ABSORPTION AND UTILIZATION OF FOLIAR-APPLIED NITROGEN IN PRUNUS SPP. Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DAVID RONALD LEECE I970 This is to certify that the thesis entitled Absorption and Utilization of Foliar-Applied Nitrogen in Prunus spp. presented by David Ronald Leece has been accepted towards fulfillment of the requirements for Ph 0 Do degree inHOI’ti culture Major professor Date AUEUSt 17, 1970 0-169 7.1" 1.- BlNDING BY , "BAG & SONS' ’ 590K BINDERY INC. .1 LIBRARY ”MOSES "II- -.- it“ .45" A ’I. 1"" M #13241wa .3”:- {.5 {m . dug-13. 1.». “it” mat.m.{ Jeremy \ ' - , to- x m gm cave L, Eu Jr *2; “,r ° '-»"'"’-"“ "$9., 'r- .or- cently < The pre as a mi | absorpt trate i would a positic Nitrate quenCe nitrate CutiCu] the pet Of You: ABSTRACT ABSORPTION AND UTILIZATION OF FOLIAR-APPLIED NITROGEN IN PRUNUS SPP. BY David Ronald Leece Foliar sprays containing nitrate-nitrogen have re- cently come into use in commercial stone fruit production. The present investigations evaluated nitrate foliar sprays as a nitrogen source for Prunus spp. and studied their absorption with a view to improving their efficiency. Although stone fruit trees normally metabolise ni- trate in their roots it was postulated that the leaves would also be able to metabolise nitrate and hence be in a position to benefit from nitrate foliar sprays if absorbed. Nitrate reductase, the first enzyme in the metabolic se- quence from nitrate to amino acids, was used to indicate nitrate metabolism, and, to avoid the complication of cuticular penetration, nitrate was supplied to the leaf via the petiole. A lSmM nitrate solution was applied to the roots of young, sand—cultured apricot, sour cherry, sweet cherry, peach the le reduct cept p active David Ronald Leece peach and plum trees. Nitrate was subsequently found in the leaves of all species and an NADH-dependent nitrate reductase was extracted from the leaves of all species ex- cept peach. The apricot enzyme was two to three times as active as the enzymes from the other species including that from an apple control. It was shown to be substrate (lOmM N03_) inducible and able to use either NADH or FMNH2 as its electron donor. Thus it would seem to be typical of nitrate reductase as commonly found in plant leaves. The enzyme extraction procedure was based on the use of insoluble polyvinylpyrrolidone (PVP.) in the extrac- tion medium to remove phenolic compounds which would other— wise have inactivated the enzyme. When nitrate reductase was extracted from oat in the presence of each of the stone fruit tissues, PVP. provided adequate protection in the presence of apricot and plum tissues, but only partial protection with sour cherry and sweet cherry, and no pro- tection with peach tissue. Hydrolyzable tannins not re— moved by PVP. were believed responsible for this inactiva- tion. Incorporation in the extraction medium of reducing agents and inhibitors of o-diphenoloxidase gig. cysteine, mercaptobenzothiazole, and dithiothreitol, at concentrations up to 10mM, gave no additional protection to the oat enzyme. Thus the failure to find nitrate reductase in peach tissue was ascribed to enzyme inactivation during extrac- tion by hydrolyzable tannins. grown a that fl nitrate foliar leaves showed ion to trees absorh David Ronald Leece Nitrate reductase was detected in leaves of field- grown apricot, sour cherry and plum trees. This indicated that field-grown leaves of Prunus spp. could metabolise nitrate and that they should be able to utilize nitrate foliar sprays, provided the nitrate ions could enter the leaves through the cuticle. Sand-culture evaluations of nitrate foliar sprays showed that K+ was likely to be the most effective carrier ion for nitrate foliar sprays when applied to stone fruit trees and that both the carrier ion and the nitrate were absorbed. Field evaluations of potassium nitrate foliar sprays were conducted on nitrogen deficient, young, commercial peach trees for two seasons. Neither three autumn (post harvest) nor three spring applications of KNO3 or a urea control at 0.2 to 0.4 9. equiv. N per litre raised leaf nitrogen significantly nor corrected tree nitrogen defi- ciency. However a soil nitrogen application (0.5 lb. N per tree post harvest) raised leaf nitrogen concentration, increased terminal growth and trunk circumference, and corrected the nitrogen deficiency. It was concluded that insufficient nitrogen or potassium were absorbed from the foliar sprays to be able to influence tree nitrogen or potassium status or growth. This was attributed to lack of cuticular penetration by KNO3 and urea. WOrking with excised leaf discs it was found that partial removal of epicuti M m3 .I using N no infl fruit I David Ronald Leece epicuticular wax doubled cuticular penetration by 0.4 M KN03. It was concluded that the commercial practice of using nitrate sprays at 0.07 9. equiv. per litre would have no influence on the nitrogen status or growth of stone fruit trees. ABSORPTION AND UTILIZATION OF FOLIAR—APPLIED NITROGEN IN PRUNUS SPP. By David Ronald Leece A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1970 G 6 7/577 ACKNOWLEDGMENTS Thanks are due to the members of my guidance committee Drs. A. L. Kenworthy (Chairman), H. C. Beeskow, M. J. Bukovac, and D. R. Dilley for guidance in the con- duct of this project and for reading the manuscript. Assistance with development of a nitrate reductase assay was given by Dr. S. K. Ries. Mr. H. Rapp, of Romeo, Midhigan, made his orchard available for the field experi- ments. This work was undertaken while the author was on leave from the New South Wales Department of Agriculture. ii TABLE OF CONTENTS. Page LIST OF TABLES . . . . . . . . . . . . . . . . . . Vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . ix INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . 4 Possible roles for nitrogen foliar sprays in the nitrogen nutrition of deciduous fruit trees . . . . . . . . . . . . . . . . . . . . 5 Fruit set . . . . . . . . . . . . . . . . . 7 Fruit and shoot growth . . . . . . . . . . 9 The site of nitrate reduction in fruit trees . . . . . . . . . . . . . . . . . . 10 The response of fruit trees to nitrogen foliar sprays . . . . . . . . . . . . . . . . . . . . l4 Urea sprays . . . . . . . . . . . . . . . . 14 Apple trees . . . . . . . . . . . . . . l4 Citrus spp. . . . . . . . . . . . . . 17 Prunus spp. . . . . . . . . . . . . . 18 Nitrogen sprays other than urea . . . . . . l9 Nitrate sprays . . . . . . . . . . . . 20 Ammonium sprays . . . . . . . . . . . . 22 Possible reasons for the anomalous response to nitrogen foliar sprays in Prunus spp. . . 22 STUDIES ON NITRATE METABOLISM IN LEAVES OF PRUNUS SPP. . . . . . . . . . . . . . . . . . 27 Materials and Methods . . . . . . . . . . . . 27 Plant material . . . . . . . . . . . . . 27 Sand culture of young trees and oats. 27 Mature tree culture . . . . . . . . . 29 Methods . . . . . . . . . . . . . . . . . 29 Root uptake studies . . . . . . . . . 29 Petiole uptake studies . . . . . . . 30 Enzyme extraction . . . . . . . . . 31 Assays . . . . . . . . . . . . . . . 31 Statistical analyses . . . . . . . . 34 iii Il'llll III I ' III: I: l I I ll SAND FOLI Page Results . . . . . . . . . . . . . . . . . . . . 34 Nitrate reductase in leaves of young trees. 34 FMNH2 as electron donor of nitrate reductase . . . . . . . . . . . 39 Extraction with Polyvinylpyrrolidone. . 47 Efficacy of enzyme extraction _ procedure . . . . . . . . . . . . . . 55 Protection of nitrate reductase during extraction from sweet cherry and peach leaves . . . . . . . . . . . . 55 Nitrate reductase in leaves of mature, field-grown trees . . . . . . . . . . . . 59 Discussion . . . . . . . . . . . . . . . . . . 68 Nitrate reductase in leaves of young trees . . . . . . . . . . . . . . . . . . 