ZINC~PHOSPHORUS ENTERACTEOMS .iN PHASEOLUS VULGARES “I. -.' . , h, L. H h mews $3.. the » egiee of Fit, u. mm Imp, .9 ”'1 “ "QH: ,tcf‘ gm” M; Uri m: 3.! c. E 33%; Ul‘éi‘i‘ !.RS‘ 3 ‘{ {ifizf’x‘i’ MAX LESSMRR 1%? IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 3 1293 105048 This is to certify that the thesis entitled Zinc-Phosphorus Interactions in Phaseolus Vulgaris presented by Gary Max Lessman has been accepted towards fulfillment of the requirements for Ph.D. degree in_S_QiLS.CJ.e' nce Major professor Date f/fl 1‘7 0-169 5:!“ I‘v‘licltigan 3&1? [IaivcnuSym L LIBRARV ”‘3 i9}.- I 4. l ABSTRACT ZINC-PHOSPHDRUS INTERACTIONS IN PHASEOLUS VULGARIS by Gary Max Lessman An investigation was conducted to: (1) study the effect of P-ZN antagonism on Phaseolus vulgaris var. Sanilac (pea or navy bean type) (2) ascertain if added Zn could overcome this effect and (3) determine the site of this antagonism and its mechanism. Field experiments were conducted for two years on the lake-plain soils of East Central Michigan where Zn deficiency was known to occur. The results indicated that high levels of applied P have a marked detrimental affect on Zn utilization. In 1963 the most effective Zn fertilizer was ZnSO4 mixed with ammonium polyphosphate (APP). Zinc ethylenediaminetetraacetate (EDTA) was nearly as effective as was ZnO incorporated into APP. Zn was definitely more effective when incorporated into APP than if incorporated into ammonium orthophOSphate (AOP). It was noted in 1963 that the 0.8 pound rate of ZnO incorporated into APP was better than the three pound rate. which deviates from a standard growth curve. The 1964 field trials were designed to study this phenomenon, and to compare APP and AOP as carriers of Zn. Studies of these fertilizers showed that Gary Max Lessman the solubility of Zn added to APP increased to a maximum with increasing Zn and then decreased with additional incre— ments. Zn incorporated into AOP had a very low water solu- bility. It is suggested that a soluble complex is formed between Zn and polyphOSphoric acid until the system becomes supersaturated. Additional Zn beyond this level is changed to an insoluble precipitate. Results of this study show that the Zn:P ratio of the fertilizers may be critical re- garding Zn availability. And the characteristics of these fertilizers might depend on their method of manufacture. A calcareous Wisner clay loam incubated with 100, 500, and 1,000 ppm P for two and one-half years and then equilibrated with Zn65 was successively extracted with four agents in the following order: H20, 0.01 M EDTA in l M (NH4)2C03. 1 M NH4OAc buffered at pH 7.0 and 0.1 N HCl. Little water soluble Zn65 was extracted and the results showed no relationship between the quantity of applied P and water woluble Zn. EDTA buffer, which is regarded as one of the best extractants for evaluating available Zn, removed more Zn from the samples highest in applied P. The remaining two extractants removed small quantities of Zn65 and the amount removed was inversely related to applied P. In nutrient culture studies, Zn65 was not translocat— ed beyond the roots of plants produced in high P solutions. Further studies showed that P in the roots of these plants could be removed by transferring them to Hoagland's solution minus P for 24 hours. After removal of this P, ten times Gary Max Lessman more Zn65 was translocated to the aerial portion of the plants than in plants which contained the P in the roots. After removing the P from the roots if the plants were once again subjected to a high P environment, Zn uptake was again decreased. Therefore, the location of the P-Zn antagonism was shown to be at or near the surface of the roots. It is postulated that the antagonism is due to the formation of a Zn-P complex which may also include other plant metabolites. ZINC-PHOSPHORUS INTERACTIONS IN PHASEOLUS VULGARIS BY Gary Max Lessman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Soil Science 1967 6. 716070! -97 [2’2 ACKNOWLEDGMENTS The author wishes to eXpress his extreme gratitude to Dr. B. G. Ellis who patiently guided this study through— out its long duration. To Dr. N. E. Good for his suggestions regarding the nutrient culture studies go my profound thanks. A deep appreciation is also extended to Janusz Czapski for performing the technical aspects of the final experiment. Finally, the cooperation and assistance of Tennessee Valley Authority in supplying fertilizers and financial aid used in this study is gratefully acknowledged. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . . Functions of Zinc in Higher Plants Geochemistry of Zinc Interaction of Soil Factors and Zinc Availability EXPERIMENTAL METHODS . . . . . . . . . . . Laboratory Studies Solution Culture Studies RESULTS AND DISCUSSION . 1963 Field Experiments 1964 Field Experiments Laboratory Incubation Study Nutrient Culture Studies SUMMARY AND CONCLUSIONS LITERATURE CITED iii 23 23 27 40 46 61 66 LIST OF TABLES Table Page 1. Yield and Zn content of pea beans as affected by carrier and rate of application of Zn and source and rate of P application -- Midland County, 1963 . . . . . . . . . . . 25 2. Yield and Zn content of pea beans as affected by carrier and rate of application of Zn and source and rate of phOSphorus application -- Tuscola County, 1963 . . . 26 f3. Chemical properties of TVA experimental fertilizers -- 1964 field trials . . . . . 29 4. Dry weight of three-week-old pea beans plants as affected by soil P level, planting time P source and Zn rate . . . . 32 5. Zn content of pea beans as affected by soil P level, planting time P source and Zn rate . . . . . . . . . . . . . . . . . . . 34 6. Zn uptake by pea beans as affected by soil P level, planting time P source and Zn rate . . . . . . . . . . . . . . . . . 35 7. P content of pea beans as affected by soil P level, planting time P source and Zn rate . . . . . . . . . . . . . . . . . 37 8. Yield of pea beans as affected by soil P level, planting time P source and Zn rate . . . . . . . . . . . . . . . . . 38 9. F ratios from analysis of variance of parameters for Arenac County pea bean. plots -- 1964 . . . . . . . . . . . . . . 42 10. F ratios from analysis of variance of parameters for Bay County pea bean pIOtS -- 1964 o o o o o o o o o o o o o o 43 11. Influence of P added to Wisner Clay Loam soil on availability of Zn . . . . . . . . 45 iv Table Page 12. Uptake of Zn65 as affected by P level and Zn content of nutrient solution cultures -- pea beans harvested at pod set . . . . 49 13. Uptake of Zn65 as affected by P level and Zn content of nutrient solution cultures -- pea beans harvested at bloom stage . . . . 50 14. Uptake of Zn65 as affected by P level of' nutrient solution and washing of roots . . 53 15. Uptake of Zn65 as affected by P level of nutrient solution and washing time of roots 0 O O O O O O I O O O O O O O O O O 56 LIST OF FIGURES Figure Page 1. Relationship between total Zn content of APP fertilizer and water-soluble Zn content . . . . . . . . . . . . . . . . . 30 2. Relationship of water-soluble and total Zn in fertilizers to yield . . . . . . . . 41 3. Effect of washing time on P removal from pea bean plants 0 O O O O 0 O O O O O O O O O O 57 4. Effect of washing time and P content of nutrient solution on uptake of Zn by pea bean leaves . . . . . . . . . . . . 58 5. Effect of washing time and P content of nutrient solution on uptake of Zn55 by pea bean roots . . . . . . . . . . . . . . 59 vi INTRODUCTION Zinc is present in most soils in adequate amounts for optimal plant growth. But mere presence alone does not foster any indication as to the availability of Zn for plants. Soil factors that influence Zn availability are: pH, P level, free CaC03 content, organic matter content, and adsorbtion by clays. Of these, the first two are regarded as the most important. Many productive soils of the Lake-plain area of East- Central Michigan are calcareous with pH values greater than seven. Added to this factor is the presence of a high level of soil P which has resulted from high levels of fertiliza- tion carried out by the area farmers to obtain maximum yields of sugar beets. Subsequent crops of pea beans grown on these soils have shown varying degrees of Zn deficiency. Pot culture studies with pea beans have substantiated the field observations thus indicating that the P level of the soil together with the effect of alkaline soil pH values intensifies Zn deficiency. Conflicting information has been published con- cerning the effect of the P level, the amount of Zn needed to correct Zn deficiency, whether the Zn deficiency was caused by precipitation of insoluble Zn compounds in the soil or if the Zn-P antagonism was of a physiological nature. These questions prompted the following objectives of this investigation. 1. To determine the effect of some carriers and rates of Zn application on yield and uptake of Zn by pea beans (Phaseolus Vulgaris). 2. To study the interaction of P and Zn in pea bean production. 