[WUHHIWNHJIHIJUIW”WI“HIWHIWWWI 122 399 THS RSITY LIBRAR llllllllllllllllllllllllllllllllllllll’lll‘ lllllllll 3 1293 01405 740 This is to certify that the thesis entitled CHANGES IN BRIX AND ACID 0F FRUITS AND VEGETABLES AFTER POST HARVEST TREATMENT WITH TRIACDNTANOL AND 9-B-L(+) ADENOSINE presented by Lynn Ellis Prlchard has been accepted towards fulfillment of the requirements for ms . degree inHQLtjgulIMne 3%46; professor Date /C%?//gi 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution T LIBRARY Michigan State University PLACE IN RETURN BOX to move this chockom from your record. TO AVOID FINES return on or More data duo. DATE DUE DATE DUE DATE DUE usu loAn Minn-am Action/Equal Opportunity III-titular: * Warns-9.1 CHANGES IN BRIX AND ACID OF FRUITS AND VEGETABLES AFTER POST HARVEST TREATMENT WITH TRIACDNTANOL AND 9-B-L(+) ADENOSINE by Lynn Ellis Prichard A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1 989 ABSTRACT CHANGES IN BRIX AND ACID OF FRUITS AND VEGETABLES AFTER POST-HARVEST TREATMENT WITH TRIACDNTANOL AND 9-B-L(+) ADENOSINE By Lynn Ellis Prichard Fruits and vegetables were treated after harvest with dips or sprays of nanomolar concentrations of 1-triacontanol (TRIA) and 9- B-L(+) adenosine to determine the effect of these growth substances on the brix, total acidity, and the brix to acid ratio. 9-B-L(+) adenosine is the second messenger elicited by TRIA. Both chemicals decreased the acidity and the sugar/acid ratio of tomatoes. Both chemicals increased the brix of strawberries in one test, but high concentrations of 9-B-L(+) adenosine decreased the brix in a second test. In one of two tests with carrots, TRIA increased the brix. Sweet cherries responded to both chemicals. TRIA increased the brix of 'ldared' apples and decreased the acidity of 'Mutsu' apples. The stage of maturity at treatment, chemical concentration applied and length of storage influenced the response of the crop to TRIA and 9-B-L(+) adenosine. Several species showed no post- harvest response to treatment. ii ACKNOWLEDGEMENTS I would like to thank Dr. Stanley K. Flies for his ideas and encouragement throughout my thesis. His constant enthusiam helped refresh my own when l was discouraged. I would also like to thank my committee members, Dr. J. Preiss and Dr. R. Herner who gave me their time, and valuable discussions. I must particularly thank Ms. Violet Wert for her invaluable technical assistance, her generous donations of time, and her friendship. I would like to thank my parents and parents-in-Iaw, whose support helped me through the tough times. Finally i wish to thank my husband Mark, whose patience and moral support made it all possible, and whose love and understanding made it all worthwhile. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES . INTRODUCTION LITERATURE REVIEW Triacontanol . Adenosine . . Brix and acidity . MATERIALS AND METHODS Obtaining and preparing samples Determination of brix and total acidity . Statistical analysis RESULTS AND DISCUSSION General response Tomatoes . Apples . Carrots . Strawberries . Sweet cherries . . . . . . Summary of non-responsive crops. Effect of external factors on the response . Concentration and cultivar Stage of maturity Length of time in storage CONCLUSIONS . LITERATURE CITED . iv Page QCDQUICDQJ< 4.1. do NM-L-A—L—L—l—L-‘d—L —& memmNOG-hh) N N 000) «bio 10 LIST OF TABLES Cultivars and general experimental conditions for all crops tested. . Response of mature field grown 'Jetstar' tomatoes to 9-B-L(+) adenosine after a one min immersion and storage at 20°C. Response of mature 'Ohio 7870' tomatoes to high concentrations of TRIA after a one min immersion and storage at 30°C. Response of mature 'Ohio 7870' tomatoes to TRIA after a 30 min immersion and storage at 30°C. Response of 'ldared apples to TRIA after a 30 min immersion and storage at 30°C. Response of 'Mutsu' apples to TRIA and 9-B-L(+) adenosine after a 5 min immersion and storage at 30°C. Response of mature 'Allstars' strawberries to TRIA, TRIM and 9-B-L(+) adenosine after a one min immersion and storage at 20°C. Response of 'Schmidt' sweet cherries to TRIA and 9- B- -L(+) adenosine when sprayed to drip and stored at 20°C. . . Response of 2 cultivars of tomato to different concentrations of TRIM after a one min immersion and storage at 20°C. Response of 'Patio F‘ tomatoes at different stages of development to TRIA and 9-B-L(+) adenosine after a 30 min immersion and storage at 30°C. .13 .13 .14 .15 .16 .17 .18 .20 .23 11 12 13 Response of 'Ohio 7870' tomatoes in 2 stages of development to TRIA after a one min immersion and storage at 20°C. . . . . 