68 The extraction procedure . . . . . . . 71 Nitrate.reductase in leaves of mature, field-grown trees . . . . . . . . . . . . 77 SAND CULTURE AND FIELD EVALUATIONS OF NITRATE FOLIAR SPRAYS FOR PRUNUS SPP. . . . . . . . . . . 79 Materials and methods . . . . . . . . . . . . . 80 Sand culture experiments . . . . . . . . . 80 Plant material . . . . . . . . . . . . 80 Treatments and experimental design. . . 81 Leaf analyses and growth measurements . 82 Field experiments . . . . . . . . . . . . . 83 Treatments and experimental design . . 84 Leaf analyses and growth measurements . 85 Statistical analyses . . . . . . . . . . . 86 Results . . . . . . . . . . . . . . . . . . . 86 Sand culture experiments . . . . . . . . . 86 Evaluation of cations as carriers for nitrate foliar sprays . . . . . . 86 Absorption and translocation of potassium nitrate foliar sprays . . . 89 Field Experiments . . . . . . . . . . . . . 92 Experiment 1 . . . . . . . . . . . . . 92 Experiment 2 . . . . . . . . . . . . . 94 Discussion . . . . . . . . . . . . . . . . . . 98 Sand culture experiments . . . . . . . . . 98 Carrier ion experiment . . . . . . . 98 Absorption and translocation experiment . . . . . . . . . . . . . 103 Field Experiments . . . . . . . . . . . . . 105 iv Page LITERATURE CITED . . . . . . . . . . . . . . . . . 109 APPENDIX A—-Methods of expressing concentration . . 119 APPENDIX B--A technique for studying nitrate penetration through intact leaf cuticles in Prunus spp. . . . . . . . . . . . . 122 LIST OF TABLES Page Nitrate reductase activity and nitrate concentration of leaves of young apple and stone fruit trees . . . . . . . . . . . . . . 35 Effect of 15mM nitrate nutrient solution on nitrate reductase activity and nitrate concentration of leaves of young apricot trees . . . . . . . . . . . . . . . . . . . . 38 Effect of 15mM nitrate nutrient solution on nitrate reductase activity and nitrate concentration of leaves of young apple trees. 40 Nitrate reductase activity of apricot and oats leaf tissue when assayed using either FMNH2 or NADH as electron donor . . . . . . . . . . 47 Nitrate reductase activity of oats leaf extracts prepared in the presence of apple and stone fruit leaf tissue compared on a percentage basis with the activity of an extract prepared in the absence of added tissue . . . . . . . . . . . . . . . . . . . 56 Effect of increasing amounts of hydrated insoluble polyvinylpyrrolidone (Polyclar A.T.) on the extraction of nitrate reductase from 0.5 g. oats leaf tissue in the presence of 0.5 g. peach leaf tissue . . . . . . . . . 60 Effect of mercaptobenzothiazole in the extrac- tion medium on the extraction of nitrate reductase from 0.5 g. oats leaf tissue in the presence of either 0.5 g. sweet cherry or 0.5 g. peach leaf tissue . . . . . . . . . . 60 Effect of dithiothreitol in the extraction medium on the extraction of nitrate reduc- tase from 0.5 g. oats leaf tissue in the presence of 0.5 g. peach leaf tissue . . . . 61 vi 12. 13. 14. 15. If). 17. 18. 10. 11. 12. 13. 14. 15. 16. 17. 18. Page Nitrate reductase activity and nitrate concentration of mid-shoot leaves of mature, field-grown stone fruit trees . . . . 67 Effect of nitrogen foliar sprays at 0.2 9. equiv. per litre on leaf injury and leaf nutrient concentration as affected by carrier form. A. Sweet cherry trees . . . . . . . . 87 Effect of nitrogen foliar sprays at 0.2 9. equiv. per litre on leaf injury and leaf nutrient concentration as affected by carrier form. B. Peach trees. . . . . . . . . . . . 88 Effect of 4 per cent potassium nitrate foliar sprays on apricot leaf nitrate reductase activity and leaf injury. . . . . . . . . . . 90 Effect of 4 per cent potassium nitrate foliar sprays on growth and nutrient content of apricot tree terminal shoots . . . . . . . . 91 Effect of certain soil and foliar applications of nitrogen on leaf nitrogen and potassium concentrations of commercial peach trees (1968-69 season). . . . . . . . . . . . . . . 93 Effect of certain soil and foliar applications of nitrogen on growth of commercial peach trees (1968-69 season). . . . . . . . . . . . 97 Effect of certain soil and foliar applications of nitrogen on leaf nitrogen and potassium concentrations and terminal shoot growth of commercial peach trees (1969-70 season) . . . 99 Approximate osmotic concentrations of foliar sprays used in the cation evaluation experiment . . . . . . . . . . . . . . . . . 102 Table for conversion from pounds per 100 Imperial or United States gallons to per cent, for the concentration range common in nutritional sprays . . . . . . . . . . . . 120 vii I‘ll-I'll IIIIIITIIII I111 I' III Page 19. Relationships among the various methods of expressing concentration for the compounds studied in this thesis, when equated on a nitrogen basis to a 0.6 per cent urea spray . . . . . . . . . . . . . . . 121 20. Induction of nitrate reductase in discs from field-grown apricot leaves, following cuticular penetration by 0.4 M potassium nitrate, as influenced by the partial removal of epicuticular wax . . . . . . . . . 125 viii LIST OF FIGURES Page Relationship between nitrate reductase activity and nitrate concentration in leaves from young apricot trees . . . . . . . 36 Time-course for the induction of nitrate reductase in leaves from young apricot trees . . . . . . . . . . . . . . . . . . . . 41 Effect of substrate concentration on induction of nitrate reductase in leaves from young apricot trees 0 O O O C C C C C O O O O C O O 43 Effect of substrate concentration on induction of nitrate reductase in leaves from young apple trees differing in initial level of enzyme activity . . . . . . . . . . . . . . . 45 Effect of increasing amounts of hydrated insoluble polyvinylpyrrolidone (Polyclar A.T.) on the extraction of nitrate reductase from apricot leaf tissue . . . . . . . . . . 48 Effect of increasing amounts of hydrated insoluble polyvinylpyrrolidone (Polyclar A.T.) on the extraction of nitrate reductase from oats leaf tissue . . . . . . . . . . . . 51 Effect of increasing amounts of hydrated insoluble polyvinylpyrrolidone (Polyclar A.T.) on the extraction of soluble protein from apricot and oats leaf tissue . . . . . . 53 Effect of increasing amounts of hydrated insoluble polyvinylpyrrolidone (Polyclar A.T.) on the extraction of nitrate reductase from 0.5 g. oats leaf tissue in the presence of 0.5 9. sweet cherry leaf tissue . . . . . 57 ix 9. Time-course for the induction of nitrate reductase in leaves from mature, field- grown, apricot trees . . . . . . . . . . 10. Time-course for the induction of nitrate reductase in leaves from mature, field- grown, sour cherry trees . . . . . . . . ll. Differential response of leaf nitrogen concentration to autumn and spring foliar sprays of 1.8 per cent potassium nitrate at different soil nitrogen levels (gig. 0 and 0.5 pounds nitrogen per tree) . . . . Page . . 62 . . 64 . . 