3. To elucidate the site and mechanism of P and Zn antagonism. REVIEW OF LITERATURE Zn has generally been recognized as an indispensable element for living organisms since 1869 when Raulin (52) showed that Zn was needed for the growth of Aspergillis niger. This observation was later confirmed by Bertrand and Javillier (6). Maze (40), in working with Zea gays in nutrient cul— ture solutions, was the first to show that Zn was essential for higher plants. The concept of Zn essentiality was not universally accepted until 1926, when Sommer and Lipmann (57) proved conclusively that Zn was an essential element for the growfii and development of several species of higher plants. After this time many workers found that physiological diseases such as white bud of corn, bronzing of tung trees and rosetting of citrus trees were corrected by Zn treat- ment. Allison gt. 4;. (2) found that ZnSO4 increased the yields of corn, peas, peanuts and millet. Later in Texas (1) pecan trees were found to respond to Zn as did citrus in California (17). It was found by these workers that the "disease" could be cured by adding Zn salts to the soil, by Spraying the tree with solutions of Zn salts, or by driving pieces of galvanized iron into the trunk. Functions of Zinc in Higher Plants In most higher plants, less than 50 micrograms of Zn per gram of dry tissue are required for normal growth (50). Even so, deficiencies of this nutrient are wideSpread across the United States and almost every economic crop has been shown to respond to Zn fertilization. One of the first visual deficiency symptoms is interveinal chlorosis of the lower leaves followed by necrosis of these mottled areas. In many plants a shortening of the internodes becomes pro- nounced. In the case of pea beans an asymmetic development of the leaf lomina causing ”sickle shaped” leaves will occur. Possibly the most well known role of Zn in higher plants is its interrelationship with auxin. Skoag (56) found that Zn deficient tomato plants were also low in auxin. And, the addition of Zn to these plants caused an increase in auxin activity within 24 hours. Because the peroxidase activity increases in Zn deficient plants, Hewitt (27) suggested that the low level of auxin in the Zn-deficient plant was a result of the destruction of the hormone rather than a lack of synthesis. On the other hand, Tsui (61) showed that Zn was implicated with auxin synthesis because Zn deficient plants that were low in auxin were also low in tryptophan. Theoretically the enzyme system which is reaponsible for the formation of indole-acetic acid from the tryptophan in healthy plants is the same as that in deficient ones. Additional support was given by Nason, 2&3.§l- (43) who showed that tryptophan Synthetase, which catalyzes the formation of tryptophan from indole and serine, is significant- ly and specifically decreased by a Zn-deficiency. The first enzyme in which Zn was established as a metal component was carbonic anhydrase (19) isolated from red blood cells. Although carbonic anhydrase activity has been observed to be present in plants (29) no one has been able to prove a functional relationship between Zn and the protein in plants. Researchers (49) have found that great increases in total free amide and amino nitrogen compounds occurred in tomato plants as a result of Zn deficiency. Reed (53) observed that Zn deficient plants possessed a higher concentration of inorganic P than normal plants. But this effect was not specific for Zn. Quinlan-Watson (51) associat- ed Zn supply with aldolase activity in subterranean clover which might eXplain, in part, the effect of Zn levels on the incrganic P status. Decreased aldolase could decrease the production of glyceraldehyde 3-phosphate and therefore cause decreased phosphorylation of adenosine diphosphate during triosephosphate dehydrogenase action. Nason and Evans (42) found that Zn deficient fungi had a twentyfold increase in DPN ase concentration, but con- centrations of other enzymes such as fumerase and hexdkinase were unaffected. During the study of Zn deficiency and its effects on metabolism, it is of importance to note that the synthesis of vitamins, amino acids, purines or pyrimidines are unaffected. Nason and McElroy (44) have presented a theory which utilizes much known information which suggest that only those enzymes which possess a relatively simple structure appear to increase in deficient cells because they can be synthesized without the occurrance of several key reactions needed for the formation of complex molecules. In this situation a polypeptide pool could be built up because a single template could function as a site for formation of a single polypeptide chain. However, the secondary and tertiary structure, which is to a certain extent under nutritional control,determines the Specificity of catalytic activity. Yeast alcohol dehydrbgenase has been conclusively shown to require Zn for activity (37). These workers believe that Zn serves to bind the pyridine nucleotide to the pro- tein portion. The Zn atoms are believed to stabilize the structure of the enzyme by forming a bridge between the apoenzyme and coenzyme. Although it can be shown that metal enzymes such as various oxidases are in lower concentration in plants de- ficient in a specific metal, it cannot be assumed that the deficiency in a given element such as Zn would decrease these enzymes. It must be emphasized that because a de- ficiency of the specific metal element caused a decrease in the concentration of the particular enzyme it does not follow that a certain enzyme contains that Specific metal. Most research today is but suggestive. More extensive research is needed in this area to illustrate a Specific need for the Zn ion in plant metabolism. Geochemistry of Zinc Zn is more uniformly distributed among rocks formed at various stages of development than other micronutrients. This fact alone perhaps accounts for the uniformly occurring Zn deficiencies in the United States. By 1962, 31 of the 50 states had reported Zn deficiencies (5). Zn is contained in most of the naturally occurring soil minerals, for example biotite and hornblende, components of acidic rocks, contains 50 ppm Zn and ferromagnesium minerals, components of basic rocks, contain approximately 100 ppm Zn (41). Other primary Zn containing minerals which occur in the soil in minute quantities are: Zn blende, ZnS; Smithsonite, ZnCO3: Willemite, Zn(Fe02)2; and calamine, ZnZSiO4°H20. A large.portion of the soil Zn is probably held as isomorphously substituted Zn in an octahedrally coordinated position nor- mally occupied by Al and Mg. Sauconite is a montmorillonite clay mineral of this type (48). Tn.the soil profile, the A1 horizon generally con- tains the greatest quantity of Zn because of the high organic matter content. But total Zn has been found to be highly variable in soil profiles and dependent on Specific soil groups (41). Interaction of Soil Factors and Zinc Availability Organic Matter There is some question as to the significance of soil organic matter in supplying Zn to the soil. Some workers (59) have found that fields which received liberal applica- tions of manure produced plants with notable Zn deficiency symptoms. They concluded that the Zn in the soil_was fixed by the organic matter. This view was supported by DeRemer. .gg..§l. (21) who found that soils became Zn deficient when incubated with ground sugar beet tops. It was concluded that microorganisms immobilize Zn while decomposing the added organic matter. On the other hand, it has been commonly observed that a soil becomes Zn deficient when the surface soil is removed, for example during the process of leveling for irrigation. Hodgsonkgg.fial. (30) found that with the 20 dif— ferent soils included in their investigation from 28 to 99 percent of the Zn in Soil solution was complexed with organic matter. Later work by Geering and Hodgson (26) showed that adding citrate to a carbonate saturated water system increased the rate of movement of Zn dramatically. The rate of increase was found to be portional to the level of citrate added to the solution. Wisconsin researchers (20), using infrared techniques, have found that soil organic materials make a Significant contribution to the Zn complexing capabilities of the soil. But others (62) have concluded that only very small quantities of total soil Zn could be attributed to the organic form. It has been reported (3) that steam sterilization of Zn deficient soils released sufficient Zn to correct the deficiency. This points to the fact that some of the Zn is fixed by microbial action, at least temporarily. But it Should.be pointed out that steam treatment may reduce soil organic matter to a more colloidal form thus yielding more organic complexing agents in the soil solution to maintain Zn in an available form. A possible explanation for these conflicting reports is that in soils with ample native Zn, the addition or removal of organic materials would not Significantly alter plant growth. But, if the available Zn is limited, addition of an organic matter source that is low in Zn may produce Zn deficiency by microbial fixation of Zn or on the other hand, removal of soil organic matter may remove sufficient Zn to produce Zn deficiency. Soil Reaction It has been well documented (16, 38, 54, 55, 65, and 68) that soil reaction is the most important factor influencing Zn availability in soils. As soil pH increases as a result of liming, Zn availability decreases. Thus, calcareous soils, which inherently have high pH values, have been noted as Zn deficient for many years. Later, Ward, .QEaél- (66) studying several factors influencing P-Zn 10 relations in plants and soils, reported that soil reaction alone was not a major factor governing P induced Zn deficiency, but the degree of K saturation was more important. Wear (68) studied the effect of Ca on Zn uptake by treating a Norfolk sandy loam soil with three different liming materials and reported that Zn uptake decreased as the calcium content of the plant increased, but this effect was due to pH alone. In fact, he found that 92 percent of the variation in Zn uptake from applied fertilizers could be attributed to changes in pH. Titratable Alkalinity Nelson, et. a1. (45) reported a method of evaluating Ithe Zn status of soils by combining acid extractable Zn and titratable alkalinity. It is a well known fact that soil pH varies little with increasing CaCO3 content. The success of their method suggest that CaCO3 content, as well as soil reaction, may govern Zn availability in Soils. Adsorption by Clays A factor closely related to soil reaction is clay fixation. Studies by Elgabaly (22) showed that Zn can re- place Mg and Al in the octahedral position of many clays. Also Elgabaly and Jenny (23) found that Zn was adsorbed partly as a monovalent complex ion after which it became a part of the inner layer of the electric double layer. However, Nelson and Melsted (46) found that after allowing 11 Zn adsorption to occur in Illinois soils the exchange capacity was not altered, thus showing in this case that Zn was not occupying cation exchange positions nor