24 Response of 'Earliglow’ strawberries to TRIA and 9-B-L(+) adenosine when given a spray to drip treatment at different stages of develop- ment and stored at 20°C. . . . . . 25 Response of 'Schmidt' sweet cherries at different stages of development to a spray treatment of TRIA or 9-B-L(+) adenosine and storage at 20°C. . . . . . . . 26 vi LIST OF FIGURES Race The change in total acidity in 2 tomato cultivars treated with different concentrations of 9-B-L(+) adenosine. An *, ** indicates a difference from control significant at the 5% or 1% level respectively. . . . . . . 22 Change in brix with time in 'Earliglow' strawberries sprayed with 1.0 ug/liter of 9-B-L(+) adenosine. The F value for the interaction of time X treatment was significant at the 5% level. . 29 Change in total acidity with time in 'Earliglow' strawberries sprayed with 1.0 ug/liter 9-B-L(+) adenosine. The F value for the interaction of time X treatment was significant at the 5% level. . 29 Change with time of % brix in 'Schmidt' sweet cherries after spraying with 1.0 ug/liter of 9-B-L(+) adenosine. The F value for the interaction of time X treatment was significant at the 5% level. 31 vii INTRODUCTION Triacontanol is a 30 carbon primary alcohol, first isolated from the cuticle of Lucerne leaves (Medicago sativa L.) by Chibnall in 1933 [3]. It has since been shown to have many plant growth regulatory effects, both in vitro and in vivo [14]. Foliar applications of micromolar concentrations of TRIA stimulated an increase in reducing sugars and total reducible nitrogen in both whole plants and cell free systems [10]. Tests with oranges (Citrus sinensis L.) showed that this increase in sugar was translated into higher brix/acid ratios in the fruit when entire trees were sprayed in the grove [23]. These increases are economically important to the citrus industry because the value and maturity of citrus fruit is partially determined by its brix/acid ratio. Brix, or % soluble solids, is used as a measurement of the sugar content since sugar is the main component. The objective of this project was to investigate the effects of treating fruits and vegetables after harvest with TRIA and its second messenger on the brix, total acidity, and brix/acid ratio. TRIM, the second messenger elicited by TRIA, had been identified as 9-B-L(+) adenosine (Ries, Wert, O'Leary and Nair, unpublished). If the brix and acid could be changed after harvest, it would be easier and more economical than applying a treatment prior to harvest. It was postulated that, as with other plant growth regulator induced responses, many factors would influence the response. The l 2 variables controlled were chemical concentration, temperature after treatment, stage of crop maturity at treatment, and the time between treatment and termination of the experiment. The experiments were conducted by obtaining samples of several species of marketable fruits and vegetables, and then treating them by immersion or spraying to drip with TRIA or 9—0- L(+) adenosine. They were stored at a constant temperature for a pre-determined length of time. After termination of the tests, the brix, (soluble solids), total acidity, and the brix/acid ratio were determined. LITERATURE REVIEW Research into increasing crop yields and crop quality is probably as old as humankind. It may have been a discussion on the relative quality of different apple cultivars that led Adam and Eve to take those fateful bites. In 1959, Crosby and Vlitos found long chain alcohols isolated from tobacco (Nicotiana tabacum L.) to have growth promoting effects on plants [4]. One of the more recently characterized of these long chain alcohols is 1-triacontanol, which has been found to have many effects on the growth and yield of plants [18]. The improvement of fruit quality through the use of chemicals has also received attention, particularly as it pertains to citrus fruits [13]. Arsenic containing compounds are widely used in grapefruit (Citrus paradisi Macfadyen) to lower acid and increase the brix/acid ratio [7]. Many other chemicals are now being tested for this purpose [20, 22, 23]. Iriacomannl The growth promoting effects of TRIA were first investigated when it was isolated from Maryland 'Mammoth' tobacco along with other long chain alcohols [4]. It was not found to be active. Small quantities of alfalfa (Medicago sativa L.) were found to be beneficial to several crops when the hay was sidedressed. This response was shown not to be due to increased nutrient availability. A crystalline substance was isolated from the alfalfa and was