foliar Application............... ............. ..10 Literature CitedOOOOOOOOO...OOOOOOOOOOOOOOOO0.0.0.015 CHAPTER 2. CALCIUM APPLICATION WITH ADJUVANTS II. III. IV. TO DETACHED LEAF PORTIONS......................20 Abstract...........................................20 Introduction.......................................21 Materials and Methods..............................22 Results and Discussion.............................34 Literature Cited...................00.0.0.0... ..... 48 iv CHAPTER 3. CALCIUM AND ADJUVANT APPLICATION I. II. III. IV. V. TO CAULIFLOWER IN THE FIELD....................50 Abstract..................... ..... .................50 Introduction................... ......... ...........51 Materials and Methods..............................52 Results and Discussion.............................60 Literature CitedOO0.0.00.00.00.00...0.0.0.0.000000072 SUMARY....0.0...OOOOOOOOOOOOOOOOOOO0.0......00.0.0000000073 LIST OF TABLES CHAPTER 2 Page 1. A list of adjuvants used in these experiments.... ..... 24 2. Calcium and adjuvant combinations applied to detached cauliflower leaves in a 5 or 2 pl drop ....... 29 3. Calcium and adjuvant combinations applied to cauliflower leaf discs in a 5 pl drop......... ..... 30 4. Ca level in various sections of the youngest full size cauliflower leaves...............................35 5. The effect of Ca concentrations on Ca penetration into detached cauliflower leaves treated with 5 pl of various concentrations of CaCl2 and O . 1% L-77 per 10 cmz leaf area OOOOOOOOOOOOOOOOOOOOOO. ..... 36 10. Ca content in detached greenhouse grown cauliflower leaves 24 hr after application of 5 pl and 2 p1 of 2M CaCl2 and Ca(NO3)2 with 0.1% adjuvants per 10 cm2 leaf area.............................................38 Ca content in greenhouse grown cauliflower leaf discs 24 hr after application of 5 pl of 2M CaCl2 and Ca (N03), with adjuvants at 2 concentrations. . . . . . . . 39 Ca content in detached field grown cauliflower leaves 24 hr after application of 5 pl of 2M CaCl2 and Ca(No,)2 with 2 concentrations of different adjuvants.............................................4O The relative spreading of the adjuvants used in this experiment...................... ......... 42 The effect of length of treatment time on Ca penetration into detached field grown cauliflower leaves with 5 pl of 2M CaCl2 and Ca (N03)2 mixed with 0.1% 02-5152 per 10 cm? leaf area.....................44 vi 11. 12. Ca level in leaf portion after treatment with 5 pl of 5M CaCLz+-0.25% Flo Mo 845 per leaf disc..........45 The effect of leaf age on Ca penetration into cauliflower leaves after 24 hrs. foliar application with 5 pl of 2M Ca(N0,)2 and 0.1% Flo No 845 per leaf disc..................................47 CHAPTER 3 1. 2. The treatments used in the first field experiment with 3.6 kg Ca/ha (0.25M Ca)......... ...... 54 The weather conditions for each treatment in the first field experiment in 1990................... ..... 55 The treatments used in the second field experiment with 4.0 kg Ca/ha (0.28M Ca)...............56 The weather conditions for each treatment of the second field experiment in 1990............. ...... 57 The effects of treatment with 3.6 kg Ca/ha, 0.1% adjuvants, and number of applications on cauliflower leaf tipburn in the first field experiment in 1990....................................61 The effects of treatment with 4 kg Ca/ha, 0.1% adjuvants, and number of applications on cauliflower leaf tipburn in the second field experiment in 1990....................................62 Ca content of leaf and curd samples after treatment with 3.6 kg Ca/ha CaCl2 and Ca(N03)2, 0.1% L-77 and x2-5309, and various number of applications in the first field experiment in 1990........................64 Ca content of leaf and curd samples after treatment with 4.0 kg Ca/ha CaCl2 and Ca(N03)2, 0.1% L-77 and X2-5309, and various number of applications in the second field experiment in 1990.......................65 The effects of treatment with 4 kg Ca/ha, 0.1% adjuvants, and various number of applications on cauliflower yield in the second field experiment in 1990...OOOOOOOOOOO0.0.00......OOOOOOOOOOOOOOOOOOOOO00.66 vii LIST OF EIGURES Page 1. The relationship of the number of tipburned leaves per plant to the Ca level of leaf tissue in 1990 field experiments. A: the first experiment, B: the second experiment..... ...... 67 2. The relationship of the percent tipburned plants to the Ca level of leaf tissue in 1990 field experiments. A: the first experiment, B: the second experiment.... ..... ...... ..................... 69 viii CHAPTER I REVIEW OF LITERATURE I. Calcium Deficiency in Cauliflower A: Introduction Cauliflower is an important vegetable crop in the United States. Leaf tipburn is a major problem in cauliflower production. Tipburn is also a problem on other vegetables such as cabbage, lettuce, and Brussels sprouts. It is similar physiologically to black heart of celery and blossom end rot of tomato and pepper, in that it is caused by lack of sufficient calcium in the affected tissue. Tip necrosis of young expanding leaves surrounding the enlarging curd is an obvious symptom of leaf tipburn in cauliflower. Necrotic leaves may lower product quality as a result of discoloration of the curd due to secondary pathogen infection (Maynard et al., 1981). Leaf tipburn is a physiological disorder caused by calcium (Ca) deficiency. Previous work on this subject indicates that tipburned tissues have much lower Ca levels than healthy tissues (Maynard et al., 1981). However, tipburn is usually not associated with Ca deficiency in the soil. Rather, there is a temporary localized shortage of 2 soluble Ca in rapidly growing tissues during periods of high requirement. The soil may have a relatively high level of Ca. However, when environmental conditions lead to rapid growth, the delivery of soluble Ca to the growing tissues may be too slow to meet the high requirement. If these conditions exist even for a short period, leaf tipburn may occur(Cox et al., 1976). B: Calcium Uptake and Transportation: Ca is an essential plant nutrient and a component of various soil minerals. Most researchers believe that Ca moves through the soil to the plant roots by mass flow and diffusion (Barber and Ozanne, 1970; Elgawhary et al., 1972). When Ca reaches the roots, it is absorbed onto exchange sites by the entire root system, but is translocated primarily from just behind the young root tip (Clarkson et al., 1968). It is believed that Ca moves to the xylem through an apoplastic route. Suberized root sections can block the apoplastic route and restrict Ca movement to the xylem (Ferguson, 1979). Therefore, the exchange capacity of the root and the length of young, unsuberized roots are important factors affecting Ca uptake and translocation. Peterson (1988) studied the relationship between root growth rate and the length of young, unsuberized root tissue behind the root tip, and indicated that rapid root growth could increase the length of the unsuberized section at the end of the root and might increase Ca uptake. 3 Burstrom’s (1954) research showed that Ca concentration surrounding the root could be used to predict root growth rate. Root growth increased when Ca concentration increased. Some other factors, such as Ca form and other divalent ions present also affect Ca uptake. Hanger (1979) found that nonionic Ca such as Ca chelate moves more freely than the ionic form (Ca“j. The introduction of other divalent ions into the xylem stream also promotes Ca movement. Ca is also present in the phloem (Bangerth, 1979; Hanger, 1979). It is possible that some Ca is transported by the phloem. But the amount is probably minimal. C. Factors Affecting Ca Deficiency and Leaf Tipburn Development: There are many factors associated with localized Ca deficiency and leaf tipburn. Collier and Tibbitts (1982) reported that 17 factors were conducive to lettuce tipburn development. 