my. a». LIBR-AR Y This is to certify that the thesis entitled Manganese Availability As Measured By Crop Uptake, Soil Extraction And Isotopic Dilution presented by Ignacio Hernan Salcedo has been accepted towards fulfillment of the requirements for Ph.D. Soil Science degree in ‘ A. " '; »' 4 r"/' -—\ I . ., ' ' 'Iv' (’7 {'4}- ‘4"/ .' C 4, ’ "Major professor Date July 9, 1976 0-7 639 ABSTRACT MANGANESE AVAILABILITY AS MEASURED BY CROP UPTAKE, SOIL EXTRACTION AND ISOTOPIC DILUTION By Ignacio Hernan Salcedo This investigation was divided in three main parts. First, the effect of 12 shaking time -- solutionzsoil ratio combinations on Mn extracted by 0.1N HCl, 0.1N H3P04, DTPA and 1N NH OAc (pH 7) from 12 4 soils was studied. Results were analyzed to determine which treatment combination gave the greatest quantities of extractable Mn; how extractable Mn correlated with un uptake by soybeans and sudangrass at each time-ratio combination; and which soil characteristics were important in determining the quantities of Mn extracted. Secondly, a greenhouse study with these 12 soils was conducted. Soybeans were grown in the soils, that had received 0, 10 and 20 ppm of Mn (as MnSO4). After harvest, soil samples were removed and analyzed for extractable Mn by six procedures, namely, the four mentioned above plus extraction with 1.5M NH4H2P04 and steam/NH4OAc. The 0.1N H3PO4 was the one giving the highest correlation with plant uptake. Soil acidity and bases ratio (Ca+Mg/K) were included in the prediction equations of these Ignacio Hernan Salcedo soil tests. Thirdly, the residual effect of the Mn applied in the prior experiment was evaluated. The check pots and those that had received 20 ppm of Mn were tagged with 54Mn and sown to sudangrass. The determination of the Mn labile pool was done by obtaining the L and E-values for each soil. These results plus those of the six chemical extractions done before were correlated with Mn uptake by the three sudangrass harvests. MANGANESE AVAILABILITY AS MEASURED BY CROP UPTAKE, SOIL EXTRACTION AND ISOTOPIC DILUTION By Ignacio Hernan Salcedo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1976 ACKNOWLEDGMENTS I wish to thank Dr. Boyd Ellis for all his help and advice during the course of these studies. This appreciation is extended to Dr. R. Lucas for his help in all the work related to the greenhouse experiments and to Dr. C. Cress for his contributions in the statistical analysis of this study. I thank also Dr. D. Warncke and Dr. M. Zabik for serving as members of my guidance committee. To D. Juchartz, B. Simone, A. Ronemus and L. Cedarstaff goes my appreciation for their help in performing soil analysis and preparation of computer cards. I am indebted to lug. I. Mizuno and his team for their silent but effective support, to the University of Buenos Aires for their financial assistance and to the Department of Crap and Soil Sciences for the research assistanship that allowed me to finish my studies. Finally, I would like to thank other fellow students, faculty and staff of this Department for their help and friendship, that made this period of my life a most rewarding one. ii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O O O O O O O O O O O O o 0 iv LIST OF FIGURES O O O O O O O O O O O O O O O O O O O O O O O O 0 v1 INTRODUWION O O O I O O O O O O O O O O O O O O O O O O I O O O 1 PART I STUDIES IN SOIL MANGANESE: 1. FACTORS AFFECTING MANGANESE EXTRACTABILITY. . . . . . . . . . . . . 3 PART II STUDIES IN SOIL MANGANESE: 2. EXTRACTABLE MANGANESE AND PLANT UPTAKE. . . . . . . . . . . . 29 PART III STUDIES IN SOIL MANGANESE: 3. MANGANESE LABILE POOL AND PLANT UPTAKE . . . . . . . . . . . . . . 48 APPENDIX 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O 68 iii LIST OF TABLES Table Page PART I STUDIES IN SOIL MANGANESE: l. FACTORS AFFECTING MANGANESE EXTRACTABILITY. 1 Main characteristics of the soils used in this study. . . 7 2 Ranges in extractable Mn by four procedures as a result of varying solution:soil ratios and shaking times . . . . 10 3 Effect of time of shaking (X ) and solution:soil ratio (X2) on the quantities of Mn extracted by 0.1N HCl, according to Equations (1) and (2). . . . . . . . . . . . 12 4 Effect of time of shaking (X ) and solution:soil ratio on the quantities of Mn extracted by 0.1N H3P04, according to Equation (1). . . . . . . . . . . . . . . . 