identified by mass 3 4 spectroscopy as TRIA. ln tests on rice seedlings grown in nutrient solution, applications of TRIA either to the leaves or in the nutrient solution increased the uptake of water and the accumulation of dry weight [18]. Similar increases in dry weight and also increases in soluble carbohydrates and soluble and insoluble nitrogen in plants grown in the dark were found by Bittenbender et. al. [2]. These responses without light were substantiated when it was found that rice seedlings within three hr of treatment with TRIA had increased their dry weight and Kjeldahl-N in the dark or the light, but that removing 002 from the atmosphere eliminated the response [15]. This dark response demonstrated that the action of TRIA was not dependant on photosynthesis. As more was learned about the action of TRIA, it was obvious that the response occurred very quickly. Whole rice seedlings showed a linear increase in dry weight, leaf area, concentration of reducing sugars, free amino acids, soluble protein and reduced nitrogen within 10 minutes of application of TRIA. Leaf disks treated for 1 min and then homoginized into cell free extracts showed a response within 2 hr [16]. The response could also be inhibited in several ways. Experiments with other long chain alcohols showed that the response of rice, maize and tomato plants to TRIA was inhibited by pre- or simultaneous treatment with other long chain alcohols, particularly octacosanol, which inhibited TRIA at concentrations as low as 2.4 x 10le [11]. Temperature, time of application, and the presence of morpholine or phthalates in the treatment can also influence the response of plants to TRIA [14]. The formulation in which TRIA is applied is also important. A 5 colloidal dispersion of TRIA was shown to be very effective at increasing the growth of seedlings [14]. This same colloidal dispersion was also shown to be effective in increasing the yield of many crops. The optimum concentration of 0.1-1.0 ug/liter resulted in average yield increases of 5-14 % [1]. TRIA has also been used successfully in tissue culture. It increased both cell number and fresh weight of haploid tobacco cell cultures [8]. It increased reducing sugars and total reducible nitrogen in cell free extracts, the response correlated with glucose levels present at the time of treatment [10]. Seed germination and early growth was adversely affected by TRIA [9]. E l . Several observations indicated that TRIA acted by eliciting a second messenger, particularly tests showing the speed with which TRIA increased the dry weight of rice seedlings [16]. In 1988, a water soluble substance called TRIM, which also increased the growth of rice seedlings, was isolated from the roots of rice plants treated with TRIA [17]. The active substance in this crude isolate was identified by IR, mass spectroscopy, melting point, nuclear magnetic resonance spectroscopy and circular dichroism to be 9-B- L(+) adenosine (Ries, Wert, O'Leary and Nair, unpublished). There have been many studies in animals on the action of adensosine as a hormone. lt regulates the actions of the heart in many ways, such as causing coronary vascular smooth muscle relaxation [19]. In all cases where its chiral structure has been determined, however, it is D(-) adenosine that is active in animals 6 and not L(+) adenosine. All L enantiomers of adenosine, AMP, ADP and others were found to be inactive as regulators of platelet function, while D(-) adenosine inhibited the aggregation of platelets [5]. ln research on the utilization of L(+) adenosine by mammals, it was found that nucleotide derivatives of L-ribo compounds went through the body unchanged, and were not acted upon by many nucleolytic enzymes [12]. This was confirmed by work which showed that L-ribonucleosides were not affected by bacterial nucleoside kinase and that deaminases were not effective on L-ribo compounds either [21]. As far as could be determined, no such studies were done on plants. The research with TRIM may be the first evidence that 9-B-L(+) adenosine was biologically active in plants (Ries et. al., unpublished). E . | . II The concentration of brix and total acidity in citrus fruit is an important measure of quality and maturity. Minimum standards defined by the Florida citrus code dictate when citrus can be legally sold, and one of these standards involves the brix/acid ratio [6]. Since the 1920's when lead arsenate was accidentally discovered to reduce acidity in citrus while being used as a pesticide to kill mediterranean fruit flies, lead arsenate and arsenical compounds in various