being ad- sorbed in the double layer as a complex ion. They further showed the following retentive relationship for the soils included in their study: Hi) Zn > Ca > ng> K. Zn adsorbed by H-Saturated soil systems could be replaced by NH4OAc, but only a portion of Zn added to a Ca-saturated soil system could be replaced by NH OAc. Also, they found that the longer 4 the period of time of contact between the Zn and the Ca—clay, the less was the Zn removed by extraction. JUrinak and Bauer (35) in studying the adsorption of Zn on carbonate materials reported about ten percent of the absorption sites on calcite are occupied by Zn where the equilibrium Zn concentration is 9 x 10-7 M at 25 C and the degree of affinity proceeded as follows: Magnesite, MgCO3 > dolomite, CaMg(C03)2 > calcite, CaCO3. Also, other researchers (10) have found that when ZnSO4 was added to a soil it became to a large extent non— extractable with 0.1 N HCl. Another investigation (14) Showed that when ZnSO4 and ZnO was added to the top inch of a soil in a column and leached with demineralized water, very 0 little downward movement occurred. Soil Nitrogen Several researchers (24) have found that Zn uptake is enhanced when a Specific Zn carrier is banded with N 12 fertilizer or incorporated into nitrogen granules. Much of the benefit of this association may be the result of an acidification effect since (NH4)ZSO4, the most acid forming N fertilizer of those tested, has been the best N material to which Zn can be added. Soil Phosphorus Perhaps no other facet of the soil-plant Zn relation- ship haS been moreopen to controversy and to a wider variety of interpretations as that of P induced Zn deficiency. One of the first reports of this effect was made by Barnette, .gt.‘al. (4) in Florida. They reported that when ZnSO4 was banded with superphosphate, corn still developed zinc deficiency symptoms but when ZnSO4 was banded without P, growth was vigorous. Other investigators (ll, 64) found no effects of phOSphoruS fertilization on Zn uptake and yield. Boawn, gt, 3;, (11) actually doubled the P content of pea beans and found no yield decrease or zinc deficiency symp- toms. A recent report from Washington (47) stated that a soil with a pH of 7.4, and fertilized with several levels of P created no Zn deficiency in wheat plants either as a visual symptom or a yield response from Zn when it was applied in combinations of N and P treatments. In spite of these reports, numerous others (12, 13, 15, 25, 33 and 34) have found that the P level of the soil and the amount of applied P are well correlated with reduced Zn utilization. 13 Evidence given by Bingham, gt, a1, (8) favors the concept of P immobilizing Cu and Zn as relatively insoluble phosphates that he concluded were external to the root. In a later report (7) Bingham found that when soils received high rates of P the total water soluble Zn increased as compared to soils which received lower amounts of P. This information led him to conclude that the mechanism of P-Zn antagonism cannot be explained on the basis of'phosphate precipitating Zn. About this same time Burleson, £E-.§l- (15) working with corn, tomatoes and beans indicated the possibi— lity of a P-Zn antagonism within the root but outlined no Specific mechanism. Nebraska researchers (66) supported this concept. They gave two reasons for this conclusion, first if the antagonism was a simple chemical precipitation outside the root, additions of P mixed with the soil should Show the greatest effects. This was not indicated by their data. Secondly, as additional Zn counteracts the detrimental effect of the P, it would indicate that an absorption phenome- non with the root cells is involved. Bingham (7) refused to support this concept. His data actually showed an in- crease in the .Zn: content of citrus leaves at the highest P level studied. Research by Bowan, gt, _1, (11) has shown that Zn3(PO4)2 may actually Serve as an effective fertilizer material. Also JUrinak and Inouye (36) in studying the solubility of Zn in P systems have found that even at a pH of 8.0 a Zn concentration of 1.02 ppm was found over solid l4 Zn3(PO This value is about 20 times the amount of zinc 4)2' required to grow healthy plants in solution cultures (28). Bowan and Leggett (9) offer an additional possi- bility. Their data on Russett Burbank potatoes does not support the view that high concentrations of P in the growth medium suppresses the movement of zinc within the plant. Instead they believe that an imbalance between P and Zn eXpressed as a critical P/Zn ratio of the tops is more valid and will give an overall indication of the metabolic imbalance within the plant. A critical P/Zn ratio of 400 is given at which Zn deficiency is detrimental to plant growth. Watanabe, gg..gl. (67) found this critical ratio to be 300 for corn. Most recently Stukenholtz, cg..§l. (58) have found a definite inhibition in the translocation process as a result of an elevated P concentration which gave them a sharp reduction in the Zn concentrations of the nodal and internodal tissues. They were unable to define a critical P/Zn ratio and stated that this ratio would change greatly depending on the part of the plant analyzed. They also concluded that the antagonism is located at the root surface or in the root cells. Although there have been variations in the results reported, some of these discrepancies might arise as a result of differences in soil properties and the kind of crop grown. 15 Soil Temperature Since root development is markedly influenced by temperature, it would seem only natural that Zn deficiency symptoms would occur in a cool, wet season, since this un- favorable environment is likely to be especially pronounced and persistant in the soil. Martin, gt. _1. (39) showed that low soil temperatures accentuated the effect of a phosphorus induced zinc deficiency. Similar results were reported by Ellis, ggsal. (25) with corn. They re— ported both Zn concentration and uptake were less at 55°F than at 75°F. EXPERIMENTAL METHODS In 1963, two experimental areas were located on calcareous lake-plain soils of Michigan, one in Midland County and one in Tuscola County. The areas were selected on the basis of (1) pH value above 7.2, (2) previous usage of high rates of P fertilizer, and (3) no previous Zn fertilization. Soil samples were collected prior to planting, air-dried, hand-crushed and analyzed. The pH of a 1:1 soil to water suSpension was determined with a glass electrode in conjunction with a potentiometer. Phosphorus was extracted with 0.025 N HCl plus 0.03 N NHAF with a 1:8 soil to solution ratio and a one minute extraction period. Phosphorus in solution was determined by the molybdenum blue reduction method. Ammonium acetate extractable K was determined by shaking one gram soil with eight m1. of l N NH4OAc, pH 7.0, filtering and deter- mining the K content of the filtrate by comparison with standards with a Coleman flamephotometer. Zinc in the soil was measured by equilibrating 5 grams of soil with 50 ml of 0.1 N HCl, filtering, and measuring the Zn content of the filtrate with a Perkin-Elmer Model 303 atomic absorption spectrophotometer. The results of the soil test from the two locations rwere: 16 17 Location pH P K Zn 4.;444-- lbs./acre --------- Midland 7.7 16 180 9.2 Tuscola 7.5 42 206 16.8 Each experimental area was prepared for planting by the cooperator using the same cultural practiées on the area as on the entire field. Beginning with planting, all cul— tural practices were performed by Experiment Station personnel and equipment. A split plot design with four replications was used. The two main plots consisted of no P applied prior to plant— ing and 350 pounds of P broadcast and disked into the soil prior to planting. Sanilac pea beans were planted during the first week of JUne with a modified three row commercial planter. All plots received a planting time fertilizer consisting of 280 pounds per acre of 9-36-18 containing three percent Mn. The Zn carriers, with the exception of the ZnO incorporated into APP and AOP, were hand-mixed with the base fertilizer at planting time. No sprays were applied to the crop during the growing season. Plant samples were collected from each eXperimental area at two stages of growth. Three weeks after emergence the total above ground portion of 10 plants was collected from each treatment in each replication, dried at 60°C in a forced air oven, ground in a Wiley mill, and analyzed for Zn content. Jast prior to bloom stage a second sampling 18 was made by collecting the terminal mature compound leaf and petiole from 10 plants in each plot. Zn content was deter- mined as before. The beans were harvested by hand-pulling, staking and then threshing with an experimental bean thresher. Plot yields were taken from one 35 foot row per plot. Using the same basis of selection as in 1963, two different locations were obtained in 1964, one in Bay County and one in Arenac County. Soil samples were col- lected ana analyzed as in 1963 with the following results: Location pH P K Zn --A--54-— 1bs./acre -------- Arenac 7.9 52 312 5.6 Bay 7.8 26 184 11.6 Cultural practices were the same as used in 1963. TVA en— countered difficulties in preparing the experimental fertili- zer in 1963 to give uniform distribution of K. Conse- quently, K was omitted from the fertilizers prepared for the 1964 season. To compensate for this, 60 pounds of K per acre was broadcast over each experimental area prior to planting- The experimental design was a Split plot with a factorial arrangement of subplots. Each treatment was repli- cated four times. Plant samples were taken approximately three weeks after emergence by collecting the above ground portion of 10 plants from each plot. Treatment of plant samples and 19 harvesting of beans were the same as in 1963. For Zn determination in 1963, plant material was dry-ashed at 500°C, the ash dissolved in HCl, and the Zn content of an alioquot determined by the "zincon” method of JOhnson and Ulrich (32). In 1964, the Zn in the dry-ashed material was dissolved in 