THE ENTRY OF NUTRIENTS THROUGH THE BARK AND LEAVES OF DECIDUOUS FRUIT TREES AS INDICATED BY RADIOACTIVE ISOTOPES By Robert Lewis Ticknor A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1953 * ACKNOWLEDGEMENT The author expresses his appreciation to all members of the Department of Horticulture at Michigan State College, especially to Dr. Harold B. Tukey who directed the study and guided the writing of this Thesis and Dr. Sylvan H. Wittwer. Appreciation is also due to the members of my guidance committee, Drs. Carter M. Harrison, Clive »v. Megee, Leo W. Meriole, and Arthur K. Mitchell. The writer deeply appreciates the financial support of the United States Atomic Energy Commission which has made this study possible. 11 TABLE OF1 CONTENTS ACKNOWLEDGEMENTS..................................... . Page i X. INTRODUCTION......................................... 1 II. REVIEW OF LETEFj'T U R E ........ ......................... III. IV. MATERIAL 2 • AND METHODS................................. 17 RESHLTS............................................... 21 Preliminary Experiments Experiment I . . . . . • • • • • • • • • • • • • 21 Experiment II.................................. 2h Experiment I I I ........................ 26 Entry of Mineral Nutrients Applied to Bark of Limbs and Branches During the Dormant Season Experiment IV.................................. 32 Experiment V ............................... 3l* Experiment VI................................35 Experiment VII .............................. 36 Experiment VIII................................ 38 Experiment IX, .............................. 39 Experiment X . . . . . . . . . . . . . . . . . . Ll Spring Growing Season Treatments Experiment X T , ......... U3 Experiment X I I .............. 1*3 Experiment XIII................................ liU Experiment XIV 1*7 > ill Experiment X V .............................. 50 Experiment XVI................. . 51 .................. • • • • • 55 Experiment XVIII........................... 56 Experiment XVII Summer Growing Season Treatments Experiment XIX.. . . . . . . . . . . . . . . . 59 Experiment X X .............. 60 Experiment XXI.. . . . . . 62 Experiment XXII . .................. ......... 6h Experiment XXIII. 65 Experiment X X I V ............................ 67 Experiment XXV. 67 ......................... Experiment X X V I ............. 69 Experiment- XXVII.......................... 71 V. DISCUSSION. . .................................. 75 VT. SUMMARY............................................. 8U VII. LITERATURE CITED...................................... 89 LIST OF TABLES Following Page Table I. Table II. Table III. Bark Area in Square Centimeters of a 3-Year-Old McIntosh Apple Tree (Tree A) as Determined by ............................. Two Methods 23 Bark Area in Square Centimeters of a 3-Iear-01d McIntosh Apple Tree (Tree B) as Determined by Two Methods . . . • • • • • • ......... . . . . . . 23 Bark Area in Square Meters and Square Feet of a 2$-Year-01d McIntosh Apple Tree ................. 23 Table IV. Retention of P^2 Phosphoric Acid by McIntosh Apple Stem Sections as Affected by Several Sticking and Wetting Agents as Indicated by Counts ’"•er Minute from P ^ 2 ......................... 2U Table V. Retention of P^2 Phosphoric Acid by McIntosh Apple Stem Sections as Affected by Several Sticking Agents as Indicated by Counts per Minute from P ^ 2 ......... Table VI. Table VII. Table VIII. Table IX. The Influence of Concentration upon Entry of . P^ Phosphoric Acid into 1—Year-Old Peach Limbs During December as Shown by Radioactivity Found in the Treated and Untreated Portions of the Limbs 25 . 36 Migration of Fadiophosphorus Applied to Dormant Horizontal Branches into Attached Vertical Limbs of the Peach at 6, 2h, and U8 Hours after Application ................................. 36 Distribution of Radiophosphorus in Tomato Plants Following the Application of Phosphoric Acid Solution to the Stem by Brush and in Cotton Gauze as Indicated by Counts per Minute ............. U5 The Effect of Temperature on the Movement of Radiophosphorus into the Developing Tomato Fruit from Phosphoric Acid Applied in Cotton Gauze to the Stem of the Plants as Indicated by Counts per Minute................................. kl V Table X. Shoot Growth Produced by Apple and Cherry Trees Under Different Nutrient Conditions Following a Dormant Spray of Radi ophoschorus or nadiocalcium..• 1*9 Table XI. Radiocalcium Content of Apple Shoots Orowinp on Two Levels of Calcium Nutrition Produced Following a Dormant Spray of Calcium Chloride as Indicated by Counts ner Minute . . . . . . . . . . . . . . . . 1*9 Analysis of Variance of Radioactive Counts from Radiocalcium in Apnle Shoots Produced by Trees Growing on Complete and Minus Calcium Nutrient Solutions Following a Dormant Spray of Calcium Chloride....................................... 1*9 Total Increase in Circumference in Centimeters of Four Tree Replications of 5-Year-Old Montmorency Cherry Trees Following Ground and Tree Application of Nutrients • • ............... . . . . . . . . . 50 Table XTT . Table XIII. Table XIV. Phosphorus and Radiophof chorus Content of Current Seasons Growth (Leaves and Stems) of Water Sprouts of Apple Following Application of Phosphoric Acid to the Basal 8 Inches of Bark of the Water Sprouts • .53 Table XV. Phosphorus and Radiophosphorus Content of Current Seasons Growth (Leaves and Stems) of Water Sprouts of Apple Following Application of Phosnhoric Acid to Bark of Adjoining 10-Year-Old Limbs........... 53 Table XVI. Phosohorus and Radiophosphorus Content of Previous Seasons Growth (Woody Stem) of Water Sprouts of Apple Following Application of Phosphoric Acid to the Basal 8 Tnches of Fcrk of the Water Sprouts . . . 53 Table XVII. Phosphorus and Radiophopphorus Content of Previous Seasons Growth (Woody Stem) of Water Sprouts of Apple Following Application of Phosphoric Acid to Bark of Adjoining 10-Y^ar-Old Limbs............. 53 Table XVITT. Retention of Phosphoric Acid by Shoots of Apple, Peach, Pear, Sour Cherry, and Sweet Cherry Following Dipping in a Solution Containing Radiophosphorus as Indicated by Counts per Minute. • 57 Table XIX. Translocation of Radiophosphorus into Untreated Shoots and Roots of Anple, Peach, Pear, Sour Cherry, and Sweet Cherry Following Dipping of One Lower Shoot in Phosphoric Acid 57 Vi Table XX, Table XXI, Accumulation of Padiophosphorus in McIntosh Aople Shoots 2, U, 6, 8, 10, and 12 Hours after the Application of Phosphoric Acid to Two Median Leaves.............. The Influence of Oirdling and Location of Shoots on the Accumulation of Radiophosphorus from an Application of Phosphoric Acid to Two Median Leaves.................. 6U . 66 Table XXIT, Comparison of Absorption of Radiophosphorus Applied to the Seventh and Eiphth Leaves of Apple and PeachQhoots During July 71 Table XXIII, Comparison of Absorption of HatH ophosphorus Applied to the Seventh and '-'ighth Leaves of Apple and PeachShoots During August........... 7U LIST OF FIGURES Following page Figure 1, Figure 2. Autoradiogram of an Apple Fruit Showing the Distribution of Rndiophosphorus in the Fruit Subsequent to Foliage Application . . . . . . . . . . . 67 Autoradiogram of a Halehaven Peroh Shoot Showing Distribution of Radiophosphorua in the Shoot Subsequent to Application of Phosphoric Acid Solution to the Seventh and Eighth Leaves of the Shoot . . . . . . . . . . . . 70 I. INTRODUCTION Although the usual path of entry into plants of mineral nutrients is through the young roots and root hairs, the above-ground portions are also capable of nutrient absorption. Interest in the application of nutrients to above-ground parts has been stimulated by the observa­ tions that several physiological diseases, induced by deficiencies of specific elements, can be corrected by sprays of the deficient elements, and that nitrogen can be supplied to McIntosh apple trees through foliar sprays of urea* However, the foliar application of urea nitrogen has raised the question, whether twigs and branches may not also play a part in ab­ sorption and utilization. Further, dormant applications of minor ele­ ments have shown nutritional value. Accordingly, this investigation deals specfically with the nature and extent of uptake and utilization of nutrients labeled with radioactive isotopes applied to the bark and leaves of certain horticultural plants during various seasons of the year. II. REVIEW OF LITERATURE Bark Application of Nutrients Forsyth (22) in the third edition of his book, "A Treatise on the Culture and Management of Fruit Trees" published in 1803, gave a formula for a dressing to be applied to pruning wounds and to cavities of trees after removal of decayed wood, as follows: 1 bushel of fresh cow dung* 1/2 bushel of lime rubbish of old buildings or hydrated lime, 1/2 bushel of wood ashes, and l/l6 of a bushel of pit or river sand. An improve­ ment of the original formula was the addition of urine and soap suds to make a thick paint so that it could be brushed on to the cleaned wounds of a tree. Before the compound dried, it was sprinkled with a powder composed of 2/3 wood ashes and l/3 ashes of burnt bone. Trees made rapid and vigorous growth after the compound was applied accord­ ing to Forsyth, who was a gardener for King George III of England. For disclosing this information to the public, he was presented a medal by the King. Gris (2U), working in France in 18U3» found that chlorosis in plants could be corrected by supplying iron sulfate either through the roots or directly to the leaves. In America, Dawning (lU) recommended a wash of 2 pounds of potash in 2 gallons of water to be used for insect control. This solution* painted on the tree during the dormant period, also promoted the growth of the tree and improved the color of the bark. Ballard and Volck (2) in 1911* reported increased growth following both foliar and dormant season sprays of sodium nitrate made to fruit trees in California, When caustic potash was added to the sodium ni­ trate * the trees bloomed earlier than when sprayed with sodium nitrate alone* A five-fold increase In yield of Yellow Belieflower apple was found when the trees were sprayed when the soil was dry. Apples and pears responded to the treatment but stone fruits did not. Work in Oregon on tree applications of sodium nitrate was reported by Lewis (35* 36, 37) in 191b. He used a spray of 135 pounds of sodium nitrate* 19 pounds of sodium hydroxide, and 135 gallons of water. This spray was applied March 17* 1913* when the plants were in the green-tip stage. Dry sodium nitrate was applied to the soil around the trees April 25* and a solution of sodium nitrate was sprinkled on the ground around the trees May 7* The trees were greener* made increased growth* and gave a higher yield when the fertilizer was applied to the tree as compared with either type of ground application and no fertilizer ap­ plication, However* no rain fell after the soil treatments were applied. The following year all treatments were made in March and no differences were noted in response to method of fertilizer application. Interest was renewed in dormant tree applications after several workers (21* 3U* 5U) showed that some physiological diseases or dis­ orders which were caused by deficiencies of mineral nutrients could be corrected by such treatments. Roach (i*9)* by using dyes* followed the movement of solution injected into a plant. To correct chlorosis* he suggested inserting tar lets contai ning the missing element into holes drilled into the tree. Bennett (3) working in California also found that injections of iron salts would correct lime induced chlor­ osis. Many workers in Australia and New Zealand have reported that dor­ mant applications are effective in curing zinc and manganese deficien­ cies. Kilpatrick (3U) in 19Ul recommended the use of 1 pound of man­ ganese sulfate in 2 gallons of water to cure manganese deficiency in peaches. Ward (6l) writing in Australia in 19l*U reported the most rapid and most enduring treatment for the little-leaf disease was found to be a 2 1/2- or 5-percent spray of zinc sulfate during the dormant season. For complete recovery it is necessary to spray 2 years in a row and in alternate years thereafter. A statement was made by Skepper C5U) in an article in 1950 on fertilizers for fruit trees that "Applications of zinc compounds to the soil do not have any beneficial effects but fortunately trees are able to absorb zinc through their leaves and bark". He recommended 10 pounds of zinc sulfate and 5 pounds of hy­ drated lime in 100 gallons of water for citrus fruits. For deciduous trees he suggested the same rates for foliar applications as for citrus trees, during the dormant season 1*0 or 50 pounds of zinc sulfate in 100 callons of water may be used on deciduous trees. With apples in New Zealand, Forester (21) has cured little leaf with 25 pounds of zinc sulfate in 100 gallons of water. Thompson (57) in a paper written in 19UU discussed methods, ad­ vantages, and disadvantages of both solid injections and dormant sprays 5 made to fruit trees. He suggested that sprays are easier to apply and also easier to use to test for a deficiency by spraying a small limb. Disadvantages are that results are often variable and that the effect may last only one season. Also, ferrous sulfate, used for iron de­ ficiency, may cause injury when used as a spray. Solid injections have the advantage that they are reliably effective in the cure of severe iron chlorosis, although considerable injury to the trunk may follow. Also injections are not well adapted to use with young trees* Thompson further suggests a dormant spray for iron deficiency of 10 pounds of ferrous sulfate in 100 gallons of water in a severe case and 5 pounds in other cases* Manganese deficiency in cherries has been cured with dormant sprays at concentrations of either U or 16 percent manganese sulfate according to Thompson and Roberts (58). The 16-percent manganese sul­ fate solution was more effective than the U-percent* Movement of radioactiv* phosphorus from a nutrient solution into the base of dormant stems of excised red maple and McIntosh apple trees was studied by Eggert (16) in New Hampshire. He found the red maple would take up P-^ from the nutrient solution but that the McIntosh did not take up rapidly until the blossom buds began to swell. Apple buds when pink c >ntained 10 times as much P^2 as the nutrient solution on a unit weight basis. The primary leaves contained 7 times as much while spurs contained only 1/5 as much as the nutrient solution* small branches contained even less P^* The Harley (30) was not able to find movement of radioactive phos­ phorus through the bark during the dormant period. However, at the time of bud swell there seemed to be some intake through uninjured bark, and when the bark was injured there was a decided increase in the absorption of phosphorus. Foliar Application of Nutrients Hamilton, Palmiter and Anderson (29) tried foliar application of various nitrogen carriers in the early 19l*0's. At that time, they found that Uramon (urea) induced darker leaf color with less damage to the plants than did the inorganic forms of nitrogen which they used. A basis for the absorption of urea or any other substance through the leaves was found by Roberts, Southwick, and Palmiter (50) who studied McIntosh apple leaves to determine the relationship of cell wall constituents to the penetration of spray solutions. Their observa­ tions indicated that the cuticle is not a continuous layer but "exists in lamellae parallel to the outer epidermal cell walls. The pecti- naceous substances form a continuous path from the outside of the leaf and extending to the walls of the v=in extensions, which also contain a large amount of pectinaceous substances.•.The amount and location of the pectinaceous substances present in the leaves account for the en­ trance of water soluble materials such as minor elements, nitrogen, hormones, and organic fungicides sprayed upon apple leaves." Fisher (20) gives three principles that underlie foliage appli­ cations of nitrogen fertiliser. The first is that yields obtained following foliar sprays are as high as from comparable soil applications. 7 The second is that a greater nitrogen effect is obtained the later the spray is applied, up to the time of the ’’second cover" spray. The third is that a foliar spray produces a "nitrogen effect" of equal magnitude more rapidly than does a comparable soil application but that the effect of the spray application is less lasting. Some factors affecting the absorption of urea by McIntosh apple leaves have been reported by Cook and Boynton (13). Lamer leaf sur­ faces absorbed urea more readily than the upper surfaces, and young leaves more readily than old leaves. A leaf which was originally high in nitrogen had a greater absorbing capacity than a leaf low in nitrogen. Low temperature, low vapor pressure, and the addition of a wetting agent increased urea utilization. A marked effect on absorption in some cases was caused by changing the pH of the solution. No effect on absorption was noted in trees kept in the dark to reduce synthesis of carbohydrates. When sucrose was added to the spray solution, the amount of injury was reduced but also the rate of uptake was depressed. The uptake was most rapid the first few hours after spraying but con­ tinued at least 1|8 hours at a measureable rate. Rodney (5l) at Ohio State worked with calcium nitrate, ammonium sulfate, and urea as foliar sprays on Rlchared apple trees. caused less leaf burning than did the other two compounds. The urea He also found that the amount of entry through the upper surface of the leaves was nearly equal to the amount which enters through the lower leaf surface. This indicates that nitrogen is capable of entering the leaf directly through the cuticle as there are no stonates on the tipper surface of the apple leaf. 8 When hydrated line and a suitable spreader were added to urea sprays, Norton (U5) was able to obtain both a color and growth response in peach trees. Starch, molasses, ammonium carbonate, ammonium citrate, and sodium bisulfite did not increase the response from a urea spray. He found that urea at 10 pounds per 100 gallons of water plus Kolofog (a bentonite-sulfur) at 6 pounds per 100 gallons, as sticking agent, was the most effective material. In Washington rotate, Bullock and others (9) concluded that Elberta peach foliare was able to absorb ur*a but that the greatest nitrogen response occurred when part of the nitrogen application was made to-the soil and part to the foliage. They found that when the leaf nitrogen was increased, the maturity of the fruit was retarded. The cure of manganese deficiency in peaches and apples by foliar applications has been reported by Woodbridge and McLarty (63). Effec­ tive spray solutions were 2 pounds of manganese sulfate in 100 gallons of water or 1 pound each of manganese sulfate, boric acid, ferrous sulfate, and zinc oxide in the same amount of water. Epstein and Lilleland (19) found that manganese deficiency in deciduous fruit trees could be cured by foliar sprays, by liquid injec­ tions into branches, and by placing dry manganese salts in holes drilled into the trunk of the tree. llottle leaf on orange has been alleviated so that trees produce larger leaves, longer intemodes and more xylem tissue when sprayed with 10 pounds zinc sulfate and 5 pounds hydrated lime in 100 gallons of water, according to Reed and Parker (1*7). 