forms have been widely used [22]. In 1957, it was found to significantly lower the acid content in 'Ruby Red' grapefruit at concentrations of between 475 mg/liter and 1.93 g/liter of spray. Since this effect was more pronounced as the season progressed, treated grapefruit could be legally harvested 7 earlier than untreated fruit [6]. Two years later Deszyck found that lead arsenate also increased the total sugars in 3 cultivars of grapefruit by increasing the non-reducing sugars by nearly 6%, accompanied by an increase in pH [7]. More recently, tests were conducted on arsanilic acid as a substitute for lead arsenate because of concerns about the safety of using inorganic arsenical compounds on food crops. In tests on grapefruit, arsanilic acid was found to increase the brix/acid ratio by decreasing the total acidity, in doses ranging 500 to 6000 mg/liter with 1000-1500 mg/liter being the most effective [23]. While arsenical compounds have improved the quality of grapefruit, they are not legal for use on oranges because they cause an extreme reduction in acidity [23]. For this reason, other plant growth regulators are now being investigated. In recent trials using foliar sprays before harvest on 'Hamlin' and 'Valencia’ oranges, TRIA reduced the total acidity by an averageof 17.5 %, increased the brix an average of 16.3 %, and increased the brix/acid ratio an average of 41.5 % using concentrations of .67 and 1.33 ppb. [23]. Other commercial plant growth regulators such as Citrus 10 and NF—10 are also being tested [20, 23]. The active compounds in these formulations are not known. MATERIALS AND METHODS DII" | . I All crops tested were marketable fruit and vegetables of standard commercial cultivars. General information about the crops and experimental conditions used are summarized (Table1). Table 1. Cultivars and general experimental conditions for all crops tested. Crop Cultivar or Sourcez Storage Sorting Type (°C) Criteria Apples ldared PFC 20 & 30 Color/Size Mutsu FR) 30 Color/Size Asparagus Viking H13 20 Size Blueberries Highbush l-FC 20 Color Carrots Regular Greenhouse 30 Size/Shape Mini Grocer 30 Size/Shape Sweet cherries Hedelfinger CES 20 Color Schmidt I-FC 20 Color Tart cherries Montmorency FR) 20 Color Grapes Concord FR) 20 Color Strawberries Allstars Local Farm 20 Color/Size Earliglow Local Farm 20 Color/Size Sugar beets Mono-hy-E4 MSU Farm 20 Size/Shape Tomatoes Jetstar SES 20 Color/Size Mountain Pride SES 20 Color/Size Patio F Greenhouse 30 Color/Size Ohio 7870 Greenhouse 20 & 30 Color/Size Z HRC (Horticulture Research Center), CES (Clarksville Experimental Station), SES (Southwestern Michigan Experimental Station) Strawberries (Frageria x ananassa Duch.) were sorted into blocks with an average of 6 strawberries per sample. Sweet cherries (Prunus avium L.) were sorted into blocks with an average of 9 cherries per sample. 'Heidelfinger' cherries were picked one day prior to use. Tart cherries (Prunus cerasus L.) were sorted into blocks with an average of 9 cherries per sample. Blueberries (Vaccinium corymbosum L.) were sorted into blocks with an average of 20 blueberries per sample. Grapes (Vitis Iambrusca L.) were broken into bunches, with an average of 10 grapes per bunch and 3 bunches per sample. Apples (Malus domesticus Borkh.) were stored at 0°C and warmed up no more than 6 hr before use. They were sorted into blocks with an average of 3 apples per sample. Sugar beets (Beta vulgaris L.) were placed in 5°C storage for 5-15 days prior to use. The beets were sorted into blocks with one beef or piece of beet per sample. Carrots (Daucus carota L.) were grown in 26 cm diameter by 28 cm deep clay pots and fertilized twice weekly. They were harvested on the day of the experiment. Mini-carrots had been chilled and stored prior to use. Carrots were sorted into blocks with an average of 3 carrots per sample. The field grown tomatoes (Lycopersicon esculentum Mill.) were picked at the MSU Southwestern Experimental Station the same day used. The greenhouse tomatoes, ('Patio F' and 'Ohio 7870'), were grown in the same size pots as the carrots and fertilized twice weekly. All tomatoes were sorted into blocks with an average of 2 tomatoes per sample. 