0.1 N HCl and analyzed on a Perkin- Elmer Model 303 Atomic Absorption Spectrophotometer. Plant material was prepared for determination of P by dry-ashing after pretreatment with a Mg(N03)2-ethyl alcohol solution. The procedures outlined by JaCkson (31) for the determination of P were used with only Slight modifi- cation. An analysis of variance was conducted on all data from field experiments and treatment means were compared by use of Duncan's multiple-range test. Laboratory Studies The affect of applied P on Zn availability was studied in the laboratory by incubating a Wisner clay loam soil for two and one-half years after being treated with three levels of P--100, 500, and 1000 ppm P. The moisture content of the Soil was held near field capacity and the temperature was 25°C during the incubation period. After the incubation period, 25 ml. of a solution containing Zn65 with an activity of 12,500 counts per minute per ml. was allowed to equilibrate with five grams of soil for eight days on a reciprocating shaker. The suSpension was 20 then centrifuged. the supernatant liquid decanted and 50 ml. of distilled water added to the soil. The soil was resuspended, shaken for one hour and centrifuged. One ml. of this supernatant liquid was analyzed for gamma activity. Subsequent extractions were carried out in the following order: 0.01 M.EDTA in 1.0 M (NH4)2CO3 as reported by Trierweiler and Lindsey (60), neutral 1.0 M NH4OAc and finally by 0.1. N HCl. The volume of all extracting agents was 50 ml. Solution Culture Studies Preliminary studies were conducted to determine if P level in nutrient solution would influence Zn uptake by pea bean plants. For all nutrient culture studies pea bean seedlings were germinated in quartz sand and watered with deionized water until the first true leaves began to unfold. They were then transplanted into two-quart polyethylene containers, two seedlings in each container. The nutrient solutions used throughout this study were basically normal Hoagland's solutions with the following modifications. Three levels of P (100, 250, and 500 ppm P) were employed, ferric citrate was the Fe source and no solutions contained Zn initially. Just after definite Zn deficiency symptoms appeared, Zn equivalent to normal Hoagland's solution was added to one-half of the containers. The plants were grown in a growth chamber and con- stantly aerated by forcing air through gas dispersion tubes that were placed in the nutrient solution. The solution 21 was changed only once during the growing period Since the high content of P created an effective buffer. 65 Two m1. of a Zn Cl solution containing 100,000 2 counts per minute per ml. was added to all containers approxi- mately one week prior to harvest. Plants were harvested at pod stage in one experiment and at bloom stage in a second experiment, separated into roots, stems, and leaves, weighed, dried in a forced air oven at 60°C and ground in a Wiley mill. Uniform weight tissue samples were pressed into a pellet in a Carver press and analyzed for gamma activity. A standard curve was developed by adding known quantities of Zn65 to ground plant tissue, drying and pressing the material into pellets. Two experiments were conducted to determine if the P that prevented Zn uptake and/or translocation to leaves could be easily removed from living plants. In the first of these experiments, seedlings were grown until the onset of flowering in normal Hoaglands minus Zn except for a 500 ppm P level. At flowering, one-half of the plants were removed from their medium, the roots dipped in distilled water and then transferred to a container with normal Heagland's minus P, minus Zn for 24 hours. Two m1. of Zn65Cl2 solution with an activity of 100,000 counts per minute per ml. were added to the containers at this time and after 30 hours the plants were harvested, divided into roots and tops, weighed and analyzed as before. In the second experiment plants were grown through 22 the initial stages as just previously outlined. Just previous to flowering, the plants were removed, dipped in distilled water for 15 to 20 seconds to remove the solu- tion adhering to the roots, and then placed in aerated Hoagland's Solution for 3, 6, 12 or 24 hours. Two treat- ments, used for controls, were not subjected to the latter washing procedure. After the required time, the plants were placed in normal Hoagland's solution or Hoagland's 65 solution plus 500 ppm P, and two ml. of Zn C1 solution 2 with an activity of 100,000 counts per minute per ml. added to each pot. After 24 hours the plants were removed, the roots washed briefly in distilled water: divided into roots, stems and leaves: dried, ground, and weighed: and counted for gamma activity. The solutions in which the plants were aerated to remove P were analyzed for P by the molybdenum blue reduction method. RESULTS AND DISCUSSION 1963 Field Experiments The results of the 1963 field eXperiments are given in Tables 1 and 2. The data show that at both locations a high rate of P applied prior to planting Significantly decreased plant content of Zn. Yield of beans was also significantly decreased by high P in Midland County but not in Tuscola County, although high P tended to decrease yield in Tuscola County also. A high level of P accentuated the yield response to Zn as compared to Zn response at a low P level. But this was not true for Zn content. At the location in Midland County application of Zn with high P did not produce as great a yield as Zn and low P. For example, with AOP addition of high P without Zn reduced yields from 22.9 to 10.2 bushels per acre. The best treat— ment with Zn and high P yielded 23.6 bushels per acre. But the comparable treatment of Zn with low P yielded 29.4 bushels. Similar analogies can be drawn with APP as the planting time fertilizer. Thus, even though the application of high levels of P is clearly inducing Zn deficiency in beans, it may be also producing other adverse effects on the plants. Of the three Zn carriers mixed with APP (ZnSO4, Zn.EDTA and Zn20804), ZnSO4 was the most effective in 23 24 supplying Zn to the plant and increasing yield. Zn EDTA was nearly as effective and it Should be noted in this regard that Zn EDTA was applied at one—fifth the rate of Zn, a quantity that would make it more competitive economical- ly. Zn20804 was clearly less effective, particularly when applied at the two pounds Zn per acre rate. When ZnO was incorporated into APP, it was Signifi- cantly superior to ZnO incorporated into AOP in increasing Zn content of plant tissue. It also gave higher yields of beans. In fact, ZnO incorporated into AOP, regardless of the rate of Zn application, was not appreciably different from no Zn. Typically yield of an agronomh: crop Should increase with increasing fertilizer application, reach a maximum yield and perhaps decrease if excessive fertilizer is applied. Some agronomists maintain that the response follows the law of diminishing returns, i.e., the yield increases at a de- creasing rate. Plant uptake of applied fertilizer should closely parallel the rate of application. But when ZnO incorporated into APP was applied to give increasing rates of Zn, the typical yield and uptake reSponse was not realized. Thus in all but one case the 0.8 pound rate yielded greater than the 3.0 pound rate but less than the 9.0 pound rate. In addition, the Zn content data closely paralleled the yield data. While the 0.8 pound treatment seldom out-yielded the 3.0 rate by more than two bushels per acre, the consistency of the data suggest that this trend is real. 25 Table 1. Yield and Zn content of pea beans as affected by carrier and rate of application of Zn and source and rate of P application -- Midland County, 1963.* Treatment Zinc Content' Yield Zn. July 2 July 18 ‘ Rate Carrier Low P** High P='= Low P** Higth* Lowa**. High Pi le./acre ppm Zn :.p—ppm Zn "~. bu/acre Source of P in Starter fertilizer:lemmonium orthophosphate 0 16.2 15.5 8.0 8.0 22.9 10.2 0.8 ZnO 19.0 15.2 9.0 10.0 29.4 23.6 3.0 ZnO 21.5 15.8 10.0 8.0 24.0 18.2 9.0 ZnO 19.0 16.8 10.0 12.0 32.0 18.5 Source of P in starter fertilizer:‘ Ammonium polyphosphate 0 -‘ 16.7 14.7 9.0 6.0 29.4 19.6 0.8 ZnO 33.7 29.2 25.0 ' 24.0 34.7 31.1 3.0 ZnO 25.2 25.5 18.0 14.0 32.9 29.6 9.0 , ZnO 57.2 45.0 26.0 24.0 35.1 33.1 2.0 Znso4 28.5 23.5 22.0 18.0 31.4 26.7 4.0 Znso4 32.9 24.2 18.0 19.0 35.4 31.3 8.0 Znso4 85.0 41.0 40.0 32.0 38.9 31.3 0.2 Zn EDTA 30.8 28.0 12.0 8.0 33.8 28.0 0.8 Zn EDTA 43.0 39.2 32.0 23.0 33.1 27.2 1.6 Zn EDTA 70.5 47.5 56.0 46.0 34.0 30.0 2.0 ZnZOSO4 19.2 13.8 17.0 13.0 22.9 13.1 4.0 2nzoso4 22.5 16.5 17.0 14.0 34.9 26.7 8.0 anoso4 22.5 17.5 22.0 14.0 34.0 28.7 *Planted June 6 and 11, harvested September 9-10. **Low P received only starter phOSphoruS. *High P received 350 pounds P per acre as 0-46-0 prior to planting. 26 Table 2. Yield and Zn content of pea beans as affected by carrier and rate of application of Zn and source and rate of phosphorus application -- Tuscola County, 1963.* Treatment Zinc Content Yield Zn. July 3 July 24 Rate Carrier Low P** High Pi Low P** High Pi Low P** High Pf 1bs./acre ppm Zn ppm Zn bu/acre Source of P in starter fertilizer: Ammonium orthophosphate 0 19.5 18.0 19.0 14.0 19.1 15.6 . ZnO 22.2 20.8 18.0 16.0 19.1 18.4 . ZnO 31.5 33.4 20.0 17.0 22.9 18.4 . ZnO 22.0 25.8 18.0 18.0 22.2 18.7 Source of P in starter fertilizer: Ammonium polyphosphate 0 19.0 21.2 10.0 17.0 18.4 19.6 . ZnO 36.0 34.8 21.0 18.0 23.1 21.6 . Zno 33.8 24.8 21.0 18.0 25.3 19.5 . ZnO 66.8 55.2 31.0 27.0 24.7 27.8 2.0 ZnSO4 29.5 29.2 20.0 18.0 21.4 20.7 4.0 ZnSO4 40.5 47.2 30.0 24.0 24.2 27.4 .0 ZnSO4 85.0 85.0 54.0 48.0 25.8 25.2 0.2 Zn EDTA 28.2 25.8 18.0 18.0 22.5 21.1 . Zn EDTA 37.0 34.2 22.0 18.0 21.3 21.1 1.6 Zn EDTA 40.5 45.6 24.0 26.0 21.3 20.0 2.0 ZnZOSO4 29.2 25.8 19.0 13.0 19.6 14.5 4.0 Zn20S04 27.5 26.0 18.0 17.0 20.4 18.9 8.0 anoso4 35.5 29.5 23.0 16.0 25.6 22.5 *Planted JUne 4, harvested September ll-12. **Low P received only starter phOSphoruS. 1*High P received 350 pounds P per acre as 0-46-0 prior to planting. 