13 5 Effect of time of shaking (X ) and solution:soil ratio (X2) on the quantities of Mn extracted by DTPA, according to Equations (1) and (2). . . . . . . . . . . . 17 6 Effect of time of shaking (X1) and solution:soil ratio (X ) on the quantities of Mn extracted by 1N NHAOAc (pfi 7), according to Equations (1) and (2). . . . . . . . 18 7 Effect of time of shaking (X ) and solution:soil ratio (X ) in the correlation between extractable Mn and plant Mn uptake, according to Equations (1) and (2) . . . 22 8 Main soil factors that relate to the quantities of extractable Mn by several procedures at different shaking time-solution:soil ratio combinations, according to Equation (3) . . . . . . . . . . . . . . . . . . . . . 24 PART II STUDIES IN SOIL MANGANES: 2. EXTRACTABLE MANGANESE AND PLANT UPTAKE. 1 Mn uptake by soybeans and soil extractable Mn by each method . . . . . . . . . . . . . . . . . . . . . . . 34 2 Relationship of Mn uptake by soybeans (Y) with several soil Mn tests, pH and bases ratio, according to Equation (1). . . . . . . . . . . . . . . . . . . . . . . 36 iv Table 3 Relationship between Mn uptake by soybeans and several soil characteristics. . . . . . . . . . . . . . . 4 Simple correlation coefficients among extractable Mn by several procedures and soil prOperties . . . . . . . . 5 Response in Mn uptake by soybeans to Mn fertilization as Mnso O O O O O O O O O O O O O O O O I I O I O O O I O 4 PART III STUDIES IN SOIL MANGANESE: 3. MANGANESE LABILE POOL AND PLANT UPTAKE. l Manganese labile pool (L-values) and Mn uptake by plants. 2 Effect of soil type and residual Mn fertilization upon measured parameters. . . . . . . . . . . . . . . . . 3 Simple correlation coefficients between Mn uptake by sudangrass and several soil tests for Mn . . . . . 4 Relationship of the average Mn uptake (three harvests) with several soil tests and soil properties . . . . . . . 5 Simple correlation coefficients among several soil tests and soil characteristics. . . . . . . . . . . . . APPENDIX II-l Soybean yield and Mn content as affected by soil type and Mr! fertilizer O O O O O O I O O O O O O O O O O O O O 0 111-1 Sudangrass yield and Mn content as affected by soil type and Mn fertilizer (lst harvest) . . . . . . . . . . . . III-2 Sudangrass yield and Mn content as affected by soil type and Mn fertilizer (2nd harvest) . . . . . . . . . . . . . III-3 Sudangrass yield and Mn content as affected by soil type and Mn fertilizer (3rd harvest) . . . . . . . . . . . . . SS 54 III-4 Results of Mn and Mn analyses of sudangrass at each harvest 0 O O O O O O O C O O O O O O 0 O O O O O O O O Page 37 40 41 53 56 58 60 61 69 7O 71 72 73 LIST OF FIGURES Figure Page PART I STUDIES IN SOIL MANGANESE: 1. FACTORS AFFECTING MANGANESE EXTRACTABILITY. 1 Effect of shaking time and solution:soil ratio in the amounts of Mn extracted by: A. 0.1N HCl; B. 0.1N HBPO ; C. DTPA; and D. IN NHAOAc (pH 7). (Average for 12 soils). 15 2 Effect of shaking time and solution:soil ratio in the correlation between 0.1N HCl extractable Mn and Mn uptake by: A. Soybeans; B. Sorghum, and 0.1N H PO extractable Mn and Mn uptake by: C. Soybeans and D. Sorghum . . . . . 21 PART II STUDIES IN SOIL MANGANESE: 2. EXTRACTABLE MANGANESE AND PLANT UPTAKE. 1 Determination of the critical level for O.lN H3P04 (A) and for steam/1N NH OAc and 1.5M NH HZPO4 (B) extractable Mn by t e Cate-Nelson procedure . . . . . . . 42 PART III STUDIES IN SOIL MANGANESE: 3. MANGANESE LABILE POOL AND PLANT UPTAKE 1 Determination of the critical level for L value and E value of Mn by the Cate-Nelson procedure . . . . . . . 62 vi INTRODUCTION There have been numerous studies related to micronutrients in recent years, among which, zinc is probably the one that has been more thoroughly studied. For soil testing purposes, some researchers have extrapolated extraction procedures well suited for this element, proposing them as tests for Mn, Cu and Fe as well. Although such a procedure would be highly desirable for any soiltesting laboratory, differences in the behavior and properties of these elements in soils raise some doubts as to the possibility of developing such a technique. Several extracting methods to estimate available Mn for plants have been studied with varying degrees of success depending on the soils and crops used by each researcher. The increasing use of chelating agents as aids for maintaining sufficient levels of soluble micronutrients