9 It has been estimated by Woodhams (61*) that in the citrus region of southern California there have been 1*02,000 trees sprayed with zinc salts, 35,000 with copper salts and 17,000 with magnesium salts. Tracer Techniques In the present problem radioactive isotopes were utilized to follow the movement of applied nutrients. Accordingly, a review of some of the methods utilized in isotopic research is included. Meas­ urement depends on the emission of radiation which either affects a n photograohic plate or actuates a Ceiger-Muller tube* Solution counting using the immersion ty e of Geiger-Muller tube has been favored by several investigators (1*0, 1*2) on the grounds that the solutions are easier to handle and show lesc variation due to position. The Geiger-Muller tube is placed in a solution cup which has a funnel side arm and a bottom drain. of solution plus the countinrr tube. solution cup is drained. The cup holds 10 milliliters After a sample is counted, the The cup need not be rinsed unless the dif­ ference in activity of the following solution is greater than 5>0 per­ cent, since less than 1 percent of the solution remains in the cup* Martin and Russell (1*0) report that if the pH of the counting solution is below 3 the phosphorus is not absorbed by the glass walls* The end window Gieg'-r-Muller tube, which is more efficient (re­ cording 25 percent of the emissions compared to 10 percent by the imIf mersion Gieger-Muller tube) is used for counting dry samples. Hall and MacKenzle (28) state that solid samples should be used because when phosphorus is in a low concentration (1-2 ppm) there is a high percentage absorption by glassware. Their method involves the precip­ itation of the phosphorus first as ammonium phosphomolybdate and a re­ precipitation as magnesium ammonium phosphate. The second precipitate, which is counted after drying, is collected on filter paper supported by a metal ring. MacKenzie and Dean (38) have described a method of makinr bri­ quettes of ground plant material so that th*' plant material has uniform sample geometry. This procedure is adapted particularly to isotopes of high energy radiation such as nhosphorus and where large amounts of plant material are available. Another technique us^ng dr^ed rround plant material has been de­ scribed by Mitchell and Linder (1*3). A weighed amount of ground plant material is placed on a disc of Scotch Tape supported by a metal ring. After the ring, is tapped to obtain uniform distribution, the excess riant material is removed for weighing. The preeeeding techniques using electronic counting are all of a rmantitative nature but the other important method, that of exposing films by means of radiation, is primarily a qualitative method. VFittwer and Lundahl (62) have described a technique by which the gross absorption and distribution of radioisotopes in young whole nlants can b«* evaluated. The young plants are dried ranidly under heat and pres­ sure before being placed in a special exposure box with 8 x 10-inch Kodak No-Screen X-ray film. After an exposure of 3 to 10 days for a phosohorus treatment, the film is developed and the distribution of the nutrient can he seen. More detailed studies of the distribution of isotopes can be made by using Eastman nuclear track plates such as types NTB and NTB2. Duggar and Moreland (15) used this type of emulsion in working with the distribution of microns thick. from in sections of hydrangea leaves 15 Localization of the radioactive carbon nearly to the exact cellular location is possible by this technique. Tracer Experiments Some of the earliest tracer exneriments using an element which occurs naturally in plants were done by Gustafson (26) in 1937 using cyclotron produced P^ , Stem cuttings with the xylem removed were found to translocate as much phosphorus as intact cuttings. In a further experiment Gustafson and Darkin (27) removed the xylem from one cutting, the phloem from another, and left one other intact, and found that either portion of the varcular system could transport the phosphorus equally well. In a later experiment, Gustafson (25) found that Bryophyllum cut­ tings which were intact were able to transport more phosphorus than a cutting with either the xylem or phloem removed. However much less phosphorus reached the top of the plant when the xylem was removed than when the nhloem was removed. Stout and Hoagland (55) using isotopes of potassium, sodium, phos­ phorus, and bromine concluded that as long as the bark and wood of the willow cuttings were in contact, the concentration of isotope was the same in both tissues regardless of the species of plant or of the ion used. Radiation was detected in the upper leaves of a rapidly trans­ piring plant one hour after P^ was applied to the soil. The movement of ohosohorus in the bean plant (Phaseolus vulgarus) has been extensively investigated by Biddulph (h). In his first work he found that phosphorus moved rapidly throughout the plant, following the transpiration stream, with the greatest concentration of phosphorus occurring in the young leaves. In an experiment in 19U1 he (5) found that there was a difference in rate of migration and in distribution at different hours of the day. The greatest downward movement took place at or near 10 A.M. and the least near 10 P.M.. The most pro­ nounced upward migration occurred near noon, but was comparatively small. Colwell (11) studied translocation from a pool of held on the leaf by a plasticine ring. solution The movement of the phosphorus was toward the nearest points of utilisation. By girdling the ^tem he found that leaf-arplied phosphorus was carried by the phloem of cotton plants (Oossypium spp.). This was verified when the phloem of the pet­ iole was destroyed by scalding. The movement of the phosphorus was then much slower than when the leaf was undamaged. The cotton olant was also used by Biddulph and Markle (6) in studies of migration of "32 solution injected into the l»af. A con­ centration gradient was found both above and below the leaf with down­ ward movement in excess of 21 centimeters an hour. The upward move­ ment varied from 0 to U0 percent of the mobile phosphate, while down­ ward movement accounted for 60 to 100 percent of the mobile phosphate* Very rapid upward movement of bromine, 15 feet in 5 minutes, was fotind in a cucurbit Diant from KBr®^ introduced into the nutrient solu­ tion vy Stout and others (56). In other experiments they found sine Z n ^ accumulated in seeds and conducting tissues of the tomato. It was determined that less than 1 percent of a soil application of phosphorus was recovered in h3 days. A study of the metabolism of elemental sulfur applied to lemons was made by Turrell and Chervenak (59) usine S ^ . Metabolic products of the sulfur were H2 S, SO2 and SO^ with a very high proportion of the sulfur in the hydrogen sulfide and SO^ being derived from the elemental sulfur. The SO*, formed was derived largely from a source within the fruit* Mitchell and Linder (l*h) studied the effects of co-solvents and surface agents on the absorption and translocation of radioactive 2—U diiodo phenoxyacetic acid when the same amount of radioactive material was applied to an equal area. A very decided incr-ase in ab­ sorption over the distilled water solution was noted with several ad­ ditives ranging up to 350 percent with Tween-20. Simultaneous movement of P^2 ancj glli in opposite directions in rhloem tissues was detected by C.hen (10). He also found that the xylem carried P^2 when applied to the roots, whereas the phloem was the channel for foliar application* Several interesting facts have come from a study of chlorosis us­ ing radioiron, by Wann and others (60). Chemical analysis of green foliage varied only slightly from that of chlorotic foliage. Resistant plants conta^ ned four times as much iron on lime-free soils as on high lime soils while susceptible Diants contain eight times as much iron. Iron from injections remains in an active state indicating that the ■vnactivation of iron must occur in the soil or in the roots. Biddulph and Woodbridge (7) have worked on the relationship of phosphorus and iron in bean plants. They found that as the amount of phosphorus in the nutrient solution was varied from low to high levels the concentration in the tissues increased but at different rates# As the -hosrhorus level was raised above that necessary for growth, there was a precipitation of iron and phosphorus in the roots interfering with the movement of both ions# Silberstein and Wittwer (53) tested various organic and inorganic phosphorus compounds for foliar application on vegetable crops# growth response was obtained with ortho-phosphoric acid. Best In a field trial, there was increased early but not total yield of tomatoes as compared to a broadcast soil application. Autoradiograms showed that P^2 had moved from the foliage of tomatoes, corn, and beans to the roots within 6 hours. In vSweden, ihrenberg and f.ranhall (18) have made injections of radioactive substances into fruit trees in an effort to secure muta­ tions. The advantage of this method is that the radiophosnhorus and radiosulfur tend to concentrate in the buds where mutations are induced without damage to the rest of the tree# The effect of exposture of the meristematic regions of barley sub­ jected to a constant, relatively high level of radiation from P^2 has been studied by Uackie, Blume, and Hagen (39). They found cell divi­ sion ceased, cells enlarged, cytoplasm became less dense, and cell walls thickened. Damage to shoot meristems was more pronounced than to root meristems when solutions of comparatively low specific activity were used# 15 Two British workers, Russell and Martin (52) have reported damage to plant tissue at levels much lower than reported by American workers* They found reduced root growth at a level of 10 microcuries per liter of culture solution. The solution rave O.li "equivalent roentgen"* per day but due to selective accumulation in the meristematic regions, the root tip received 300 "equivalent roentgen"* per day* Blume (8) studied the effect of different ratios of P32 to P^1 on the growth of plants in soil. The only reduction in growth of barley seedlings occurred at the highest concentration of 12,500 microcuries of P^2 per gram of P ^ * The movement of calcium into tomato, alfalfa, red clover and wheat from a soil application has been studied by Ririe and Toth (1*8). They found greater amounts of Ca^5 in the lower leaves than in the upper leaves. Split root studies with tomatoes indicate little movement of C a ^ through the roots in a solution containing Ca^5 to other roots in solutions of varying levels of calcium* The relative absorption of hsophorus by apple trees was studied by Eggert et al (17) working in New Hampshire. They have shown con­ clusively that ohosphorus in water solutions sprayed on the foliage and small branches of apple tr^es can be absorbed and translocated to other parts of the tree. Over half the phosphorus sprayed on the plants plants was found to have been translocated to the roots while 2 to 3 percent of the total phosphorus in the plant came from the foliar spray* * A Hequivalent roentgen* (er) is' essentially the same as a flRoenigen-equivalent-physical" (rep) which is defined as "That dose of any ionizing radiation which produces energy absorption of 93 ergs per gram of tissue"* A device for the measurement of the rate of urea hydrolysis has been reported by Hinsvark, Tukey, and Wittwer (31)* of The relative ratee production were studied for several vegetable and woody plants and it was ascertained that the rate of urea hydrolysis as indicated by 0 ^ 2 evolution was closely correlated with the amount of injury pro­ duced by the spray. The greater the rate of hydrolysis the less toler­ ant a olant was found to be to a given concentration of urea. Fraser and Mawson (23) found a uniform distribution in a narrow spiral band to a height of 15 feet following an injection during the g/ growing season of Rb below th<- surface of a trough holding potassium chloride solution which was attached to the trunk of the tree. The rate of movement into the olant from the solution decreased after the initial rapid intake. Upward movement of Rb®^ ceased in October when leaves became senescent but downward movement continued. In healthy trees, the movement was confined to a narrow path, while in diseased trees, the movement was over a fan-shaped area. III. MATERIALS AND METHODS All radioactive materials, which were used in these experiments, were obtained from the Atomic Energy Commission at Oak Ridge, Tennessee. Radioactive calcium (Ca^), radioactive phosphorus (P^) and radioactive Potassium (K1*2 ) were used to study the rate and extent of absorption and subsequent translocation following foliar and bark application of nutrient elements. Radioactive calcium (Ca^), which was received as a solution of calcium chloride, was added to a 2 percent solution of stable calcium chloride* Radioactive phosphorus (P^), which was received as a solution of phosphoric acid, was prepared in most experiments as a 0.3 percent solution of stable ohoschoric ac^d to which radioactive ^hosrhorus (p32) was added to make concentrations ranging from 1 to 10 microcuries per milliliter. In certain experiments, concentrations of stable phos­ phoric acid ranging from 0,2$ to 8.0 percent were used, while in these same experiments the number of microcuries per milliliter was varied from 0.01 to 53.3. Radioactive potassium (K^), which was received as irradiated units of potassium carbonate, was prepared as a 0.3 percent solution of potassium carbonate. Various procedures were used for applying the radioactive mater­ ials depending upon the nature of the plant material under study. leaf application, the leaves were dipped into a 600 For -milliliter beaker containing the desired solution, the leaves being forced into the sol­ ution by means of a glass stirring rod. When the solution was applied to limbs or branches, paint brushes were used, a 3-inch brush being used on the old limbs and a l/8-inch brush for water sprouts and small limbs. For some of the dormant ap­ plications, a measured amount of solution was pipetted onto cotton gauze wrapped around a limb. Spraying with a Shur-Shot sprayer at 60 to 100 pounds pressure was another method used for young dormant trees. This work was done in the middle of an open field, mask and protective plastic cover being worn by the operator. Samples that were collected from experimental treatments were placed in a drying oven at 70°C. Because of the large volume of some of these sanples, principally those in the out-of-doors experiments with ohosphorus and calcium sprays, the samples were dried at 120°F, in a large circulating air dryer such s used for food dehydration. To reduce the shielding effect of the tissue and to prepare the samples for chemical analysis, the tissue was reduced to ash in $0 milliliter porcelain crucibles in the muffle furnace at 600°C, Phos­ phorus samnleg were handled as described by the A.O.A.C. (1)* adding first ,?N magnesium nitrate, then concentrated hydrochloric acid. Magnesium nitrate will form magnesium pyronhosphate with the phosphorus compounds present in the plant tissue, a compound which is thermally stable at 600° C, The hydrochloric acid partially digests the plant material which permits close contact of the magnesium nitrate and ths phosphorus compounds. Before ashing, leaves and current-season*s shoots were ground to 20 mesh in a Wiley mill. Watersprouts and other older tissues were reduced to small pieces, then excess hydrochloric acid added to insure penetration of the magnesium nitrate. Ash from foliage treatments was dissolved in 2N hydrochloric a d d then transferred to a 25-milliliter volumetric flask and made to vol­ ume. An aliquot of 5 milliliters was removed for counting. Ash from woody samples was dissolved in acid at the rate of 5 milliliters of acid to 1 gram of dry plant material. Five milliliters of the acid solution of the woody material was evaporated to dryness on an electric hot elate and used as a sample for counting. A Tracerlab autoscaler equipped with a shielded chamber was used for counting the samples. Autoradiograms were used to evaluate the cross intake and distri­ bution of foliar and bark applied nutrients. Autoradiograms were pre­ pared by the method outlined by Wlttwer and Lundahl (62) with one mod­ ification. The treated leaves were removed from the shoot to prevent blurring of the autoradiogram rather than washing the leaves to remove the excess phosphorus or -otassium which causes the blurring. The steps in the preparation of autoradiograms are as follows* 1. Place plant nart between two sheets of botanical specimen paper and dry under pressure with infra-red heat lamps. 2. Remove botanical specimen paper from both sides of plant part and replace with a she«»t of pliofilm on one side and a new sheet of absorbent paper on the other. 