95 —— —. —-— —. POL in roc On Ca as INTRODUCTI ON Nitrogen is the principal nutrient required by fruit trees, Which, in commercial practice, has to be supplied as a fertilizer (Johnston and Larson 1965). As sudh it constitutes an important cost factor in fruit pro- duction and thus warrants investigations leading to its more efficient use. SuCh investigations have received impetus recently from the rising public concern over fer- tilizer contamination of water supplies, particularly by nitrate ions which can be toxic to both animals (Lewis 1951) and humans (Altman and Dittmer 1968). Commercially, nitrogen is normally supplied to fruit trees as a soil fertilizer application. Up to one pound of elemental nitrogen is given to eadh mature tree in autumn or winter (Johnston and Larson 1965) to build up root and rootstock storage reserves which the tree relies on for spring growth (Taylor 1967). A supplemental appli- cation of up to half a pound may be applied in spring to assist fruit development (Keatly §E_§l, 1968). Soil appli- cations of nitrogen are subject to losses due to leaching (principally as nitrate ions), soil fixation, denitrifica- tion, and to bad placement in relation to tree roots. These losses, particularly those due to leaching, have cont: watei turai gen plet of n contributed to the build up of nitrate ions in the ground water (Tisdale and Nelson 1966) and subsequently in agricul- tural and domestic water supplies. One approach to improving the efficiency of nitro- gen fertilizer applications has been to partially or com- pletely replace soil applications with foliar applications of nitrogen in the form of urea sprays. This has proved successful for apple trees (Fisher 1952) and Citrus spp. (Jones and Embleton 1965) but not for stone fruit (Prunus spp.) or pear trees (Benson 1953). Foliar applications of urea in spring to apple trees have permitted more efficient regulation of tree nitrogen supplies, than have correspond- ing soil applications. As a result problems of insufficient nitrogen for fruit development or of excess nitrogen as the fruit approaches maturity have been more easily avoided (Fisher 1952). Other advantages of these foliar sprays have been that trees low in vigor due to low temperature (winter) injury or root damage have responded more rapidly to them than to soil applications (Benson 1953), and the losses associated with soil applications have been avoided. However, there are some disadvantages associated with foliar sprays. The natural way for fruit trees to absorb nutrients is via their roots not their leaves. Leaf absorption:hasometimes minimal, particularly as the leaf ages (Wittwer and Teubner 1959). Losses may occur during application through excess spray drifting to adjac subse trate fert. may ' thes Ipri beir Sou‘ are. tflia adjacent plantings or dripping to the ground (it may subsequently be absorbed by feeder roots which are concen- trated at the periphery of trees - an example of good soil fertilizer placement), and rainfall soon after application may wash or leach spray material from the leaves (again these losses would be well placed for root absorption). Recently commercial interests have marketed nitro- gen foliar sprays containing urea and nitrate-nitrogen (principally as potassium nitrate), and these are now being used in commercial fruit production in Michigan, South Africa (Bester §t_al, 1965) and other fruit growing areas. Growers and industry representatives have claimed that potassium nitrate sprays aid recovery of sweet cherry, peach, and plum trees from potassium deficiency and winter injury (Kenworthy 1965). It is well established that urea has little value as a nitrogen foliar spray for Prunus spp., but no field evaluations of nitrate sprays have been reported. The investigations now reported were carried out to evaluate nitrate foliar sprays as a nitrogen source for Prunus spp. and to study their absorption with a view to improving their efficiency. REVIEW OF LITERATURE Nitrogenous fertilizer was first applied to fruit trees in the form of a foliage spray when Hamilton 23 31, (1943) sprayed apple, cherry and peach trees in the spring with solutions of urea, sodium and potassium nitrate, and ammonium sulphate at concentrations from 0.6 to 1.2 per cent.1 The only favorable response obtained was an increase in apple tree nitrogen resulting from the urea sprays. A great many field evaluations of nitrogen foliar sprays for all commercially important fruit tree species rapidly followed. It soon became apparent that urea was the best form of nitrogen for foliar sprays, to Which apple and cit- rus trees responded very favorably, While stone fruit trees failed to show any response. In this review the nitrogen nutrition of fruit trees will first be examined to see at what points in the annual rhythm of root absorption, storage and mobilization of nitrogen, foliar nitrogen sprays might prove beneficial. 1In this thesis, in all reports of field evalua- tions of foliar sprays, concentration has been expressed on a per cent basis. Appendix A gives a more complete explanation. Next tion frui rhyi fru hit; and thi sto the bar COP S'wg tis tr; 0n C>f tr- Next the results of the many field and greenhouse evalua- tions of nitrogen foliar sprays will be studied and finally the possible reasons for the anomalous behavior of stone fruit trees will be considered. Possible Roles for Nitrogen Foliar Spraysgin the Nitrogen Nutrition of Deciduous Fruit Trees Taylor (1967a) has recently reviewed the annual rhythm of storage and mobilization of nitrogen in deciduous fruit trees. The cycle commences with the creation of nitrogen storage reserves in woody tissues during autumn and winter. The nitrogen absorbed by the fine roots at this time is stored principally in the larger roots or root- stock wood and from 20 to 80 per cent of the nitrogen in the leaves migrates to woody storage tissue, mainly the bark of scion wood, in autumn prior to leaf fall. The mobilization phase commences at bud swell and continues until growth ceases in mid-summer. From bud swell to flowering protein hydrolysis occurs in the storage tissue of older shoots and branches and soluble nitrogen (principally aspartic acid, asparagine and glutamine) is translocated from these tissues to the developing meristems. Once the air temperature rises to 40° to 45° F mobilization of root and rootstock reserves commences followed by its translocation to the shoots. These root and rootstock reserves quickly replace the reserves of the older shoots half main absc to t rese oped nit: muct as the main source of nitrogen for the developing meristems. Next, prior to the abscission of the flowers which have failed to develop (only a small portion of the total number of flowers per tree develop into fruit), one-third to one- half of their nitrogen is translocated back into the shoots mainly in the form of free amino acids. Finally nitrogen absorbed by the roots during the spring may be translocated to the shoots to supplement that available from the storage reserves. This is more apparent in trees with poorly devel- oped reserves than in trees that have been well supplied with nitrogen. Under conditions of adequate nitrogen supply, much of the nitrogen absorbed by the roots during the spring appears to be used to replenish depleted reserves rather than be directly involved in growth. Three aspects of the annual nitrogen rhythm in deciduous fruit trees will now be considered in detail in relation to the use of foliar sprays, namely fruit set in relation to summer, autumn and spring sprays; fruit and shoot growth in relation to spring sprays; and the site of nitrate reduction in fruit trees in relation to nitrate sprays. A fourth aspect, whether soil applications which are essential to the build up of root and rootstock nitro- gen reserves, can be partly or wholly replaced by foliar- applied nitrogen, will be considered in the review of tree response to foliar-applied nitrogen. I‘CI ra at- Fruit Set The role of nitrogen in fruit set (i;g;_in the growth of young fruit immediately after fertilization-— growth which occurs by cell division only) is well estab- lished in the literature (Taylor 1969). Nitrogen is essen- tial to the cell division process in deciduous fruit and this nitrogen comes from the reserves accumulated in the woody storage tissue the previous season, especially in the autumn. As examples of the influence of nitrogen on cell division Reeve and Neufeld (1959) found with the Elberta peach that high nitrogen fruit of a given size had more but smaller parenchyma cells than low nitrogen fruit and Hosoi and Ishida (1964) found that cell division was mark- edly