in soils for plant uptake, have prompted the use of these substances as extractants for soil testing purposes. Published information in the use of some of these chelating agents for determining soil available Mn is still scarce. In spite of all the methods available for testing soil Mn, little work has been done concerning the effect of extraction conditions upon their ability to predict Mn deficienty situations. In the case of procedures which extract sufficient soil Mn to be measured without analytical difficulties, the selection of solution:soil ratios and lengths of extraction should be based on that combination giving the highest correlation with plant uptake or yields. The objectives of this investigation were, therefore, to study the effect of different experimental conditions upon several extractants, relating them to: a) the quantities of Mn extracted from the soil, b) the sources of the extracted Mn and c) the correlation of extracted Mn with plant uptake. Also, six extracting solutions including a chelating agent were compared in the effectiveness to predict Mn uptake by plants. Furthermore, radioactive Mn was used to assess the residual effect of Mn fertilizer, and to determine the labile Mn of the soils used (E and L values) correlating these results with plant Mn uptake. PART I STUDIES IN SOIL MANGANESE l. FACTORS AFFECTING MANGANESE EXTRACTABILITY Introduction Several extracting procedures to evaluate available Mn in soils have been developed. Reviews in this subject have been published by Cox and Kamprath (7) and by Viets and Lindsay (13). The selection of a certain solution:soil ratio and time of shaking arises in most cases from practical considerations. Few detailed studies have been reported establishing the relationship between these two variables, extractable Mn and plant uptake. Boken (2) studied the influence of these parameters on the quantitiy of Mn extracted by 1M Mg(NO3)2 and Ca(N03)2 as a percentage of total Mn. Sorensen, et a1., (12) used 0.1N HCl at various solution:soil ratios and shaking periods to characterize the release of Mn from several Nebraska soils, but did not correlate this information with plant uptake. Using this same extractants, Dolar and Keeney (9) correlated the Mn, Zn and Cu extracted from several soils with uptake by oats, using one solution:soil ratio and two extraction periods. Hoff and Mederski (11) used 0.1N H PO as 3 4 an extractant for soil Mn. Using a fixed solution:soil ratio they studied the influence of acid concentration and time of shaking on the quantities of this nutrient extracted from soils of different textures and with varying degrees of Mn insufficiency. Although Mn extracted by 1N NH4OAc (3) and DTPA extractable Mn (13) have been considered to correctly predict deficiency situations in soils, no studies on the effect of the time and ratio variables have been published for these extractants either. The purpose of this study was to characterize the release of Mn from soils from Michigan and Indiana by four extractants, as affected by several solution:soil ratios and shaking time combinations and, furthermore, to see how these variables affect the relationship between extractable Mn and plant uptake by soybeans and sorghum. An attempt to characterize the main Mn fractions and soil properties that closely related to the quantities of extracted Mn was also done. Materials and Methods Soils Surface soil samples from 12 different soil types were collected from various locations in Michigan and Indiana. The soils were selected to give varying levels of available Mn for plant growth. The following subgroups were included: Typic Haplaquolls (Maumee and Granby series), Typic Argiaquolls (Brookston series), Mollic Haplaquets (Toledo and Parkhill series), Arenic Hapludalfs (Metea series), Aquic Arenic Hapludalfs (Selfridge series), Aeric Ochraqualfs (Fulton series), Udollic Ochraqualfs (Conover series), Typic Hapludalfs (Miami series), Typic Medisaprists (Houghton series) and Typic Udipsamments (Plainfield series). The main characteristics of these soils are given in Table 1. Methods of Analysis All soil samples were air dried and then ground to pass a 10 mesh sieve. Small subsamples for total nutrient determination were finely ground with mortar and pestle. Tbtal C: 0.1 g of the finely ground soil was analyzed by the dry combustion method (Ovejera Belo, 1970)1 with a Leco carbon analyzer. Inorganic C: 3.0 g of the finely ground soil was analyzed following the procedure described by Bundy and Bremner (4). 