3. Press plio-film side against 8 x 10 inch, No-screen Kodak X-ray film with steel plates and leave in the dark (photo— graphic) room 5 to 7 days for exposure. lu Process films, using X-ray developer and fixative. Information Is given on wither conditions when they were unusual and had a probable affect on the results of an experiment. More detailed information on materials and methods will be de­ scribed in each experiment. IV. RESULTS The research undertaken in this project may be divided into pre­ liminary experiments not using radioactive materials and experiments using radioactive materials which are grouped according to season (a) applications to the bark during the dormant season, (b) applications to both bark and leaves during the early spring season, and (c) appli cations to the leaves durirg the mid-summer and late summer season* Preliminary Experiments Experiment I Object: To determine the total bark area of twigs, branches, and trunk of a dormant apple tree, so as to gain some appreciation of the surface exoosed to nutrient applications. Part 1 Materials and Methods: Two 3-year-old McIntosh apple trees were used in preliminary measurements to determine a practicable means of de­ termining the surface area of a dormant tree. Two methods were com­ pared (a) computation from measured diameters, and (b) computation from volume as determined by actual water displacement. Both methods depended on finding the surface area and weights of stem sections 80 millimeters in length. These stem sections were cut into eight diameter classes, namely 3, 1*, 5, 6, 8, 11, 13, and 15 millimeters which represented all the diameter sises found on the 3— year-old McIntosh apple trees. In the first method, it was assumed that the stem section was a perfect cylinder. Since the diameter and the length of each section was known, the surface area of the cylinder could be calculated by using the formula for the surface area of a cylinder, TYD!. D was the diameter of the section, and L was the length of the section. In the second method, the 80-millimeter sections were immersed in graduated cylinders and measurements made of the volume of water dis­ placed. It was then necessary to determine the radius of a stem 80 millime+ers in length which would have this measured volume. This was done by substituting the value obtained for the measured volume Into the formula for the volume of a cylinder, Volume ■ TrR^L or Radius^ » Volume . This calculated value for the radius was used to determine fr Length the surface area of the 80-millimeter stem sections. A oroportion was then used to find the total surface area of stems of a "iven diameter class, as follows: A1 a2 - *1 ' Wj °r A, ’ Alw2 »! = calculated surface area for a 80 millimeter section of * diameter D (calculated by either of the 2 methods) k-2 z total surface area of wood of diameter D — weight of a 80 millimeter section of diameter D W 2 s total weight of wood of diameter D Results: The b^rk areas of the two 3-year-old McIntosh apple trees are given in Tables I and II. These data show that the surface area obtained by the two methods are similar, with the value computed by the measured diameter method being the more conservative estimate* 23 Part 2 Materials and Methods: During January 1952, the total surface area was determ»ned of a 25-year-old McIntosh apple tree with a limb spread of 26 feet 8 inches and a height of 20 feet 10 inches by the measured diameter method. The tree was cut down and the wood grouped into 19 diameter classes, ranging from 5 millimeters to 305 millimeters, as shown in Table III, 80 Branches up to 8,5 centimeters in diameter were cut into -millimeter lengths and the average weight of 10 sections was de­ termined, This average weight and calculated surface area were sub­ stituted in the proportion used in part 1 of this experiment. For limbs with a diameter greater than 8,5 centimeters, the area was cal­ culated directly assuming the limb to be cylindrical. It will be seen from the accompanying table (Table III) that the surface ar*a of the 25-year-old McIntosh apple tree was 85,99 square meters. Interestingly enough, wood of 6 millimeters or less in dia­ meter constituted slightly over one-third (35,7 percent) of the entire surface ar»-a, and over one-half (53.7 percent) was 10 millimeters or less in diameter. Thus a large portion of the surface area of the tree occurred in wood most adapted for the intake of mineral nutrients because of the relatively thin layer of periderm. TABLE I BARK AREA IK SQUARE CEKT7V2TERS OF A 3-Y EA R-O LD McINTOSH APPLE TREE (TREE A) AS DETEHMTKED BY TWO METHODS Ag« 1-yr. Wt. of 80-mm. Section Vol. of 80-om. Section Total Weight Total Area by Diameter Method Area of 80-am. Section Volume Method Total of Area by Volume Method (Oms.) (Ml..) (Oms.) (sq. cm.) (Sq. Cm.) (Sq, Cm.) .8 .7 1.2 1.0 9.97 1*1.6 10.06 10,06 1.81 1.81 2.72 2.26 11.78 Area of Aver, Diam, 80-nun, Section of Branches by Diameter Method Oh.) (Sq, Cm.) 7.55 7.55 1*7.8 1*7.6 8.97 8.1*2 11.00 10.03 Wood 3 3 1* 1* 2-yr. k Wood 6 8 10.06 15.10 21.00 2.72 3.62 5.1ib 1.1* 3.0 i*.5 5.W* 8.15 16.77 21.5 31.9 61*.7 11.87 17.36 21.30 23.8 39.1 62.6 11 13 15 27.63 32.61 37.71 9.51* 13.13 16.77 9.0 11.9 15.0 1*2.20 1*1*.80 32.60 122.1* 111.1* 73.1* 30.50 3U.62 38.90 135.0 118.2 75.7 3-yr. Wood Total 51U.3 1*9.7 551.9 TABLE II BARK ARKA IN SQUARE W T T K E T E R S OF A 3-YEAH-OUD McINTOSH APPLE TREE (TREE B) AS DETERMINED BY TWO METHODS Age 1-yr. Wood 2-yr. Wood 3-yr. Wood Total Aver. Area of Diam. 80-mm. of Section Branches by Diameter Method (lb.) (Sq. Cto.) Wt. of 80-ram. Section Vol. of 80-mm, Section Total Weight Total Area tjr Diameter Method Area of 80-mm. Section Volume Method Total Area by Volume Method (Gms.) (Ml,l (Gms.) (So. On.) (Sq, On.) (Sq. Cm.) .7 .85 1.29 1.50 2.2 2.U 9.51 39.6 8.15 30.2 16.32 61*.7 1.08 3 3 1* 1* 5 5 10.06 10.06 12.57 12.57 1.81 1.81 2.72 2.72 3.18 3.18 5 6 8 12.57 15.10 21.00 3.18 3.63 5.1*1* 1.9 2.3 U.6 11 13 15 27.63 33.61 37.71 9.08 12.68 17.63 8.7 13.5 17.0 7.55 7.55 8.1*2 9.26 11.39 12.23 H*.91 15.56 1*6.1* 12.68 16.2 35.9 1*9.0 13.1*7 15.22 21.30 17.3 36.0 1*9.5 23.55 39.1*0 72.5 ioii.5 iio.7? 89.3 29.55 36.10 1*1.80 76.9 109.0 99.2 8.60 501.9 35.1* 78.5 TABLti III BARK AR: A IN SONAR-' METERS AND S0UARJ5 FEET OF A 25—YEAR-OLD McINTOSH APPLE TREE Aver. Diam. of Branches (Mm.) Area of 8 0 -mm. Section (Sq. Cm.) Total Area Total Area (Sq. M.) (Sq. F.) Portion of Tree % Av*r. tit. Total of 8 0 -mm,, Weight Section (Gms.) (Oms.) 5 7 9 12.57 17.59 22.62 30. 7l* 8.78 6.73 330.76 914.L7 72. Ul 35.7 10.2 7.8 1.81* 3.39 5.60 1*1*,990.68 16,925.71 16,669.89 11 11* 27.61* 35.39 1*5.21* 5.01 7.70 5.38 53.91 82.85 57.89 5.8 9.0 6.3 8.21* 13.73 21.99 11*,91*0. 35 30,050.79 26,167.61 6.57 3.87 2.92 70.69 1*1.61* 31. U2 7.6 1*.5 3.h U2.79 85.27 139.68 1*1*.735.31 37,535.93 36,088.98 1.5 1.8 .9 208.1*9 291.20 387.70 19,1*1*8.58 28,066.79 16,1*99.78 1*97.97 622.01* 759.88 13,1*38.06 18 25 35 16 62.83 87.96 113.10 55 65 75 138.23 163.36 188.50 1.29 1.57 .80 13.88 16.89 8.61 85 95 105 213.63 238.76 263.89 .58 .38 1.21 .2 I4 U.09 13.02 .7 •U l.h 9.01* 1 .1*0 U. 6 3 1 1 .U1 1.0 — .1 .5 1.2 ___ _ ___ __ — — _ _ _ _ ----- ----- 925.25 99.8 ----- ----- 115 135 205 305 Grand Total I. .13 .1*3 • --— - --- 8 1 1.06 85.99 6 --_ Experiment II Object: To test the effectiveness of various sticking a; ents when used with nutrients ar.plied to the bark of fruit trees. Part 1 Materials and Methods: A $ percent solution of phosphoric acid con­ taining 1/100 microcurie of P^ per milliliter was used as the basic solution to which were added the following sticking agents at a con­ centration of 2 ounces to 100 gallons of water: Triton B-1956, Ortho, Dupont Sticker-Spreader, Atlas G-772, Atlas Tween-20, and Methooel Uooo c.p.s. Other sticking arents used were Plant Spray Spreader at 1 ounce to 8 gallons of water, Kolofog at 6 pounds to 100 gallons of water, and Oood-rite PE—PS at 6 rounds to 100 ballons of water. To test the effectiveness of the sticking agents, uniform sections of water sprouts from McIntosh apnle trees 6 millimeters in diameter and 8 centimeters in length were immersed in solutions containing the sticking agents. Five stem sentions were used with each agent, A hook-shaped piece of wire was inserted into the pith of each section, jn order that the sections could be immersed in the solutions and later hung up until dry. Standard procedure for nhosphorus analysis according to the A.O.A.C. (l) was followed, except that instead of being ground the sections were cut into small pieces before being treated with the re­ agents in preparation for ashing. The ash wss dissolved in 5 milliliters of 2N hydrochloric and then transferred to metal sample boxes for counting the radiation. TABLE IV RETENTION OF P32 PHOSPHORIC ACTp BY McINTOSH APPLE STEM SECTIONS AS AFFECTED BY SEVERAL STICKING AND WETTING AGENTS AS INDICATED BY COUNTS PEP MTNUTE FPOT/ P32 Sticking and/or Wetting agent Retention Count/min. per sq. cm, of surface Grains deposited for entire mature apple tree** Atlas G-772 6.93 179.5 Carbowax 1500 6.3k 16k.? Kolofog 5.U3 lko.7 Dupont S.S. 7.58 196.3 Methocel U000 8.03 200.1 Ortho 6.35 16U.5 PE-PS 5.5 lk?. 5 Flant Spray 6.29 162.9 Triton 1956 6.33 16k. 0 Tween-20 7.16 185.5 •^Coirruted on the basis of the 5 perrent solutions used for these tests and the surface ar»a of a 25-year—old McIntosh apple tree as determined in exneriment I. 25 Results{ The results of this experiment f'iven in Table IV are the av'rrpcF of the amount of radioactivity found on the five sections used for each solution. Both Kolofog and Ooodrite PE-PS are known to be pood sticking a,1ents but in this t<*st they showed the lowest figures for retention, A possible explanation is that during the ashing process a compound may be formed with nhosnhorus which is volatile below 600°C. Phos­ phorus di-, tri-, pents-, and heota-sulfides, having boiling points ranging from 33?°C. for the disulfide to 523°C. for the heptasulfide, could have been the compounds formed. Part 2 Materials and Methods: A second series of tests was conducted using Tupont Sticker-Spreader and Methocel 1:000 c.p.s, at the same rates as were used in the first tests, ?lus solutions of Methocel U000 c.p.s, and Methocel 1500 c.p.s. at the rate of U pounds of sticker to 100 gallons of solution. The ends of th» stem sections were dioped in paraffin so as to prevent absorption of solution through the '-ut ends. After the ctem sections were dry, the ends were cut and discarded, lea ing a piece of shoot 6 centimeters in length. The stem sections were prepared for analysis in the same manner as described in Part 1. Results: The results of the second series of tests are shown in Table V. The values obtained confirmed the results obtained in the first test. Methocel 1:000 c.p.s, wan *»gain found to be the most effective TABLE V RETENTION OF P32 PHOSPHORIC AOTF BY McINTOSH APPLE STEM SECTIONS AS AFFECTED RY SR’ /FPAL STICK INC ACENTS AS INDICATED BY COUNTS PER MINUTE FROM P32 Sticking and/or Wetting apents Retention Count/min. per Sq. Cm. of surface Grams deposited for entire mature apple tree* Dupont S.S. 2 oz./lOO gal, 1.68 Methocel 1*000 2 oz./lOO gal. 1*.33 232 6 Methocel 1*000 1* lbs./lOO gal. 3.99 211*.38 Methocel 1$00 1* lbs./lOO pal. 3.18 170.86 90.26 .$ ♦Computed on the basis of the $ percent solutions used for these tests and the surface area of a 25-year old McIntosh aople tree as determined ■ ’n experiment I. sticking atrent, but increasing the amount of sticking agent did not result in a corresponding increase in the amount of phosphoric acid retained on the stem section. Experiment III Object: To determine the tolerance of McIntosh apple and Montmorency cherry trees when dormant and when in the green tip stage to sprays of mineral nutrients at different concentrations. Materials and Methods: One year-old Montmorency cherry trees (KnightEowd strain) and 2-year-old McIntosh aoole tre*s were obtained from the Greening Nurseries of Monroe, Michigan, in February, 195>2, for tiiis experiment. The trees were nlanted in lard cans which held ap­ proximately one cubic foot of coarse quartz sand. Tap water alone, without any added nutrient material, was supplied to the plants. Solutions or slurries of calcium chloride, phosphoric acid, p o ­ tassium nitrate, NuGreen (urea), and a 20-20-20 fertilizer were pre­ pared at concentrations of 2, U, 8, 16, and 32 percent by weight and sprayed onto the plants with a small hand sprayer until the plants were thoroughly wet with the solutions. Potassium nitrate was not used in Part 1 of his experiment but it was used in Parts 2, 3, and U because a peculiar marginal chlorosis developed on the trees sprayed with NuGreen and the 20—20—20 fertilizer which both contain urea ni­ trogen. Two trees were sprayed at each concentration of the five materials and two trees, which were not sprayed, served as check trees for each part of the experiment. This experiment was divided into four parts: Part 1- Dormant sprays on cherries, Part 2- Greentip sprays on cherries, Part 3Dormant sprays apples, and Part h- Greentip sprays on apples. Cherry trees, whi^h were sprayed in he dormant state, were planted, and were sorayed February 28 in the greenhouse. Cherry trees, which were sprayed in the greentip stare, were planted March 28. These trees remained outside the greenhouse until April 11th, when they were sprayed and then were taken inside the rr» nhouse. Apple trees, which were sprayed in the dormant state, were planted and were sprayed March 26th. On March 27th these trees were moved to an unheated building for 10 days. Acple trees, which were sprayed in the green- tir stage, wer» planted March 2cth and then were left 10 days out-ofdoors. The gr-entio state of bud growth was net reached till after the trees had been in the greenhouse seven days. Results: The effects of the different materials on the development of the trees in each of the four parts of this experiment will be presented separately. Part 1 Dormant Spray on Cherry Inhibition of the rate of development of the buds was shown by trees snrayed with 2-, U-, and 8—percent s 'xutions of calcium chloride* The inhibition had been overcome by the time final observations were made four w»eks after planting and these plants were average in de­ velopment. The two highest concentrations of calcium chloride killed seme buds and caused Inhibition of others. Approximately 20 percent of the buds sprayed with the 32-percent solution were dead and another 35 percent were not growing but were still rreen Inside when cut open* Approximately 5 percent of the buds, most of which were terminal, were killed by the l6-percent solution while another 10 percent of the buds acpeared to be inhibited* No killing of the buds was noted with any of the concentrations cf NuOreen used. The 16- and 32-percent solutions did cause a white or yellow marginal chlorosis of the leaves of the developing shoots* No »ffect on the development of the buds was noted when concen­ trations of phosphoric acid 8-percent or less were sprayed on the trees 3niy a few lateral buds were destroyed by the 16-percent solution. The 32-percent solution of nhosohoric acid proved to be the most toxic s lution used in part 1 of this »xreriment as 75 percent of the buds sprayed with it were killed. The lateral buds were destroyed by this s lution in contrast to the terminal buds which were destroyed by the calcium chloride solutions. Sprays of 20-20-20 fertiliser produced the same results as those described for the NuOreen* Part 2 Oreen Tip Spray on Cherry Calcium chloride caused no inhibition of the rate of development of the buds when used at a concentration of 8 percent or less* At first all the buds appeared to have been destroyed by the 32-percent solution and only one bud on each tree sprayed with the l6-percent solution was growing. Later three buds developed on each tree sprayed with the 32-percent solution while only one bud developed on one of the trees sprayed with 16 percent calcium chloride* 29 NuGreen had no effect on the trees which were sprayed with a sol­ ution of 8 percent or less# About SO percent of th<» buds were destroy­ ed by the 32-percent solution. Marginal chlorosis started toappear 18 days aft«»r soraying with the 16— and 32-percent solutions# Phosphoric acid at concentrations up to 8 percent did appear to be toxic# At 16 percent about $0 percent of the buds were killed and at 32 percent all buds were killed althourh one shoot was produced from an adventitious or latent bud of one of the trees before the final measurements were taken. Potassium nitrate did not cause injury at any of the concentra­ tions used in this experiment# Nc ^njury in the form of bud killing was found but marginal chloro­ sis of the leaves occurred following the application of the 16 - and 32- percent concentrations of the 20-20-20 fertilizer# Leaves of shoots, affected by chlorosis, were painted with 0*3— percent solutions of manganese chloride, magnesium sulfate, magnesium nitrate, potassium sulfate, and potassium chloride 21 days after they had been sprayed, to find whether a deficiency of magnesium, manganese, or potassium was responsible for the chlorosis. Each solution was painted on all leaves of a single shoot but no recovery was noted on subsequent observations* Part 3 Dormant Spray on Appla While the apples, which had been sprayed in the dormant condi­ tion, were being held in the unheated storage building, it was ob­ served that certain of the spray materials were sufficiently hygro­ scopic to accumulate water which ran down the stems# Calcium chloride, 30 phosphoric acid, NuOreen, and to a certain extent 20-20-20 showed this property while potassium nitrate apparently was not hygroscopic. The effects of the materials durirg th* growing season are described as follows: Calcium chloride had no inhibiting *ff*ct at concentrations up to 8 percent. However the terminal buds of the trees sprayed with the 16— and 32-percent solutions did not develop, NuOreen had no effect when applied at a concentration of 8 percent or less. r Some buds were killed by the 16-percent concentration and ost buds by the 32-percent- concentration. No chlorosis was noted following application of any concentration of NuGreen to the apple trees. Some lateral buds were destroyed oy the 8-percent solution of nhosohoric acid but none were injured by the 2- or li-percent solutions, 4t first only on* bud developed on one tree and two on the other spray­ ed with 16—percent nhosohoric acid. tively were produced by the trees. Later two and four shoots respec­ These shoots and those produced by the trees sprayed with 32-percent solution, which had appeared to be dead, were of adventitious origin. No effect on the development of the shoots was noted following the use of notassium nitrate at any concentration. Sprays of 20-20-20 fertiliser of a concentration of 8 percent or less had no effect on the growth of the apple trees. At 16 percent some of the lateral buds failed to develop and most of the growth was confined to term*nal regions of the shoots. Host of the lateral branch- 31 es of one plant were killed by the 32-percent solution while most of the buds were killed on the other plant but in a more scattered manner* Part 2* Green Tip Spray on Apple The effects of the materials during the growing season are de­ scribed as fallows: Calcium chloride at 8 percent killed some of the buds but no effect was noted at the lower concentrations. Only a comparatively f-w buds developed on trees sprayed with the l6-percent solution. The trees sprayed with the 32—percent calcium chloride at first ap­ peared to be dead but later several buds on each tree developed. NuGreen and 20-20-20 sprays were without effect. Phosphoric acid solutions of 8 percent or less concentration had no effect on the growth of the plants. About $0 percent of the buds were killed by the l6-peroent solution; all were lat*ral buds. Both tr*es sprayed with 32-percent phosphoric acid at first appeared to be dead but later thr*e adventitious buds produced shoots. Potassium nitrate snrays had no effect on the development of the plants at any concentration. Entry of Mineral Nutrients Applied to Bark of Llmba and Branches during the Dormant Season Experiment IV Object: To determine whether potassium from potassium carbonate enters the plant when applied to the bark oflimbs and branches of an apple tree during the late winter. Materials and Methods: A 0.3 percent solution of potassium carbon­ ate to wh ch had been added Triton B-1956 at a concentration of 0.1 percent was used. The activity of the K**2 was approximately 2 micro- curies per milliliter at the time of treatment on February 6, 19$1« Potted 2-year-old McIntosh apple trees growing in the greenhousa and mature dormant Rhode Island Greening apple trees in the orchard were treated with this solution. The application was made at selected positions on the t r ^ s by wrapping cotton gauze around the limb and then saturating the gauze. A U-inch band of gauze was wrapped around a lateral limb of one of the young McIntosh trees and around the trunk of the other 6 inches above the soil line. After 28 hours, samples * were taken from the trees. On the dormant Rhode Island Greening trees in the orchard, the solution was painted directly on 1- and 2-year bark as well as ont# a 6-inch cotton gauze band which had been wrapped around a 10-year-old limb. As a precaution, a dam of cheese cloth was placed at the base of the 2-year-old shoot growth to prevent any contamination by the solution beyond this point. Sampling of the dormant wood in the field was done after 2U and U8 hours, at erch harvest one limb was sawed off and sections removed both at 6 and 18 inches above and below the pause* Sections were re­ moved 6 and 18 inches basipetal to the treated 2-year-old shoots at 2k hours and again U8 hours after treatment. The samples at the various distances were divided into bark (phloem and periderm) and