1. Favorable Growing Conditions are Associated with Ca Deficiency: Cauliflower leaf tipburn usually occurs during the period from when heads start to form until harvest. Growth rate is an important factor affecting tipburn incidence. Previous work has demonstrated that rapid, luxuriant growth can promote leaf tipburn (Cox, et al., 1976). Such environmental factors as temperature, light, humidity, and nutrition not only affect growth rate but also influence the 4 availability and translocation of this relatively immobile element within the plant. a. The Effects of Temperature on Leaf Tipburn: Temperature plays an important role in controlling plant growth and development. Changes in temperature are related to changes in other environmental factors. Therefore, the effects of temperature on the development of leaf tipburn must be considered in conjunction with all of these factors. Generally, high temperature increases growth rate and transpiration, and results in more tipburn. Cox and McKee (1976) reported a closer correlation between air temperature and tipburn of lettuce in the field than with any other single factor. Tibbitts and Bottenberg (1971) induced tipburn within one day by transferring lettuce plants from a growth chamber at 21°C to one at 29°C, while the plants maintained at 21°C did not show tipburn symptoms for several days. b. The Effects of Moisture on Leaf Tipburn: Numerous reports suggest that soil moisture, low humidity, and high humidity are responsible for leaf tipburn. Palzkill et al. (1980) pointed out that high humidity promoted tipburn on young cabbage. Both Bottenberg and Tibbitts (1968), and Thibodeau and Minotti (1969) reported that subjecting lettuce to high humidity could increase tipburn incidence. High humidity during the day increases plant growth rate, and as a result, causes more 5 leaf tipburn. High humidity at night, however, may increase Ca uptake and decrease tipburn development (Bottenberg and Tibbitts, 1968). Water shortage may also cause leaf tipburn. Under hot and dry conditions plants cannot take up enough water and Ca from soil. The transpiration from the leaves may be greater than the amount of water taken up by the roots. Therefore, water may be withdrawn from the growing tissues, which may cause tipburn in young leaves. It is difficult to determine the effects of soil moisture on the incidence of leaf tipburn because soil moisture interacts with many other factors, some of which are usually uncontrolled. Adequate soil moisture raises soil osmotic potential and thus promotes root pressure and Ca uptake. As the soil becomes dryer, the soil osmotic potential is lowered. Haber et al.(1983) studied the effect of osmotic potential on Ca uptake by peach seedlings, and found that root growth, shoot growth, water uptake, and Ca uptake decreased when the seedlings were stressed by reducing the osmotic potential. A water-logged soil can stop root growth by becoming anaerobic in the root zone, and as a result, less Ca is taken up by the roots. When the soil becomes anaerobic, on the other hand, the Ca content of the soil solution may rise as dissolved C02 concentration increases the solubility of Ca (Smith et al., 1989). 6 c. The Effects of Light on Leaf Tipburn: Light intensity and light duration are related to the incidence and severity of Ca deficiency. Tibbitts and Rao (1968) pointed out that increasing light intensity and/or extending light duration could accelerate tipburn development in lettuce. It is well known that leaf tipburn frequently occurs in the field following a period of bright sunny days. An increase in light level may promote Ca deficiency by enhancing shoot growth more than root growth. The plants with a low root to shoot ratio may not take up enough Ca to meet the high requirement in the growing tissues. d. The Effects of Nitrogen on Leaf Tipburn: High nitrogen (N) fertilization favors leaf tipburn. This could be due to increased growth rate. Both Shafer and Sayer (1946), and Nieuwhof (1960) reported that high rates of nitrogen fertilizer were correlated with the incidence and/or severity of leaf tipburn in cabbage. Nitrogen may affect Ca nutrition by altering the root to shoot ratio. Nitrogen stimulates shoot growth more than root growth (Kuchenbuch et al., 1988). Marschner (1989) pointed out that with high levels of N in the rooting medium during the early growth, shoot elongation was enhanced and root elongation inhibited. Because increasing N fertilization decreases root to shoot ratio, Ca uptake by the root may not keep pace with the increased demand for Ca by the larger 7 shoot. Therefore, more leaf tipburn results. 2. Nutrient Imbalance Affects Ca Deficiency: The antagonistic interaction between Ca and other cations is well known (Ashkar and Ries, 1971, Bunemann and Ludder, 1970). When other cations (e.g. NH“ K, Mg, Na) are relatively high in the soil, Ca uptake by the plant will tend to be lower. When salt concentration increases, Ca uptake decreases. Hori, et al.(1960) reported that increasing the salt concentration of culture solutions induced leaf tipburn in Chinese cabbage. Increasing salt concentrations may increase Ca deficiency by reducing Ca uptake through competitive absorption. The effectiveness of various cations in reducing Ca uptake through competitive absorption is in the order NH, > K > Mg > Na (Shear, 1975) . High salt concentrations may also induce Ca deficiency by decreasing root pressure. 3. The Relationship Between Root Pressure Flow and Transpiration Pull Affects Ca Deficiency: If the weather is hot, dry, and windy, the transpiration pull may play a greater role than the root pressure flow. Under such conditions, more tipburn will result. During mild, humid conditions, on the other hand, root pressure flow becomes a more critical factor, and little, if any, tipburn will develop. Cox and McKee (1976) reported that a high root to shoot ratio could enhance nutrient uptake and decrease the occurrence of leaf tipburn of lettuce. In 8 cases of high root to shoot ratio, the transpiration pull becomes smaller, while the root pressure flow is greater. Ca uptake increases, and no or little tipburn appears. That may explain the effects of transpiration pull and root pressure flow on tipburn development. It may also explain why young leaves have a Ca deficiency problem in such leafy vegetables as cabbage, lettuce, Brussels sprouts, and cauliflower. 4. Genetic Differences: Different cauliflower cultivars have different sensitivities to Ca deficiency. Some cultivars appear somewhat resistant to leaf tipburn, while others are susceptible (Rosen, 1990). Hochmuth (1984) studied the variations in Ca efficiency between cauliflower strains, and indicated that the most Ca efficient strain produced 14 times more dry matter than the least efficient strain did under low Ca conditions. Genetic differences in the amount of Ca absorbed and accumulated by different plant species of tomato has also been reported (Greenleaf and Adams, 1969). Different plant species have various lengths of root tip which is an important parameter affecting Ca uptake and transportation (Perumalla and Peterson, 1986). This may, in part, explain the genetic differences in Ca uptake. II. ADJUVANTS AND FOLIAR SPRAYS A. Introduction: According to Chow et al. (1989), an adjuvant is "a thing (an additive) that assists". For agrichemical purposes, it is an ingredient in a pesticide or other agrichemical mixture, which aids or modifies the action of the active ingredient. A surfactant is a "surface active" adjuvant whose primary function may be as a wetting agent or as an emulsifier. Adjuvants used to increase the efficiency of foliar applications have various physical and chemical properties. The functional group of a adjuvant is the active ingredient which usually contains a polar (hydrophilic) and a non-polar (lipophilic) group. The active ingredient of the adjuvant allows polar solutions, such as most nutrient salt solutions, to contact the leaf surface, which is non-polar. Adjuvants do not always enhance uptake (Darlington and Barry, 1965), and their effects may also vary with the concentration of the active ingredient (Midgeley, 1982). However, increased absorption is the general result and has been associated with various properties of adjuvants, including wetting of the leaf surface (Sands and Bachelard, 10 1973), penetration of the surface waxes and their disruption or solubilization (Bukovac et al., 1979). The ability of adjuvants to enhance uptake, particularly of polar chemicals at low humidity, may be related to their hydroscopic nature and their ability to solubilize the active ingredient (Stevens and Bukovac, 1987b). B. Factors Affecting the Efficiency of Foliar Application 1. Wetting Ability of the Adjuvant: Sands and Bachelard (1973) indicated that adjuvants* increased foliar absorption significantly. The effects of different adjuvants and different adjuvant concentrations on foliar uptake depended largely on the degree of wetting by the applied solution on the leaf surface. Stevens and Bukovac (1987a) worked with octylphenoxy adjuvants and found that the wetted area of the leaf surface was inversely related to the surface tension of the adjuvants. They also found that the surface tension of the octylphenoxy adjuvants increased with their hydrophile : lipophile balance (HLB) which was a function of oxyethylene content. HLB is the balance of the size and strength of the hydrophilic (water- loving or polar) and the lipophilic (oil-loving or apolar) group of an emulsifier (Chow et al., 1989). Organosilicone adjuvants which have low surface tensions (Neumann and Prinz,1974b) are especially effective in wetting leaf surfaces and are sometimes used to increase efficiency of foliar applications. Neumann and Prinz ll (1974a) found that organosilicone adjuvants such as Silwet L-77 were effective and relatively non-phytotoxic for treating iron chlorosis of citrus trees, and for increasing bean leaf absorption of iron and phosphate. 