10 Samples were treated either by spraying to drip with a hand sprayer or an immersion of a pre-determined length. Controls were always treated with distilled water. All strawberries, sour cherries, 'Schmidt' sweet cherries, blueberries, grapes, and carrots and greenhouse grown tomatoes were picked no more than 4 hr before use. DI 'I' II' |||l 'I'I Fruit were sorted into samples according to size and stage of ripeness. The samples were treated for 1-30 min with either TRIA, TRIM, or 9-B-L(+) adenosine. TRIA was applied as a colloidal dispersion [14], in concentrations ranging from .1-1000 ug/Iiter. Crops that were subject to dessication after treatment were stored in plastic bags sealed with twist ties. These included strawberries, cherries, grapes, blueberries and carrots. After treatment, samples were stored at the desired temperature for 24 hr, or the duration of the time course study. They were then homogenized in a blender, (Osterizer) for 30 sec and centrifuged, (IEC Centra - 7R) at 2800 RPM for 20-40 min, the time required to pellet the solids on the bottom of the tubes. The supernatant was decanted and frozen for subsequent analysis. To determine total dissolved solids, 4-5 drops of the supernatant were placed on a zeroed digital brix meter (Atago PR-I) and the values recorded. To determine total acidity, 5 ml of the supernatant were added to 95 ml distilled water that had been boiled and cooled to expel dissolved gasses. It was then titrated with .1N NaOH to a final pH of 8.1-8.2, and the total ml recorded. 11 All sugar beet, carrot, asparagus and cherry samples were ground with 1 mil/g of distilled water to facilitate homogenization. Apple samples were squeezed through a cheese cloth after homoginization to obtain clear juice. All sugar beet experiments were titrated with .01N NaOH since they were low in acid content. Incubation times were between 16 and 3200 min in time course experiments. SI I' I' I I . Randomized complete block designs were used for all single factor tests. In experiments with two or more factors, split-plot ’ designs were used with treatments as subplots. The number of blocks per experiment ranged from 3-6. The results of all tests were subjected to analysis of variance, including trend analysis when applicable. Mean separation was by Fisher's LSD test or by relevant F tests with single degrees of freedom. RESULTS AND DISCUSSION fieneraLresmnse Tomatoes Seventeen experiments were conducted with 5 different cultivars of tomatoes grown in either the greenhouse or the field, and 10 of them responded to treatment with TRIA and/or 9-B-L(+) adenosine. Two experiments with 'Smallfry' tomatoes and one experiment with 'Mountain Pride' showed no response to treatment (data not shown). The 20% higher brix and 44% higher acid concentration in controls of 'Smallfry' tomatoes compared to other cultivars studied may explain why they did not respond to treatment. The most common result was a decrease in total acidity and a corresponding increase in the brix/acid ratio using either TRIA or 9- B-L(+) adenosine (Table 2.3). The change in brix varied with the application of both chemicals. Tomatoes immersed in TRIA and stored at 30°C showed an 11.2% decrease in brix and a 16.6% decrease in acidity (Table 4). Regardless of a storage temperature of 20°C (Tables 2,9) or 30°C (Tables 3,4), the tomatoes responded to treatment with both chemicals. Seven experiments similar to the ones presented had no statistically significant differences in either the brix or total acidity (data not shown). There is no obvious explanation for this discrepancy. 12 Table 2. Response of mature field grown 'Jetstar' tomatoes to 9-8- L(+) adenosine after a one min immersion and storage at 20°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 4.67 3.16 1.66 L(+) adenosine 1 4.50 2.80 1.84 L(+) adenosine 10 4.65 2.73 2.02 LSD 5% NS .22 .17 LSD1% NS .32 .25 Table 3. Response of mature 'Ohio 7870' tomatoes to high concentrations of TRIA after a one min immersion and storage at 30°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 5.38 3.68 1.462 TRIA 1 5.43 3.40 1.60 TRIA 100 5.23 3.38 1.55 TRIA 1000 5.38 3.85 1.41 zF value for quadratic trend with concentration significant at the 5% level. Table 4. Response of mature 'Ohio 7870' tomatoes to TRIA after a 30 14 min immersion and storage at 30°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 6.45 4.15 1.56 TRIA 0.1 5.73 3.46 1.67 TRIA 1.0 6.30 3.85 1.64 TRIA 10.0 6.65 4.17 1.60 LSD 5% .63 .62 NS Apples Four experiments were conducted with 'ldared' apples, 3 of them treated for 30 min and one treated for one min. In one of these tests the brix was increased by TRIA treatment an average of 6.8% (Table 5). Two others showed similar increases with TRIA and 9-B-L(+) adenosine that were not statistically significant, but with an average increase in brix of 6.1% (data not shown). In the fourth experiment there was an average decrease in brix of 1.8% that was not statistically significant. Since the zero time controls were higher than either the controls or treatments, this increase in brix in three experiments can be attributed to slowing the decrease in brix normally associated with storage. 