wood (xylem) as nearly as was possible* These were olaced in crucibles and ashed in the muffle furnace at 600°C, The sample was counted directly in the crucible with a Tracerlab Autoscaler* P.esuits; The amount of radioactivity found in the untreated portions of the young trees in the greenhouse was small but in general it was greater than twice the radioretive count of background. However, samdes taken 6 inches below the treated area on the lateral limb and also root samples from this same tree did not contain this amount of radioactive material* All samples from the tree, where the solution was applied 6 inches above the soil line, were above twice background in radioactive count. Greater radioactivity was found in the bark samnles than in the wood smnoles, except in the roots where the wood contained more radioactivity than the bark. The amount of radioactivity found in the tree in the orchard was small as it was not large enough in most samples to produce a radio­ active count twice that of background, in general greater radi oactivity was found in the bark tissue than in the wood portions. The only counts recorded in the 10-year-old limbs, which were twice that of background after 2h hour?;, were samples of the bark and wood in the treated area. The samole of bark 6 inches above the treated area as well as the bark and wood in the treated area of the 1*8—hour treatment of the 10—year-old limb showed radioactive counts over twice that of background. The amount of radioactivity found in the 3—year-old por­ tion of the limbs 6 and 18 inches below the treated 2-year-old limbs was not large enough to produce a radioactive count of twi^c back­ ground. IShilc the radioactive cotint levels were too low to be con­ sidered positive, a definite trend towards a higher count value was noticed for the bark sample than for the corresponding wood sample. Experiment V Object: To determine the value of a lime slurry as a means of in­ creasing entry of potassium carbonate through the bark of apple and peach trees. Materials and Methods: A stock solution of ft** potassium carbonate was made up by adding 11 grams of water. potassium carbonate to 3 liters of The stock solution was divided into two portions: agricultural lime was added to one oortion to make a slurry and Dreft was added to the other nortion to improve the wetting properties of the solution. These solutions were applied to the limbs with a paint brush. Six uniform 8— to 10-year-old limbs of~Sfouth Haven peach and four sim'lar limbs of Rhode Island Greening apple tree were used, half the number fceinr used with each material applied. The treatments were made to the limbs at U P.M. on April 3# 1951* The limbs were cut from the trees after 23 hours and thin longitudinal sections were sawed through area which had both treated and untreated bark. After these sections were dried, autoradiograms were made from them. Trimmings from the pieces of the limts from which the thin longitudinal sections were cut were ashed and the radioactive count was measured. Results: No potassium was found either in the portions of the wood which were ashed and cotmted or which were used for autoradiograms. A clear line of radioactive material was shown by the autoradiograms to be present on the epidermis but none was evident in the area of the nhloeril. However, these results may be questionable because the radioactivity of the samcle may have dropped below a detectable level due to the short half life of K ^ . Kxperiment VI Object: To determine the relation between concentration of phosphoric acid solution and the rate of entry into 1-year-old peach shoots in early winter. Materials and Methods: Three concentrations of phosphoric acid, 3, 20 and 80 milligrams per milliliter with the same relative specific ac­ tivity of 0.21 microcuries ner milligram of phosphorus were applied to 1-year-old Halehaven oeach limbs on December 16, 19^2. The amounts of P32 per milliliter of solution were 2.0, 13*3, and 93.5 microcuries per milliliter, respectively. Vertical limbs about 2 feet long were selected for this treatment. In order that transloc-ition within the limb could be studied without TABLE VI THE TNFT IINCE O'^ C GNOENTR;VfTON UPON ENTRY OF P^2 PHOSPHORIC ACID INTO 1-YEAR-OLD PEACH LIMBS PUPTNO DECEMBER AS SHOWN BY RADIO— ACTIVITY FOUND : IN THE TREATED AND UNTREATED PORTIONS OF THE LIMBS 8 Hours Dry WT. Radioactivity per 1 gram Dry Wt. (Gms.) (Count/min.) ll* Days Dry Wt. Radi oactivity per 1 gram Dry Wt. (Gms.) (Count/min.) 0.3$ A Autoradi ogram Shavings Treated Portion 1.UU25 U.U623 20.1* Hi,091*.0 1.1025 9.9589 3.8 2,172.9 0.3$ B Autoradiogram LShavings Treated Portion 1.0362 3.7726 20.8 20,652.0 1.1686 U.6201 16.3 7,019.7 3.1939 5.9 925.8 1.01*72 3.7779 I*.7 82.9 2.0$ B Autoradiogram Shavings Treated Portion 1.1691 3.9278 7.0 61*9.2 1.011*1 3.3627 8.1* 268.9 8.0$ A Autoradiogram Shavings Treated Portion 1.1*602 5.1*730 9.2 35,571*.0 0.8532 5.1*1*38 130.9 11*,260.3 8.0$ B Autoradiogram Shavings Treated Portion 1.0661 U.37SU 58.1** 39,282.0 1.0167 5.11*56 33.2 13,293.0 Samole 2.0$ A Autoradiogram Shavings Treated Portion 1.0006 Check A Untreated Limbs 1.1*123 Check B Untreated Limbs 1.186$ 3.5 9.6 1/100 ml. of the 3 mg/ml treating solution 1/100 ml. of the 20 mg/ml treating solution 1/100 ml. of the 80 mg/ml treating solution ■^Contaminated 723.0 1*,900.0 17,652.0 interfering contamination, only the basal 10 inches of the limbs were painted with the solutions. Four limbs were used for each concentration, two beinr cut after 8 hours and the other two after ll* days. The weather during the 8- hour period was clear with a low temperature of I*2°F. Rain did not fall dur*ng the lU day period until the evening of the fourth day after which some precipitation fell for the followinr 6 days. This was followed by clear weather until the limbs were cut. After removing the shoots from the tree, a l/l6—inch section was removed from the terminal 9 inches to make autoradiograms. The pieces that were trimmed off the shoots were dried, ashed, and counted, and were designated ”autoradiogram shavings”. Results: Radioactive count in treated and untreated portions of peach shoots shown in Table VI indicated that very little entry occurs in the late fall or early w ;nter regardless of concentration of acid. A and B in Table VI refer to reolicate limbs for each concentration. The autographs made of the 8-hour treatment showed no evidence of exposure to radioactive material except for the base of the 8—h mr B treatment. However, a faint image was made on all the films exposed to the lU—day treatment with the strongest image being found with the 8 percent treatments. Experiment VII Object: To determine the accumulation of P^2 at 6-, 21*— > and l*8-hour intervals following a bark application of haven peach trees. phosphoric acid to Hale— 37 Materials and Methods: Nine horizontal limbs, from which vertical branches arose, were selected on nine U-year-old Halehaven peach trees. A 0.3 percent solution of phosphoric acid, with Dreft as a wetting agent, was applied to the horizontal branches only. The solution froze to the limbs as it was applied with a paint brush at 10 A.M. on March 21, 1951. The vertical limbs were cut at 6, 2h, and U8 hours after treatment and examined to determine whether any radioactive phosphorus had been translocated into them from the application to the horizontal limbs. The vertical branches were separated ■’ "nto untreated buds, xylem, and wood, and the horizontal limbs into treated buds, xylem, and wood. Intact stem sections were designated "wood” while alternate sections which were scraped to remove the rhloem tissues were designated "xylem”. Results* The results are riven in Table VII. To calculate the figure for the total solution applied, the radioactive count from each hori­ zontal limb and attached vertical branch were totaled and were con­ verted to milliliters of solution. To find the radioactivity of the discarded phloem, it was assumed that this tissue had the same activity as the corresponding wood sections minus the activity of the xylem sections. The percent of material translocated was determined dividing the total activity found in t.he plant into the activity found in the untreated l-*mb. Table VII shows that six hours after application of P32 phosphoric acid to horizontal limbs, radioactivity of 169.5 to 222 counts per TABLE VII MIGRATION OP' RADIO ’DSrIMRUS APPLIED To DCK'ANT HORIZONTAL BRANCHES INTO ATTACHED VERTICAL LIMB? OF THE PEACH AT 6, 2i|, Aim U8 HOURS AFTER APPLICATION Time of Harvest Tree 1 MI. of Radio­ phosphorus Solution Applied 6 Hours Tree 2 Tree 3 3.^3 0.60 Tree 1 1.76 1.78 13,987.3 l6,53h.2 9,385.7 29,613.3 8,996.8 U,887.1 Hi,791.5 Radio­ activity Translocated Count/min. 187.I 222.0 169.5 138.9 25ii,5 77.7 117.1 1I16.9 Percent of Radio­ activity Trans­ located 1.3li 1.3U 1.81 2.83 1.59 0.79 0.85 0.U7 1.10 Tree 3 liS Hours Tree 2 1.50 Radio­ activity of Solution Applied Count/min. 1.01 Tree 1 2li Hours Tree 2 2.05 Tree 3 1*63 17,311.3 13,698.8 l56.Ji l.lli 38 minute war detected in the 12- to 2l*-inch vertical branches which arose from the treated horizontal limbs. Only one limb of the three cut 21* hours after treatment and no limbs cut 1*8 hours after treatment showed a radioactive count hicher than the minimum count found 6 hours after treatment. Possibly injury in the treated area, which permitted greater entry, had occurred in the one limb cut 21* h>urs after treat­ ment whi.rh showed a radioactive count of 25U.S counts per minute. A possible explanation for the lower radioactive counts found in the untreated vertical limbs at 21+ and 1*8 hours after treatment than were found 6 hours after treatment, may be that equilibrium had not been reached 6 hours after treatment between the count of phosphorus which was entering the vertical limb from the treated horizontal limb and th* amount whi -'h was being translocated to other parts of the olants. Experiment VIII Obiect: To determine the accumulation of rhosnh irus within 8— to 10— year-old peach limbs after 6, plication of 1 6 , 21*, 1*1*, and 72 hours following ap­ phosphoric acid to cotton gauze wrapped around the Admbs in early spring. Materials and Methods: A solution of phosphoric acid contained O.Ol* microcuries ner milligram of ohosohorus was applied April 19 to limbs of Pouth Haven peach trees 1 1/2 to 2 inches in diameter. A continu­ ous supply of phosphorus was provided on the surface of the limbs by using several thicknesses of cotton gauze, which were wrapped around the limbs in a band U to 5 Inches wide. The gauze was held in place by waterproof tape which also prevented movement of the solution beyond the cause. 39 Longitudinal sections 1/16- to l/8-inch thick were cut from two limbs at each of the following time intervals of 6, 72 hours, using an electric table saw. 16 , 2i*, Ul*, and These sections were placed on botanical drying paper and dried between hot steel plates. The outer bark war removed on all but one of the sections at each harvest so that an indication of the amount of P*32 ^ which had been applied to the bark and also the amount of P^2 which had entered the limbs could be seen in the autorad5ograms made from the limbs. The solution with the exception of the 6-hour samples, which were treated at 9 A.M. of April 20th, was applied at 1**30 P.M. on April 19th. The weather at the time of treatment showed broken clouds and a tem­ perature of 55°F. on Aoril 20th. Limbs for the 16-hour samples were cut at 8*30 A.M. The 2l*-hour samples were cut at 1**30 P.M. on April 20th. During the day, the weather was clear and the temperature was 60°F. limbs were cut at 1*1* hours or at 12*30 P.M. on April 21st. Two Final samples were sectioned at 5*00 F.M. on April 22nd after a night of heavy rain. Results* Activity in the phloem area was seen in the autoradiograms, with the heaviest line at 6 and 16 hours. The density of the line de­ creased with time so that at 72.5 hours no activity was visible in the phloem tissue. From this information, it would seem that after the initial entry, the phosphorus is moved to other parts of the plant* Experiment IX Object* To determine whether P-^ phosphoric acid from a bark applica­ tion would enter dormant 3-year-old apple limbs in February* Materials and Methods: Fifteen vertical limbs of a WinesaD apple tree, which were 3 years old and 8 feet tall, were used in this experiment, which w^s started February 2ht 19!?3. They were cut from the tree, all the side limbs removed, and 5 layers of pause 3 inches wide wrapped around the limb 18 inches, from the base. The limbs were laid hori­ zontally over saw horses and 5 milliliters of a solution, which con­ tained 0.10 microcurie per milligram of phosphorus, were pipetted onto the rauze. A piece of plastic was fastened over the gauze with masking tape to prevent drying. After all the limbs had been treated, the base of each was slashed and placed in a bucket of water. The treatments were held inside an unheated storage building on the college farm where no freezing occurred until the> time that the 96-hour samples were taken. At time intervals of 12, 2U, U8, 96, and 192 hours, three limbs were removed from the bucket and samnles l/li-inch long ware removed at 3, 6, and 12 inches below and 3 and 12 inches above the treated area. Because of the smaller diameter of the wood, a 1-inch section was used at U8 inches above the treated area. Results: Rather high values of radioactive count were found in the samples immediately above and below the treated area, however the variability between samples was great within the limb replications. For the 12-hour samples, this statement is not true as no treatment gave a radioactive count exceeding twice background* The most interesting fact is that the average minimum count values, although low, showed an increasing value at each successive 1*1 harvest. However the only time at which the minimum amount of radio­ activity, usually the sample 1*8 inches above the treated area, exceeded twice the rad-’nactive count of background was at 192 h >urs after treat­ ment. Intake and movement of a large amount of phosphorus over ll* inches ’n a basal direction can be di scounted in th* s experiment. Only a small amount of radioactivity was found in the water in wh-ich the shoots wer^ rlaced during the experiment. Experiment X Object: To determine whether pruning wounds in the treated area affect the rate of entry of phosshorus from P^ phosphoric acid as well as to observe the effect of sucrose on the rate of entry of phosphorus from nhosphoric acid. Materials and Methods: This experiment, which was started April 23, 19!?3, usinc a phosphoric acid solution containing 0.06 microcurie per milligram of nhosnhorus, followed the same experimental procedure as Experiment IX with some modif cations. years 'Id were used. Black Twig apple limbs 3 to 5 Since the distribution of ohosphorus within the limb was the main consideration, only one period of sampling, 1*8 hours, was used. cations. This made It possible to use an increased number of repli­ A side shoot or spur was removed from the area which was to be treated of half the limbs. Sucrose was added to part of the orig­ inal s lution to make a £ percent solution. Each solution was pipetted onto cotton gause wrapped around the limbs on four of the eight limbs. Each limb was placed upright in a bucket of water immediately after treatment, so as to prevent contamination by horizontal spread of the solution. 1x2 Results: The levels of radioactivity in this experiment were much lower than those encountered in the previous experiment, but the dis­ tribution appeared to follow the same trend. The greatest amount of radioactivity was found 3 inches above the treated area where 6 of the 8 limbs had an amount of radioactivity preater than twice the back­ ground count level. On the other hand, at 3 inches below, only 2 of the 8 limbs had an amount of radioactivity preater than twice that of background and in one of the limbs this radioactivity was due to sur­ face contamination. The other larger amount of radioactivity was on one of the injured limbs which in general travc evidence of preater entry. Sucrose did not seem either to increase or decrease entry of chosnhoric acid. However, the values recorded are much too close to background to be regarded as conclusive. m Spring Growinc Season Treatments Experiment XI Object: To determine whether movement of phosphorus from bark appli­ cations of nhosphorir acid will take place at the time of bud swell. Materials and Methods: The basal foot, of eight intact 2-year-old Halehaven peach shoots approximately 2 foot long, was painted with phosphoric acid solutions which contained O.OU microcurie per milli­ gram of nhoschorus on April 3, 1953. Four limbs were painted with a solution containing phoschoric acid only and four with a solution con­ taining phoschoric acid plus 5 percent sucrose. The temperature war about 50°F. during the 2U-hour period allowed for absorption. Rain fell after the solution had been in place for 7 hours. When the limbs were cut, the basal or treated portion of the limb was separated from the apical or untreated portion. The two portions of each limb were arhed then counted separately. Results: An amount of radioactivity greater than twice the background level was found in the untreated portion of only two of the eight limbs, both these limbs had been treated with the solution containing no sucrose. A11 the radioactive count values were lower on the treated portions of the limbs when sucrose was included in the solution. Experiment XXI Object: To determine whether increased intake of phosphorus from phosphoric acid occurs when the bark of an apple shoot is scraped. Materials and Methods^ The bark of 1-year-old water sprouts of Ked Astrachan aople was scraped lightly with a knife, co as to roughen the bark but not remove much of it. The basal 8 inches of four of the shoots were so scraped, but four others were left untouched. On April ?3, 1953, all were painted with a solution of phosphoric acid containing 0.10 microcurie per milligram of phosphorus. The solution dried rapid­ ly and twenty-four hours after application, rain fell for I* hours. forty-eight hours after application samples were collected, the shoots being cut 2 inches above the treated area in order to avoid possible contamination. The swelling buds were removed from the un­ treated portion and were considered as a separate sample for counting purposes. Results: Only two of the e;rht samples from the untreated portion gave counts twice that of backgr und and they were buds from scraped limbs. Approximately five times as much "hosphorus remained on the scraped shoots ns on the unscraped, suggesting th-t the greater entry into scraned shoots may have been due in part to the ,reater amount of solution retained by the ccmred bark. Experiment XIII Object: To determine whether phosphoric acid will enter the tomato plant from applications made to the sides of the stem. Part 1 Materials and Methods: Seeds of Michigan State Forcing variety of tomato were planted September 23, 1952, and the seedlings transplanted to 3-inch pots on October 30, and to 12-inch pots on November 28. In a preliminary experiment, intake from a continuously moist source of phosphoric acid was compared to a single application by brush. This treatment, which ran from December It to 8, utilized a phosphoric acid solution which on November 12, 19^2, had an activi ty of 2 microcuries per