reduced in nitrogen-deficient pear fruit. That the source of this nitrogen for fruit set is the shoot and root storage reserves created the previous season was well demon- strated by Hill-Cottingham and Williams (1967). Nitrogen- ous fertilizer was applied to apple trees only during spring or summer or autumn. The following spring only summer and autumn treated flowers had ovules that remained viable for six days after anthesis; this was the minimum period found necessary for the pollen tubes to effect fertilization. An earlier experiment (Hill-Cottingham 1963) had shown that fruit set on young apple trees was dependent on the level of nitrogen supplied to the root- stocks in the previous year. One possible role for foliar applications of nitro- gen then is to increase the amount of nitrogen available to the fruit during fruit set. As indicated above this nitro- gen would normally come from shoot storage tissue and as these reserves are depleted it is drawn from root and root- stock reserves. Spray applications during and just after bloom (two pink, a calyx, and a first cover spray) have been advocated by Fisher and co-workers (Fisher gt_§l. 1948; Fisher and Cook 1950; Fisher 1952) to supplement tree re- serves during fruit set. Difficulties with spray applica- tions at this stage were that there was very little leaf area available for absorption and spray concentrations had to be kept low to avoid injury to buds and young leaves. Oland (1960) recommended one or more nitrogen foliar sprays be applied between fruit harvest and leaf fall, to build up the nitrogen stored in both shoots and buds in the autumn for the following spring. He found sprays could be applied in autumn at seven to eight times the concentration possible in spring. However great varia- tions, from 20 to 80 per cent (Taylor 1967a), may occur in the amount of nitrogen which migrates from the leaves back into the shoot prior to leaf abscission. The foliage of a mature apple tree may contain up to one-half of the nitrogen content of the tree at the end of the growing season (Murneek 1942), and translocation from leaves to woody shoots commences three to four weeks prior to abscis of the windy be cor post-l leaf 2 post; ture appli durir avai tku abscission (Oland 1963b). 0n the average about 40 per cent of the nitrogen is reabsorbed (Murneek 1930), but under windy conditions causing premature leaf abscission this may be considerably reduced (Oland 1963b, c). In one study post-harvest sprays increased the incidence of premature leaf abscission (Delver 1966). Another disadvantage of post-harvest applications, rarely referred to in the litera- ture (2434 Murneek 1930), is that much of the nitrogen applied as a spray and subsequently stored may be lost during pruning. In a third approach to increasing the nitrogen available for fruit set in apple trees, Williams and Rennison (1963) advocated summer foliar applications made once shoot growth had ceased. An added advantage of such sprays might be the forcing of undifferentiated buds to differentiate into flower rather than leaf buds where this might be desirable (943;; when wishing to force a young tree into production). They suggested two possible dis- advantages of the method, gig. that if applied too early the shoots may be forced into new growth subject to winter injury and more importantly that fruit maturity may be delayed. Fruit and Shoot Growth Boynton and Oberly (1966) have explained the effect of nitrogen on fruit growth as follows. Where nitrogen 10 has caused bloom and set to increase in greater proportion than vegetative growth and leaf efficiency, nitrogen fer- tilization may cause a decrease in average fruit size at harvest, but Where there is no great increase in bloom or set or where the increase in vegetation and leaf efficiency more than compensate for the increase in bloom and set, there may be a marked positive effect of nitrogenous fer- tilizer on fruit size. Thus in seasons of heavy fruit set the nitrogen reserves of a tree may be inadequate to provide sufficient vegetative growth to ensure sizing of the crop. If the additional fertilizer necessary is applied as a foliar spray it should be available to the meristems more rapidly and its effects should be of less duration (thus reducing the danger of delaying fruit maturity) than it would if applied to the soil (Fisher 1952). The use of nitrogen foliar sprays in conjunction with the cover sprays in late spring and early summer has been the only approach to foliar spraying with nitrogen widely accepted commercially. The Site of Nitrate Reduction in Fruit Trees It is generally accepted that the site of nitrate reduction in deciduous fruit trees is mainly in the roots (Nightingale 1937, Bollard 1953a), which immediately raises the question whether fruit trees would be able to metabolise and assimilate in their leaves foliar sprays 11 containing nitrate ions. However a detailed examination of the evidence strongly suggests that they would. Thomas (1927a, b) assayed for nitrate and nitrite in various parts of a mature apple tree through an annual cycle and While finding nitrate ions principally in fine roots also found them in leaf buds as they were opening. Eckerson (1931) determined the nitrate reductase activity of various parts of a mature Stark apple tree at weekly intervals for one year. She found high nitrate reductase activity during autumn and winter in the fine roots and during spring in both the fine roots and the buds and adjacent bark. Nitrate ions were always found in buds at bud swell but not in the leaves when they opened. However there was a very low but detectable level of nitrate reduc— tase in the leaves throughout their cycle except for three weeks in late spring. Activity in the leaves reached a peak at the end of June and again at the end of July. She also found that, although apple trees do not ordinarly con- tain nitrate, two trees which had received heavy nitrate applications shortly after the flowers had opened contained nitrate ions in leaves at the five foot level after three days. Within a week all the nitrate had disappeared and no more was found in the leaves that season. Stuart (1932) reported similar results. When sodium nitrate was supplied in extremely high amounts to the roots of small apple trees, nitrate ions appeared in the leaves (0.037 per cent on a dry weight basis). 12 In summary this early evidence indicated that in apple trees reduction of nitrate and synthesis of amino acids takes place mainly in the roots, however nitrate ions are translocated, unreduced, to the buds during bud burst or to the leaves in the exceptional case of excess nitrate application. The nitrate reduction data together with the rapid disappearance of nitrate from the apple leaves was evidence of the ability of apple leaves to metabolise ni— trate. This latter point was not conclusive though, for as Beevers and Hageman (1969) have pointed out, the nitrate reduction data may have represented non-enzymatic conver- sion of nitrate to nitrite in Eckerson's (1931) study, because even boiled plant extracts effected the conversion. More recent research has however confirmed the early evidence. Bollard (1953a, b, 1957a) failed to find any nitrate ions in tracheal sap extracted from one—year— old shoots of mature apple trees at any time during the annual rhythm. This also held true for other commercial stone and pome fruit trees (Bollard 1957b). Subsequent work on the nitrogen constituents of, and storage and mobiliza- tion of nitrogen in apple (Oland 1959) and peach trees (Taylor 1967b; Taylor and May 1967; May and Taylor 1967; Taylor and van den Ende 1969) confirmed the absence of nitrate ions in scion tissues under normal conditions. In confirmation of root reduction Grasmanis and Nicholas (1967) extracted an active nitrate reductase from apple roots but 13 were unable to determine its physiological electron donor, then Klepper and Hageman (1969) extracted from apple roots a typical NADHl-dependent nitrate reductase using insoluble polyvinylpyrrolidone in the extraction medium. Klepper and Hageman (1969) also added excess ni- trate ions to the root medium of apple seedlings and sub- sequently found nitrate ions and both nitrate and nitrite reductase enzymes in the leaves and nitrate ions and nitrate reductase in the stems and petioles. They found trace amountsmm mcwmmmuocw mo mHmom oalo C so pmumu hancHx o mm.o m mo.a m moo.o m 4m.o pm Hm.H e . . . . . «+62 pm mm.o n ¢~.