15 Table 5. Response of 'ldared' apples to TRIA after a 30 min immersion and storage at 30°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 12.882 5.36 2.41 TRIA .01 13.50 5.58 2.43 TRIA 1.0 13.90 I 5.93 2.36 TRIA 100.0 13.88 5.73 2.43 zF value for linear trend with concentration significant at the 5% level. Two experiments were conducted with 'Mutsu' apples, under similar conditions. In one test, treatment of the apples with TRIA decreased the total acidity by 6.9% (Table 6), but another test showed no response to either chemical (data not shown). This response is very different from that of the 'ldared' apples because there was no change in brix and the total acidity decreased. The condition of the apples at treatment may have affected the response since these 2 cultivars would be expected to ripen similarly. Both cultivars had been stored before use, but the 'ldared' apples showed more storage disorders such as bruises and fungal infections than the 'Mutsu' apples. 16 Table 6. Response of 'Mutsu' apples to TRIA and 9-3L(+) adenosine after a 5 min immersion and storage at 30°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 16.18 2.74 5.97 TRIA 1.0 16.07 2.552 6.45 L(+) adenosine 0.1 16.20 2.59 6.46 L(+) adenosine 10.0 16.30 2.78 5.93 zF value for difference from control significant at the 5% level. Carrots In a single test, mini-carrots showed a 7.2% decrease in brix when treated with 1 ug/liter of TRIA. The F value for the difference from control was significant at the 5% level. Two other experiments with carrots were conducted under similar conditions. One experiment showed a significant increase in weight loss for the treatments over the controls at the 5% level, although the magnitude of thechange was small, less than 1% of the total weight. In the other experiment there were no significant differences between treatments. Strawberries In an experiment conducted with 'Allstars' in 1988, there was a 7.3% decrease in brix and a 10.6% decrease in the brix/acid ratio after treatment with a high concentration (100 ug/liter) of 9-B-L(+) adenosine, and a 6.2% increase in the brix/acid 17 ratio with TRIA (Table 7). This is in contrast to an experiment conducted in 1989 with 'Earliglow' showing an increase in brix of 11.6% with 9-B-L(+) adenosine (Table 12). Since the fruit of both cultivars were fully ripe when tested and had approximately the same brix concentration, other factors may have influenced the response. Table 7. Response of mature 'Allstars' strawberries to TRIA, TRIM and 9-B-L(+) adenosine after a one min immersion and storage at 20°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 6.58 2.59 2.55 TRIA 10 ‘ 6.70 2.48 2.71 2 TRIM 20,000 6.55 2.56 2.57 L(+) adenosine 10 6.52 2.71 2.42 L(+) adenosine 100 6.102 2.69 _ 2.282 LSD 5% .58 NS .28 ZF value for the difference from average of all treatments significant at the 5% level Sweet Cherries All sweet cherry experiments were conducted with 'Schmidt' cherries except one, conducted with 'Hedelfinger', which Showed no response (data not shown). In the experiment with 'Schmidt' cherries in 1988, the brix and the brix/acid ratio 18 decreased when the samples were treated with 9-B-L(+) adenosine (Table 8). Experiments conducted in 1989 show a change in acid dependant on the stage of maturity of the fruit at treatment and are presented later (Table 13). Table 8. Response of 'Schmidt' sweet cherries to TRIA and 9-I3-L(+) adenosine when sprayed to drip and stored at 20°C. Concn Brix Acid Brix/acid Chemical (pg/liter) (%) (ml) ratio Control 0 7.80 1.02 7.95 TRIA 1.0 7.73 1 .03 7.77 TRIA 10.0 7.68 1 .00 8.03 L(+) adenosine 1.0 7.552 1 .05 7.492 L(+) adenosine 10.0 7.512 1.05 7.432 ZF value for the difference from control significant at the 5% level. Summary of non-responsive crops There were no statistically different responses in the brix, total acidity or the brix/acid ratio in experiments with any of the following crops: asparagus, sour cherries, grapes, blueberries and sugar beets. Both TRIA and 9-8- L(+) adenosine were applied by immersion of all of these crops. Two experiments were conducted with asparagus, both with a one min treatment of 1.0 ug/liter of TRIA. Two experiments were conducted with blueberries which were treated for one minute with 1.0 ug/liter TRIA, 1.0 mg/liter TRIM or 9-B-L(+) adenosine at 1.0 and 19 10.0 jig/liter . One experiment