27 Increasing rates of ZnSO4, Zn EDTA or Zn20804 increased Zn content and yield in the eXpected manner. Maximum yield with ZnSO was reached with a 4.0 rate, 4 whereas, 8.0 pounds of ZnZOSO4 were required to give maximum yield. Nearly maximum yield was obtained with 0.2 pounds of Zn as Zn EDTA. Yield increases due to application of Zn were much greater at Midland County as compared to Tuscola County. This agrees well with the soil test information and with the Zn content of the plants from the no Zn treatments. It has been suggested that the critical level of Zn in bean tissue is between 15 and 20 ppm. The value of from 14.7 to 16.7 obtained in Midland is in the lower part of this critical range. But the values of 18.0 to 21.2 from Tuscola County are near the upper part or even exceeding this range. 1964 Field Experiments In the 1963 field trials ZnO incorporated into AOP did not appear to be a satisfactory source of Zn for pea beans. But Zno incorporated into APP was as good as any Zn Source tested. Since the response curve in the low Zn rates did not appear normal with ZnO incorporated into APP, eXperimental fertilizers were prepared by TVA with varying Zn:P ratios to allow study of that part of the growth reSponse curve that is between zero and three pounds of Zn per acre. 28 The ZnO was added to the fine fertilizer material being recycled and added to the melt as it was transferred from the reaction chamber to a rotary granulator. The Zn was therefore uniformly distributed through the granule but was not in contact with the hot, liquid melt for ex- tended periods of time. The properties of the fertilizers supplied by TVA are given in Table 3. The Zn:PZO5 ratio varied from 1:202 to 1:7 for APP fertilizers prepared with Zn. The cor- responding ratios for AOP fertilizers were 1:196 to 1:9.5. As the percentage of Zn in the APP fertilizer increased the percentage of added Zn that remained water soluble increased until the Zn:P205 ratio of 1:67 was reached. Increasing the Zn beyond this level, i.e., lower Zn:P205 ratios, markedly reduced the percent of the added Zn that remained water soluble. The percent of the added Zn that was water soluble remained very low at all levels of Zn addition to AOP suggesting that the new compounds formed were even less soluble than the ZnO added. The total water soluble Zn in the AOP fertilizer was less than 0.63 percent in all cases. As the Zn content increased, the percentage of water soluble Zn in the APP fertilizer increased until a Zn:P205 ratio of 1:47 was reached. At this point the percentage of water soluble Zn decreased markedly but increased again at the highest Zn level. This discussion is summarized in Figure 1. This figure clearly shows that in the regions of low content of 29 om.0 v.0 0.0 No.0 No.0 0.0 n.0ua 0.m 0|5.5¢I¢.HH 00¢ n0.m 0.H ~.H no.0 No.0 0.m 0.0Nua 5.H 010.0mlm.HH 00¢ «5.0 0.H 0.H no.0 «0.0 0.m m.moua H.H 01¢.0ml5.aa 00¢ N5.m m.a m.n 00.0 no.0 0.m n.00ua 05.0 olv.omlm.aa 00¢ 0m.m 0.H m.a no.0 no.0 H.m m.mmuH mm.0 0|N.Hmla.aa 00¢ mm.m v.H m.HH 50.0 no.0 H.m n.0oaua 0N.0 0:0.Hm10.aa 00¢ mm.0 m.oa 0.0 00.0 nm.0 5.0 H.5«H 0.0 0|0.0¢|5.HH 000 00.0 5.HH n.ma H0.0 nm.0 0.0 0.¢nua m.a 01¢.mmlm.ma 00¢ om.0 0.H 5.00 0H.0 50.0 ~.m n.5eua H.H 0|N.leo.na 00¢ 0H.0 m.na «.00 «5.0 0n.0 mum H.50ua 05.0 0|n.~mln.na 00¢ no.0 m.HH 0.00 H0.o 0H.0 n.m m.nnHuH 0v.0 01v.nmlm.na 00¢ 0H.0 N.na 0.0n 05.0 00.0 n.m 5.N0mua 0m.0 0:5.Nm|¢.na 000 s. an x x. mumuuaau as an as as c: mommucn cm 06600 0089060 no mm uomnmum ucmfimam 0H0Emm Hmuou mo a0>0a ~33 60 x :33.” 8038 x... .6336». o m 0.033.383: uomwnuocouoflz .mamwuu paowm eoma :1 mumuaafluumm kucmeflum0x0 «>8 «0 mmfluum0ou0 donnedco .n manna 3180108 HEIlVM UZ 0300\7 :10 % 0N oo 00 .ucmuooo cm mesHOm numums can nmuflflauumm 00< no 0000000 on Hmuou cmmaumb dwsmcowuwflma .0 ouowwm mMNICmmE 004. z_ cN o\o 0.0 0.m 0 _ \< )\ _ _ _ 2 a _ .CIII 0% III .0 II.\\.\\\\ .111. .k\ -mo 0 ll \I \O .0 6 led \ Cs“.\ 10 Lec HBZI'IIlHEH ddV NI 02 3180103 HEIlVM °/o 31 Zn in APP the water soluble Zn present goes through a maximum correSponding to about one percent Zn in the fertilizer. This phenomenon may be due to the formation of different compounds with increasing level of Zn in the fertilizer. Thus, when Zn is present in low amounts in relation to polyphOSphoric acid, a relatively stable complex is formed that is water soluble. But as increasing quantities of Zn are added to this system, it becomes saturated or even supersaturated with respect to this complex. A new Zn-P compound is then formed which is much less soluble than the complex formed at low Zn levels. Once this compound is formed it may actually ”mine" Zn from the complex first formed and thus total water soluble Zn may decrease for a short range. The variables measured in the field experiments in 1964 were dry weight of young bean plants, Zn content and uptake and P content by the same plants, and yield of beans. For convenience, statistical analysis for all data are presented in Tables 9 and 10 at the end of the discussion. The data for dry weight are given in Table 4. Without exception the 8.0 pound rate of Zn incorporated with APP caused the greatest amount of early growth of any treatment at either location. Ammonium polyphOSphate appeared to be the better P source at the location in Arenac County as every Zn treatment with APP produced greater plant growth than the correSponding Zn treatment with AOP as the P source. These differences did not appear at the location in Bay 32 Table 4. Dry weight of three-week-old Pea beans plants as affected by soil P level, planting time P source and Zn rate. Treatment Arenac County Bay County Planting time 1 . 2 . P source Zn rate Low P ngh P Low P ngh P lbs/acre gms/lO plants Ammonium Ortho- 0 22.7 23.2 24.1 23.7 phOSphate 0.4 27.6 28.7 24.5 26.7 0.7 28.0 29.2 27.6 23.8 1.2 28.3 30.0 28.8 27.7 1.6 27.9 27.9 26.0 24.7 2.7 29.8 31.4 25.3 26.0 8.0 32.3 32.7 26.5 26.3 Ammonium Poly- 0 23.7 23.3 22.5 24.9 phosphate 0.4 27.8 27.7 26.0 26.1 0.7 32.2 30.2 26.3 27.4 1.2 29.4 31.1 26.2 26.4 1.6 33.5 31.9 26.8 24.4 2.7 32.6 32.1 28.3 25.8 8.0 35.8 33.9 34.2 30.2 1Zn was applied as ZnO incorporated into either AOP or APP. 2 Low P was zero P prior to planting: high P was 350 pounds P per acre as 0-46-0 applied prior to planting time. 33 County. Early growth was slowed at the latter location be- cause of dry weather. Application of high P did not decrease plant growth at this stage of growth. Zinc content generally followed a similar trend. The 8.0 pound rate with APP was significantly better than every other treatment. The data in Table 5 Show that in Arenac County the 0.7 pound rate with APP produced plants with a higher content of Zn than any Zn rate with AOP. Results from Bay County, although somewhat inconsistent, Show that plants receiving the highest rate of Zn-APP possessed the highest Zn content of any on that location. On both high and low P plots at Arenac County the Zn content was less for the 2.7 pound Zn-APP treatment than at the 1.6 pound Zn-APP rate. The P level caused a significant decrease in the Zn content at location A, and even at the highest level of applied Zn the plant content never reached the level attained on the low P plots. High P level appear- ed to have reduced Zn content at the Bay County location but the effect was not significant. The 8.0 pound Zn-APP rate was again significantly better in promoting zinc uptake than the other treatments (see Table 6). Ammonium polyphOSphate was the better Zn carrier on both locations, the P level appeared to have a detrimental effect on Zn uptake but the difference was insufficient for statistical significance. The AOP source did not produce much uptake at the lower rates and only the higher rate of Zn in the fertilizer made a noticeable 34 Table 5. Zn content of pea beans as affected by soil P level, planting time P source and Zn rate. Treatment Arenac County Bay County Plgnzizgcgime Zn rate1 Low P High P2 Low P High P lbs/acre ppm Zn Ammonium Ortho- 0 21.4 14.4 19.2 16.9 phosphate 0.4 16.4 17.7 20.3 17.7 0.7 18.3 16.1 17.9 19.2 1.2 18.0 15.7 19.9 17.4 1.6 17.4 17.7 17.4 18.7 2.7 19.5 20.1 18.9 17.9 8.0 19.5 21.6 20.1 20.1 Ammonium Poly- 0 916.2 16.7 19.6 18.5 phosphate 0.4 19.2 17.4 19.0 18.9 0.7 20.1 21.8 21.5 16.5 1.2 21.4 20.8 22.8 18.7 1.6 23.9 24.9 20.6 21.8 2.7 22.3 20.9 23.3 21.3 8.0 31.0 30.1 26.9 24.4 lZn was applied as ZnO incorporated into either AOP or APP. 2 Low P was zero P prior to planting: high P was 350 pounds P per acre as 0-46-0 applied prior to planting time. Table 6. 35 Zn uptake by pea beans as affected by soil P level, planting time P source and Zn rate. Treatment Arenac County Bay County Planting time Zn ratel Low P High P Low High P source lbs/acre mg Zn/lO plants Ammonium Ortho- 0 0.14 0.08 0.16 0.14 phosphate 0.4 0.20 0.23 0.18 0.19 0.7 0.21 0.22 0.21 0.15 1.2 0.24 0.28 0.27 0.21 1.6 0.20 0.21 0.18 0.17 2.7 0.27 0.32 0.18 0.18 8.0 0.32 0.37 0.22 0.21 Ammonium Poly- 0 0.13 0.12 0.12 0.17 phosphate 0.4 0.23 0.21 0.20 0.25 0.7 0.31 0.32 0.23 0.22 1.2 0.30 0.35 0.24 0.23; 1.6 0.40 0.40 0.22 0.19 2.7 0.36 0.34 0.30 0.22 8.0 0.63 0.55 0.51 0.36 1Zn was applied as ZnO incorporated into either AOP or APP. 2 Low P was zero P prior to planting: high P was 350 pounds P per acre as 0-46-0 applied prior to planting time. 36 difference. Again a decrease in Zn uptake occurs at the 2.7 pound Zn-APP rate at the Arenac County location on both P levels. This was consistent with the data on Zn content. Phosphorus content was inversely affected by zinc rate (Table 7). In fact, the 8.0 pound rate of Zn-APP which was significantly better in increasing Zn uptake and content produced significantly lower P contents than any treatment at either location. One result which might have been anticipated was the significant increase in P content produced by increasing the P level. Both locations Showed a direct relationship between P level and content. Here the APP proved to be different from the AOP, the latter seemingly a better P source. The reason for this may not be because AOP is a better source of P but as Zn was shown to be less available from the AOP, Zn deficiency might result which in turn might cause a build-up of an unusable P pool in the plant. This was discussed earlier in the literature review. Much of the information that was found concerning seedling weight, plant content and uptake of Zn corresponds quite well when considering the yield data in Table 8. The P level caused a significant decrease in yield at both locations but the difference was more noticeable in Arenac County. A response was obtained from Zn in Bay County but with the exception of a response over the check and the highest rate correlating well with the highest yield the 37 Table 7. P content of pea beans as affected by soil P level, planting time P source and Zn rate. Treatment Arenac County Bay County Planting time , 1 . 