2. The Interaction Between the Adjuvant and Ca Forms: Reduction of surface tension with adjuvants is an important factor in foliar absorption and translocation, but the relationship of molecular interaction between the adjuvant and Ca forms may be equal to or more important than that of lowering surface tension (Stevens and Bukovac, 1987a). Stevens and Baker (1987) found as wetting ability of leaf surface increased (surface tension decreased), 2,4-D uptake by leaf surface decreased. Therefore, understanding the chemical and physical natures of the adjuvant and the highly specific requirement for adjuvant formulation to fit Ca is important in order to achieve maximum effectiveness (Stevens et al., 1988). 3. Chemical Forms: McFarlane and Berry (1974) measured the penetration rate of several cations through isolated leaf cuticles, and found that the smaller the ionic radius, the faster the ion penetrated the cuticle. Calcium, which has a relatively large ionic radius, penetrated the cuticle slowly. The form of Ca applied also has a significant effect on foliar uptake. Glenn and Poovaiah (1985) measured the rate of Ca penetration from five different Ca forms [CaClu 12 Ca(N0g2, calcium acetate(Ca(Cfi§C0g2), and two commercial Ca formulations] in 'Golden Delicious’ apple fruit. They found that CaCl2 penetrated the cuticle of the fruit significantly faster than other Ca forms tested. 4. Plant Species: Different plant species may respond differently to foliar Ca sprays. The cuticle of the leaf surface and the thickness of wax play an important role in foliar absorption. The cuticles of Braggiga spp. are covered with a relatively thick epicuticular wax. This wax works as a barrier to the movement of surface-applied nutrients, especially in polar solutions. For example, water without a adjuvant usually beads and rolls off the cauliflower leaf surface. Cabbage leaves absorbed manganese (Mn) only when a adjuvant was used, or the formation of surface wax was inhibited by EPTC (Cantliffe and Wilcox, 1972). However, some other researchers believed that the barrier to the movement of surface-applied Ca did not appear to be the surface itself. Bukovac and Wittwer (1957), working with beans, found that the absorption rate of 4"‘Ca into leaves was comparable with that of 32P. Chen (1964) even showed that tomato foliage was more efficient at absorbing Ca than the root. 5. Growing Conditions: Growing conditions may affect wax thickness, morphology, 13 and chemistry. Stevens and Bukovac (1987b) reported that the thickness and general state of the cuticle and the attendant stomata vary markedly with growing conditions. Baker (1974) studied Brussels sprouts, and discovered a relationship between wax formation on leaf surfaces and several environmental factors. According to him, the size and number of crystalline wax formations increased with increased radiant energy, and a relatively more horizontal crystalline growth habit was produced with rising temperature. Reed and Tukey (1982) found that rubidium (Rb+ ) and phosphate (HZPO; / HP04') penetration through cuticles of detached Brussels sprouts leaves was faster at 15° C than that at 25° C, while Glenn and Poovaiah (1985) found that decreased temperature could reduce the rate of Ca penetration through apple cuticles. In addition, van Goor (1973), showed that the efficiency of foliar uptake of Ca could be increased by high relative humidity (up to 90%). Soil moisture content also affects leaf wax deposition. Hunt and Baker (1982) showed that wax deposits on pea (Eigum satiyum) leaves not only increased as irradiance increased and humidity decreased, but also as soil moisture decreased. In spite of the apparent immobility of Ca within leaf tissue, translocation of foliar-applied Ca has been recorded by Biddulph et al. (1959) in beans, and by Millikan and Hanger(1969) in Brussels sprouts. In addition, Ca applied as a foliar spray has been successfully used to reduce the 14 severity of Ca deficiency disorders in some plants, e.g. blackheart of celery (Geraldson, 1954), tipburn of lettuce (Thibodeau and Minotti, 1969), and blossom—end rot in tomato (Evans and Troxler, 1953). LITERATURE CITED Ashkar, S.A. and S.H. Ries. 1971. Lettuce tipburn as related to nutrient imbalance and nitrogen composition. J. Amer. Soc. Hort. Sci. 96:448-452. Baker, E.A. 1974. The influence of environment on leaf wax development in Braseisa_elerasea var. genuifera- New Phytologist 73:955-966. Bangerth, F. 1979. Calcium-related physiological disorders of plants. Ann. Rev. Phytopath. 17:97-122. Barber, S.A., A.D. MacKay, R.0. Kuchenbuch, and P.B. Barraclough. 1988. 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Effects of surfactant on phenoxyalkaneic herbicides: A preliminary report. Aspects of applied biology. 1:193-200. Millikan, C.R. and B.C. Hanger. 1969. Movement of foliar- applied‘5Ca in Brussels sprouts. Aust. J. Biol. Sci. 22:545-557. Neumann, P.M. and R. Prinz. 1974. Evaluation of surfactant for use in the spray treatment of iron chlorosis in citrus tree. J. Sci. Food Agr. 25:221-226. Neumann, P.M. and R. Prinz. 1974. The effect of organosilicone surfactant in foliar nutrient sprays on increase absorption of phosphate and iron salts through stomatal infiltration. Israel. J. Agr. Res. 23(3-4):123- 128. Nieuwhof, M. 1960. Internal tipburn in white cabbage. Euphytica 9:203-208. Palzkill, D.A., T.W. Tibbitts, B.E. Struckmeyer. 1980. High relative humidity promotes tipburn on young cabbage plants. HortScience 15(5):659-660. Perumalla, C.J. and C.A. Peterson. 1986. Deposition of Casparian bands and suberin lemellae in the exodermis and endodermis of young corn and onion root. Can. J. Bot. 64:1873-1878. Peterson, C.A. 1988. Exodermal casparian bands; their significance for ion uptake by roots. Physiol. Plantarum 72:204-208. Reed, D.W. and H.B. Tukey Jr. 1982. Permeability of Brussels sprouts and carnation cuticles from leaves developed in different temperature and light intensities, pp. 267- 278. In: D.F. Cutler, K.L. Alvin, and C.E. Price (eds). The Plant Cuticle. Acad. Press, London. Rosen, C.J. 1990. Leaf tipburn in cauliflower as affected by cultivar, calcium sprays, and nitrogen nutrition. HortScience 25(6):660-663. Sands, R. and E.P. Bachelard. 1973. Uptake of picloram by Eucalypt leaf discs. 1. Effect of surfactant and nature of the leaf surfaces. New Phytol. 72:69-86. Shear, C.B. 1975. Calcium-related disorders of fruits and vegetables. HortScience 10(4):361-365. 19 Shafer, J. and C.B. Sayer. 1946. Internal breakdown of cabbage, as related to nitrogen fertilizer and yield. Proc. Amer. Soc. Hort. Sci. 47:340-342. Smith, M.W., F.N. Wazier, and S.W. Akers. 1989. The influence of soil aeration on growth and elemental absorption of greenhouse-grown seedling pecan trees. Commun. Soil Sci. & Plant Anal. 20 (3-4):335-344. Stevens, P.J.G. and M.J. Bukovac. 1987a. Studies on octylphenoxy surfactants. Part 1: Effects of oxyethylene content on properties of potential relevance to foliar absorption. Pestic. Sci. 20:19-35. Stevens, P.J.G. and M.J. Bukovac. 1987b. Studies on octylphenoxy surfactants. Part 2: Effects on foliar uptake and translocation. Pestic. Sci. 20:37-52. Stevens, P.J.G. and E.A. Baker. 1987. Factors affecting the foliar absorption and redistribution of pesticides; 1. Properties of leaf surfaces and their interactions with spray droplets. Pestic. Sci. 19:265-281. Stevens, P.J.G., E.A. Baker, and N.H. Anderson. 1988. Factors affecting the foliar absorption and redistribution of pesticides; 2. Physicochemical properties of the active ingredient and the role of surfactant. Pestic. Sci. 24:31-53. Thibodeau, P.G. and P.L. Minotti. 1969. The influence of calcium on the development of lettuce tipburn. J. Amer. Soc. Hort. Sci. 94 (4):372-376. Tibbitts, T.W. and G.E. Bottenberg. 