~ m «40.0 pm mo.H am mo.~ a . . . . . ~+mo pm mm.o m no.a a mam.o pm Ho.H Am Ho.~ m . . . . . .+mz no mm.o m om.o m mHo.o o oo.~ m «m.H H . . . . . . +x n hm.o m «N.H m eao.o n Hm.a pm em.H m . . . . . vmz "mhmumm mwmnuaz w m~.o m mo.H m oao.o n nm.a n Hm.m m . . mmnmm mmus up H¢.o m om.H m nHo.o a mm.H m >R.H o . . nmmmummcn umHOHuCOU lav Axe as Axe xx. oz mu mz M z MuzncH Show wannmu cowumnucmocoo ucmwuusc mmmq .mmmuu hunmno ummBm .¢ .NEuom nmwuumo an pmuommmm mm hCOAHMHuchcoo ucmwuusc mama paw thsncfl mama so muuwa mom .>Hdvm .m m.o um mmmumm Hmeom cmmouuwc mo uommmmll.0H magma 88 .Amo.ouvmv Maucmoflmacmflm Hmmwflp muouuma mxflacz ha po3oHHom CESHOU Sumo CH mammzu .mwmmn usufim3 who m co commoumxo cowumuucmocooh .huflum>om mcflmmouocfl mo mamom OHIO C so pmpmn huoncHx n ma.a mHo.o m mm.H m sm.m m . . . . . . . m+mo m HH.H NvH.o m mm.H m mm.m m . . . . . . . +mz gm om.a sao.o m mo.m m om.m o . . . . . . . +z nu m~.H mao.o m m>.H m mo.m o . . . . . . . +¢mz ”mmmnmm mumuuflz m cm.a Hao.o m mh.a m sa.m o . . . . mmumm mmus am om.H oHo.o m mm.H m H~.m o . . . . commummcs "maouucou $3 Axe Axc 33 mo mz M z hasncH Ehom nowunmo coflumuucmocoo ucmfluudc mmmq o mwmhu. 3.060% .m .NEHom umfluumo an omuowmmm mm wcowpmuucmocoo ucmwuusc mama pom xwunmcfl mmoa co ouuwa mom .>H:Uo .m m.o um mwmumm unwaom zmmouuflc mo uommmmnl.aa manna 89 some marginal necrosis. The slight injury caused by K+, 4+ and the urea spray control on sweet cherry leaves was NH principally a tip necrosis which appeared to be caused by external spray residues being higher at the leaf tip. The nitrate sprays, regardless of carrier ion, failed to significantly increase the leaf nitrogen concen- tration of either the peach or sweet cherry trees (Tables 10 and 11). In contrast, the urea spray control increased sweet cherry leaf nitrogen concentration. However the carrier ions tended to raise the con- centration of their respective elements in the sprayed leaves. In sweet cherry, leaf potassium, sodium, calcium, +2 2 and magnesium were increased by K+, Na+, Ca , and Mg+ sprays respectively, although the Mg+2 spray did not in- crease leaf magnesium significantly above the unsprayed con- trol value. Also, apparent urea x magnesium and potassium x magnesium antagonisms were found, both the urea and K+ sprays decreasing leaf magnesium. In peach, the Na+ spray increased leaf sodium and the Ca+2 spray tended to increase leaf calcium (though not significantly above the unsprayed control value), however the K+ spray did not increase leaf potassium. Absorption and Translocation of Potassium Nitrate Foliar Sprays.--When 4 per cent KNO3 foliar sprays were applied at weekly intervals for 3 weeks to the leaves of l-year—old apricot trees in the greenhouse during the early 90 period of spring growth, leaf nitrate reductase activity was increased within 24 hours (Table 12). This indicates that nitrate absorption and initial metabolism occurred in the leaves. However the final spray application caused extensive tip and marginal leaf necrosis and reduced leaf photosynthetic area by 22: 50 per cent. Table 12.--Effect of 4 per cent potassium nitrate foliar sprays on apricot leaf nitrate reductase activity and leaf injury.X —7 Nitrate reductase activity Treatmenty Day 2 Day 9 Day 16 Injuryz (mu, moles N02 forTed, g. fr. wt. '1, hr. Untreated control . . . 275 a 467 a 209 a 0 Soil nitrate control . . 292 a 1271 b 457 ab 0 Foliar nitrate . . . . . 450 b 1300 b 975 b 5 XMeans in each column followed by unlike letters differ significantly (P< 0.05). YTreatments were applied on Days 1, 8, and 15. zInjury rated on a 0—10 scale of increasing severity Which occurred within 24 hours subsequent to the last spray application. Terminal shoot growth made in the 3 weeks subsequent to the final spray application (Table 13) was stunted by the foliar nitrate treatments but the associated reductions in dry weight and shoot potassium content were not signifi- cant. Both the soil and foliar nitrate treatments produced a higher terminal shoot nitrogen concentration than the 91 .Amo.o vmv hHquoHHHCmHm “CHMHU mnmuuwH oMHHCC >9 Um3oHHom CECHOU nomw CH .mCmmxscfl .CoHumoHHmmm wmnmm HMCHH may on uCosvomQCm mxwm3 omnflu may CH ome Cn3ouwx m omo.o m hm.m m mmo.o Q NH.N m No.m Q ¢.n .mumuuHC HmHHom m mCH.o m mm.m Q OOH.o Q hm.m m mm.v m n.~m . . . . HouuCoo oumnuHC HHom m me.o m oo.¢ w m¢o.o m mN.H m om.m m v.0H . . . . HouuCoo poummHuCD :3 xx; :3 CE :3 EB HCmuCoo CoHumuquoCoo HCmHCoo CoHumuquoCoo HCWMM3 SumCmq HCmEummuB ECHmmmuom Comouqu .b >.muoonm HMCHEHCH own» HOUHHmm mo quuCoo quHHuCC UCm xC¥3onm Co mhmumw HMHHom oumuuHC ECHmmmuom quU Com C mo uommmmnu.MH mHQme 92 untreated control but only the soil nitrate treatment in- creased shoot nitrogen content. Thus, no evidence of nitrogen or potassium translocation from foliar nitrate treated leaves to their subtended terminal shoots was found. Field Experiments Experiment l.--In the 1968-69 season the influence of an autumn soil nitrogen application on the nitrogen status of moderately nitrogen deficient commercial peach trees was compared with that of autumn and spring foliar sprays of KNO3. An October soil application of 0.5 lb. nitrogen per tree (as 1.5 lb. of NH4NO3) increased the nitrogen concentration of both young spring leaves and mature leaves in mid-summer (Table 14). On the basis of Kenworthy's (1950) standards for peach trees in Michigan, this leaf nitrogen increase represented a Change from a nitrogen shortage to a normal leaf nitrogen level. The soil nitrogen treatment also reduced the mid-summer leaf potassium concentration, however the concentration remained "normal". Three post-harvest sprays of 1.8 per cent KNO3 increased both the nitrogen and potassium concentrations of the Sprayed leaves prior to leaf fall (Table 14) but had no effect on either the leaf nitrogen or potassium concentration of the following season's growth. There was, 93 .Amo.o vmv hHquonHCmHm HmmmHU mumuumH mxHHCC an meoHHom CECHoo nomm CH umm quEumeu Sumo Com mCmmE Howmmm CHMZx m mb.H m mm.m . . . . . popmmuuCD m mb.H m o¢.m . . . . . . pmumous mCHummlCmmouuHC umHHom m m>.H m vH.N NC.H m mm.m m @H.m Q «H.N . . . . . ooummuucb m mm.H m mo.m hm.H m mm.m m o¢.m m mm.m . . . . . . pmummue CECqu I ComonuHC HMHHom Q mm.H m mm.m oo.H Q mm.m Q mo.m m om.m . . . . . pmummHuCD m mm.H m hm.H m¢.H m on.m m om.m m mm.m . . . . . . pmummue CECquIComouuHC HHom axe axe Axe Axe 68V axv HmEECm mCHHQm HHmm HwEECm oCHumm HHMH IUHS mama IUHE Hmmq quEpmmue ECHmmmuom Comouqu x.ACommom molmmmHv moon» nommm HCHonEEoo mo mCoHumuquoCoo ECHmmmuom ow ComouuHC mmoH Co ComouuHC Ho mCoHHmUHHmmm umHHom pCm HHOm CHmuumo mo uommmmll.