2 . P source Zn rate Low P High P Low P High P lbs/acre 1% P Ammonium Ortho- 0 0.46 0.59 0.55 0.74 phosphate 0.4 0.45 0.58 0.46 0.62 0.7 0.42 0.56 0.51; 0.65 1.2 0.46 0.50 0.46 0.67 1.6 0.44 0.54 0.47 0.63 2.7 0.44 0.44 0.48 0.61 8.0 0.36 0.42 0.41 0.56 Ammonium Poly- 0 0.50 0.61 0.53 0.61 phosphate 0.4 0.43 0.49 0.40 0.51 0.7 0.34 0.43 0.40 0.57 1.2 0.34 0.42 0.40 0.52 1.6 0.33 0.42 0.42 0.62 2.7 0.32 0.40 0.43 0.47 8.0 0.29 0.33 0.38 0.43 1Zn was applied as ZnO incorporated into either AOP or APP. 2 Low P was zero P prior to planting: high P was 350 pounds P per acre as 0-46-0 applied prior to planting *time. 38 Table 8. Yield of pea beans as affected by soil P level, planting time P source and Zn rate. Treatment Arenac County Bay County Planting time 1 . 2 w. P source Zn rate Low P High P Low P High P _F— lbs/acre bu/acre Ammonium Ortho- 0 16.9 6.2 32.0 31.1 phOSphate 0.4 19.6 10.7 40.0 33.8 0.7 20.5 15.1 38.3; 32.0 1.2 22.2 19.6 34.7 29.4 1.6 24.9 13.3 38.3 34.7 2.7 24.0 19.6 38.3 32.0 8.0. 29.4 19.6 42.7 33.8 phosphate 0.4 24.0 16.0 39.2 36.5 0.7 25.8 17.8 40.9 37.4 1.2 22.2 16.9 40.9 37.4 1-6 29.4 19.6 41.8 40.0 2.7 28.5 18.7 40.0 36.5 8.0 32.0 21.4 44.5 41.8 1 2 Zn was applied as ZnO incorporated into either AOP or APP. Low P was zero P prior to planting: high P was 350 pounds P per acre as 0-46—0 applied prior to planting time. 39 results seemed to be inconsistent. The superiority of APP over AOP as a P source in the planting time fertilizer was again Significant at both locations. At.Arenac County with APP a 14.6 percent increase was obtained over AOP and a 12.7 percent increase was obtained at Bay county. These data show that at both locations the yield from the 2.7 pound rate of Zn-APP was lower than the highest yield reached with lower applications of Zn. This is consistent with the results obtained with Zn uptake and Zn content. Throughout this study it is noticed that more Signifi- cant results were obtained on the Arenac County experimental area. This is undoubtedly because of the higher amounts of extractable Zn obtained from the analyses of the soil from the Bay County plot. This would be indicative of the natural ability of the soil to supply Zn to the plant. A lucid explanation of a consistent decrease in Zn content, Zn uptake and yield with the 2.7 pound rate of Zn-APP might relate directly to Figure 1. As the percentage of water soluble Zn in the APP fertilizer increased, the values mentioned above also increased. As the water solu- bility decreased at the 2.7 pound rate, the measured values also decreased. Throughout the experiment APP was a Signifi- cantly better carrier of Zn than AOP. In Table 1 we can see that the water solubility of the Zn in the AOP fertilizer is at a low level. From this information one might con- clude that the percent water soluble Zn in a given fertilizer is a very important criteria in determining the effectiveness 40 of the fertilizer as a supplier of micronutrients to the plant. But a direct plot of water-soluble Zn applied against yield gave little indication of a good relationship. However, if some weight is given to the total Zn present in the sample, a better relationship is obtained. An example of such a relationship is given in Figure 2. This indicated that the total value of Zn in a fertilizer is a function of both total and water-soluble Zn in the fertilizer but water-soluble is the more important factor. Laboratory Incubation Study From field studies of 1963 and 1964, it was clearly shown that applied P had a significant effect in inducing Zn deficiency in pea beans. As previously discussed in the review of literature, there has been considerable controversy regarding the availability of Zn in a soil high in applied P. In an attempt to clarify this enigma of whether the Zn is rendered unavailable in the soil by high P levels, or if this antagonism is a physiological effect, a calcareous Wisner clay loam was incubated with three levels of P (100, 500, and 1000 ppm) for two and one-half years. To five grams of this soil 25 ml. of a ZnGSCl2 solution containing 10,000 counts per minute per ml. was allowed to equilibrate for one week. Following this treat- ment the sample was successively extracted with four extracting solutions in the following order: H20, 0.01 M EDTA in l M (NH 1 N NH OAc buffered at pH 4’2C03' 4 (Bu/A) YIELD OF BEANS 35 01 O 25 20 41 - o o /e o o o o o o APP o O 0 AOP o I 0 1 l l l l J l l l llj 0 IO 20 zoxuzo SOLUBLE ZN + TOTAL ZN Figure 2. Relationship of water-soluble and total Zn in fertilizer to yield. 42 .Hcsma as 06 HCMOHMACmHm cents a .Hm>mH am um unmoamaemamnw mmxmxam 440.4 aao.c se~.ma 0.H 0.0 c coauomuwuca mmxm ssm.0 ssv.¢ ssm.moa 040.5 m.N 0 GOHHOMH0HCH A000 mohoom 0 s¥¢.¢a ssm.N0 ssm.omv ssm.m0 sso.mH H mafia mcflucmflm .ss0.mm ssn.m~ ssm.~mm ssm.HN sso.mn 0 Amv Oumm CN aee.mc sea.mm c.c 45.ea «.6 H lame Hw>ca 6 names ucwucoo e 0x600: an ammucoo an passes 06840 .0.0 mouoom down 000 >9G500 .8060 I: anode omcou< mom numumfimumm mo woGMHHm> mo manhawcm Scum monumu 0 .0 manna 43 .Hm>oH.xH um uccoamammam ase .amaca an em ucmoamacmam a mmxmxnm N.H N.H ss¢.m ssm.m ssm.m 0 :OfluOMH0HCH mmxm a.H v.0 «so.m c.~ a. c coauomumucH A000 wuuoom 0 «4m.mm 44c.am 440.00 44m.cm m.m H 0889 meanness s¥¢.m ssm.0 ssm.m ssm.m so.n 0 Amy 090% SN e.m ate.cae.a m.oi e.m am. a lame Hm>ma 0 came» uccueoo 0 6x600: cm ucmucoo cw ucmams 0:640 _ , I. 0.0 mousom oaumm 0 .ccma u: muoam down 000 musoou 500 How mumumeonm0 mo mononum> mo nflmwamom Eoum mOAHMH 0 .0H manna 44 7.0, and 0.1 N HCl. Each of these solutions have been used to characterize available soil Zn. The extracting order was selected to give a sequence of decreasing suspension pH. 65 were found in the water Very small quantities of Zn extract. In fact this fraction included less than two percent of the Zn65 which was removed by the four extractions. AS shown in Table 11, there is little indication that applied P was influencing the water-soluble Zn level. Certainly, if P and Zn are precipitating in the soil, the water soluble Zn65 Should have decreased with increasing level of P. Extraction of the soil with EDTA buffer solution removed greater than 85 percent of the recovered Zn65. In addition, the quantity of Zn65 recovered increased with increasing P level. Thus, an additional 11,000 counts per minute were recovered in this fraction where 1,000 ppm P were added as compared to 100 ppm P. Colorado researchers (60) have shown that this extracting solution is one of the best for estimating available soil Zn. Consequently, this data Show that addition of high levels of P increases rather than decreases the availability of Zn in soils. The third extraction with 1N NH4OAc removed about 10 percent of the Zn65. Because of the lower pH of this solution it should be expected to remove more difficultiy available Zn. AS the amount of P applied prior to incubation increased, the quantity of Zn extracted by this reagent de- creased consistently. This result can be explained because of the effect the applied P has on making Zn more easily 45 Table 11. Influence of P added to Wisner Clay Loam soil on availability of Zn. P l Extraction Extracting evel (ppm) number solution 100 500 1,000 1, H20 2,000 3,350 2,550 2. Na-EDTA- 193,500 197,400 204,500 (NH4)2C03 3. NH4OAc 23,200 20,350 17,300 4. HCl 8,450 6,700 6,250 Total --- 227,150 227,800 230,600 46 available and thus extractable with the EDTA buffer. The quantity of Zn present in the more difficultly available forms is less. Finally, the sample was extracted with 0.1 N HCl which should remove Zn which had precipitated as a basic compound when the equilibrium was established between the Zn65C12 and the soil. This fraction contained less than four percent of the Zn65 which was extracted. This fraction was similar to that extracted by NHQOAC in that the quantity extracted decreased with increasing P level. AS the EDTA extracting agent is assumed to give an estimate of the readily available Zn, the results of this eXperiment indicate that when the P level of the soil is increased by heavy fertilizer applications, the amount of available Zn is also increased. The data actually Shows a slight increase in the total Zn65 replaced when high levels of P had been applied, but this increase is well within experimental error. Collectively, this is strong evidence that the Zn-P antagonism does not occur in the soil due to formation of insoluble Zn phosphate compounds as has been suggested by some early work. Nutrient Culture Studies The hypothesis was made that the antagonistic effect of P on Zn was of a physiological nature. Solution culture studies were initiated to test this hypothesis under a controlled environment, permitting more efficient observa- tions of P effects without interference from adsorption 47 of P and Zn by soil. Pea bean seedlings, which were germinated in quartz sand, were transplanted into two-quart polyethylene containers that held two seedlings each. The treatments were three levels of P (100, 250, and 500 ppm). One half of each level contained Zn in a concentration recommended by Hoagland and the other half received no Zn. Each treatment was established in duplicate. The main objective of the research at this time was to determine if P prevented Zn uptake from solution culture and if so, the location within the plant where the Zn was blocked and made unavailable for use. The plants were allowed to set pods since it was believed that at seed formation the plants requirements for Zn was increased putting added Zn stress on the plants. The plants which were Zn deficient were extremely chlorotic and in some instances necrotic. At this time Zn65Cl2 was added to all pots for 48 hours. The plants were constantly aerated during this period as well as during the entire growing period by forcing air through gas dispersion tubes which were in each container. The plants were harvested, roots washed, separated into roots, stems and leaves, dried in a forced air oven at 60 C, and ground in a Wiley mill. The tissue was then weighed into one gram portions, pelleted by a Carver hydraulic press and analyzed for gamma ray activity by a scintillation crystal and counter. 