1971. Effects of temperature increases on tipburn injury of lettuce. HortScience 6:306. Tibbitts, T.W. and R.R. Rao. 1968. Light intensity and duration in the development of lettuce tipburn. Proc. Amer. Soc. Hort. Sci. 93:454-461. Van Goor, B.J. 1973. Penetration of surface applied ”Ca into apple fruit. J. Hort. Sci. 261-270. CHAPTER 2 CALCIUH APPLICATION WITH ADJUVANTS TO DETACHED LEAP PORTIONS I. ABSTRACT Detached cauliflower (Braeeisa_elerasea var. betrxtie) leaves and leaf discs were treated with nine adjuvants and two salt sources of calcium to evaluate Ca penetration and uptake. Calcium concentration of the leaf portions was determined by atomic absorption spectrophotometry. An ethoxylated alkyl phenol adjuvant (Flo Mo S45) gave moderate spreading and maximum penetration. Three organosilicone adjuvants (L-77, 02-5152, and x2-5309) and an ethoxylated alcohol adjuvant (Flo Mo 6T) gave maximum spreading and intermediate penetration. Other adjuvants (Flo Mo 8100, AG- 98, LI 700, and crop oil concentrate) gave minimal spreading, and did not increase Ca penetration. There was no significant difference between calcium chloride (CaClfl and calcium nitrate (Ca(N0g2) as Ca sources to increase penetration. 20 II. INTRODUCTION During periods of rapid growth and during hot dry weather, insufficient calcium is transported to developing cauliflower leaves, resulting in leaf tipburn (Cox et al., 1976, Cox and McKee, 1976). Previous work has demonstrated that if the plants take up sufficient Ca, leaf tipburn can be reduced (Geraldson, 1954, Thibodeau and Minotti, 1969). Foliar sprays have been moderately effective in providing cauliflower leaves with more Ca. Some adjuvants have given increased Ca penetration (Sands and Bachelard, 1973). However, there are many adjuvants with various chemical and physical properties. It is difficult to predict the effectiveness of an adjuvant in improving penetration and absorption of a chemical into a plant. However, if an effective adjuvant is found, it may be possible to determine the mode of action and thus predict the effectiveness of other related compounds. In this section, we studied 1. the effectiveness of various adjuvants in improving penetration and absorption of Ca into cauliflower; and 2. the effectiveness of other physical factors in Ca absorption, including Ca form and concentration, time of application, and volume of application. 21 III. MATERIALS AND METHODS A. General Plant Production and Laboratory Practices: The general information below covers all the experiments in this chapter. Additional information is described in each individual experiment. 1. Plant Production in the Greenhouse: Cauliflower (fl;a§§19a_glgra§ga var. bgtzytig) seedlings were planted and grown in 200 cell flats in the Plant Science Greenhouse (PSG), Michigan State University with temperature 24° C day and 21° C night, 70-85% related humidity (RH), and natural sunlight with an average 14 mol. n3. day'1 photosynthetic photon flux (PPF). Each cell had a surface area of 1.8 cm2 and a volume of 4.6 cm’. The seedlings were watered daily. The plants were fertilized with 400 ppm 20N-8.6P-16.6K (Peters 20-20-20) weekly after emergence, and twice a week after 6 weeks. Four weeks after the seedlings emerged, the seedlings were either transplanted in 5 liter (22 cm tall and 23 cm diameter) pots in the same greenhouse or to the field at the Horticultural Teaching and Research Center (HTRC), Michigan State University, East Lansing. 22 23 2. Leaf Selection, Leaf Tip Portion, and Leaf Discs: The experiments were performed with detached recently expanding full-size leaves from the plants 7 weeks after transplanting. Leaf discs, 3.6 cm in diameter, were cut from one third of tip portion of the leaves with a cork borer, and placed in 14.5 X 1.5 cm petri-dishes containing deionized water. The midrib and larger veins were avoided when cutting discs. Distal portions (one third of tip portion) of detached leaves were also placed in containers containing deionized water. Both detached leaf tip portions and leaf discs were treated and maintained after treatment at 26-28°C temperature, 80-95% RH, and a light intensity of 96 p mol. s4. ma. 3. Adjuvants: Nine adjuvants (Silwet L-77, X2-5309, Q2-5152, Flo Mo 6T, Flo MO 845, F10 Mo 8100, Triton AG-98, LI 700, and Herbimax COC) were applied at a concentration of 0.1% v/v. Some adjuvants (Flo Mo S45, Flo Mo 8100, Triton AG-98, LI 700, and Herbimax COC) were also applied at 0.25% v/v. The name, type, and sources of all the adjuvants used in this experiment are listed in Table 1. The most effective adjuvants were selected for further study. 4. Ca Analysis on AA: Cauliflower leaf tissue was prepared for Ca analysis by the following method (Adler and Wilcox, 1985): The samples with wax removed were forced air dried at 70°C and ground in 24 Table 1. A list of adjuvants used in these experiments. Name Type Source Silwet L-77 Organosilicone Union Carbibe x2-5309 Organosilicone Dow Corning 02-5152 Organosilicone Dow Corning Flo Mo 6T Ethoxylated alcohol DeSoto Flo Mo S45 Ethoxylated alkyl phenol DeSoto Flo Mo 8100 Ethoxylated octyl phenol DeSoto Triton AG-98 LI 700 Herbimax COC Alkyl poly oxyethylene glycols Nonionic [acidifier Crop oil concentrate Rohm 8 Haas Loveland Loveland 25 a Wiley mill to pass through a 1.3 mm screen. The ground tissue was redried at 70°C for at least 24 hours. 0.1 g of ground tissue of each sample was digested in 0.5 ml of 30% hydrogen peroxide (th) and 1 ml of perchloric acid (HCloa in a 100 ml digestion tube for 5 min on a Tecator Digestion System 40,1016 digester at 300°C. The digestion tubes were then removed from the digestion block, and cooled for about 5 min. Another 1 ml of 30% H45 was added to each sample. The digestion tubes were then returned to the digestion block for 30-35 min. After the digestion tubes were cool (about 5 min), they were filled one-third full with deionized water. Then the samples were reheated slightly for 1-1.5 min to dissolve crystals that formed when the samples cooled. Then 5% w/v lanthanum (La) was added to equalize ionization among the samples and standards. Lanthanum also acts as a releasing agent, preventing the formation of Ca compounds which are not atomized by an acetylene (CHE, flame. The La stock solution was prepared by mixing 117 g lanthanum oxide (Lagh) with a small amount of deionized water and adding about 180 ml HCl to dissolve the Laph before bringing the solution up to 1 liter with deionized water. The samples were analyzed with an Instrumentation Laboratory Video 12 atomic absorption-atomic emission spectrophotometer at a wavelength of 422.7 nm and a 26 bandwidth of 1.0 nm by acetylene (Cfifi) flame, atomic absorption spectrophotometry with an Instrumentation Laboratory hollow cathode-Ca lamp. 5. Statistical Analysis: A completely randomized design (CRD) with four replications was used in this study. A replication consisted of eight leaf discs placed in the same petri-dish or four detached leaf portions with two treated spots on each leaf portion placed in one container. Both leaf discs and detached leaves in one replication were from the same plant except the leaf age experiment. An analysis of variance was done on all data. LSD values and Orthogonal comparisons were computed for the relevant levels of significance. The statistical analyses were performed using MSTAT. B. Experiments with Detached Leaf Portions: 1. Tissue Ca Concentration within Cauliflower Leaves: This experiment was performed using leaf discs cut from recently expanded full-size leaves of field grown cauliflower 7 weeks after transplanting in 1989. Ten leaf discs were cut from five different areas (tip, next to tip, middle, next basal, and basal area) of each leaf sample, and four same age leaves were removed from one plant. Eight leaf discs from the same area of four leaves of the same plant were combined together as a replication for Ca analysis. A completely randomized design (CRD) with four 27 replications was used. The samples were forced air dried at 70° C and ground for Ca analysis. 2. Ca Salt Source and Concentration Selection: We initially tested several Ca sources including CaCl” Ca(NO,)2, Ca(H2PO,)2, CaSO,, and chelated calcium. However, because of limited solubility of the other Ca forms, CaCl2 and Ca(NOQ, were selected for further study. Leaf tip portions were used in this experiment. The leaf samples were treated with CaCL,in.various concentrations and 0.1% L-77 by using a microsyringe applicator. One droplet (5 pl) of CaCl2 solution was applied to the upper surface of each treated spot (10 cm2 leaf area) of the detached tip portions. After 24 hours, treated leaf tissues were rinsed with deionized water to remove residual chemical deposits. After drying, the epicuticular wax was removed from the treated leaf surface with a cellulose acetate strip. A 5% w/v cellulose acetate solution was prepared by dissolving cellulose acetate powder in acetone according to the method described by Silcox and Holloway (1986). The solution was applied with a Freon powered microsprayer to cover the region of the leaf surface required. When the film became opaque and whitish in color (about 2 min at room temperature), it usually began to peel away from the leaf surface around its edges, and could be removed easily with a tweezers. 