milliliter of P-^. A 1 1/2—inch band of solution was painted onto one plant, while a 1-inch gauze which was soaked in solution was wrapped ar und the stem of another. A piece of plastic film prevented the gauze from drying during the experiment. The two plants were cut into sections consisting of a piece of the stem and an attached leaf. weight determined for erch. These sections were dried and the dry Each section was ground to a fine powder v.ith a mortar and pestle before the radioactivity in each section was determined on December 17. Results: The entry of radiophosphorus as indicated by counts per minute from VTII. in different parts of the plants is given in Table The greatest concentration of nhosphorus appeared to be in the youngest leaf tissue, as mi^ht be expected from previous experiments with tree fruits. The amount that entered the plant was much greater from the gauze treatment than from the single brush application. How­ ever, the percent of the total phosphorus that was translocated was less from the gauze treatment than from the single brush application* Only 21+.1 percent was translocated during the U day period when applied in rauze as comoared to 31.U percent translocated from a single brush application. TABLE V I I I DISTRIBUTION 0* RAPTOP'tOSPHORUS IN TCUATO PLANTS FOLLOWING THE APPLICATION OF PHOSPHORIC AOIF SOLUTION TO THE STEM BY BRUSH AND IN COTTON GAUZE AS INDICATED BY COIfNTS PER MINUTE Gauze Areplication nadioactivity Dry ift. (Gms.) 1 Cm. Dry Wt* (Counts/min.) Location of Plant Part Brush Application Dry Wt. Radioactivity 1 Gm. Dry Wt. (Gms.) (Counts/min.) Tip .151*9 739.2 .1665 9,1*93.7 Leaf 5 above .2257 361.1 .2552 1*,367.9 Leaf I* above .2870 30U.7 .3900 2,637.7 Leaf 3 above .3071 135.5 .5030 1,031.2 Leaf 2 above .3321 150.9 .5333 993.2 Leaf 1 above .3859 132.9 .1*185 933.6 Treated Stem .1019 11,185.5 .2228 83,337.9 .3609 1*5,269.3 .2755 689.7 Treated Gauze Leaf 1 below .3658 117.6 - Leaf 2 below .3353 110.6 .21*67 971.2 Roots .31*50 100.9 .1*303 1,1*79.7 The number of radioactive counts per minute measured in 1/200 of a milliliter of the treating solution was 21*6.8. Part 2 Materials and Methods: Aft~r December 20, only distilled water was supplied to the tomato plants so that flowering would be induced. On January lli, 1953» flowers of six plants were sufficiently developed to set fruit artificially when the flowers were dipped into a solution of 50 ppm of alpha (p-chlorophenoxy)-propionic acid. On January 18, two plants were moved into each of three greenhouse rooms, which were held at 50°, 65°, and 80°F. constant temperatures* One day was allowed for the plants to become acclimated to the new environment before the plants were treated by the gauze method with a phosphoric acid solution which contained 0.21 microcurie per milligram of ohosphorus. A 6-inch oiece of gauze, 1—inch wide, was wrapped around the stem of each plant 17 inches below the developing fruit. Fifteen drops, which was approximately 3/h of a milliliter, .of the solution were applied to each gauze. gauze to prevent drying of the solution. Plastic film covered the An additional piece of cotton was placed at the basal end of the plastic to absorb any excess solution. Entry of phosohorus from nto the plant was determined by the activity which was found in the developing fruit. To obtain a record of the rate of entry of phosphorus into the fruit, a Gieger—Muller tube was placed next to the fruit. The radiation pulses received by the Gieger-Muller tube were recorded by an Esterline Angus chart recorder which was attached to a Tracerlab count rate meter according to the method described by Hinsvark, Tukey, and Wittwer (31)* A, 1*7 record of the temperatures in each room was obtained by means of thermographs* At each temperature, the largest fruit was placed next to the Gieger-Muller tube to obtain a continuous record of the amount of radioactivity in the fruit. and than dried. After four days, all fruit were picked The dried fruits were pulverized with a mortar and pestle, then a record of the actual counts per minute per gram of dried fruit tissue was obtai ned with a Tracerlab Autoscaler* Results: Intake of P ^ into the developing tomato fruit following a stem application of pendant on temperature. phosphoric acid in gauze was found to be de­ The results expressed on the basis of 1 gram of dried plant material in Table IX show that 176 and 300 counts per minute were found in iruit grown at $0°, 572 and 663 counts per minute when grown at 65°, finally 657 and 738 counts per minute in fruit grown at 80°. Similar results were found by Hinsvcrk and flittwer (32) who were studying absorption o'1 phosphorus from foliage applications at these same temperatures* The size of fruit was influenced by the temperature. Large fruits 5.9 and U.l grams were produced at the high temperature, one large U.l grams and one smell fruit 0.5 grams were produced at the intermediate temperatures, and small fruit 1.2 and 0,3 grams were produced at the low temperature'* Experiment XIV Object: To determine whether a spray application of mineral nutrients applied to the bark can sunply a portion of the mineral requirements of developing shoots* TA B L K I X THE EFFECT OF TEMPERATURE OH THE MOVEMENT OF RADIOPHOSPHORUS INTO THE DEVELOPINO TOMATO FRHTT FROM PHOSPHORIC ACID APPLIED IN COTTON GAUZE TO THE STEM OF THE PLANTS AS INDICATED BY COUNTS PER ?.!TNUTE Temperature Green Wt. (Gma.) 50° 65° 80 ° Dry Wt. Total Radioactivity (Gms.)_____ (Counta/min.) Radioactivity 1 Gm. Dry Wt. (Counta/min.) 1.222 0.1028 30.8 300.0 0.276 0.0290 5.1 175.8 U. 137 0.3250 158.9 572.0 0.502 O.OU98 32.3 663.0 5.869 O.U127 30U.5 738.0 U.105 0.3029 197.8 657.0 1*8 Materials and Methods: Dorntnt 1-year-old Montmorency cherry trees and 2-year-old McIntosh ap; le trees from cold storage were planted in 12-inch pots filled with nuartz sand on June 11, 1952. These trees were sorayed June ll*th with a 2 percent solution of phosphoric acid which contained 0.22 microcurie per gram of phosphorus and which util­ ised Methoeel 1*000 c.p.s. as a sticking acent at the rate of 1/1* pound per 100 gallons of water. The sr>ray was applied at a pressure of 100 rounds per square inch with a Sure-Shot sprayer. Twenty trees of each variety were sprayed with the solution and four non-spray»d trees served as controls. Dormant 1-year-old Montmorency cherry trees and 2-year-old McIntosh apple trees from cold storage were planted in 12-inch pots filled with quartz sand on June 18th. These trees were sprayed June 19th with a 2 percent solution of calcium chloride which contained 0.2 0.20 microcurie per gram of calcium and which utilized Methocel 1*000 c.p.s. ac a sticking a rent at the rate of l/l* pound per 100 gallons of water. The spray was applied at a pressure of inch with a ^:ure-?hot snrayer. 60 pounds per square Sixteen trees of each variety were sprayed with the solution and four non-snrayed trees served as controls. As the trees were grown out-of-doors it was necersary to fasten "la-t-vc sheet 18 by 1*2 inches around the tree and around the pot to prevent the entry of rain water, which might carry same of the spray material into the root medium. A piece of cotton, which was placed under the plantic, absorbed any water which seeped between the plastic and the stem. A budding rubber held the plastic and cotton tightly to the stem. The plastic was fastened to the pot with twine, leaving a covered opening to permit watering of the plant. The bosic nutrient solution was 1/2 Hoagland's (33 )• Half of the tree^ of the phosphorus bark spray treatment received complete Hoag­ land's solution while the other half received a minus phosphorus Hoarland's solution. The same pattern was followed with the calcium treatments, half receiving complete solution and the other half minus calcium. On August 7, 195>2, all the new shoot growth was removed from each tree. After drying, these shoots were ground to 20 mesh in a Wiley mill. One gram of the dried material from each tree was ashed fol­ lowing the procedure described in the A.O.A.C. ( l) for calcium or phosnhorus analysis depending upon which spray the tree had received. Results: Numerical data from this experiment are shown in Tables X, XI, and XII. More new growth was produced by the trees receiving the complete nutrient solution than by those r^^eiving the deficient solutions. No valid count data were obtained from the phosohorus spray be­ cause the phosphorus passed through too many half-lives before analysis was made. Root initiation did not occur in many of the cherry trees, pos­ sibly beeause of the high temperatures at the time of planting. little new growth was made by the tops of these plants. data obtained were very variable. Very The count TABLE X SHOOT GRG.VTH PRODUCED BY AhPLE AND CHERRY TREES UNDER DIFFERENT NUTRIENT CONDITIONS FOLLOWING A DORMANT SPRAY 0* RADTOFHOSPHORUS OR RADTOCALCTUM Treatment (Dry weight in grams)* Apples Cherries -P Nutrient Solution 10.63 2.38 ■fP Nutrient Solution 1L.66 3.03 -P Nutrient Solution 8.09 1.52 •fP Nutrient Solution 7.57 1.82 -Ca Nutrient Solution 6.8U 1.51 ■fCa Nutrient Solution 9.01 2.35 -Ca Nutrient Solution 6.56 1.87 fCa Nutrient Solution 8.L8 2.17 Phosphorus Spray No Spray Calcium Spray No Spray Mean values for the treatments TABLE X I RADTOCALCIUM CONTENT OF APPLE SHOOTS GROWING ON TWO LEVELS OF CALCIUM NUTRITION PRODUCED FOLLOWING A DORMANT SPRAY OF CALCITM CHLORIDE AS INDICATED BY COUNTS PER MTNUTE Nutrient Level -Ca fCa Radioactivity Radioactivity 1 Gram Dry Wt* 1 Oram Dry Wt. (Counts/mln.) (Counts/min.) 1 672 31*8 2 838 281* 3 1*72 267 1* 602 21*1 5 561 230 6 1*1*5 318 7 275* 390 8 U73 753* Total 1*063 2078 580.1* Mean 296.! Differences necessary for significance: 5 percent level l8l,7 1 percent level ♦These values were not used in the statistical analysis 275.1 TABLE X I I ANALYSTS OF VARIANCE OF RADIOACTIVE COUNTS FROM RADIOCALCIUM IN APPLE SHOOTS PRODUCED BY TREES GROWING ON COMPLETE AND MINUS CALC TUT/ NUTRIENT SOLUTIONS FOLLOWING A DORMANT SPRAY OF CALCIUM CHLORIDE Sourc® of Variation Total Decrees of Freedom Sum of Squares Mean Square F 1U 1*19*039 Treatment 1 281,U3U 28l,U3l* 25,6^ Replication 6 60,631 10,105 0.919 Error 7 76,97k 10,996 A highly pirn’''leant difference was found in the radioactive "intent of the current season shoots of anrle tre«s which r^ce’ved comrlete nutrimt solution and those whJeh received minus ’a1~iun nutr’ent solution, more radioactive calcium beine found in the ”larrts which had received the minus calcium solution. Experiment XV Cbject: To d e t * m :.r.» whether mineral nutrients applied to semldormar.t sour o*” rry tree would induce a response in growth. Materials and ~^»t .ods; Two srrays, NuGreen and 15—3r:“l5 fertiliser, w°re supplied by spraying the solution at the tree and by spraying the solution at the ground. applied to each tree. An ecual am'unt of nitrogen, .07 pounds, was This amount, which is equivalent to 1/2 pound of sodium n ’trate, was contained in 1/2 gallon of spray, which the sprayer delivered in 2 seconds. The NuGreen spray contained 17 pounds of NuGreen in 50 gallons of water. The complete fertilizer spray was composed of 20 rounds of ronro 10-52-17 and 25 pounds of Rar>id-Gro 23-21-17 in 50 gallons of wa*»r. Siethocel 1*000 c.p.s. wrs used as a r ticking a~ent for both sprays at a rate of l/U pound per 100 gallons of water. Eighty Montmorency cherry trees, 5 years old, were used for this experiment which was started Aptil 18, 1952. At that time, the circum­ ferences of the trunks 1 foot above the ground were measured and re­ corded. The sprays were applied to the trees, which were in the green tip stage, on April 20th. TABLE X I I I TOTAL INCREASE IN CIRCT11FERFNCE IN CENTIMETERS OF FOUR TREE REPLICATIONS OF 5-YEAR-OLD MONTMORENCY CHERRY TREES FOLLOWING GROUND AND TREE APPLICATION OF NUTRIENTS Replication Check N-G N-S NPK-G NPK-S 1 15.5 16.1* 21.0 18.1 15.9 2 18.0 18.7 17.1 19.5 20.9 3 18.6 18.3 19.2 19.7 20.3 52.1 53.1* 57.3 57.3 57.1 Total Average per tree 1*.1*25 U.117 Diffprences necessary for sign!finance Footnote N-G - NuGreen sprayed on ground N-S - NuGreen sprayed on tree NPK-G — 15-30-15 sprayed on ground NPK-S - 15-30-15 sorayed on tree I*.775 •U.775 U.758 5 percent level - *275 Four spray treatments containing four trees each were replicated four times. The check treatment of four replications of four trees received no fertilizer. Results: The increase in circumference during a 1-year period was used for evaluation of this experiment. As one replication each in the NuGreen application to the ground and complete spray to the tree were very low as compared to the other replications, it was decided to run the analysis of variance on the three highest replications. As would be expected on trees that had not been fertilized pre­ viously, a sirnificatn response to fertilizer application, which is shown in Table XIII, was noted. However, the NuGreen ground treatment was significatnly lower than the check, partly because two repli­ cations made less than average growth. The important point is that no difference? in response were noted from the two methods of ap­ plication. Response was about the same whether the solution was sprayed at the tree or on the ground around the tree. Experiment XVI Object: To determine whether greater nutrient intake from an appli­ cation of phosphoric acid to the bark of twigs and branches occurs during the period of rapid growth than during the dormant period* Materials and Methods: Water sprouts and 10-year-old limbs of a single Jefferis apple tree were used in this experiment. With the water sprouts, four treatments were used, with two water sprouts per treatment. In the first treatment, the solution was painted directly 52 on the basal 8 inches of an undamaged water sorout. In the second treatment, the solution was painted on the basal 8 inches of a water after the bark had been scraved lightly with a knife. In the third treatment, the solution was applied to a 6-inch gauze wrapped around the basal 8 inches of on undamazed water sprout. In the fourth treat­ ment, the solution was applied to a 6-inch pause which had been wrapped around a sh >ot after the water snrout had been scraped. Plastic film was used to cover the pauze to prevent rapid drying. To study the movement of phosphorus into water sprouts following application to the bark of 1-year-old limbs from which the water sprouts arose, three treatments were used. In the first treatment, the solution was painted on the undamaged bark of a limb from which two water sprouts arose. In the second treatment, the solution was minted onto scraoed bark from which three water sprouts arose. In the third treatment, the solution was anplied to a piece of rauze 6 x 36 inches which had been wrapped around an area of the limb which had been scraoed. The treated area carried two water sprouts. A sheet of plastic film was used to cover the gauze to prevent rapid drying. A 0.3 percent solution of phosphoric acid containing 0.53 microcurie per milligram of phosphorus was applied to the limbs at noon on May 23rd. The water sprouts were removed for sampling 1*8 hours later. No rain fell during the period. Current seasons shoots (leaves and stem) and the untreated portion of the previous seasons shoot (stem) were collected from each water sprout for sampling. Water sprouts that had been treated directly 53 were cut two inches above the treated area while shoots that had been growing in a treated area of older bark were cut one inch above the treated area. After drying, the samples were ashed according to the procedure outlined for phosphorus analysis in the A.O.A.C. (1). Radioactive counts were measured on the total ash of each sample on June 11th. The count recorded on June 11th for several of the samples was higher than the mechanical ability of the scaler to record efficiently. These samples were counted ars^n on July 6, 1953, after being diliited 1 to 20. Total phos'horus in each sample was determined "’ccord'inr to the micro method given in A.C.A.C. ( 1 ) on June 30th. Results: The numerical data obtained in this experiment is presented in Tables XIV, XV, XVI, and XVII. A greater amount of rndiophosphorur entered limbs when the bark was scraped lightly, when cotton rauze saturated with 32 phosphoric acid solution was wrapped around the limbs, and when cotton gauze saturated with phosphoric acid solution was wrapped around lightly scraced limbs than when the solution was applied to a limb with intact bark. When the radioactive counts found in current seasons growth of the various treatments were compared to those found in the water sprouts with intact bark (195—339 counts/min.), a 10-fold increase was noted with scraped berk (2,086-5,li37 counts/min.), a 30-fold in­ crease followinr absorption from gauze over intact bark (7,£0U-10,9<5l T A B L E X IV PHOSPHORUS *NB RADIOPHOSPHORUS CONTENT OF CURRENT SEASONS GROWTH (LEAVES ANT STEMS) OF WATER SPROUTS OF APPLE FOLLOWING APPLICATION OF PHOSPHORIC ACID TO THE PASAL 8 INCHES OF PARK 0* THE WATER SPROUTS Replication Total Dry Wt. of Leaves and Stems (Gms.) Radioactivity Per 1 gram Dry Wt. of Leaves and Stems (Counts/min.) Total Phosphorus per 1 gram dry wt. of Leaves and Stems (_M£s .) _ Solution Applied to Intact bark 1 2 1.6005 1.6875 339.0 19U.6 U.327 a .222 Solution Applied to Cotton Gauze over Intact Bark 1 2 1.1787 1.0113 7,8oU.2 1C,961.2 a .666 6.180 Solution Applied to Lightly Scraped Park 1 2 1.3868 1.U811 5,U37.8 2,086.2 a. 507 a. 6 9 2 Solution Applied to Cotton Gauze over Lightly Scraped Bark 1 2 0.9322 0.91U2 U23,815.0 368,177.0 ia.321 15.31a The phosphorus content of 1/100 milliliter of the treating solution was 0.508 milligrams and the radioactive count of this amount was 3 7 ,8 8 0 , 0 counts rer minute. T A B L E XV PHOSPHORUS and raptophosphonus content of current SEAS::MS CROVfTH (L AVES AMP STEMS) OF WATER SPROUTS OF APPLE FOLLOWING APPLICATION OF PHOSPHORIC ACID TO BARK OF ADJOINING 10-YEAR-OLD LTMbS Replication Total Dry Wt. of Leaves and Stems (Gms.) Radioactivity per 1 gram Dry Wt. of Leaves and Stems (Counts/min.) Total Phosphorus per 1 gram dry Wt. of Leaves and Stems (Mgs.) Solution Applied to Intact Bark 1 2 1.3300 0.9030 21.lt 17.2 3.308 3.267 Solution Applied to Heavily Scraped Bark 1 2 3 0.8955 1.1»789 l.lliOO 11*5.5 751*.1* 917.9 3.238 3.01*3 3.1*91 Solution Apllied to Cotton Gauze Over Heavily Scraped Bark 1 2 1.6831* 1.3532 80,511.0 62,716.0 8.091* 6.836 The phosphorus content of 1/100 milliliter of the treating solution was 0.508 millirrams and the radioactive count of this amount was 37*880.0 counts per minute. TABLE XVI PHOSPHORUS AND R<‘DTOrlK^PP.iORlIS CONTENT OF PREVIOUS SEASONS GROWTH (WOODY STEM) OF WATER SPROUTS OF APPI.E FOLLOWING APPLICATION OF PHOSPHORIC ACID TO THE BASAL 8 INCHES OF BARK OF THE WATER SPROUTS Replication Total Dry Wt. of Woody Stem (Gms. ) Radioactivity Per 1 gram Dry Wt. of Woody Stem (Counts/min.) Total Phosphorus per 1 gram dry wt. of Woody Stem (Mgs.) Solution Applied to Intact Bark 1 2 2.6717 3.5557 83.7 50.2 1.192 1.159 Solution Applied to Cotton Gauze over Intact Bark 1 2 1.77U7 U.20U5 2,196.1 1,326.9 1.155 1.11*6 Solution Applied to Lightly Scraped Park 1 2 2.97U5 U.1C16 881.7 328.2 1.150 0.959 Solution Applied to Cotton Gauze over Lightly Scraped Bark 1 2 1.1096 2.1U03 15U,1*82.0 80,718.0 8.562 11*.601 The phosphorus content of the 1/100 milliliter of the treating solution was 0.508 milliprams and the radioactive count of the amount was 37,880.0 counts per minute* TABLE X V II PHOS PH'HUS ANT RAPTQPHOSPHORUS CONTENT OF PREVIOUS SEASONS GROWTH (WOODY STEM) OF WATER SPROUTS Ob AiTLF. FOLLOWING APPLIGATTON OF rHOSPHORIC ACID TO PARK OF ADJOINING 10YEAR—OLD LIMBS Replica tion Total Dry Wt. of Woody Stem (Gms.) Radioactivity per 1 gram Dry Wt. of Woody Stem (Counts/min.) Total Phosphorus per 1 gram Dry Wt. of Woody Stem (Mgs.) Solution Applied to Intact Bark 1 2 3.27W* 2.21(55 3.2 8.5 0.803 —— Solution Applied to Heavily Scraped Bark 1 2 3 2.1(200 U.hlhl U.6828 28.1 183.1 202.1 0.791 0.768 0.722 Solution Applied to Cotton Gauze Over Heavily Scraped Bark 1 2 5.0736 U.1297 8,336.0 5,881.0 1*375 1.170 The phosphorus content of the 1/100 milliliter of the treating solution was 0.508 milligrams and the radioactive count of this amount was 3 7 ,8 8 0 . 