48 The results in Table 12 Show that regardless of prior Zn treatment, most of the added Zn65 was not trans— located further than the roots. And this was also true to a large degree at all P levels. The data do indicate, 65 were absorbed into however, that greater quantities of Zn the roots at higher P levels. Plants which were produced in normal Hoagland's solution held more Zn65 in their roots as compared to the Zn deficient plants. Hewever, these differences were small except for.the 250 ppm P level. The total activity found in the stems and leaves was so low that any differences between treatments may be due to experimental error. Mere total Zn65 was adsorbed by plants produced under high P levels, but this reflects little more than the data for the roots. The experiment was repeated as before except that the plants were given Zn65Cl2 at the onset of flowering and at a level 10 times the previous dosage. This was done to: (l) ascertain if the age of the plant affected Zn trans- location and (2) permit a more accurate measurement of the translocation pattern because of the greater activity supplied to the plants. As in the previous experiment, the largest portion of the added Zn65 was blocked in the roots (see Table 13); however, a greater amount of the Zn65 moved from the roots and into the stem and leaves, than in the previous experiment. In this experiment, from 25 to 37 percent of 65 the Zn absorbed was found in the stems and 8 to 13 percent 49 Table 12. Uptake of Zn65 as affected by P level and Zn content of nutrient solution cultures--pea beans harvested at pod set. P levelvof'nutrientlsolution' Zn level of Plant Part nutrient solution 100 ppm 250 ppm 500 ppm Zn65 in counts/minute/gm* Roots none 4853 5203 6853 Roots Normal Hoagland 5453 6753 7053 Average 5153 5978 6953 Stems None 153 503 I 53 Stems Normal Hoagland 78 103 3 Average 115 303 28 Leaves None 13 213 18 Leaves Normal Hoagland 28 13 3 Average __;Q _113_ .__;g Total Zn65 ' . Absorbed None 5019 5919 6924 Normal Hoagland 5559 6869 7059 *Each value reported is a mean of two replications. 50 Table 13. Uptake of Zn65 as affected by P level and Zn content of nutrient solution cultureS--pea beans harvested at bloom stage. Zn level of P level of nutrient solution Plant Part nutrient solution 100 ppm 250 ppm 500 ppm Zn65 in counts/minute/gm* Roots None 11,500 15,500 11,650 Roots Normal Hoagland 8,500 8,500 11,500 Average 10,000 12,000 11,575 Stems None 6,300 6,300 8,500 Stems Normal Hoagland 3,600 3,300 5,500 Average 4,950 4,800 6,500 Leaves None 1,700 2,100 2,730 Leaves Normal Hoagland 1,200 1,250 2,650 Average 1,450 1,675 2,690 Total Zn65 Absorbed NOne 19,500 23,900 22,880 Normal Hoagland 13,300 13,050 19,650 *Each value reported is a mean of two replications. 51 in the leaves. With the older plants in the previous experi- ment from 88 to 99 percent of the Zn65 remained in the roots. This may have been because of a greater metabolic activity of the younger tissues. In this experiment the plants growing in normal Hoagland's solution absorbed less Zn65 than the Zn deficient plants. This may be explained 65 with nonradioactive Zn in the normal Hoagland's solution which reduced Zn65 uptake. by the competition of the Zn This would be expected to be more evident in the younger, more metabolic plants of the latter experiment. Results from the prior two eXperiments Show that Zn is held in the root cells or at the surface of the cells in pea bean plants produced under high P conditions. To more closely pinpoint the exact location of this Zn-P antagonism an experiment: was performed to ascertain if the Zn was precipitated within the cell or at the cell surface. Pea bean plants were grown as before with the exception that none of the seedlings were given Zn. The plants were allowed to grow until budding when the plants from one-half of the containers at each P level were removed from their medium, their roots dipped into deionized water for 15 seconds to wash away excess P from the nutrient solution and then transferred to a normal Hoagland's solution minus P. After 24 hours, Zn65Cl2 was added to all containers. Forty hours later all plants were removed, dipped into deionized water for 15 seconds and divided into roots and tops. The tissue samples were treated as before. The 52 nutrient solutions were analyzed for P at the completion of the eXperiment to ascertain the quantity of P which had moved from the plants to the containers. The data from this experiment are given in Table 14. The roots that were washed for 24 hours had less Zn65 present in them than those which were not washed. But the most striking results are those from the tops. The Zn65 activity from the tops whose roots were washed was nearly ten times greater than the activity from the tops whose roots were not washed free of P. These results clearly indicate that as the P is removed from the roots by washing, the Zn is free to be adsorbed and translocated to the top of the plant. If the P remains concentrated in the roots, the Zn65 accumulates and very little is able to move from the root system. From these results it is postulated that an insoluble Zn-P complex is formed in the root cortex or at the membrane surface itself making translocation of Zn difficult if not impossible. If this solid phase is dissolved because of the absence of orthophosphate ions in the medium, the Zn65 is released from the complex and is accumulated by the plant. Table 14 Shows that roots produced in high P solutions re- leased approximately 9 ppm P to the solution during 24 hours washing: whereas, the roots produced in low P Solutions released less than 1 ppm P to the washing solution. This does Show that the P accumulated by the pea bean roots in high P solutions is free to move to external solutions and 53 Table 14. Uptake of Zn65 as affected by P level of nutrient solution and washing of roots. Treatment P in solution -Zn65 Uptake Fresh weight at completion P level Washing of Experiment Roots Tops Roots Tops time* ppm hours ppm Counts/min/gm gm/pot 100 none n.a. 5,139 318 27.7 29.7 . 100 24 < 1 4,745 3,103 23.0 21.3 ;.250 none 50 6,207 408 25.9 24.5 250 24 1 4,634 3,221 24.3 26.7 500 none 110 6,804 453 23.3 22.3 500 24 9 3,834 2,827 22.7 26.9 *Washing was accomplished by placing plants in aerated, Hoagland's minus P and Zn nutrient solution for 24 hours. 54 thus, is assumed to be external to the cell interior. Two questions that remain unanswered from the pre— vious experiment are: (l) was the Zn65 able to translocate as a result of new growth which may have been stimulated by washing and (2) does the P in solution have an effect in Zn utilization? To answer these questions, a final experi- ment was conducted. Pea beans were grown in high P (500 ppm) nutrient solution until one week prior to bloom stage. Zn was added at the end of the first week at a 0.01 ppm level to prevent Zn deficiency from becoming so severe as to prevent growth. The plants were Showing marked Zn deficiency during the entire experiment. Plants were washed as in the prior experiment by placing them in Hoagland's solution minus P and Zn for varying periods of time (0, 3, 6, 12, and 24 hours). At the end of the washing period the plants were transferred to nutrient solution containing either 60 or 500 ppm P and Zn65C12. After a period of 36 hours the plants were removed, dipped into deionized water, separated into roots, stems, and leaves, and analyzed as before. It was hypothesized that if new growth accounted for the increased uptake of Zn65, the effect should uniformly increase with length of washing period. If on the other hand the antagonism is due to the P in existing roots as previously hypothesized, the greatest effect should be seen during the early washing periods because the rate of dissolution of the suggested complex would be greatest at 55 that time. By precisely controlling the level of P in the nutrient solution at the time of uptake, the effect of P in the solution on uptake may be ascertained. The results of this experiment are given in Table 15. The results for the P removed during the washing period are summarized in Figure 3. A rapid loss of P occurred from 0 to three hours, followed by a linear loss. When expressed on a percent basis (mg PXlOO/mg dry roots), the P lost varied from 0.6 percent in three hours washing to 1.4 per- cent in 24 hours washing. With increasing periods of washing up to six hours 65 in the root there was a decrease in the amount of Zn system when the plant was placed in a high P solution during the uptake period. And there was a corresponding increase in the translocation of Zn into the tops. Increased wash- ing beyond six hours had little effect on the Zn65 content of any plant part. Little weight was given to the 12 hour points since one replication at this treatment had to be discarded because one plant in each container was quite small compared to other plants in the experiment. Despite washing of the roots the beans placed in a normal P environment did not change in their Zn65 uptake. But there was a marked increase in the Zn65 in the tops with increased washing time with the greatest change occurring up to 12 hours washing time. Evidence strongly indicates that as the P is washed from the root system, Zn is free to translocate to the tops. 56 .ncofiumowa0mu 03» 00 some n mum nooam> Hmnuo .haco 0H0Emm 0:0 nwooaocHss . COH “3 HON “£0.