28 3. The Effects of Adjuvants on Ca Penetration into leaves: Nine adjuvants were selected using greenhouse leaf samples and field samples. Three similar experiments were done, two times by using greenhouse grown cauliflower samples, and one by using field grown cauliflower samples. L-77, X2-5309, 02-5152, and Flo Mo 6T have greater wetting ability which is inversely related to the surface tension (Stevens 8 Bukovoc, 1987a; Stevens et al., 1988). The solutions with these four adjuvants spread very quickly. In order to avoid the sprayed solution spreading over the edge of leaf discs, the studies were done using tip portions of detached leaves. The leaf samples were taken from the plants 7 weeks after transplanting. Four leaves were removed from one plant, and two treatment spots were made on each leaf sample. One droplet (5 pl or 2 pl) of CaClzcn: Ca(NO3)2 solution was applied to the upper leaf surface in a treated spot of about 10 cmfl A.completely randomized design (CRD) with four replications arranged as 2 X 4 factorial plus an untreated control was used. The treatments used on detached leaves are listed in Table 2. The studies with Flo Mo S45, Flo Mo S100, AG-98, LI 700, and COC were done using leaf discs. Four leaves were taken from each plant 7 weeks after transplanting. Two leaf discs were made from the tip portion of each leaf sample. One droplet (5 pl) of CaCl2 and Ca(NO;.,)2 solutions with the 29 Table 2. Calcium and adjuvant combinations applied to detached cauliflower leaves in a 5 or 2 pl drop. 1. CaClz 214 0.1: L-77 2. CaCl, 2M 0.1% x2-5309 3. CaClz 2M 0.1% Flo Mo 6T 4. CaCl, 2M 0.1% 02-5152 5. Ca(NO3) 2M 0.1% L-77 6. Ca(NO3) 2M 0.1% x2-5309 7. Ca(NO3) 2M 0.1% Flo Mo 6T 8. Ca(NO3) 211 0.1% 92-5152 Untreated Table 3. Calcium and adjuvant combinations applied to 3.6 cm cauliflower leaf discs in a 5 p1 drop. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. CaCl2 CaC12 CaCl2 CaCl2 CaCl, Ca (N03) Ca (N03) Ca (N03) Ca (N03) Ca (N03) CaCl2 CaCl2 CaCl2 CaClz CaClz Ca (N03) Ca (N03) Ca (N03) Ca (N03) Ca (N03) 2M 2M 2M 2M 2M 2M 2M 2M 2M 2M Untreated + Flo Mo S45 F10 MO 8100 AG-98 LI 700 COC Flo Mo S45 FlO MO 8100 AG-98 LI 700 COC F10 MO S45 Flo Mo AG-98 LI 700 COC Flo Mo Flo Mo AG-98 LI 700 COC 8100 845 S100 31 adjuvants at two concentrations (0.1% and 0.25%) were applied to the upper surface of each leaf disc. A completely randomized design (CRD) with four replications arranged as 2 X 2 x 5 factorial plus an untreated control was used. The treatments used on leaf discs are given in Table 3. Twenty four hours after treatment leaf tissues were washed with deionized water to remove residual chemical deposits. After drying, the epicuticular wax was removed from the treated leaf surface with a cellulose acetate strip as described above. 4. The Effects of Treatment Duration on Ca Penetration: The effect of treatment duration on Ca penetration was studied by leaving the treatments on the leaves for 6, 12, or 24 hours. The leaf samples used in this experiment were taken randomly from field grown cauliflower 7 weeks after transplanting (the plants had just started curd formation). Tip portions of detached leaves were treated with 5 pl of 2M CaCl2 or Ca(N03)2 mixed with 0.1% 02-5152 per 10 cm2 of leaf area. Four leaves were taken from each plant, and each leaf sample was treated with two drops of solution at different spots. The leaves from the same plant were combined together as a replication for Ca analysis. A completely random design (CRD) with four replications arranged as 2 x 3 factorial plus an untreated control was used. Other treatment processes were the same as described above. 32 5. Ca Uptake and Penetration: For the purposes of our study, we defined penetration as entering the epidermis of the leaf tissue. Ca remaining in the leaf wax was not considered to have penetrated the leaf. We made no effort to determine the amount of Ca that was absorbed or translocated within the leaf. In this study, 5 pl drops of 2M CaCl2 mixed with 0.25% Flo Mo S45 were applied to the upper surface of leaf discs taken from full size cauliflower leaves. The leaves were taken from greenhouse grown cauliflower as curd began to form. Four leaves were removed from each plant, and two leaf discs were cut from the tip portion of each leaf sample. The leaf discs from the same plant were combined together after treatment as a replication for Ca analysis. A completely random design (CRD) with four replications was used. Other treatment processes were the same as described above. Stevens and Bukovac (1987b) reported the method of determination of “C uptake and penetration as follows: “C uptake = “C in the leaf tissue + 1‘C in cellulose acetate strip. l“C penetration = 1‘C in the leaf tissue. “C in the wax = 1‘C in the cellulose acetate strip. In our study, Ca uptake and penetration were determined as: Ca uptake = Ca in treated leaf tissue with wax minus Ca in untreated leaf tissue with wax. 33 Ca penetration = Ca in treated tissue without wax minus Ca in untreated tissue without wax. Ca in the wax - Ca in the cellulose acetate strip. 6. The Effect of Leaf Age on Ca Penetration: The leaves used in the leaf age experiment were obtained from greenhouse grown cauliflower just as curds began to form. The young wrapper leaves, recently expanded full size leaves, and old mature leaves were removed from the same plant. Two leaf discs were made from each leaf sample. Eight leaf discs made from four same-age leaves from two plants were put in one petri-dish, and treated with 5 pl of 2M Ca(NO3)2 mixed with 0.1% Flo M0 845 in 5 drops for 24 hours. The treated leaf tissues in the same petri-dish were combined together as a replication for Ca analysis. Ca penetration into the leaf was determined by the method described above. IV. RESULTS AND DISCUSSION A. Experiments with Detached Leaf Portions: 1. Tissue Ca Concentration within Cauliflower Leaves: An investigation of Ca concentration in different portions of leaf tissue is shown in Table 4. The results indicated that there were Ca concentration differences within cauliflower leaves, with lower Ca concentrations near the leaf tip and higher concentrations near the leaf basal area. This agrees with results reported by Rosen et al. (1987). 2. Ca Salt Source and Concentration Selection: The effect of different Ca concentrations of applied solution on tissue Ca concentration in leaves is shown in Table 5. When the Ca concentration of applied solution was less than 1.0M, the Ca levels in treated leaf tissues were not significantly different from untreated. However, when Ca concentration increased above 1.5M, the Ca level of the leaf tissues was significantly higher. There was no evidence of phytotoxicity of Ca solution mixed with L-77, X2-5309, 02-5152, or Flo Mo 6T, even at 2M Ca concentration. The leaf tissues treated with 2M Ca concentration and AG-98, Flo Mo 8100, LI 700, or COC, turned yellow within one day. 34 35 Table 4. Ca level in various sections of the youngest full size cauliflower leaves. Areas Ca conc. (% dry wt) Tip area of the leaves 1.87 Next tip area of the leaves 1.90 Middle area of the leaves 1.99 Next basal area of the leaves 1.98 Basal area of the leaves 2.00 F * LSD (0.05) 0.10 c.v.(%) 3.29 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 36 Table 5. The effect of Ca concentrations on Ca penetration into detached cauliflower leaves treated with 5 pl of various concentration of CaCl2 and 0.1% L-77 per 10 cm? leaf area. Ca Conc. Ca cone. of solution (% dry wt) Untreated 1.94 0.1M 1.95 0.3M 1.98 0.5M 1.99 1.0M 2.01 1.5M 2.13 2.0M 2.18 F * LSD (0.05) 0.14 c.v.