0 counts per minute. Sh count s/min.), and a 1000—fold increase following' absorption from pause over scraped bark (368,177-1*23,81? counts/min.). Tt thus appears that one of the limiting: factors in bark application is the small amount of material that is retained by the bark if an aqueous solution of mater­ ial is ncolied. Also, exposure of li’^'nr cells within and beneath the bark aids entry. It is interesting in this connection to observe that reports in old horticultural literature of beneficial results from nutrient applications to the woody portions of trees, usually stress both serening of the nr^a where the application is to be made, and application of the material in a thick slurry or paste. Drying of the leaves at the edges, shriveling of the bark, and p*nk discoloration of the phloem were observed in the shoots which were serened and then covered with gau?.e. This was possibly due to phosphorus toxi.ri.ty as shriveling and oink disc loration of the ohloem were obs^-ved in cxcr riment III when 2-year-old McIntosh anple trees were s rayed with rhos"hori o acid solutions at- c ~ncentrations ranging from 8 to 32 percent. More radioactive phosrhorus w:s found in the expanding shoots of the current season than in the noody shoots of the previous season. For example in Tables XIV and XVI , the amount of P^ absorbed through the intact bark of water sprouts gave 19? and 339 counts per minute in the current seasons growth (leaves and stem) and ?0 and 81* counts per minute in the previous se sons growth (stem) whereas the amount of absorbed through lightly scraped bark of water sprouts gave 2,086 and ?»1*38 counts per minute in the current seasons growth and 328 and 882 counts per minute in the previous seasons growth. As the current season shoots were still in active growth, more phosphorus metabolism could be exoected there than in the previous season shoots where only the cambial area was in active growth. Greater entry of radioactive phosphorus occurred following appli­ cation of the solution to the bark of water sprouts than occurred fol­ lowing application of the solution to the bark of 10-year-old limbs. Thus in Tables XV and XVI counts ner minute found in current seasons growth were l?5and 339 when the solution was brushed on the intact bark of watersprouts and 16 and 28 when the solution was brushed on the intact bark of a 10-year-old limb in an area on which two water r;.routs arose. Experiment XVII Object; To comnare the entry and subseauent movement of radiophos^horus noplied to leaves alone, bark alone, and leaves and bark to­ gether of young neach trees. Materials and Methods: Three 1—year-old Elberta peach trees growing in rots in the greenhouse, and two U-year-old Halehaven peach trees growing in the orchard were used. £t the time the experiment was begun, February 17, 1951, the leaves were starting to unfold on the trees in the greenhouse, /.n eoual amount of a phosphoric acid solu­ tion, which cont.a;ned 0*07 microcurie per milligram of ohosphorus, was used on each tree. In the greenhouse, the solution was applied to the expanding leaves of one tree, to the bark of another, and to both the leaves and bark of a third. greenhouse was U0°F, The night temperature of the 56 Tn the orchard, most of the solution was applied to the buds on the brsal cortion of 1-yecr-old twi^s. Th° temperature remained above freezing during the night and was in the UO's F. during the day. Results: No intake of radioactive phosphorus was observed from the application to the dormant buds of peach twigs out-of-doors. However, entry and movement occurred in some cases from applications to the trees growing in the greenhouse. Following an application to either the leaves and bark or to the bark alone, movement to other parts was detected* However, when the application was made solely to the ex­ panding leaver, no movement occurred away from this area, probably because rhosohorus was being metabolized ■>n the region of application. Experiment XVIII Object: To compare the retention of rad-> ophosphorus solution by ‘ leaves of aonle, peach, near, s">ur cherry, and swe^t cherry. Vat*-rials and Methods: One tree each of McIntosh apple, Flberta peach, Bartlett near, Montmorency sour cherry, and Windsor sweet cherry were used. The trees were actively growing in pots in the "rfpnhouse where they had V>een in active growth for approximately 3 weeks before the treatments were made. A solution of 0.3 percent phosphoric acid containing 0.11 microcurie rer milligram of nhorohorus and containing Dreft as a wetting agent was applied to one of the lower growing shoots of each plant on March 3, 1951. The entire shoot was submerged in a beaker of the radioactive solution after each potted tree had been laid horizontal on the greenhouse bench. The trees were left horizontal until the S7 anolied solution had dried on the shoots so that contamination of the surrounding limbs by lateral movement of the radioactive solution would be eliminated; because any excess solution on the horizontal trees dripped onto paper snreed on the greenhouse bench. After the solution dried, the trees were placed upright. Samples of treated and non-treated shoots, and new roots were collected 6 days after treatment and the radiophosphorus in each part was counted utilizing a Traoerlab Autoscaler. Thus, the total amount of nhosnhorur solution ai'olied and the amount translocated to the shoots and t- the roots vrere deterrrd ned. F>suits: Data obta’ned are shown in Tables XVIII and XIX. A relation «vas observed between adherence and retention of material and the Physical cjhcracters of the most Pubescence, leaves. Thus, apple leaves, which have the showed the greatest retention of the solution, whereas reach leaves, which are*glabrous, retained the least. Translocation of phosphorus was not correlated with the amount whieh had adhered to the leaves '•f the different fruits. The dvta show that the pear and sour cherry were more efficient in absorption as indicated by the percent of the solution translocated of that which was anplied. The data also indicate that there may be a difference in direction of movement of the absorbed Phosphorus in the different plants. On a unit weight basis, the radioactive count found in the tops of pear and sour cherry was greater than the anount found in the root tissue. In the apple, neach, and sweet cherry on a unit weirht basis, the greatest amount of radiorctive count from was found in TABLE X V I I I RETENTION OF iHOSFHORI : ACID BY SHoOTS OF APPLE, PEACH, PEAR, SOUR CHERRY, AND StfEST CHERRY !OLLCWINC PIPPING IN A SOLUTION CONTAINING RADTOPHOSPHORUS AS INDICATED BY COUNTS PER MINUTE Fresh Wt, of Shoot Growth (Gms,) Total Activity (Counts/min.) Amount of Solution (Ml.) Apple 3.655 131,532.9 2,91 Peach 3.593 28,59^.2 0.633 P^ar 1.389 39,332.0 0.87 Sour Cherry 3.761 UU,076.8 0.976 Sweet Cherry 7.573 63,661.8 1.1*1 TABLE X JX TRANS L O ’AT TON OF P'•' TOPM OS 11IORUS 'NTO UNTREATED SHOOTS AND ROOTS OF APPLE, PEACH, PEAR, SOUR CHERRY, AND SWEET CHERRY FOLLOWING DIPPING OF ONE LOWER SHOOT TH PHOSPHORIC ACID Radloantivity in Counts per Minute per 1 pram Fresh Tissue Shoot Root Total Percent P ^ Translocated to Shoots and Roots Ap^le 88.7 162.0 0.52 Peach 12.7 1*6.9 0.39 Pear 186.3 loh.o 2.65 fG'ur Cherry 206.3 3l.o 3.37 Sweet Cherry 12 .h 0.36 the roots. Thus the movement, after entry of into the more ef­ ficiently absorbinp plants, was apparently different than m efficiently absorbinp plants. the less 59 Summer Growing Sep son Treatments Experiment XIX Object: To determine whether greater entry of potassium occurred when the amount of potassium carbonate on the leaf was increased by the use of a lime slurry# Materials and Methods: The potassium solution was prepared by adding 11 grams of ’•'•otassium carbonate contrining to 3 liters of water# To half the solution, Dreft was added as a wetting agent and to the rest of the solution hydrated lime was added to form a slurry. Median leaves of McIntosh anole, “lberta peach, and Montmorency f.cur cherry shoots on trees growing in pots in the greenhouse were dicped into the solution using one tree of each variety. The pro­ cedure was repeated with the lime slurry using one tree of each variety. The dipping of the median leaves was done at 9 A.M. on April 1*, 19^1, and the shoots were cut after 7 hours. The shoots were dried under heat lamps for 16 hours aft^r harvest. Autoradiograms were made by exposing X-ray film W ’th these dried shoots. Results: Intake of with both carriers. by the actively growing apple shoots occurred The intake appeared to be slightly greater in the shoot dipped into the solution. Perhaps this occurred because the wetting agent in the solution wet the oubescent apple leaf more easily, whereas the slurry was prevented by the epidermal hairs from coming in contact with the epidermis of the leaf. Some injury to the leaves was noted where the slurry and leaf epidermis were in contact# Potassium 60 was found to be concentrated in the stem of the apple with a diffusion gradient extending in both directions from the treated leaves. Greater intake of K^ by the actively growing peach and sour cherry shoots occurred when th» leaves were dipped into the slurry. The reach and sour cherry leaves were not wetted by the solution but the slurry was in contact with most of the epidermis of the leaf. Some burning of the leaves was caused by the slurry. Peach leaves, other than the treated leaves, as well as the stem were visible in the autorcrii ogrcm made from the slurry treatment wh* le only the stem was visible in the autoradiogram made from the solution treatment. Petioles and midribs of sour cherry leaves, other than the treated leaves, as well a' the stem were visible in the autoradiograms where 'oth treatments were used. The autoradiogram made from the slurry treatment was darker indicating a rreater accumulation of K1**. Contact of the mater1al with the epidermis of the leaf was ap­ parently necessary for entry. The nature of the leaf surface was the factor which controlled contact. Thus different resnonses, were ob­ tained with the pubescent aprle leaves and the glabrous peach and sour cherry leaves to the solution and the slurry methods of application of potassium carbonate. Experiment XX Object: To determine whether the distribution of potassium and phos­ phorus within limbs differed follow'ng foliage applications of rotassium carbonate and ohosphoric acid. Methods and Materials: Four potted 2-year-old McIntosh trees, which were in active rrowth, were used In a greenhouse experiment. The tips of shoots of two trees were dipped,' and the median leaves of shoots of the other two trees were dipped in the solutions at 9:30 A.M. on May 9, 19^1. The shoots were cut after 8 hours. The potassium solution was prepared by adding 11 grams of potas­ sium carbonate containing to 3 liters of water. The phosnhorus solution was a 0.3 percent solution of phosphoric acid which contained 0.03 mtcrocurie oer milligram of phosphorus. Autoradiograms were made from the shoots after they were dried. Results: Very little movement of either phosphorus or potassium was seen in the autoradiograms of shoots in which the tip leaves were dipped. A faint indication of movement of potassium was seen in the stem immediately below the treated leaves, and the movement of phos­ phorus was indicated by a faint outl’ne of the leaves immediately below the treated leaves. More movement occurred when the median leaves of the shoots were dipped. Both elements moved acropetally and basipetally in the stem from the treated leaves. Concentrations of phosphorus appeared in the stem immediately above and below the treated leaves while distribution of potassium appeared to have been more uniform along the stem. Mayberry (ill) working with beans and squash found potassium to be uniformly distributed in the olant while phosphorus accumulated in the actively growing regions. 62 Experiment ZX1 Obiect: *• To determine the movement of nhosDhorus within shoots for a 2ii-hour period following application of P-^ phosphoric acid to two leaves of each shoot. Materials and Methods: Two median leaves of one shoot on each of five 2-jear-old McIntosh apple tre*=*s were dipped into 0,3 percent solution of phosphoric acid, whi^h contained 0,11 microcurie per milligram of phosphorus on May 18, 1951, at 10:15 A.M. in the greenhouse. All shoots were not in the same stage of rrowth-, as som^ had matured while others were st'll in active rrowth. One shoot was cut after the elapse of each of the following time intervals after treatment: 2 , 6, 12, 18, and 2U hours. Results: Results of this experiment are explained on the basis of the amount of exoosure wh^ch was seer, in the autoradiogram made from each shoot. Increased exnosure results from a higher concentration of radioactive phosphorus in the plant material. At 2 hours aft^r treatment, the terminal leaves of this shoot, which was still growing, were visible in the autoradiogram. More of the phosphorus had apparently moved acropetally in the shoot than had moved basioetally. At 6 hours after treatment, the terminal leaves were faintly visible although the shoot was more mature than the shoot used for the 2-hour treatment. The st^m of this shoot was also visible in the autoradiogram although it was not as dark as the stem of the 2—hour treatment. 63 At 12 hours after treatment, the terminal leaves of this shoot, winch vrrs still grow’n^, were more clearly outlined in the autoradio— pram than were those from the previous two treatments. Also visible were the leaf petioles and a fa^nt outline of some of the leaves above the treated leaves. At 18 hours after treatment, the terminal leaves of th^s shoot were clearly vi'-itlc in the autoradiogram although the shoot was more r.stuir than those used *n both the ?-h mr and 12-hour treatments. The stem was clearly visifcle as were some of the leaves above those treated. At 2h h-jurs after treatment, the terminal leaves of tins shoot were clearly v^s-’ble in the autoradiogram although this was the most mature one in the experiment. The stem and some of the leaf petioles were clearly visible in th* s experiment even though they were not as dark as the 18-hour treatment, Continued accumulation of phosphorus in the shoots after entry through the leaves occurred duri np the m r bient. The ar • rt of 2h hours covered by this ox— accuiuilnl ton of radi ophosphorus in the term­ inal l®av<->s was apparently dependant upon the maturity of these leaves and upon the amount of time allowed for entry. The rad *onhoschorus content of the shoots appeared to be greater at each successive harvest. However the less mature shoots at 2 hours and 13 hours showed increased accumulation of phosnhorus over the 6—hour and 2l».— hour-treated shoots, respectively. E x p e r im e n t X X II Object; To detombne the accumulation 'n ihe rhoots of rariiophosphorus at 2, U, 6, o, 10, and 1? hour inter'als, following a leaf application of P^2 T^hor'hDric ac*d to McIntosh aprle trees. I/atorials and Methods: The sixth and growing the "-ci s e v e n t h expanded leaves below nt. of 30 shoots iverc diored June lL, 1951* at 11:00 b'l, *nto a 0.3 percent solution of ohosphorie acid which con+ained ■'''.16 microcurie ~er milligram of 12 h ours hosphorus. At 2, U, 6, 8, 10, and oft r dip' ini , five of the shoots ween cut. Two shoots at each srsir linr: were vertical shoots and three were horizontal shoots. Aft^r the shoots we-e out, two were used to make autoradiograms ■vcd were ashed then r mnteri to record the total activity in the shoots. In the shoots that were counted, an effort was made to avoid contact nation of the untreated ^ortn ‘on by remov*nr the section of the stem c ~nta vnbnr leaves numbers 5 to 8 before ashing. The portion of the shoot above leaf number 5 was designated the tip, while that sortion of the shoot below leaf number 8 was designated the base. Temperature at the time of trertment was 70°F. and a maximum tecmeraturc of 75°F. was reached during the day. Fesults: The autoradiograms showed a progressive accumulation of phosphorus during the period from 2 to 12 hours after treatment, as ‘ndicatod by increasingly darker images the shoots on the X-ray film negatives. of the untreated areas of However, the negatives were not suitable for reproduction because surface contairn nation was not removed from the treated leavog and considerable blurring resulted. T A B L E XX ACCUMULATION OF RAOTOPUOSl-HypUS IN Me TNTOSH APPLE SHOOTS 2, ii, 6, 8, 10, and 12 HOURS AFTER TIfl- APPLICATION OF PHOSPHORIC ACTD TO TWO MEDIAN LEAVES (Average Values for Three Shoots) Time of Harvest (Hours) Radioactivity per 1 Oram Pry Wt. (Counts/min.) Tip Base Two 325. 9 68.2 Four 1*920.3 123.7 792.1 188.0 Eirht 1,181.9 225.5 Ten 2,862.8 371.U Twelve 2,625.2 1,517.0 Si x Radioactive counts in one milliliter of treating solution were 6h$978.1 counts per minute. 65 Data obtained from the shoots that were ashed and then coanted are shown ir. Table XX. Considerable variability is shown between the replications, but a trend of increasing accumulation at each suocrsive 2-hour interval of sampling Is evident. Also it a-roars in most cases that the tin area of the shoot contains mo^e radioactive '■hoschorus than does the ba^e area of th° shoot. Experiment XXIII Object: To determine the effect of girdling on the distribution of phosrhorus within a shoot follow^ne leaf application of phosphoric ac Id. Materials and Methods : Five sets of three limbs each, on the south side of mature Delicious a;-ole trees, were used in th:s experiment. Median leaves of the shoots were dipped on June 5, 1951* at U P.M. into a 0.3 oerrent solution of phosphoric acid containing approximately 0.11 microcurie oer milligram of phosphorus. Each set of limbs was divided into three sections: A, used for -.utoradiographs; B, ashed and counted; and G, girdled, then either ashed or use for an "utoradioerram. The limbs were located on the trees as follows: set 1, terminal shoots of primary limbs; sets 2, 3^, and U, terminal shoots of second­ ary limbs; and set 5, water sprouts from the interior of the tree. Girdling was done by drawing a knife completely around the shoot at the base, so as to cut through to the secondary xylem, thus severing the phloem tissue. 