¢H oHQme 94 however, a tendency for the post-harvest sprays to increase the leaf nitrogen concentration of the following season's growth of trees which had not received any soil nitrogen in the autumn (Figure 11a); however, this increase was not significant. Three spring sprays of 1.8 per cent KNO3 had no effect on either the nitrogen or potassium concentration of mid-summer leaves (Table 14). There was again, however, a non-significant tendency for the spray treatment to increase the nitrogen concentration of trees which had not received any soil nitrogen the previous autumn (Figure 11b). Neither soil nor foliar post-harvest applications of nitrogen had any effect on terminal shoot growth prior to winter in the season (1968) of application (Table 15). The soil nitrogen treatment increased both terminal shoot growth and trunk circumference during the 1969 growing season; however, the foliar nitrogen treatments did not affect either. Experiment 2.——In the 1969—70 season Experiment 1 was essentially repeated except that the foliar spray concentration was doubled, the spray x soil nitrogen inter- action was not examined and a urea spray control was added. Additionally, the spring spray treatments were superimposed on the autumn spray treatments. As in the 1968-69 season, an October soil applica— tion of 0.5 lb. nitrogen per tree increased the nitrogen 95 Figure ll.--Differentia1 response of leaf nitrogen concentration to autumn and spring foliar sprays of 1.8 per cent potassium nitrate at different soil nitrogen levels (gig. 0 and 0.5 pounds nitrogen per tree). (a) Response to autumn foliar sprays by mid—spring (Interaction F value not significant; (P< 0.05). (b) Response to spring foliar sprays by mid-summer (Interaction F value not significant; (P‘<0.05). ‘96 A N. N N N m m. w m. o .5. o m u A R P S F O R E B w 1 m d "TFON - 4 d d "L o 5 . s o o .... o s c 6 5 5 4 4 3 3 at 0x. zo_.rH . . . . . . . . . . . pmumouuCD m mo.m m m.mH . . . . . . . . . . . . pmpmmua mCHHmm I ComouuHC umHHom m b.mH . . . . . . . . . . . CoummuuCD m hm.m m m.mH m h.mH . . . . . . . . . . . . cmummue CECqu I ComonuHC HMHHom Q Hm.w Q ¢.mH m o.mH . . . . . . . . . . . topmoupCD m ov.m m 0.0m m ¢.mH . . . . . . . . . . . . topmoua CECqu I CmeHUHC HHom Apocflc lgocflc laocflc onlmmmH onlmmmH mmlmmmH umuCHB umuCHz MquHz quEummuB moCmummECouHo xCCHB QHBOHm COOQm HMCHEumB x.ACommmm mmlmmmHv moon» Qommm HmHouoEEoo mo Qu3oum Co ComOHUHC Ho mCoHumoHHmmm CMHHOH pCm HHom CHmuuoo mo HomHHMII.mH mHQma 98 concentration of both young spring leaves and mature leaves in mid-summer (Table 16). Neither leaf potassium concen- tration,. nor terminal shoot growth immediately following treatment, were affected by this treatment. Three post-harvest sprays of 4 per cent KNO3 again increased both the nitrogen and potassium concentrations of the sprayed leaves prior to leaf fall (Table 12), but had no effect on either the leaf nitrogen or potassium concen- tration of young spring leaves the following season. Similarly three post-harvest sprays of 1.2 per cent urea increased the nitrogen concentration of the sprayed leaves prior to leaf fall but had no effect on the nitrogen con- centration of young spring leaves the following season. Further, neither set of post—harvest sprays had any effect on terminal shoot growth immediately following treatment. The combination of three autumn plus three spring sprays either as 4 per cent KNO or as 1.2 per cent urea 3 failed to influence mid—summer leaf nitrogen or potassium concentrations (Table 16). Discussion Sand Culture Experiments Carrier Ion Experiment.--The evaluation of cations as carriers for nitrate foliar sprays at the same nitrogen concentration revealed interesting differences in phytotoxicity. Salt injury (Ehlig and Bernstein 1959) may 99 .Amo.o vmv hHquonHCmHm HmHmHU mumuumH mMHHCC hQ UCBOHHOH CECHoo Qomm CH mCmmEx m N.Hm m om.H m mm.H Q om.o Qm mm.¢ Qm o¢.¢ Q m¢.m . . . . mozx ComouHHC HMHHom m m.m~ m Hm.H m >©.H m Hm.o Qm mm.v Qm o¢.v Q mm.m . . . . moms CmmOHuHC HMHHom m o.mm m mo.H m Ho.H m Hm.o n 44.4 n Ho.4 m mo.m . :wmouuHc HHOm m b.mm m mm.H m mm.H m m¢.o m mo.¢ m Cm.v m mo.m . . . topmoHuCD 30ch xx; 1x; xx; xx; xx; Ax; onlmmmH HOEECm mCHumm HHmm nmEECm mCHHmm HHMH HmuCHS ICHS . mmmq Isz mmoq quEummuB Qu3ouw ECHmmmuom Comouqu x.ACommmm onlmmmHv mo Qu3oum HOOQm HCCHEMmu UCm mCoHumuquoCoo mme Co ComouuHC mo mCOHumoHHmmm umHHom pCm moon» Qommm HmHonEEoo ECHmmmuom pCm ComouuHC HHOm CHmuuoo mo QUCMHMII.0H oHQwB 100 occur either from external salt residues accumulating on the foliage which cause injury because of the very high osmotic concentration that develops when the residues re— dissolve (243; in dew), or the injury may occur from foliar absorption when the spray contains one or more ions to which the species is specifically sensitive. Stone fruit trees are sensitive to salinity on both counts (Benson 2; El: 1966). They have low tolerance to high osmotic concen- trations and Na+ and Cl- have specific toxic effects. It is considered that the severe injury observed in these studies was due to absorption rather than external residues. If injury was caused by external residues, then the injury should have taken the form of necrotic spots or patches spread at random over the leaf surface with injury in the leaf tip region especially. Instead, the injury took the form of severe marginal necrosis with accompanying leaf roll. SuCh symptoms are characteristic of salt toxicity in stone fruit trees following root absorption (Benson 2; 31. 1966) and Ehlig and Bernstein (1959) reported similar symp— toms following sprinkler irrigation of stone fruit trees with water high in sodium and calcium chlorides and sul- phates. They found that injury in plum trees occurred with a leaf sodium concentration of 0.2 to 0.7 per cent and Lilleland (1951) reported injury in peach at 0.5 per cent. Thus the sodium levels found in the present study of 0.39 (sweet cherry) and 0.14 (peach) per cent are consistent 101 with the view that injury occurred following absorption. The author could find no description of calcium or magnesium toxicity symptoms in the literature though one might expect them to be similar to other salt toxicities. The relatively low calcium and magnesium levels attained in the leaves on the basis of published standards (Benson 2; 21, 1966; Leece 1967) however are not consistent with the view that toxicity followed absorption, although some absorption clearly occurred. On the other hand, consideration of spray osmotic concentration values for the various treatments tends to rule out external residues as the cause of injury. On this basis (Table 17) injury should have occurred as follows: K+ = Na+ >Ca+2= Mg+22>NH22>Urea. However, the observed order in sweet cherry trees was Na+>’Ca+2:>Mg+2>»NH+ = 4 + + +2 . Urea:»K . Thus K and to some extent Mg did not cause the degree of injury predicted on the basis of osmotic con- centration. It might be argued that injury was caused by 2 which external residues except in the case of K+ and Mg+ were partially absorbed. However this contention is not supported by the visual symptoms of injury. If then it is concluded that carrier ion absorption occurred and that this in turn produced the observed leaf injury, the absence of injury with K+ sprays must be attrib- uted either to little K+ absorption or to the less toxic nature (or greater metabolic need for) K+. The latter 102 Table l7.