(%) 4.78 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 37 3. The Effects of Adjuvants on Ca Penetration into Leaves: In these studies, the results of two greenhouse experiments were very similar, so they were combined together as replicates over time for statistical analysis. The results in Table 6 indicate that Ca plus L-77, X2- 5309, 02-5152, and F10 Mo 6T in a 5 pl drop increased Ca concentration of the leaf tissues compared with the untreated controls. There were no differences in Ca concentration between the leaf tissues treated with 2 pl Ca solution and the untreated control. There were no differences between L-77, X2-5309, 02-5152, and Flo Mo 6T in increasing Ca penetration. Table 7 shows that among Flo Mo S45, Flo Mo 8100, AG- 98, LI 700, and COC, only Flo Mo S45 increased Ca concentration over the untreated control. The results of the experiment using detached field grown cauliflower leaves (Table 8) also revealed that there were significant differences among nine adjuvants in increasing Ca penetration. Flo Mo S45, L-77, x2-5309, 02- 5152, and F10 Mo 6T caused significant increases in Ca penetration into cauliflower leaves compared with untreated control. Flo Mo S45 gave moderate spreading and maximum penetration. L-77, X2-5309, 02-5152, and F10 Mo 6T gave maximum spreading and intermediate penetration. Other 38 Table 6. Ca content in detached greenhouse grown cauliflower leaves 24 hr after application of 5 pl and 2 pl of 2M CaCl2 and Ca(NO,)2 with 0.1% surfactants per 10 on2 leaf area . Ca Conc. (% dry wt) Treatment 5 pl 2 pl Ca salt CaCl, 2.14 2.04 Ca(NO3)2 2.17 2.04 F NS NS Adjuvant L-77 2.13 2.04 x2-5309 2.14 2.04 02-5152 2.18 2.05 Flo Mo 6T 2.18 2.05 F NS NS Untreated 1.96 1.96 Orthogonal comparisons Ca vs Untreated *** NS L-77 vs Untreated ** NS X2-5309 vs Untreated ** NS 02-5152 vs Untreated *** NS Flo Mo 6T vs Untreated *** NS C.V.(%) 6.53 6.27 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 39 Table 7. Ca content in greenhouse grown cauliflower leaf discs 24 hr after application of 5 pl of 2M CaCl2 and Ca(NO,)2 with surfactants at 2 concentrations. Treatment Ca Conc. (% dry wt) Ca salt CaCl2 2.03 Ca(N03)2 2.07 F NS Adjuvant Flo Mo S45 2.23 Flo Mo 8100 2.03 AG-98 2.03 LI 700 1.98 COC 1.98 F *** LSD(0.001) 0.11 Adjuvant concentration 0.1% 2.04 0.25% 2.06 F NS Untreated 1.98 Orthogonal comparisons Ca vs Untreated NS Flo Mo S45 vs Untreated *** Flo Mo 8100 vs Untreated NS AG-98 vs Untreated NS LI 700 vs Untreated NS COC vs Untreated NS c.v. (%) 5.90 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 40 Table 8. Ca content in detached field grown cauliflower leaves 24 hr after application of 5 pl of 2M CaCl2 and Ca(NO3)2 with 2 concentration of different surfactants. Treatment Ca conc. (% dry wt) Ca salt CaCl2 2.23 Ca(NO3)2 2.27 F NS Adjuvant 0.1% L-77 2.39 0.1% X2-5309 2.37 0.1% 02-5152 2.34 0.1% Flo Mo 6T 2.33 0.25% Flo Mo 845 2.53 0.25% Flo Mo S100 2.09 0.25% AG-98 2.09 0.25% LI 700 2.02 0.25% COC 2.07 F *** LSD(0.001) 0.18 Untreated 2.03 Orthogonal comparisons Ca vs Untreated *** L-77 vs Untreated *** x2-5309 vs Untreated *** 02-5152 vs Untreated *** Flo Mo 6T vs Untreated *** Flo Mo S45 vs Untreated *** Flo Mo 8100 vs Untreated NS AG-98 vs Untreated NS LI 700 vs Untreated NS COC vs Untreated NS C.V.(%) 4.75 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 41 adjuvants, including Flo Mo S100, Triton AG-98, LI 700, and crop oil concentrate, gave minimal spreading and did not increase Ca penetration compared with the untreated control. The spreading ratings of these nine adjuvants are listed in Table 9. Solutions containing L-77, X2-5309, 02-5152, and F10 Mo 6T have a lower surface tension, which was inversely related to the wetting area of leaf surface (Neumann and Prinz, 1974b). Therefore, adjuvants like L-77 are often used to increase penetration of nutrients or herbicides into plant foliage (Neumann and Prinz, 1974; Lee and Dewey, 1981). Stevens and Bukovac (1987a) reported that the maximum solubilities of the adjuvants were observed with adjuvants having intermediate hydrophile : lipophile balance which was correlated with the surface tension. Flo Mo S45 has an intermediate surface tension and large solubility. In addition, the calcium solution containing Flo Mo S45 took a longer time to dry than the solutions containing L-77, X2- 5309, 02-5152 and Flo Mo 6T. There is some evidence that plants absorb salts from solutions faster than from dry deposits (Stevens et al. 1988). There were no significant differences between CaCl2 and Ca(NOQ2 as Ca sources to increase penetration into cauliflower leaves as shown in Tables 6, 7, and 8. Both CaClz and Ca(NO3)2 significantly increased Ca penetration compared with the untreated control. 42 Table 9. The relative spreading of the adjuvants used in this experiment. Adjuvant Spreading rating L-77 5 X2-5309 5 02-5152 5 Flo Mo 6T 5 Flo M0 845 3 Flo MO 8100 2 AG-98 2 LI 700 l COC 1 Water 0 Spreading was evaluated visually size similar to Ca in water; 5 = over the leaf surface. on a scale of 1-5: 1 = drop rapid spreading of solution 43 4. The Effect of Treatment Duration on Ca Penetration: The amount of time between application and leaf sampling is also an important parameter affecting Ca uptake and penetration. Table 10 shows the effects of the length of treatment on Ca penetration. There was a significant increase in Ca levels of leaf tissues treated for 12 and 24 hours. There was no difference between CaCl2 and Ca(NO3)2 as Ca sources. Both CaClz and Ca (N03), increased Ca penetration into cauliflower leaves compared with the untreated control. 5. Ca Uptake and Penetration: Table 11 shows the difference between Ca uptake and penetration by leaf tissues after the same amount of Ca was applied. Ca uptake was the Ca concentration in treated leaf tissues washed in 0.15N HCl solution minus Ca concentration in the untreated control, while Ca penetration was determined as the Ca concentration in the leaf tissues with wax removed minus the Ca concentration in the untreated control. In general, Ca penetration increased with Ca uptake, and both increased with the addition of 0.25% Flo Mo $45 to the Ca solution. However, there was a small difference between uptake and penetration. The difference between Ca uptake and penetration should equal the Ca concentration in the wax strip removed from the leaf surface (Stevens and Bukovac, 1987). The results of our experiment showed that the Ca concentration in the surface wax was 44 Table 10. The effect of treatment time on Ca penetration into detached field grown cauliflower leaves with 5 pl of 2M CaCl2 and Ca(NO,)2 mixed with 0.1% 02- 5152 per 10 cm2 leaf area. Treatment Ca Conc. (% dry wt) Ca salt CaCl2 2.31 Ca(NO3)2 2.33 F NS Time on leaf 6 hrs 2.24 12 hrs 2.33 24 hrs 2.39 F ** LSD (0.01) 0.12 Untreated 2.15 Orthogonal comparisons Ca vs Untreated ** 6 hrs vs Untreated NS 12 hrs vs Untreated ** 24 hrs vs Untreated *** C.V. (0.05) 3.69 NS,*,**, *** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 45 Table 11. Ca level in leaf portion after treatment with 5 pl of 2M CaCl2 + 0.25% Flo Mo S45 per leaf disc. Treatment Ca Conc. (% dry wt) Untreated 2.06 Whole unwashed leaf discs 2.55 Leaf discs washed in 0.15N HCl 2.30 Leaf discs with surface wax removed 2.18 Cellulose acetate strip 0.07 F *** LSD(0.05) 0.09 C.V.(%) 2.70 NS,*,**, *** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 46 less than the difference between Ca uptake and penetration. This may be the result of experimental error and the accuracy (1 0.05%) of the atomic absorption spectrophotometer. 6. The Effect of Leaf Age on Ca Penetration: Leaf age was also an important factor affecting Ca penetration. Ca penetration through the leaf surface decreased as the leaf age increased. The absolute Ca level was higher in the older leaves (Table 12). Ca is a relatively immobile element within the plant, and old cauliflower leaves have a much higher Ca level than young expanding leaves. This may be a part of the reason that cauliflower leaf tipburn occurs in young expanding leaves. However, the cuticle of young leaves is covered with a thinner epicuticular wax than that of the old leaves, so there is more potential for Ca penetration into young leaves. 47 Table 12. The effect of leaf age on Ca penetration into cauliflower leaves after 24 hrs. foliar application with 5 pl of 2M Ca(NO3)2 and 0.1% Flo Mo S45 per leaf disc. Leaf Age Ca conc. Increased Ca (% dry wt) conc.(% dry wt) Treated young wrapper leaves 1.42 0.50 Untreated young wrapper leaves 0.92 Treated youngest full size leaves 2.32 0.32 Untreated youngest full size leaves 2.00 Treated old mature leaves 2.40 0.12 Untreated old mature leaves 2.28 F *ee *** LSD (0.001) 0.40 0.07 C.V. (%) 7.48 11.05 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. LITERATURE CITED Adler, P.R. and G.E. Wilcox. 1985. Rapid perchloric acid digest methods for analysis of major elements in plant tissue. Commun. Soil Sci. & Plant Anal. 16(11):1153- 1163. Cox, E.F. and J.M.T. McKee. 1976. A comparison of the tipburn susceptibility in lettuce under field and glasshouse conditions. J. Hort. Sci. 51:117-122. Cox, E.F., J.M.T. McKee, and A.S. Dearman. 