66 Eighteen hours after the leaves of the shoots wer® dipped* all shoots were harvested. The shoots upon which the radioactive count was to be determined were ashed immediately after the fresh weight of the untreated portions of the shoots had been determined. Auto­ radiograms were made from the shoots to be used for th.ot purpose after the shoots were dried. Results: The stem as well as the terminal leaves were visible in the autoradiogram made from the water sprout. Only a faint outline of the stem was seen in the autoradiograms made from the four non-girdled shoots. The auotradiograms made from the girdled shoots, one of which was the terminal shoot of a primary limb and the other, the terminal shoot of a secondary limb, showed leaf petioles as well as the stem but the terminal leaves were not visible. The amount of radionhosphorus in the untreated portion of the shoots as indicated by counts per minute is shown in Table XXT. Two non-^irdled shoots had a level of rad^ oactivity considerably above the rest of the limbs. One was the terminal shoot of a prl.mary limb while the other was the terminal shoot of a secondary limb. In the autoradiograms, it appeared that girdling might have in­ duced accumulation of phosphorus in the shoots but this was not shown in the count data. Because the effect of girdling on accumulation was questionable it was not used in further experiments. No distinction between terminal shoots of primary limbs and of secondary limbs war made in further experiments because there is a comparatively small number of primary limbs on the tree and because T A B IE XXI THE INFLUENCE OF GIRDLING AND LOCATION OF SHOOTS ON THE ACCUMULATION OF h ADIQRHOSPHGRUS FROM AN APPLICATION OF PHOSPHORIC ACID TO TWO MEDIAN LEAVES Limb Fresh Weight (Gins.) Radioactivity 5 grams Fresh Wt. (Counts/mln.) 1 B Primary terminal shoot U.3U1 U71.2 2 B Secondary terminal Shoot U.oUl 56.2 ? G Secondary terminal shoot, girdled 3.175 80.3 3 8 Secondary terminal shoot 5.880 228.3 B Secondary terminal shoot 6.036 U?.U It G Secondary terminal shoot, girdled 5.715 liO.2 5 B Water sprout 2.786 121.1 4 Radioactive counts per minute in one milliliter of treating solution were 6U>978.1 counts oer minute. no conclusive evidence was found to show that there was a difference between the two types. No consistent difference was noted in the amount of absorption of radionhosohorus or in the subsequent distribution between the terminal shoots of primary limbs and of secondary limbs. Therefore no distinction was made between the two types in further experiments. Experiment XXIV Object: To determine the distribution of nhosnh >rus in the apple fruit following leaf application of p32 phosphoric acid. Materials and Methods; Two median leaves of two secondary shoots on a spur containing two apples were dipped June 21, 19^1* into a beaker oonta^nlng a solution of phosphoric acid whose radioactive strength was 0.08 microcurie per milligram of phosphorus. shoots were harvested July 1, 19?1. One fruit was cut into longitudinal sections and the other into transverse sections. the fruit were made. These fruits and Aut©radlograms of See figure 1. Results: Phosphorus was found to move into adjacent fruit from spur leaves to which it was applied. The greatest concentrations occurred in the seed and vascular system with a second high concentration at the periphery of the fruit. Fruit on spurs both above and below the treated spur showed no radioactivity. These facts agree with other finding's that phosphorus moves to the nearest ooint of mobilisation* Experiment XXV 1Ji 1 ■— — — Object: To determ^ne whether di fferent varieties of apples absorbed p3? from p32 phosphoric acid at different rates following foliar application. Figure 1 Autorndiogram of an apple fruit showing the distribution of radiophosohorus in the fruit subsequent to foliage application. Materials and Methods: The sixth and seventh expanded leaves below the growing point of five shoots each of Delicious, Jonathan, McIntosh, and Northern Spy apple trees were dipped June 27, 19J>1, at 12s00 P.M. into a 0.3 percent solution of phosphoric acid which contained microcurie per milligram of phosphorus. 0.08 Terminal buds of the Delicious and Jonathan shoots were still growing at the time of treatment while the terminal buds of McIntosh and Northern Spy shoots had ceased growth. The temperature was about 80°F. when the median leaves were dipped. A maximum of 85°F. was reached at U:00 P.M. before the onset of a r?in storm wh^ch lasted 3 hours. Because the ra'n occurred soon after treatment, only two of five shoots of each variety were harvested and were used to make auto­ radiograms to see whether the rain had caused contamination of the untreated leaves with radioactive material. Results: The autoradiograms made from the shoots rave no evidence that radioactive phosphorus had been washed from treated to untreated leaves. Differences between the amount of absorption of phosphorus between the different varieties were noted but they appeared to be related to maturity of the shoots rather than variety. Both of the Delicious shoots and one of the Jonathan shoots showed a concentration of phos­ phorus *n the exoand^ng terminal leaves. Neither of the matured Northern Spy shoots showed evidence of phosphorus accumulation in the autoradiograms. One of the autoradiograms of the matured McIntosh shoots showed phosphorus concentrations in the stem and leaf petioles. These results indicated the need for using comparable plant material for an experiment because the age of the nlant material apparently affects the amount and manner of accumulation. Experiment XXVI Object: To determine whether leaves of different varieties of apples and peaches absorbed radiophos horus at different rates. Materials and Methods: The seventh and eirhth expanded leaves of five shoots each of Delicious, Jonathan, and McIntosh apple trees and Elberta, Halehaven, and fouth Haven peach trees were dipped July 7, 1951, at 10:00 A.M. in a 0.3 percent solution of phosphoric acid containing 0.21 microcurie oer milligram of phosphorus and no wetting arent. The fourth and fifth leaves of five shoots of Northern Spy ancle trees were dipped in the same solution. The shoots were cut after 26 hours. The temperature was 80°F. at the time the leaves were dipped. A maximum temperature of 85°F. was reached before rain started falling at 3:30 P.M. The rain continued till 5:30 P.M. and intermittent showers fell durlnr the nir:ht. Two shoots of each variety were used to make autoradiograms and three shoots were ashed then counted to obtain the true amount of radioactive phosphorus which had been absorbed. Before the shoots were ashed, leaves six through nine and the corresponding section of the stem were removed. The section of the shoot above the sixth leaf was designated the tip and the section below the ninth leaf was 70 designated the base. In a Wiley mill. After the shoots were dr^ed, they were rround A O.Of? pram sample was ashed accord’m- to the pro­ cedure given for phosnhorus analysis in the A.Q.A.C. (1). Results: Results shown by the autoradiograms made from the shoots are described for each variety of fruit. Maturation of the terminal buds of the Delicious shoots had occur­ red but the terminal leaves were not expanded. The stem, leaves, and aphids on the young leaves were visible in the autoradioprams made from both shoots. Phosphorus was more concentrated in the bodies of the aphids than in the shoots. The terminal buds of the Jonathan shoots had matured and the terminal leaves were fully expanded. Autoradiograms of both shoots showed the stem, and the leaf petioles. A fa’nt outline of all the leaves above the treated leaves was visible in one of the autoradio­ grams but only a faint outline of the youngest leaf was visible in the other autoradiopram. The terminal buds of the McIntosh shoots had matured and the terminal leaves were expanded. Only a portion of the stem near the treated leaves aopeared in the autoradiogram of one shoot while the outline of the stem and some of the leaves near the treated ones ap­ peared to have been contaminated. Northern Spy shoots were completely mature at the time of treat­ ment. The stem and the leaf petioles were visible in autoradiograms made from both shoots but leaves above the treated leaves were visible in only one autoradiogram. Fijerure 2. Autorad iorran of a Halehaven peach shoot showinr distribution of radiophos'horus in the shoot subsequent to application of phosphoric acid solution to the seventh •and eighth leaves of the shoot. 71 A large accumulation of radioactive phosphorus occurred in the young expanding leaves of the growing Klberta shoots and a lesser concentration occurred in the older leaves and stems according to the autoradiograms* More phosphorus had accumulated in the expanding shoot tips of Halehaven and in the stem than had accumulated in the same areas of the Klberta stems. An outline of the older leaves was also visible in the autoradiogram. See figure 2. South Haven shoots were still rowing at the time of treatment but the rate of growth was not as vigorous as the preceding two vari­ eties of peaches. The autoradiograms showed the terminal leaves and stem but not as clearly as did the autorediograms of Blberta and Hale­ haven. From the autoradiograms it can be concluded that a growing peach shoot absorbs more phosnhorus than does a mature apple shoot. How­ ever, it is possible that the direction of translocation after absorp­ tion may be different in a mature shoot than in a growing shoot* Count data from the shoots that were ashed are presented in Table XXII. These results also showed that the growing peach shoots in gen­ eral absorbed more rhos^horus than did the mature apple shoots* The phosnhorus content from the foliage application was found to be higher in the younger tin area than in the older base area* Experiment XXVII Object: To determine whether the accumulation of P^ from .phos­ phoric acid by different fruit varieties varied with the stage of maturity of the shoots* TABLE XXII COMPARISON OF ABSORPTION OF RADIOPHOSPHORUS APPLIED TO THE SEVENTH AND EIGHTH LEAVER OF APPLE AND PEACH SHOOTS DURING JULY (Average Value of Three Shoots) Variety Radioactivity in Counts oer Minute per 1 gram Dry Aft. Tip Base Delicious 1*50.9 107.7 Jonathan 123.9 61*.1* McIntosh 332. U 321*.7 Northern Spy 516.7 217.3 Klberta 81*0.5 280.5 Halrhaven 51*9.9 1 65.2 South Haven Q5U.2 21*0.3 * Radioactive counts per minute in one milliliter of treating solution were l5»l*8i*.6 counts oer minute. Materials and Methods: The seventh and eighth expanded leaves of five shoots each of delicious, Jonathan, and McIntosh apple trees and El— berta, Halehaven, and South Haven reach trees were dipped August 3# 19^1, at 12:00 P.M. in a 0.3 percent solution of phosphoric acid con­ taining 0.10 microcuries per milligram of phosphorus and no wetting agent. The fifth and sixth leaves of Northern Spy were dipoed in the same solution. day. A high temperature of 80°P. was reached during the A strong wind was blowing while the limbs were being dipped. The shoots were cut after 12 hours. Two shoots of each variety- were dried under heat lamps, and then used to make autoradiograms. Three shoots of each variety were ashed and then counted to obtain the true amount of radioactive nhosphorua which had been absorbed. Before the shoots were ashed, they were cut above the sixth leaf and below the ninth leaf to remove the treated leaves and other possible contamination. The portion of the shoot above the sixth loaf was designated the tip while the portion of the shoot below the ninth leaf was considered the base. The tip and base pieces were dried in a drying oven, and then ground in a Wiley mill. A 0.50—gram sample was ashed according to the procedure given for phosphorus analysis in the A.O.A.C. (I). Results: Maturation of the terminal buds of the apple shoots of all varieties had occurred when th-fs experiment was started. All leaves of the shoots were fully expanded at this time. Similar results were visible in all the autoradiograms produced from the apple shoots. A faint outline of the stems was visible In 73 all the autoradiocrams while an outline of the leaves was visible in some autoradiograms. Black spots on the leaves, indicating contamina­ tion may have been caused by the wind whipping the shoots after they were dipped. Maturation variety at this of theterminal buds of the peach shoots varied with time. No maturation had occurred in the Elberta peach and the shoots were still growing. The yotmg leaves and stems were clearly outlined in the autoradiograms. Terminal buds of the Halehaven peach trees had matured but elong­ ate on of the stem was occurring in most shoots. All of the younger leaves, midribs of older leaves, and all of the stem were visible in theautoradiogram made and from one shoot. The other shoot had matured the younger leaves were only **a'ntly visible in the autoradiopram. T'either the stem nor the older leaves were visible except near spots indicating contamination. iShoots of South Haven peach were completely matured. One shoot was visible in the autoradiogram near a dark spot indicating contam­ ination. The youngest loaves of the other shoot were visible in the aut orad iogram• These results suppest that the entry of radioohosphorua into peach shoots was influenced by the maturity of the shoot, as was found with shoots of different aprle varieties in previous experiments. Thus the greatest accumulation of radiophosphorus occurred in the Tlberta shoots which were still in active grcwrth at the time of treat­ ment. Radioactive count data for the shoots that were ashed are pre­ sented in Table XXIII. Considerable variability existed among the rep­ licates. Translocated phosphorus from a foliar application in August into apple and peach shoots appeared to be similar in amount in the terminal recion. The amount of phosphorus, which had been translocated :nto the base of the apple shoots, appeared to be greater. TABLE XXIII COMPARISON OF ABSORPTION OP KADIOPHGfli HOl'IJS APPLIED TO THE SEVENTH AND EIGHTH LEAVES OF APPLE AND PR CH SHOOTS DURING AUGUST (Average Value of Three Shoots) Variety Radioactivity in Counts per Minute per 1 gram Dry Wt. Tip Base * Delicious 91 6 . 6 Jonathan 1,275.0 McIntosh 1 Northern Spy F.lberta ,2 1 3 . 8 699.0 1,263.U 1 ,31*6 .1* 1 ,1*0 9 . 8 771.6 * 1*19.2 Halehaven 610.1 350.1 South Haven 920.6 1*30.0 *Not enough lant material* of base to weigh and count Radioactive counts per minute in one milliliter of treating solution were 231*180. counts per minute. V. DISCUSSION During the course of this research, it was found that calcium, phosphorus and potassium w~>uld enter the above-ground portions of fruit trees. Some of the factors which may influence the entry of mineral nutrients into the leaves, twigs, and branches of fruit trees were studied. These factors were: season of the year, length of ab­ sorption period, concentration of nutrients, differences between vari­ eties, and method of application* Some entry of mineral nutrients occurred followinp an application of phosphoric acid or k U2 potassium carbonate to bark of twigs or branches of fruit trees durinr the dormant period. The amount of entry, dur:nr this dormant period as indicated by radioactive counts ’r. the untreated portions of the plants, was small as these counts were very seldom twice background. Entry of rad iophos uhorus during May after new rrowth occurred, was very large ranging up to several thousand t^mes background count in the untreated portions of plants when the solution was applied to injured bark, when the solution was applied to cotton gauze wrapped around the limb or when the solution was applied to cotton gauze over injured bark. Negligible entry oc­ curred at this time following application of P^2 phos horic acid solu­ tion to uninjured 10-year-old berk but sufficient entry to produce counts 10 to 1*> times background occurred when the solution was painted on uninjured 1-year-old bark* 76 Others who have worked with entry of mineral nutrients into dor­ mant and growing trees have obtained similar results. Harley and Jef­ ferson (30) found that almost no entry occurred during the dormant period but entry did occur after active growth resumed in the spring. Ercert (l6) reported that entry of a solution containing radioactive phosphorus *nto the excised stem of a McIntosh apple tree occurred only after the buds of the stem swelled. Movement of rubidium Rb®® injected into the trunk of a yellow birch tree was toward the leaves during the growing season and toward the roots during the dormant season accordinr to Fraser and Mawson (23). This directional movement with the seasons nay be the reason activity was not found in some dormant season experiments as in most instances sampling was done above the treated area rather than toward the roots* The distribution of phosphorus following foliar application and possibly the amount of entry were influenced by the season of the year* Differential mobilization of phosphorus during active growth occurred; thus a concentration of ohosphorus was found in the shoot tips. Mobilization of nhosphorus after maturation of the shoots was apparently not selective*within the shoots; thus concentrations of phosphorus in a particular ree.ion of the shoot did not occur. How­ ever, at the time of shoot maturation the developing fruit is rapidly metabolizing phosphorus and movement of phosphorus which enters the plant is towards the fruit. But it is possible that less entry of nhosphorus rather than direction of movement after entry explains why there was less phosphorus in a mature shoot tip than in young shoot tip. This idea is supported by the fact that several workers (13* 51) have found that young leaves absorb urea more readily than do mature leaves. A difference in distribution of phosphorus occurred when applied to different tissues of plants. I’hosphorus applied to expanding peach leaves did not move out of the leaves while phosphorus applied to the bark at this time did move to the leaves and to the roots. Continued absorption of foliar apnlied phosphorus was found up to a period of 2U hours, the longest interval used in the experiments in this project. period. However phosphorus absorption continues over a longer Mayberry (Ul) found that absorption was still occurring after 160 hours following a rapid initial intake. Absorption continuing over a 30—day period following a folia*e application of radioactive 'hosphorus was reported by ",ggert (1 6 ). Results obtained concerning entry following bark application of phosnhoric acid were of a conflicting nature. Decreased entry, no effect, and increased entry with increasing time intervals occurred in different experiments. Phosphorus applied to 10-year-old limbs of South Haven peach trees showed in autoradiograms the highest radio­ activity in the phloem tissue after 6 and 16 hours following treatment. The amount of radioactivity decreased as indicated by the amount of exposure of the film between each successive sampling at 2h, I*lt, and 72 hours. The level of radioactivity found in 1—year—old portions of Halehaven peach limbs was nearly the same when samples were taken at 6, 21*, and 53 hours following an application of solution to the 2-year- old portions of the limbs* Increased entry from 12 to 192 hours after treatment occurred in apple limbs* Rapid intiial intake followed by movement of the phosphorus to areas of mobilization may be the explanation of these results* Rapid intake was shown bv the high levels of radiophosphorus which occurred 6 hours after treatment of 2-year-old Halehavon limbs and of 10-yearold South Havon limbs. Translocation followine this entry would explain why the radioactivity level in samples which were adjoininp the treated area dropped in the 10-year-old peach limbs. However, the radio­ activity in the 1-year-old limbs of