was conducted with 'Montmorency' sour cherries. They were treated with 10.0 ug/Iiter TRIA and 10.0 ug/liter 9-B-L(+) adenosine for 2 min. Two experiments with 'Concord' grapes were conducted using TRIA and 9-B-L(+) adenosine at 1.0 and 10.0 ug/liter, and TRIM at 1.0 mglliter. Three experiments with sugar beets were performed. The beets were treated with 1.0 ug/liter TRIA and 1.0 and 100.0 ug/liter of 9-8- L(+) adenosine. The difficulty in accurate sampling of beets may have contributed to the lack of significant results. W In addition to studying the response of different fruits and vegetables to TRIA and 9-B-L(+) adenosine under similar conditions, other factors such as concentration, stage of maturity, and length of storage were considered. Concentration and Cultivar The response of fruits and vegetables to different concentrations of TRIA and 9-B-L(+) adenosine was variable. In some experiments, there was no clear response to dose. In two tomato experiments using different cultivars, 9-B-L(+) adenosine applied in concentrations from .001 - 100 ug/Iiter decreased the total acidity, but not in a consistent pattern (Fig. 1). In other experiments there was a consistent response to concentration. In two tests with ’Ohio 7870' and 'Jetstar' tomatoes, a crude extract of the TRIA second messenger, (TRIM), at 1.0 mglliter was more effective than other 20 concentrations in increasing the brix (Table 9). The tests may have been complicated by other factors, such as chemicals penetrating the cuticle of the samples to a variable extent, or there may be no optimum concentration of chemical for the response, but rather a range of concentrations over which it is effective. Table 9. Response of 2 tomato cultivars to different concentrations of TRIM after a one min immersion and storage at 20°C. TRIM Brix Cultivar (mg/liter) (%) Statistical analysis 'Jetstar' 0 4.98 LSD 5% - .28 1 5.36 10 5.26 'Ohio 7870' 0 4.922 2F value for quadratic 1 5.15 trend with concentration 100 4.92 Significant at the 5% level. 21 Figure 1. The change in total acidity in 2 tomato cultivars treated with different concentrations of 9-B-L(+) adenosine. An *, ** indicates a difference from control significant at the 5% or 1% level respectively. 96 of Control acidity 22 —-9— “Ohio 7870' —0—- 'Patio F‘ .001 .01 .1 1 10 100 1000 Concentration of L(+) Adenosine (pg/liter) 23 Stage of Maturity The response of all crops to both chemicals was related to the maturity of the fruit at the time it was treated. The magnitude of the response to the chemical was correlated with the stage of maturity in studies with tomatoes. The less mature fruit was not as responsive to treatment with either TRIA or 9-8- L(+) adenosine (Table 10, 11). Table 10. Response of 'Patio F‘ tomatoes at different stages of development to TRIA and 9-B-L(+) adenosine after a 30 min immersion and storage at 30°C. Concn Acid Chemical (pg/liter) (ml) 100% Red 40-60% Red Control 0 3.94 3.74 TRIA .1 3.642 3.66 TRIA 1.0 3.80 3.68 L(+) Adenosine 1.0 3.53Y 3.66 L(+) Adenosine 10.0 3.44)! 3.67 2.)! F value for the difference from control significant at the 5% and 1% level respectively. 24 Table 11. Response of ‘Ohlo 7870' tomatoes at 2 stages of development to TRIA after a one min immersion and storage at 20°C. Concn Brix/acid ratio Chemical (pg/lite r) 100% Red 5-25% Red Control 0 1.49 1.49 TRIA 1 1.562 1.53 TRIA 100 1.47 1.46 zF value for difference from control significant at the 5% level. In strawberries, the altered response at different stages of maturity was more dramatic. Treatment with either chemical raised the brix, but with differing magnitudes at different stages of maturity (Table 12). TRIA increased the brix in the less ripe, orange strawberries, while 9-B-L(+) adenosine increased the brix in the more mature, red ripe strawberries. TRIA increased the sugar/acid ratio only at one stage of maturity. 25 Table 12. Response of 'Earliglow’ strawberries to TRIA and 9-B-L(+) adenosine when given a spray to drip treatment at different stages of development and stored at 20°C. Maturity Concentration Orange Red Dk. Red Orange Red Dk Red Chemical (pg/liter) Brix (%) Brix/acid ratio Control 0 7.5 7.4 7.3 .938 1.08 1.09 TRIA 1.00 8.3 7.8 7.6 1.04 1.07 1.07 L(+) adenosine 0.01 7.2 8.0 7.7 .923 1.13 1.21 L(+) adenosine 1.00 7.6 8.2 7.6 .981 1.14 1.11 L(+) adenosine 100.00 7.6 7.7 7.5 .958 1.07 1.19 Brix: LSD 5% for means within same maturity - .52 Brix/acid ratio: the interaction of TRIA X maturity is significant at the 5 % level An experiment with 'Schmidt' sweet cherries showed a decrease in the total acidity when the cherries were treated with either TRIA or 9-B—L(+) adenosine. Both chemicals lowered the total acidity, but the magnitude of the change was different depending on the stage of maturity of the fruit at treatment (Table 13). 