1976. The effect of growth rate on tipburn occurrence in lettuce. J. Hort. Sci. 51:297-309. Geraldson, C.M. 1954. The control of black heart of celery. Proc. Amer. Soc. Hort. Sci. 63:353-358. Lee, J.J.L. and D.H. Dewey. 1981. Infiltration of calcium solutions into 'Jonathan’ apples using temperature differentials and surfactant. J. Amer. Soc. Hort. Sci. 106(4):488-490. Neumann, P.M. and R. Prinz. 1974. The effect of organosilicone surfactant in foliar nutrient sprays on increase absorption of phosphate and iron salts through stomatal infiltration. Israel. J. Agr. Res. 23(3-4):123- 128. Rosen, C.J., H.J. Buchite, and G.G. Ahlstrand. 1987. Cauliflower response to gypsum on a coarse textured soil: Relationship between tipburn and leaf nutrient distribution. J. Plant Nutr. 10:1925-1934. Silcox, D. and P.J. Holloway. 1986. A simple method for the removal and assessment of foliar deposits of agrochemicals using cellulose acetate film stripping. Aspects of Applied Biology. 11:13-17. Stevens, P.J.G. and M.J. Bukovac. 1987a. Studies on octylphenoxy surfactants. Part 1: Effects of oxyethylene content on properties of potential relevance to foliar absorption. Pestic Sci. 20:19-35. Stevens, P.J.G. and M.J. Bukovac. 1987b. Studies on 48 49 octylphenoxy surfactants. Part 2: Effects on foliar uptake and translocation. Pestic. Sci. 20:37-52. Stevens, P.J.G., E.A. Baker, and N.H. Anderson. 1988. Factors affecting the foliar absorption and redistribution of pesticides; 2. Physicochemical properties of the active ingredient and the role of surfactant. Pestic. Sci. 24:31-53. Thibodeau, P.O. and P.L. Minotti. 1969. The influence of calcium on the development of lettuce tipburn. J. Amer. Soc. Hort. Sci. 94 (4):372-376. CHAPTER 3 CALCIUM AND ADJUVANT APPLICATION TO CAULIFLO'ER IN THE FIELD I . ABSTRACT In field experiments, CaCl2 and Ca(NO,)2 with two adjuvants (L-77 and X2-5309) at 0.1% v/v concentration were applied on 'White Rock' cauliflower as foliar sprays. Foliar application of Ca with adjuvants significantly increased the Ca concentration of leaf tissues and reduced leaf tipburn in cauliflower. There was no significant difference between CaCl2 and Ca(NO,)2 as Ca sources or between L-77 and X2-5309 as adjuvants in increasing Ca penetration and reducing leaf tipburn. As the number of applications increased, leaf tipburn decreased. At least three applications of Ca were required to significantly reduce tipburn. 50 II. INTRODUCTION Leaf tipburn is a serious problem in cauliflower production in the United States, especially in areas that have hot dry weather during the cauliflower production season. In severe cases, leaf tipburn may not only decrease cauliflower quality by discoloring the curd, but also may decrease cauliflower yield due to rot of the curd as a result of secondary pathogen infection such as soft rot (Maynard et al., 1981). Previous research on tipburn was mainly concerned with determining the causes of the problem and the factors that affect the incidence of tipburn. The causes of leaf tipburn are complex and many factors are associated with it. Therefore, there is not a successful method to control leaf tipburn in the field. Rosen et al. (1987) tried to prevent cauliflower leaf tipburn by application of CaSO, to the soil, but it was not successful. In this experiment, we tried to develop methodology to prevent leaf tipburn in cauliflower by foliar application of Ca with adjuvants. 51 III. MATERIALS AND METHODS Two field experiments were conducted at the Horticultural Teaching and Research Center (HTRC), Michigan State University, East Lansing, Michigan in 1990. ’White Rock' cauliflower was used in all experiments. The seeds were planted in the Plant Science Greenhouse (200 plants per flat) under conditions as described in the second chapter. Four weeks later the seedlings were transplanted to the field. In the first field experiment, the seedlings were planted in the greenhouse on 7 April, and transplanted to the field on 10 May. Before transplanting, the fields were broadcast with 500 kg/ha 10N-8.6P-16.6K fertilizer, 34 kg/ha Solubor, 1.7 kg/ha Treflan, and 6.8 kg/ha Lorsban. The plants were set with a single row mechanical transplanter and watered immediately after transplanting. Irrigation was applied whenever the plants needed water during the season. The field was sidedressed with 186 kg/ha urea (45-0-0) 4 and 7 weeks after transplanting. The plants were set 50 cm apart in the row, with 1.2 m between rows. Rows were 28 m long, containing 45 plants each. Guard rows were planted between treatment rows and around the field 52 53 edges. The experiment was arranged as a randomized complete block with three replications. Foliar treatments were applied with a hand carried, carbon dioxide (C02) powered sprayer at a pressure of 200 Kpa, with a TJ60-8006E spray tip, and a spray volume of 370 liter/ha (Gruesbeck, 1990). Two organosilicone adjuvants (L-77 and x2—5309) at a concentration of 0.1% v/v were added to the tank mixes. Ca was applied as CaCl2 and Ca(NO3)2 at a concentration of 3.6 kg Ca/ha (0.25M Ca). Calcium treatments were applied 1, 2, or 3 times. The treatments in the first field experiment are listed in Table 1. The first foliar treatment was applied on 9 July, at 10:00 am. At that time, most plants had just started curd formation. A few tipburned leaves were found. After that, foliar treatments were applied weekly at about the same time in the morning. The weather conditions for each treatment time are listed in Table 2. On 17 July, tipburn appeared on about 10% of the plants. The plants were harvested on 30 July. Before harvesting, the number of tipburned plants and the number of tipburned leaves per plant were recorded. The second field experiment was a repeat of the first field experiment. The seedlings were transplanted in the field next to the first field experiment on 2 July. In this experiment, the cauliflower was sidedressed with 186 kg/ha urea 3, 5, and 8 weeks after transplanting. The actual Ca concentration for foliar application was increased to 4 kg 54 Table 1. The treatments used in the first field experiment with 3.6 kg Ca /ha (0.25M Ca). Ca salt CaCl2 (30.2% Ca) 12.0 kg/ha Ca(N03)2 (22% Ca) 16.5 kg/ha Adjuvant L-77 0.1% X2-5309 0.1% Water No. of applications 1 2 3 Untreated control 55 Table 2. The weather conditions for each treatment in the first field experiment in 1990. Date Time Wind Soil Air RH (miles/hr) temp. temp. (%) 9 July 10:00 am 2-4 N 28° C 29° C 64 17 July 9:00 am 5-8 SW 24° C 27° C 80 23 July 9:00 am 4-6 W 24° C 27° C 68 56 Table 3. The treatments used in the second field experiment with 4.0 kg Ca [ha (0.28M Ca). Ca salt CaCL,(30.2% Ca) 13.2 kg/ha Ca(NO3)2 (22% Ca) 18.7 kg/ha Adjuvant L-77 0.1% X2-5309 0.1% Water No. of applications 2 3 4 Untreated control 57 Table 4. The weather conditions for each treatment of the second field experiment in 1990. Date Time Wind Soil Air RH (miles/hr) temp. temp. (%) 28 Aug. 12:30 pm 6-9 N 27° C 31° C 72 4 Sep. 9:10 am 3-5 SW 21° C 20° C 80 11 Sep. 10:30 an 5-7 NE 20° C 21° C 69 17 Sep. 2:00 pm 4-6 N 17° c 16° c 69 58 /ha for both CaC12 and Ca(NO,)2 (0.28M Ca), and one more foliar spray was applied. The treatments used in the second field experiment are listed in Table 3. Foliar sprays began on 28 August, and were applied weekly for 4 weeks. The weather conditions for each treatment are listed in Table 4. The plants were harvested on 20 September. The number of tipburned plants and the number of tipburned leaves per plant were recorded before harvest. Tissue analysis: Leaf and curd samples were collected at harvest from at least six plants in each plot for mineral analysis. Two tip portions of non-tipburned wrapper leaves and one piece of curd were taken from each plant. The harvested samples were dipped 6-8 times in four separate containers which contained deionized water, 0.15 N hydrochloric acid (HCl), and two deionized water containers. The samples were shaken to remove excess water or solution. The samples were forced air dried at 70°C and ground in a Wiley mill to pass through a 1.3 mm screen. The ground tissues were redried at 70°C for at least 24 hours and weighed for ashing. The methods of tissue digestion and Ca analysis were the same as described in chapter 2. Statistical Analysis: The experiment was designed as a Randomized Complete Block (RCB) with three replications. The treatments were 59 arranged as a 3 X 3 X 2 factorial plus an untreated control. An analysis of variance was done on all data. LSD values were computed for the relevant levels of significance. Orthogonal comparisons were computed for comparison of treatment means. Statistical analyses were performed with MSTAT. IV. RESULTS AND DISCUSSION The