“ NUDE CN 0:0 0 modes n.0cmHmmom .omumumm on mucma0 0:000H0 an 00:0«H0Eooom 003 0:03003s «5.0 «0.0 0n.0 000.0H 0H0.0H 000.0« 000 0« 00.H 50.0 00.0 00H.na 5H0.«H 000.0« 00 0« ss55.0 ss«0.0 ss«0.o ssom0.«a ssna0.oa ss«0a.0« 000 «a «s00.H s««5.0 0000.0 ssoo0.0a «*0«0.na_ ssna0.0« 00 «H 05.0 «0.0 0n.0 000.0H 00«.na 0H5.0« 000 0 00.0 00.0 0«.0 000.HH 000.«H 00n.0« 00 0 00.0 H0.o 0n.0 00«.0 5«0.na 0H«.©N 000 n 0«.H «0.0 00.0 0H0.0 005.0H n«0.0« 00 n 00.0 «0.0 00.0 «0n.0 000.HH 000.5« 000 0:0: 05.0 00.0 0n.0 n«a.0 00«.«H H50.0« 00 woos uo0\E0 #00\0uocwa\musooo E00 mason 00>mmq 08000 muoom mm>mmn mawum 00000 000u00 oxmu0o 0050» came? 930003 awn 0xmuao 00:0 0owuoo H0>0H 0 monsmma .nuoou 00 many 0cannm3 0cm cowuoaom usmwuuoc mo H0>0H 0 an nmuommmm mm QN mo 0xmu0D .0H manna 00 57 3.0 — E o. .9: O c .9. p 2.0 _ :3 H o 0) c o -a 0 7’ I I I 1.0 .. .' I l I I l I I I I 0 l I I I 0 6 12 24 Washing time (hours) Figure 3. Effect of washing time on P removal from pea bean plants. 58 Leaves 16‘ 12 4.) C m 'a.‘ \\ 3 5 8 I: 'E' \x n 4.) S 8 0—0 60 ppm P in solution 4 - H 500 ppm P in solution 0 l I I J 0 6 12 24 Washing time (hours) Figure 4. Effect of washing time and P content of nutrient solution on uptake of Zn55 by pea bean leaves. 59 28 T‘ g Roots \ P 4.) a m 'a'. E 20 ,. 0—0 60 ppm P in solution ‘3 'g O———O 500 ppm P in solution \ ,n 4.) s o 0 U 4 - 0' 1 I I o 6 12 24 Washing time (hours) Figure 5. ,Effect of washing time and P content of nutrient solution on uptake of Zn55 by pea bean roots. 60 This is strong support for the hypothesis that at or near the cell membrane an insoluble Zn-P complex forms either alone or with other components of plant metabolism. A subsequent removal of this P then frees the Zn for plant use. P content of the nutrient solution at the time of uptake was found to have a definite influence on Zn trans- location. Data Shown in Figure 4 indicated that up to a 65 translocated to the leaves washing time of 6 hours the Zn was similar under both P levels. However, at the conclusion of the experiment there was a large difference between 65 translocated to the leaves. Thirty quantities of Zn percent more was translocated to the leaves of the plants in normal Hoagland's solution at this time as compared to the plants grown in solution containing 500 ppm P. This might be expected since the longer the plants were grown in a high P environment the more likely they would be to revert to the high P condition that existed prior to the washing. The insoluble complex would certainly have adequate time to reform after a six hour period in this unfavorable environ- ment. It does indicate that the effect of the high P in the nutrient solution is to produce high P roots, rather than to precipitate Zn3(PO4)2 solution. SUMMARY AND CONCLUSIONS Zinc deficiency has long been associated with calcareous soils. More recently reports indicate that a high P soil level is usually detrimental to Zn utilization. Some reports indicated that the nature of the antagonistic effect of P was the formation of an insoluble precipitate in the soil itself while other showed indirect evidence that P-Zn antagon- ism is physiological in nature and that Zn is precipitated at or very near the surface of the root cells or possibly inside the root cells themselves. The latter seemed quite feasible as previous reports have indicated the presence of an abnormally large inorganic P pool as a result of an inadequate supply of Zn. Recently Michigan farmers have found that pea beans are very susceptible to P induced Zn deficiency. Farming practices on the calcareous soils of the lake-plain region of East Central Michigan are generally characterized by a sugar beet—pea bean rotation. This resulted in high P levels that are required for high sugar beet yields and there- fore in wideSpread Zn deficiencies. Field experiments conducted in 1963 confirmed the fact that high P levels do accentuate a Zn deficiency in pea beans. Of the several materials used to correct the deficiency, ZnSO4 mixed with APP was the most effective in 61 62 both supplying Zn to the plant and in increasing yield. This was followed by Zn EDTA (applied at one-fifth the rate for inorganic carriers), and finally by Zn20804 which was not an efficient Zn source. Zn0 incorporated into granular APP was superior to ZnO incorporated into AOP. A trend was also noted which indicated that increasing increments of Zn in the APP fertilizer gave yield increases which did not follow a normal growth curve. Results also confirmed that the critical level of Zn content in pea bean tissue is 15 to 20 ppm. Because of the striking differences obtained between ZnO incorporated into AOP as compared to APP and because the response curve with.Z00-APP appeared to be atypical in the low Zn rates, the 1964 field studies were initiated to investigate these pheonomea. Fertilizers were supplied by TVA with variable Zn:P ratios. Water solubility studies showed that Zn0 incor- porated into AOP was no more than 0.03 percent water soluble at any ratio indicating that Zn is transformed into compounds less soluble than Zn0. ZnO incorporated into APP showed an increasing solubility up to a value of one percent Zn in the fertilizer and then decreased suggesting that at high Zn:P ratios a soluble complex is formed between the Zn and polyphosphoric acid. This system becomes super- saturated at the lower ratios and an insoluble precipitate is then formed. Most of the additional Zn added beyond this point is converted into an insoluble compound. 63 Results from 1964 conclusively showed that Zn supplied at an eight pound rate as ZnO incorporated into APP was superior to all other treatments as measured by early growth, Zn content, uptake and yield. For APP, yield and uptake data reflected the water solubility curve. The dramatic effect of the soil P level was again noticed. High amounts of P were so detrimental to plant utilization of Zn that even at the highest rate of applied Zn, the yield at the high P level never attained the level equal to that on low P soil. These experiments indicated that water solubility of Zn in the applied fertilizer is important in estimating fertilizer efficiency. But, a rather close relationship is obtained if the total Zn in the fertilizer is considered along with water solubility, the latter being the more important. To clarify the question of high soil P levels affecting Zn availability in soils, a Wisner clay loam was incubated with three P levels and then allowed to equili- brate with a Zn65C12 solution. The soil was successively extracted with four agents (H20, 0.01 M EDTA in l M (NH4)2C03, l N NH4OAc buffered at pH 7, and 0.1 N HCl) to characterize the availability of soil Zn. very small quantities of Zn65 were found in the water extract. There was, in fact, little indication that applied P had any influence on the water- soluble Zn level. Extraction with EDTA buffer solution removed greater than 85 percent of the recovered Zn65. 64 And the Zn removed increased with increasing soil P levels. This extracting solution had previously been found to be one of the best for estimating available soil Zn. The final 65 two extractions removed Zn in an inverse relationship to the soil P level. The NH OAc extracts the difficulty avail- 4 able Zn and the 0.1 N HCl extracts the Zn which precipitated as an insoluble compound. The results indicate that with increasing levels of soil P the amount of easily available Zn also increases. This would then leave less in the fractions that represent the difficultly available and insoluble portions. This evidence points to the fact that P-Zn antagonism does not occur in the soil. In nutrient culture studies, Zn65 was not'translocated beyond the roots of plants produced in high P solutions. To pinpoint the exact location of the Zn-P antagonism plants were grown in Ibagland's solution with high P (500 ppm) minus Zn. Before harvest one-half of the plant roots were washed with normal Hoagland's solution minus P for 24 hours and then all plants were given Zn65. Although the 65 roots that were washed had less Zn in them than those not washed, the most dramatic effect was that the tops from the washed roots contained ten times more Zn65 than tops from roots which were not washed. This is strong evidence that an insoluble Zn-P complex is formed in the root cortex or at the membrane Surface making Zn translocation very difficult. To answer two additional questions--(l) was the Zn65 65 able to translocate as a result of new growth which may have been stimulated by washing and (2) does the P level in solution have an effect in Zn utilization,--a final experiment was conducted. Pea beans that were grown in high P nutrient solution until one week before budding were washed in Hoagland's solution minus P and Zn for varying lengths (0, 3, 6, l2, and 24 hours) and then one-half were transferred back to a normal Hoagland's solution and the remainder to Hoagland's with 500 ppm P. All cultures received Zn65Cl2 for 36 hours. It was found that washing time had a direct influence on Zn translocation up to six hours under both P levels, i.e., the greater the P removed 65 translocated. At the end of 24 hours there was the more Zn 30 percent more Zn65 translocated to the leaves of plants grown in normal Hoagland's as compared to the solution con- taining 500 ppm P. From these results it is concluded that an insoluble complex is formed near the surface of the cell membrane involving Zn, P and possibly other plant metabolites. If the P is removed the complex is dissolved and then Zn is free to move into the plant. But if P is again increased in the solution the complex will again reform to its previous state. 10. LITERATURE CITED Alben, A. O., J. R. Cole and R. D. Lewis (1932). New development in treating pecan rosette with chemicals. Phytopath. 22:595-601. Allison, R. V., O. C. Bryan, and J. H. Hunter (1927). 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