26 Table 13. Response of 'Schmidt' sweet cherries at different stages of development to a spray treatment of TRIA or 9-B-L(+) adenosine and storage at 20°C. Maturity Concentration Purple Purple & Red Red Chemical (pg/liter) Acid (ml) Control 0 2.32 2.34 2.33 TRIA 1 2.19 2.25 2.19 L(+) adenosine 1 2.12 2.28 2.29 LSD 5% for the interaction of treatment X maturity: 0.14 Sweet cherries responded similarly to strawberries and tomatoes in that immature fruit treated with the chemicals did not show the same change in brix, total acidity, or the brix/acid ratio as mature fruit. This similar response for 3 different fruit species indicates that there is probably some basic ripening process common to most fruits affected by the chemicals. The process must change as the fruit matures, resulting in the observed differences in response at different stages of maturity. Length of time in storage In order to investigate how the response changed over time, experiments were conducted with both strawberries and sweet cherries, with the length of time in storage ranging from 16 min to 48 hr. The response varies over time and is 27 not the same for all fruits. In strawberries, the brix and total acidity decreased over time in the control fruits, but treatment with 9-B-L(+) adenosine slowed the rate of loss of both parameters (Fig. 2, 3). In 'Schmidt' cherries the brix increased more in the 9-B-L(+) adenosine treated fruit after 160 min , and showed no significant change thereafter (Fig. 4). In these tests, using 9-B-L(+) adenosine on strawberries resulted in long term increases in brix and total acidity over the controls, while in cherries the only significant increases in brix were over the short term. Figure 2. Figure 3. 28 Change in brix with time in 'Earliglow' strawberries sprayed with 1.0 ug/llter 9-l3-L(+) adenosine. The F value for the interaction of time X treatment was significant at the 5% level. Change in total acidity with time in 'Earliglow' strawberries sprayed with 1.0 ug/liter 9-B-L(+) adenosine. The F value for the interaction of time X treatment was significant at the 5% level. 96 BRIX ACID CONCENTRATION (ml of (MN) 29 7.6 . —I— Control —0— L(+) Adenosine l ‘ I f I ‘ 0 500 1000 1500 2000 TIME (min) 9.0 * 8.5 —l— Control —0— L(+) Adenosine I 6-0 T I ' l f l V 0 500 1000 1500 2000 TIME (min) Figure 4. 30 Change with time of % brix in 'Schmidt' sweet cherries after spraying with 1.0 pg/liter of 9-8-L(+) adenosine. The F value for the interaction of time X treatment was significant at the 5% level. % BRIX 31 —-|I'— Control ——0— L(+) adenosine U I I T 1000 2000 TIME (mln) V I 3000 IF 4000 CONCLUSIONS Both triacontanol and 9-B-L(+) adenosine applied after harveSt change the brix (soluble solids) concentration and the total acidity of several horticultural crops, however the results of these studies varied widely depending on the crop and the condition or quality of the samples. Factors which affected this response were species, stage of crop maturity at the time of treatment, chemical concentration applied, and length of time between treatment and termination of the experiment. The stage of maturity of the crop when the chemical was applied influenced the magnitude of the response. In some experiments, TRIA was more active at an earlier stage and 9-B-L(+) adenosine was more active at a later stage of maturity. This indicates that the two chemicals may have different modes of entry into the fruit, particularly since they vary widely in their water solubility. The response to concentration seems to be more complex than a standard dose response curve. Either there is no optimum concentration for achieving a response with most crops, or the tests were complicated by other factors, such as the chemicals passing through the cuticles in varying amounts. The optimum concentrations may not have been discovered, since ten-fold dilutions in concentration were used in all tests. 32 33 The time dependant effects of the chemicals, which occured when the fruits were treated and then stored, was the most complex response. In strawberries which lose soluble solids in storage over time, 9-B-L(+) adenosine slowed down the loss. In sweet cherries, where soluble solids increased during storage, the 9-B-L(+) adenosine increased the brix more compared to controls at the earliest sampling time. 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