Halehaven peach came from trans­ location of phosphorus following entry and not from entry of phosphorus alone thus this radioactivity mirht be less subject to further translocation than would be radioactivity from radiophosphorus which had entered the plant but which had not been translocated from the right of entry* The radioactivity level in the parts of the apple limbs most distant from the noinb of application increased because of translocation to these rerions* In order to determine whether the concentration of the phosphoric acid w uld influence the rate of entry, 0.3, 2*0, and 8.0 percent sol­ utions of phosphoric acid, which had the same relative specific activ­ ity of radioactive phosphorus, were applied to 1-year-old limbs of Halehaven peach trees* tration* Very little entry occurred with any concen­ This response probably occurred because this experiment was done durinr early December. However, no increased entry occurred with the higher concentrations so they were not used in further experiments* 79 Several experiments were conducted to determine whether there wa6 a difference in response to foliar application of phosphoric acid by different fruit varieties. Varieties used were Delicious, Jonathan, McIntosh, and Northern ^py apples and Flberta, Halehave, and South Haven peaches. In experiment XVIII, it was found that the retention of phosphoric acid by the leaves of apple, peach, pear, sour cherry, and sweet cherry, differed. Differences observed in absorption of phosphorus by the different varieties appeared to be more closely correlated with maturity of the shoot than with variety. This result was more clearly illustrated in the autoradiograms than it was in count data, Autoradiorrams made from shoots which were still prowing produced a much darker ' ‘mage on the X-ray negative than did mature shoots. Effective means for increasing the amount of nutrients which en­ tered the plant were (a) injuring the plant, (b) using a cotton pause to hold the solution, and (c) using hydrated lime to obtain an increas­ ed deposit. Harley and Jefferson (30) reported entry into stems and branches which were mechanically injured. Results obtained in some of the experiments of this project confirm those obtained by Harley and Jefferson but the influence of the season of the year and the activity nf the plant were found to be greater. During the dormant season, in­ creased entry was not noted following mechanical injury although more material adhered to the branches. Increased entry following mechanical injury was noted in the spring after active growth of the tree had oc­ curred. Variations in results obtained between replications in some 80 bark absorption studies may have occurred because of growth cracks or insect and disease damage. A very effective method for increasing the amount of phosphorus that entered a plant war to saturate a strip of cotton gauze which had been wrapped around the stem. This procedure proved to be more ef­ fective than scrapinp or injuring the shoots of apple trees to stimulate entry. Increased entry of ohosphoric acid to the extent that toxicity was produced, occurred when a gauze was wrapped around an area of a shoot winch had been previously scraned. A pink discoloration of the phloem tissues of the shoots occurred, whi^h was a symptom of toxic concentrations of phosnhoric acid. . While the procedure of scrapinp and providing a reservoir for continued absorption was only of an experimental nature, it may ex­ plain why Forsyth's compound was effective. One of the first points stressed by Forsyth for the use of his compound was the need to scrape down to livin? tissue before making an application. Another reason why the compound may have been successful was that in the moist climate of England the manure and wood ash mixture might stay almost con­ tinuously wet. Thus the two conditions which promoted the greatest entry in Experiment XVI were fulfilled by Forsyth's method. Entry of radiopotaesium into leaves ao-eared to be controlled by the amount of contact between the applied !^2 potassium carbonate and the leaf epidermis. Thus increased absorption of potassium by peach and sour cherry leaves occurred when hydrated lime was added to a sol­ ution alone. Greater absorption of potassium by apple leaves occurred when the solution rather than the lime slurry was used. The pubescence of the apple leaves prevented contact of the lime slurry with the epi­ dermis. Conversely, the pubescence of apple leaf was easily wetted by the solution which contained Dreft as a wetting agent while the peach and sour cherry leaves were not easily wetted. Concentrations of chemicals higher than the 0,3 percent solutions used for foliar application would be necessary if the mineral nutrient requirements of a fruit tree were to be supplied by a dormant spray. Therefore a series of experiments were conducted to determine the tolerance of McIntosh apple and Montmorency sour cherry to different levels of nutrient sprays while the trees were dormant and while the buds were in the gr®en tip stage. The chemicals used ranged widely in their phytotoxicity concentration, nlant material, and rrowth stage. with No injury was found with the 2 or 1* nercent c ncentrations and only rarely with the 8 per­ cent concentration of any of the materials. 11>wever, the 16 and 32 percent concentrations of the chemicals other than potassium nitrate caused varying degrees of injury. It was determined that the injury produced by 32 percent NuGreen was related to the tcme of application to the apple. Buds and some small limbs of 2-year-old McIntosh trees were killed when the trees were sprayed while dormant. This same solution when applied to the trees when the buds were in the green tip stage was not injurious. same effect was produced by a spray of a 20-20-20 fertilizer at the percent concentration but to a lesser degree. The 32 This injury was probably 82 caused by urea toxicity since NuOreen is a commercial preparation of urea, Hinsvark, Wittwer, and Tukey (31) have determined that apple twigs are capable of raoid hydrolysis of urea. Rapid hydrolysis of urea durinc the dormant period when utilisation of nutrients was low nay have resulted ' “n toxic levels of nitrogen compounds accumulating within the plant. During the rrowinp season, these materials would be utilised and so r.ot a^umulate to toxi~ levels. Urea injury of the sour cherry showed as a form of chlorosis of the leaves in whi-h the marrins turned yellow. This injury occurred where 16 and 32 percent solutions of NuDreen or 20-20-20 fertiliser were used. The 16 and 32 percent solutions of calcium chloride and phosphoric acid were the most phytotoxic. The injury produced by the two higher concentrations of calcium chloride a peared to be a form of desiccation. When the buds of the sour cherry were examined, multiple layers of what ameared to be bud scales were ^ound to te dehydrated Immature leaves. Terminal buds were destroyed or inhibited by the high concentrations of calcium chloride. Phosphoric acid estroyed the buds and the tissues near the buds when applied as a 16 or 32 percent solution. Shrunken areas around the buds and leaf scars were the typical injury produced in the McIntosh trees. stroyed. Puds on the sour cherry trees were also de­ In both apple and sour cherry, lateral buds were destroyed while buds in the term?nal position were not as readily destroyed. After entry, distribution of phosphorus and potassium was found to differ. Phosphorus tended to accumulate in the rapidly growing reeions wh*le rotas-Hum tended to have even distribution throurhout the shoots. Similar results were found by Mayberry (Ul) working with beans and squash* From the results obtained ’n this project, it would se*cn that sprays of urea or other mineral nutrients would be most successful ^f they were applied as a delayed dormant snray. At this time, the danger from urea hydrolysis without utilization, has passed. Also, before this time it was found that very little absorption of mineral nutrients occurred. If the nutrient sorav is combined with the regular pest control sprays, the oost of annlioation is ne-lirible. It would save time and money by eliminatenq a special trir through the orchard to distribute fertiliser. Even though the percent of nutrients absorbed directly by the tor of the tr»p may be small, the mat'-rial not absorbed would be washed to the rround by rain and thus become ava'ilable to the plants through the roots. One of the major reasons for dormant arrays mi.^ht be to promote recovery of w'nter injured trees. Partridge (1*6) observed that peach trees wh;ch had been damaged by cold often exhibited symptons of po­ tassium deficiency. This injury results from an impaired conductive system rather than lack of ootassium in the soil, dere an application of potassium to the above-ground parts of the tree, by relieveing the potassium deficiency, may uromote faster recovery of the tree* The rreatest effect on the recovery probably would occur following a delayed dormant spray. VI. SUMMARY 1. In a series of experiments, the leaves and the bark of shoots and branches of several tyr>es of fruit trees were treated with sol­ utions containing radioactive calcium (Ca^), radioactive phosphorus (p32)> an(j radioactive potassium (K^) and with solutions of other materials. Most of the experiments were undertaken to study the rate and extent of absorption and subsectuent translocation. 2. Methods of applying the solutions varied with the material under study. Foliar treatments were containing the solution. made by dipping the leaves into a beaker Bark treatments were made by (a) painting the solution on the limb with a brush, (b) soaking a piece of cotton gauze wrapped around the limb with the solution, or (c) spraying the solution onto the olant. 3. Leaf and mowing shoot samples were dried, ground, and prepared for analysis by the methods outlined in the A.O.A.C. (l). samples were cut into small pieces and then dried. Woody To insure the penetration of the magnesium nitrate used in phosohorus analysis, extra hydrochloric acid was added to the woody sample. U. Radioactivity in the samples was measured directly by the use of a Tracerlab Autoscaler, or it was measured indirectly by the use of Autoradiograms* The bark surface area of a 2*>—year—old McIntosh aople tree was found to be 86 square meters. Of this total area, 35.7 percent occurred on limbs of 6 millimeters or less in diameter and 53»7 per­ cent occurred on limbs of 10 millimetors or less in diameter, 6. Methocel 1*000 c.p.s. was the most effective of ten sticking or wetting agents whi rh were tested by the amount of s $ percent solution of phosphoric acid w h :ch adhered to sections of McIntosh water sprouts, 7. Calcium chloride, nhosrhoric acid, potassium nitrate, NuGreen, and a 20-20-20 fertilizer as 2, 1*, 8, 16, and 32 oercent solutions were sorayed on 1-year-old Montmorency cherry trees and 2-year-old McIntosh arole trees. Spraying was done either when the trees were dormant or when the buds were in the rreen tip stage. Injury to the trees was found to depend upon concentration of solution, stage of growth, and type of tree but injury rarely resulted from any material used at a concentrat-'* on of 8 percent or less, 8. Some evidence of entry of radi o^otassium through 6- to 10-year- old bark of apnle and peach limbs during the dormant oeriod. On the other hand, much greater entry of radiopotassium occurred through bark ofactively crowing 2—year-old apple trees in 9* the greenhouse, Radionhosphorus entered 2-year—old peach limbs following a dormant application of phosphoric acid. Approximately the same amount of radiophosphorus was found in untreated portions of the limbs whether cut 6, 21*, or 1*8 hours after treatment. No appreciable difference was found in the amount of entry from 0,3* 2,0, or 8,0 percent phosphoric acid applied to the bark, 10, Radiophosphorus entered 8— to 10—year—old South Haven peach limbs when a solution of phosphoric acid was applied in cotton gaume to the bark during the dormant season. Autoradiograms showed that the great­ est am*>unt of phosphorus was in the phloem tissues near the p iint of application 6 and 11. 16 hours after the solution was applied. Radiophosnhorus was found to have entered 3-year-old a^ple limbs following application of phosphoric acid to gauze wrapped around the l^mbs during the dormant season. Injury in the treated area did not seem to stimulate entry during the dormant period. 12. Radiophosphorus entered through the sides of a tomato stem in a larger amount wh»n the solution was applied to gauze wraroed around the stem rather than when the solution was brushed on the stem. At the three temperatures used in this experiment, 50°, 65° j and 85°F.» greater entry occurred with each increase in temperature. 13. Radiocalcium entered 2-year-old McIntosh apple trees when calcium chloride was sprayed onto the bark while the trees were dormant. er entry occurred Great­ nto trees grown on minus calcium nutrient solution than into trees grown on complete nutrient solution. lit. NuGreen and a 15-30-15 fertilizer were sprayed onto Montmorency trees which were in the green tip stare. A greater increase in circum­ ference was made by the trees receiving fertilizer than was made by the non-fertilized check trees. 15. Radiophosphorus entered 1—year—old water sprouts and 10—year—old limbs of Jefferis apple trees when a solution of phosphoric acid was applied to the bark during the growing season, in May 1953. The an ount of entry as indicated by radioactive counts per minute in untreated new growth was 195-339 when the solution was applied to undamaged shoots; 9*199-11,085 when the solution was applied to cotton gauze over intact bark; 3,090-7,5Ul when the solution was applied to scraped bark; and 368 ,177-1*23,8l5 when the solution was applied to cotton gauze over ■craped bark* 16 . Radiophosphorus did not move from the expanding Elberta peach leaves that had been treated with phosphoric acid* Movement was found when the solution was applied to both the expanding leaves and the bark or to the bark al->ne* 17. The amount of r^dioohosphoric acid, which was retained by the leaves of apple, peach, pear, sour cherry, and sweet cherries, was found to differ with the greatest amount being retained by apple leaves and the smallest amount by peach leaves. Efficiency of trans­ location also varied with the greatest percentage of translocation occurring in the sour cherry and the smallest percentage in the sweet cherry* 18. Radiopotassium entered peach and sour cherry shoots from a foliage application in a preater amount when the potassium carbonate was ap­ plied to the leaves in a lime slurry rather than as a 0.3 percent soltition. The reverse was true of anple shoots as the greater entry oc­ curred when the leaves were dipped in solution rather than in the slurry. 19. Distribution following entry of foliar applied radiophosphorus and radiopotassium was found to differ. Phosphorus accumulated in the rapidly growing areas of the shoot while the distribution of potassium was characteristically more uniform in the shoots* 2C. Radiophosphorus entered McIntosh apple shoots for at least 2k hours after a solution of phosphoric acid was applied to the leaves. This was the longest period of time for foliar entry in these experi­ ments. PI. Radiophosphorus entry and subsequent distribution was found to be influenced very little by whether the shoot was girdled or not girdled, whether the shoot was terminal on a primary or a secondary branch, or whether the shoot was ^n a horizontal or vertical position. 2?. Radiophosnhorus entered McIntosh arple fruit after the application of rhosphoric acid to the leaves of the sour on which the fruit was growing. Concentrations of rediorhos'-horus were visible in the auto­ radiogram in the seeds., in the vascular system, and at the perphery of the fruit. 23. To find whether different plants absorbed radiophoschorus at dif­ ferent rates, collations of phosphoric acid were applied to the leaves of shoots of Delicious, Jonathan, McIntosh, and Northern Spy apples and Rlberta, Halehaven, and South Haven peaches. Maturity of the shoots rather than variety of the shoots appeared to be the orinciple cause of variation. 89 VII. LITERATURE CITED 1. 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Citrograph 32:112-112. 19U7* THE -sNTPY OF NUTRIENTS T H R V f ' H THE S„RK AKD I.SAVES VF DRCIPU ,us FRUIT TREES AS ITPIC-.TKD B*' RADIO ACTIVE ISOTOPES ty Robert Lewis Ticknor AN A K ’TRACT Submitted to the School of Craduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements > for the degree of DOCTOR OF HJTLOSOPHT Department of Horticulture 1953 Several factors which may influence the entry of mineral nutrients into the above—ground portions of fruit trees were studied by applying solutions containing r-diocnloium, r«diophos: horus, radlopot&eslmp, and also some non—radioactive materials to the trees. These factors were: season of the year, length of absorption period, concentration of nu­ trients, differences between varieties, and affect of method of appli­ cation, Mineral nutrients were round t > have entered through the bark of fruit trees following the application of mineral nutrients during the dormant season in limited ouanlty. Greater entry occurred following the application of the mineral nutrients to the bark after foliage growth had been produced in the spring, bntry of mineral nutrients applied to the foliage occurred more readily while the new shoots were still expanding than after the shoots had matured. Evidence was found that entry was still occurrirg 192 hours after application of radiophosphorus to hark of 3-year—old arple limbs. the case of folia e application, entry was still occmrring 2 In U hours after the application of radiophoschorus. Solutions of 2, 1** 8 , 16, and 32 percent concentration of calcium chloride, MuGreen, phosohoric acid, potassium nitrate, and a 20—20330 fertiliser were sprayed on 1-year-old Montmorency sour cherry trees and 2-year-old McIntosh apple trees wh le the buds were dormant and while the buds were in the green tip stage. Very little injury was found when the trees were sprayed with any solution of less in concentration. 8 percent or Primarily, terminal buds were destroyed by calcium chloride and lateral buds by phosphoric acid when used at concentrations of 16 and 32 percent on either apnle or cherry trees* The effects produced by 16— and 32—percent NuGreen and the 20-20-20 fertiliser which contained urea were different on apple and cherry trees* Many of the buds of the apple trees were killed when sprayed while dormant with these materials but few were killed when sprayed whi le in the green tip stage* A marginal chlorosis or variegation of the cherry leaves was found following the use of either NuGreen or the 20-20-20 fertiliser at both growth stages* Potassium nitrate had no effect at any concentration or at either growth stare* Variations in maturity appear to be the principle cause for the differences in the intake of rad* ophosphorus noted when the leaves of several varieties of apple and p^ach shoots were dipped into radio— phosphorus solution at any calendar date* Entry following the applications of the radiophosphorus solution to a mechanically injtired area or following the use of a continuously moist source of the radioohosphorus solution (a saturated cotton gause) was greater than when the solution was applied by brush to the int.aet bark.