-—Approximate osmotic concentrationsx of foliar sprays used in the cation evaluation experiment. # . Ionization Osmotic compound Molarity Factor concentration (M) (1) (bar) Urea 0.1 l 2.47 NH4N03 0.1 2 4.95 KNO3 0.2 2 9.89 NaNO3 0.2 2 9.89 Ca(NO3)2 0.1 3 7.42 Mg(NO3)2 0.1 3 7.42 xEstimated using the van't Hoff equation (Salisbury and Ross 1969): n = miRT where: osmotic concentration (bars) molality of solution degree of ionization of compound universal gas constant (0.083 litre bars/mole degree) absolute temperature. a ”.132: "II" n For the estimation, molarity was substituted for molality, it was assumed that the ionic compounds were completely ionised, and a temperature of 25° C (T = 298° A) was used. 103 seems the more likely explanation as the leaf analysis data indicated considerable K+ absorption. As none of the nitrate sprays raised leaf nitrogen concentration significantly and as K+ was the carrier ion causing least injury, subsequent evaluations of nitrate foliar sprays were confined to KNO and at higher concen- 3 trations than used in this experiment. In light of the many reports of the effectiveness of urea sprays on crops other than stone fruit @331; Wittwer gt 21, 1967) it is noteworthy that the urea control was the only treatment to increase leaf nitrogen concentra- tion in sweet cherry trees. It is also interesting that peach leaves were less susceptible to injury than sweet cherry leaves. This suggests cuticular differences between the species, the peach cuticle being more resistant to spray penetration than the sweet cherry cuticle, although it could also mean that the peach was less sensitive to salt toxicity than the sweet cherry. Both, in fact, may be true: however, the leaf analysis data, which showed in- creased leaf nitrogen and potassium concentrations produced by the urea and KNO3 treatments respectively in sweet cherry trees in contrast to peach trees, support the penetration hypothesis. Absorption and Translocation Experiment.--Having failed to demonstrate nitrate absorption in the carrier ion experiment this aspect of the experiment was repeated 104 using double the concentration of nitrate under more con- trolled conditions (greenhouse). A more sensitive indicator of absorption than leaf nitrogen concentration gig. induc- tion of leaf nitrate reductase activity, was employed, and apricot trees were used as they were more sensitive to enzyme induction than other Prunus species (see the pre- ceding section). This time nitrate absorption and initial metabolism occurred, however the increased spray concentra- tion produced leaf injury the nature of which indicated salt toxicity following absorption. This injury was considered responsible for the stunting of subsequent terminal shoot growth and the failure to demonstrate translocation of nitrogen or potassium from the sprayed leaves to the new growth. In summary, the sand culture experiments showed that K+ was likely to be the most effective carrier ion for nitrate foliar sprays when applied to stone fruit trees and that absorption of both the carrier ion and the nitrate occurred. Depending on the carrier ion and the species injury occurred at a nitrate concentration of 0.2 to 0.4 g. 2 caused the most injury at equivalent nitrogen concentrations, Mg+2 was equiv. per litre. Sodium ions and Ca+ intermediate and K+ caused least injury. Apricot seemed to be the most sensitive species to injury, sweet cherry was intermediate and peach was least sensitive. This 105 range of decreasing sensitivity may reflect increasing cuticular resistance to foliar penetration. Field Experiments A major question left unresolved by the sand culture experiments was whether sufficient nitrate could be absorbed from nitrate foliar sprays by the leaves to influence tree growth and fruit development. Field evaluations of nitrate foliar sprays were undertaken in an attempt to resolve this point. It was necessary to conduct these evaluations concurrently with the sand culture studies and to some ex- tent this was unfortunate. In retrospect apricot or sweet cherry trees may have been more sensitive indicators of absorption and utilization than peach trees, and the initial nitrogen level chosen of 0.18 9. equiv. per litre seems too low. Nevertheless this rate was three times the rate that was being used commercially and in the second season the rate was increased to 0.4 9. equiv. per litre. The selec- tion of K+ as the carrier ion (on the basis of preliminary phytotoxicity studies) proved fortunate. In both seasons autumn soil nitrogen applications corrected the nitrogen deficiency of the young commercial peach trees used in this experiment without, at the same time, breaking terminal bud dormancy in autumn and causing unwanted terminal growth susceptible to winter injury. In contrast autumn and/or spring nitrogen foliar sprays (either 106 as KNO3 or as urea) at concentrations six times that used commercially did not affect the nitrogen status of the trees. Further the soil nitrogen treatment increased both tree terminal growth and trunk circumference the following season, which again were not affected by the sprays. It is thus concluded that insufficient nitrogen was absorbed from the foliar sprays to be able to influence tree nitro- gen status or growth. Similarly with the carrier ion K+, insufficient K+ was apparently absorbed to influence tree potassium status or growth. On the basis of these findings, it would seem doubtful that nitrogen foliar sprays would benefit other Prunus species. It would seem almost certain that the commercial practice of using nitrogen foliar sprays on stone fruit trees at a nitrogen concentration of 0.075 per cent (i;§;_equivalent in nitrogen to a 0.6 per cent KNO3 spray) would have no influence on tree nitrogen status or growth. It also follows that further studies directed towards evolving a nitrate foliar spray suitable for stone fruit trees should center on examining surfactants and other spray additives that may improve leaf absorption of the spray. A sensitive assay system is provided by the induction of nitrate reductase in apricot leaves, either as used in the greenhouse in the sand culture studies reported herein or by modifying the greenhouse assay for 107 laboratory use. The nitrate reductase assay is rapid and avoids the criticism of nitrate or Kjeldahl nitrogen analyses that surface adsorbed nitrate, not removed by waShing, may have been included in the analysis. During the course of these studies, a modification for laboratory use of the greenhouse absorption assay was developed, based on the leaf disc technique of Sargent and Blackman (1962). It is presented as Appendix B. This leaf disc technique would permit a detailed study of the factors affecting spray penetration as well as the initial evaluation of spray additives which might enhance absorp— tion. Preliminary studies undertaken using the leaf disc technique (Appendix B, Table 20) showed that partial re- moval of epicuticular waxes from field-grown apricot leaves enhanced cuticular penetration by 0.4 M KNO This adds 3. to the evidence presented in the Review of Literature that epicuticular waxes may reduce cuticular penetration by nutrient sprays in Prunus spp. The finding in the sand culture studies that urea was the only compound to increase sweet cherry leaf nitro— gen concentration, together with the finding in Field Experiment 2 that urea and KNO produced similar increases 3 in leaf nitrogen immediately following application in autumn, suggests that renewed studies of urea as a nitro- gen foliar spray for stone fruit trees are warranted. The nitrogen concentration of urea is 3.5 times that of 108 KNOB, and, now that a low biuret form of urea is available for spray purposes, urea could probably be applied to peach trees at more than double the concentration used in the second field experiment. In contrast, KNO3 was being used close to its phytotoxic limit. LITERATURE CITED LITERATURE CITED Abdelal, A. F., and A. L. El-Tomi. 1965. A comparative study of urea sprays upon some deciduous fruit trees. Ann. Agr. Sci. Cairo. 10:333-349 (Hort Abstr. 39:4233). Altman, P. L. and D. S. Dittmer. 1968. Metabolism. Federation Amer. Socs. Exp. Biol., Bethesda, Md. pp. 91-92, 418-422. Anderson, J. W. 1968. Extraction of enzymes and subcellu- 1ar organelles from phant tissues. Phytochemistry 7:1973-1998. Anon. 1951. "Science and the Land". Ann. Rept. New Jersey Agr. Exp. Sta. 72:49-50. Bandurski, R. S. 1965. Biological reduction of sulfate and nitrate. lp_Plant biochemistry. J. Bonner and J. E. Varner (Eds.). Acad. 