results from the two field experiments show that the addition of L-77 or X2-5309 to Ca solution decreased leaf tipburn (Table 5 & 6). There was no significant difference between L-77 and X2-5309 in reducing leaf tipburn. Adding adjuvants to Ca solutions decreased both the number of tipburned plants and the number of tipburned leaves per plant. The decrease in the number of tipburned plants was not statistically significant in the first field experiment compared with water, but was highly significant compared with untreated control. There was no difference between Ca in water and untreated controls in either first or second field experiments. There was no significant difference between CaCl2 and Ca(NOfl, in reducing leaf tipburn (Table 5 & 6). Both reduced tipburn significantly as compared with the untreated control. CaCl2 was always slightly more effective than Ca(NO,)2 even though the differences were not statistically significant. In the field, tipburn is associated with many factors. Among these factors, rapid growth rate may be the main cause of tipburn (Cox et al. 1976). Since Ca(NO_.,)2 is a good source of nitrogen, plants 60 61 Table 5. The effects of treatment with 3.6 kg Ca/ha, 0.1% adjuvants, and number of applications on cauliflower leaf tipburn in the first field experiment, 1990. Treatment Tipburned No. of tipburned plants(%) leaves per plant Ca salt CaCl2 78.5 5.2 Ca(NO,)2 81.5 5.7 F NS NS Adjuvant 0.1% L-77 78.9 5.1 0.1% X2-5309 77.8 5.1 Water 83.3 6.1 F NS ** LSD(0.01) -- 0.9 No. of applications 1 85.6 6.6 2 81.7 5.6 3 72.8 4.2 F *4 *** LSD (0.05, 0.001) 10.1 1.1 Untreated 91.9 8.5 Orthogonal comparisons Ca vs Untreated ** *** L-77 vs Untreated ** *** X2-5309 vs Untreated ** *** Water vs Untreated NS ** T1 vs Untreated NS ** T2 vs Untreated ** *** T3 vs Untreated *** *** C.V.(%) 13.3 17.7 NS, *, **, *** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 62 Table 6. The effects of treatment with 4 kg Ca/ha, 0.1% adjuvants, and number of applications on cauliflower leaf tipburn in the second field experiment, 1990. Treatment Tipburned No. of tipburned plants(%) leaves per plant Ca salt CaC12 60.2 2.7 “(1:03), 63.7 2.9 F NS NS Adjuvant L-77 55.4 2.5 X2-5309 58.2 2.5 Water 72.4 3.5 F est *** LSD(0.001) 10.3 0.5 No. of applications 2 71.9 3.3 3 61.8 2.7 4 52.2 2.4 F *** *** LSD (0.001) 10.3 0.5 Untreated 77.3 3.8 Orthogonal comparisons Ca vs Untreated *** *** L-77 vs Untreated *** *** X2-5309 vs Untreated *** *4* Water vs Untreated NS NS T2 vs Untreated * ** T3 vs Untreated *** *** T4 vs Untreated *** *** C-V-(%) 13.2 14.3 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 63 treated with it may have had an increased growth rate. The growth rate of shoot may be greater than the growth rate of root. Under dry conditions that may result in increased tipburn. Increasing the number of foliar Ca applications significantly reduced the number of tipburned plants and the number of tipburned leaves per plant in both experiments. One application of Ca did not reduce tipburn over the control, but two or more foliar applications reduced both the number of tipburned plants and the number of tipburned leaves per plant. Foliar application of Ca with adjuvants significantly increased Ca concentration in both leaf and curd tissues (Table 7 & 8). The Ca concentration in curd tissue is very low and it probably is not an indicator of the incidence of leaf tipburn. The Ca concentration in leaf tissue is negatively correlated with the incidence of leaf tipburn (Figure 1 8 2). There was no effect on cauliflower yield from any of the foliar Ca treatments (Table 9). The results of two experiments were similar, only the Ca level in leaves and tipburn level were different. The Ca level of the leaf tissues was higher and tipburn incidence was lower in the second field experiment than in the first experiment. One more foliar application was added in the second experiment, and the Ca concentration was increased 64 Table 7. Ca content of leaf and curd samples after treatment with 3.6 kg Ca/ha CaCl2 and Ca(N03)2, 0.1% L-77 and X2-5309, and various number of applications in the first field experiment, 1990. Treatment Ca conc. (% dry weight) Leaf Curd Ca salt CaCl2 0.53 0.22 Ca(NO,)2 0.51 0.22 F NS NS Adjuvant L-77 0.57 0.23 X2-5309 0.55 0.23 water 0.43 0.20 F 44* *** LSD(0.001) 0.09 0.02 No. of applications 1 0.46 0.21 2 0.52 0.22 3 0.57 0.23 F *** mu LSD(0.001) 0.09 0.02 Untreated 0.40 0.20 Orthogonal comparisons Ca vs Untreated ** *** L-77 vs Untreated sea *** X2-5309 vs Untreated *** *** Water vs Untreated NS NS T1 vs Untreated NS * T2 vs Untreated *** *** T3 vs Untreated *** see C-V-(t) 13.84 6.19 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 65 Table 8. Ca content of leaf and curd samples after treatment with 4.0 kg Ca/ha CaCl2 and Ca(NO3)2, 0.1% L-77 and X2-5309, and various number of applications in the second field experiment, 1990. Treatment Ca conc. (% dry weight) Leaf Curd Ca salt CaClz 1.31 0.26 Ca(NO3)2 1.28 0.26 F NS NS Adjuvant L-77 1.38 0.26 X2-5309 1.30 0.26 water 1.21 0.25 F *** NS LSD(0.001) 0.11 -- No. of applications 2 1.22 0.24 3 1.31 0.26 4 1.36 0.26 F *** * LSD (0.01, 0.05) 0.11 0.02 Untreated 1.14 0.22 Orthogonal comparisons Ca vs Untreated *** ** L-77 vs Untreated *** ** X2-5309 vs Untreated ** ** Water vs Untreated NS ** T2 vs Untreated * * T3 vs Untreated ** ** T4 vs Untreated *** 44* (IN-(%) 7.27 9.04 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 66 Table 9. The effects of treatment with 4 kg Ca/ha, 0.1% adjuvants, and various number of applications on cauliflower yield in the second field experiment, 1990. Treatment Yield (kg/plot) Ca salt oac12 15.8 Ca(NO3)2 15.7 F NS Adjuvant 0.1% L-77 16.1 0.1% X2-5309 15.7 Water 15.4 F NS No. of applications 2 15.6 3 15.7 4 16.0 F NS Untreated 15.5 Orthogonal comparison Ca vs Untreated NS C.V. (%) 10.2 NS,*,**,*** Nonsignificant or significant at 0.05, 0.01 or 0.001 level, respectively. 67 Fig. 1. The relationship of the number of tipburned leaves per plant to the Ca level of leaf tissue in 1990 field experiments. A: the first experiment, B: the second experiment. 68 C; be 5 one»: use. .0 .06. oo 00; 3..— OJ.— 3.- 3.6 3.6 00.0 03000.2 x 3.0..506 pl» .6 Comgocluz XGDdIG —.O «I; .< zuogd Jed some] 0.10qu )0 0N 69 Fig. 2. The relationship of the percent tipburned plants to the Ca level of leaf tissue in 1990 field experiments. A: the first experiment, B: the second experiment. 70 9; be 5 03mm: “:00. Co .26. so 3; a... b and 3.0 0v...- 3008.: x0560 Tun-don...» .0 :.o$.o...¢ . o xnndnuooso...» 4 #3 lg (%) ezuold pewnqdn to 0N 71 from 3.6 kg/ha Ca to 4 kg/ha in the second experiment, which may have accounted for part of the increased Ca level. However, the untreated plants were also lower in Ca in the first field experiment, which indicates that the environment was also a factor associated with relative Ca deficiency and leaf tipburn incidence in cauliflower. LITERATURE CITED Cox, E.F., J.M.T. Mckee, and A.S. Dearman. 1976. The effect of growth rate on tipburn occurrence in lettuce. J. Hort. Sci. 51:297-309. Maynard, D.N., D.C. Warner, and J.C. Howell. 1981. Cauliflower leaf tipburn: A calcium deficiency disorder. HortScience 16:193-195. Gruesbeck, R.V. 1990. Soil and foliar applications of calcium and molybdenum to improve broccoli yield and cauliflower quality. Thesis for the degree of M.S. Michigan State University, East Lansing, MI. Rosen, C.J., H.J. Buchite, and G.G. Ahlstrand. 1987. Cauliflower response to gypsum on a coarsetextured soil: Relationship between tipburn and leaf nutrient distribution. J. Plant Nutr. 10:1925-1934. 72 SUMMARY Previous research indicates that temporary localized deficiency of soluble Ca in rapidly growing tissues leads to leaf tipburn in cauliflower. Our results indicate that foliar applications of Ca plus adjuvants directly on the growing tissues increased Ca penetration through leaf surfaces and reduced tipburn. The following conclusions summarize our research: 1. Foliar application of Ca with adjuvants could significantly reduce leaf tipburn in cauliflower. There was no significant difference between CaCl2 and Ca(NO3)2 as Ca sources to increase Ca penetration and uptake. Adding adjuvants could improve Ca penetration. However, the effects of different adjuvants were significantly different. Flo M0 845 was the most effective adjuvant to enhance Ca penetration into cauliflower leaves, and should be used at 0.1% volume. Weekly foliar application of Ca with adjuvants starting 40 days after transplanting reduced leaf tipburn under field conditions. Three or more applications were required to effectively reduce tipburn. 73 ‘lllllllfilllllllli