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European workers express concentration on a per cent basis (i;g;_g. per 100 ml.). Most British workers use pounds per 100 Imperial gallons and most Ameri- can workers use pounds per 100 United States gallons. The relationship between the three units is as follows: 1 per cent 5 10 pounds per 100 Imperial gallons 8.345 pounds per 100 U.S. gallons. As these methods, particularly the pounds per 100 gallons systems, lead to confusion When used interchange- ably, reports of all field experiments have been expressed on a per cent basis in this thesis. In reviewing other work the author's units of concentration have been converted to per cent Where necessary. Table 18 provides a quick 119 120 reference for conversion from pounds per 100 gallons (either Imperial or united States) to per cent, for the concentra- tion range common in nutritional sprays. Table 18.--Table for conversion from pounds per 100 Imperial or United States gallons to per cent, for the concentration range common in nutritional sprays. Pounds per Per cent 100 gallons from Imperial gallons from U.S. gallons 1.0 0.10 0.12 5.0 0.50 0.60 10.0 1.0 1.2 20.0 2.0 2.4 30.0 3.0 3.6 40.0 4.0 4.8 50.0 5.0 6.0 While the above weight per volume system of con- centration expression is very convenient as far as the commercial preparation of sprays is concrened, it tends to be misleading when two or more sprays are being compared unless they happen to contain the same percentage of the active ingredient in question, in this case nitrogen. To facilitate comparisons on an active ingredient basis, con- centration in the reports of the greenhouse and laboratory experiments has been expressed either on a molar or a gram-equivalent basis. The relationships among the various methods of expressing concentration for the compunds studied in this thesis are given in Table 19. 121 ¢.H« m.m« om.« m¢m.m« H.o «.o o«mo.«1mozcmz «.ma o.m« mm.« mam.m« H.o «.o o«m¢.«1mozvmo «.4H o.«a o«.H mmm.mH «.o «.o mozmz m.oH «.o« «o.« «««.o« «.o «.o «02x «.6 o.m om.o moo.m H.o «.o m02¢mz o.m 0.6 om.o ooo.o H.o «.o mmus .Hmm .m.D .Hmm .QEH o mHuHH .z 00H Com .QH 00H Com .QH a mom .6 2 .>HCU0 .m UCComEoo .hmumm moms uCoo Com 0.0 m 0» mHmMQ ComouuHC C CO pwumsvm CmQB .mHmeQ mHQQ CH pmesum mpCComEoo oQu How CoHumHUCOOCoo mCHmmmumxm mo mUOQumE mCoHHm> mQu mCOEm mmHQmCOHQMHCMII.mH mHQme APPENDIX B A TECHNIQUE FOR STUDYING NITRATE PENETRATION THROUGH INTACT LEAF CUTICLES IN PRUNUS SPP. It became clear during the foregoing studies that stone fruit trees were able to metabolise nitrate in their leaves yet were unable to benefit from nitrate foliar sprays. It was concluded that insufficient nitrate was entering the leaves to be able to influence growth. A technique for studying nitrate pentration through intact leaf cuticles was developed, then used to test the hypothesis that epicuticular waxes were reducing cuticular penetration by nitrate ions. Materials and Methods Plant Material Mature, mid-shoot leaves were obtained from 5-year- old apricot trees (Prunus armeniaca L. cv. "Curtis") growing at the Horticulture Research Center, Michigan State University, during the summer of 1970. The trees were in good vigor and had received 0.5 lb. nitrogen fertilizer per annum. The leaves were transferred to the laboratory in polyethylene bags at 00 to 40 C. 122 123 Methods The technique developed for studying nitrate pene- tration through intact cuticles was based on induction of nitrate reductase in excised leaf discs following cuticular penetration. Using nitrate reductase induction as an index of penetration was preferred to using nitrate-nitrogen or Kjeldahl-nitrogen determinations because nitrate and Kjeldahl determinations would include not only that nitrate which had penetrated the cuticle but also that adsorbed on the surface. WaShing techniques may not remove all the adsorbed nitrate. Leaf discs from the apricot leaves were prepared for penetration studies using the technique of Sargent and BlaCkman (1962) as modified by Schdnherr (1969). Glass cylinders (height 10 mm: external diameter 24 mm; internal diameter 21 mm) were attached to leaf discs (diameter 25 mm) using RTV ll liquid silicone rubber (General Electric Company, waterford, New York) hardened with Harter Tl catalyst (wacher Chemie GMBH Company, Munich, Germany). The discs were placed in 9 cm. petri dishes lined with moist filter paper, 1.0 m1 of the appropriate treating solution was added to eaCh disc, then the dishes were incu- bated in a water bath at 25° C, with a light intensity of 800 ft. candles (fluorescent) at leaf level. At the end of the incubation period the glass cylinders were peeled off the discs, the discs were rinsed with distilled water, 124 blotted dry, then assayed for nitrate reductase activity as described earlier. Penetration was examined through the upper, astomatous surface only. The standard enzyme induction solution was 0.4 M KNO3 and the control was 0.4 M KCl, each solution containing 0.1 per cent X77 surfactant (alkylarylpolyoxyethylene glycols, free fatty acids, and isopropanol--Chevron Chemical Company, Ortho Division, San Francisco, California). The standard induction time was 15 hours. Where required, partial removal of epicuticular waxes was achieved either by brushing the leaf with a camel's hair brush, or by wiping the leaf with a cloth dipped in 80 per cent (v/v) aqueous acetone, then drying the leaf immediately with an untreated cloth. Use of chloroform to remove the wax proved unsuitable in this system as it produced leaf disc senescence during the 15- hour incubation period. Normally four discs could be obtained from each leaf without including major veins. This permitted four treatments to be compared at the one time. Each treatment comprised four discs (23; 0.4 g. fresh weight tissue), one from eaCh of four leaves, such that each leaf was represented by one disc in each treatment. Data were analysed by stan- dard statistical procedures (Steel and Torrie 1960) and treatment means were compared by Tukey's cu-procedure. 125 Results and Discussion Nitrate reductase was induced in discs excised from field-grown apricot leaves by 0.4 M KNOB, using the tech- nique described above (Table 20). When leaves were pre- treated, either by brushing to re-orient the epicuticular wax, or by wiping with an acetone-soaked cloth to partially remove the epicuticular wax, enzyme activity induced by 0.4M KNO3 was double that of non-pretreated leaves. Table 20.--Induction of nitrate reductase in discs from field-grown apricot leaves, following cuticular penetration by 0.4 M potassium nitrate, as influenced by the partial removal of epicuticular wax.X Induction Nitrate System reductase activity (mu moles N02' formed,) (g. fresh weight'l, hour'l) 0.4 M KCl . . . . . . . . . . . . .-. . . . 217 a 0.4 M KNO3 . . . . . . . . . . . . . . . . 629 ab 0.4 M KN03--1eaves brushed . . . . . . . . 1246 bc 0.4 M KNO3--leaves acetone washed . . . . . 1454 c xMeans followed by unlike letters differ significantly (P< 0.05) . It is concluded that the leaf disc technique would be a suitable system in which to evaluate spray additives in relation to cuticular penetration by nitrate ions. It 126 is also concluded that epicuticular waxes were reducing cuticular penetration by nitrate. It is possible that the action of acetone was not restricted to the epicuticular waxes but that some of the acetone penetrated the cuticle and reduced the resistance of embedded waxes to nitrate penetration. In the Review of Literature the available evidence indicated that the failure of peach trees to re- spond to urea sprays was caused by cuticular waxes reducing urea absorption. The data just presented indicate that cuticular waxes may also cause the lack of response eXhibited by Prunus spp. to nitrate sprays.