1 Determination of total C by dry combustion and its relation to forms of soil N as measured in the laboratory and in the greenhouse. Ph. D. Thesis, Michigan State University, East Lansing. 0.0 0.00 0.00 0.00 00.0 00.0 0.0 0.0 -- 00.0 0.0 0.0 0.0 0.00 0000 000.00 00 0.00 0.00 000 000 00.0. 00.0 0.00 0.00 00.0 0.00 0.0 u- -u n- 00:3 0000000: 00 0.00 000 000 0.00 00.0 00.0 0.0 0.00 .. 00.0 0.0 0.00 0.00 0.00 330 00000 00000000 00 0.00 000 000 0.00 00.0 00.0 0.0 0.0 n. 00.0 0.0 0.00 0.00 0.00 e000 0200: 0 0.00 000 000 0.00 00.0 00.0 0.0 0.00 -- 00.0 0.0 0.00 0.00 0.00 3000 00:00 00>ocou 0 0.00 000 000 0.0 00.0 00.0 00.0 0.0 n. 00.0 0.0 0.0 0.00 0.00 0000 09000 0000: 0 0.0 0.00 000 0.00 00.0 00.0 0.0 0.00 .. 00.0 0.0 0.00 0.00 0.00 a000 000000000 0 0.0 0.00 000 0.0 00.0 00.0 0.0 0.0 00.0 00.0 0.0 0.0 0.0 0.00 0000 000000000 0 0.00 0.00 000 0.00 00.0 00.0. 0.0 0.00. 00.0 00.0 0.0 0.00 0.00 0.00 0000 00000 000000 0 0.00 0.00 000 0.00 00.0 00.0 0.0 0.00 n. 00.0 0.0 0.00 0.00 0.00 0.00 0000. 000000 0 0.0 0.00 000 0.00 00.0 00.0 0.0 0.0 00.0 00.0 0.0 0.0 0.0 0.00 00.. 005:0: 0 0.0 0.00 000 0.0 00.0 00.0 0.0 0.00 00.0 00.0 0.0 0.0 0.0 0.00 0000 0000000000 0 sea 0000000a 0 0 0 000mw000 00mm00 0hmwa 000 02 0 0: 00 00000 hummmmw 0umm 0000 0000 0:00 0000 0000 «Haw .muaua «0:0 :0 one: oawou 050 no uuuuawuuuouuanu can: .H manna Organic 0: It was found by difference between total and inorganic C, except for the Houghton muck in which organic C was obtained by the loss of weight after ignition for 4 hours at 600 °C. thal Mn: 0.2 g of the finely ground soil was weighed into a platinum crucible, 5 m1 of 70% HNO3 added and the crucible heated on a hot plate until the residue was dry. After cooling to room temperature, 5 ml of 49% HF and 1 m1 of 712 HClO4 were added; heat was applied again and the digestion carried to dryness. This last treatment was repeated if there was evidence that the decomposition was not complete. After the crucible cooled, the residue was dissolved in 5 m1 of 6N HCl, diluted to 50 m1 and Mn determined by atomic absorption spectrophotometry. The Houghton muck was first ignited for 4 hours at 600 °C and then the procedure as described above was followed. All analyses were done in duplicate. Chelated Mn and'Mn oxides: 1.0 g of the finely ground soil was successively extracted with 10 m1 of 0.005M DTPA twice, shaking for 60 min each time and separating the supernatant by centrifugation. The sum of the Mn from both extractions was called chelated Mn to differentiate it from the DTPA extractable Mn, as defined in the Experimental Section. This denomination, however, refers to the way the Mn was extracted (chelating agent) and not to the chemical status of this Mn fraction in the soil. The soil remaining from the second extraction with DTPA was extracted twice with 20 m1 of 0.1N hydroxylamine hydrochloride (pH 2) (5), for 30 min each time. The sum of Mn from both extractions was considered Mn oxides. Other soil analysis: Soil pH was determined with a glass electrode in a 1:1 soil:water ratio. Saturation with 1N NH OAc (pH 7) (6) was used to 4 measure the cation exchange capacity and exchangeable bases were determined in the NHAOAc filtrate. Texture was determined by the pipet method (8). Experimental Four extracting solutions for Mn were used: 0.1N HCl, 0.1N H3P04, 1N NH OAc (pH 7) and DTPA (0.005M diethylenetriaminepentaacetic acid, A 0.01M CaCl2 and 0.1M triethanolamine - pH 7.3). Extraction periods were 10, 30, 60 and 120 min combined with three solution:soil ratios (volume/air dry weight): 5 (25:5), 10 (20:2) and 25 (25:1), resulting in a 3 x 4 factorial arrangement. For DTPA only, a ratio of 2 (10:5) was also included since this was the one suggested in the original procedure developed by Lindsay and Norvell (1969)?. Statistical Analysis Multiple regression was used to study the functional relationship of extractable Mn (Y) with time of shaking (X1) and solution:soil ratio (X2), by fitting the data with a second order model of the type: 2 2 Y ‘ 80 + 81x1 + 32x2 + B11X1 + B22x2 + 312x1x2 + E (1) where the estimates b1 of the parameters 81 were obtained by the method of least squares. A set of estimates was calculated for each soil and extractant. For soils not showing any significant effect (except interaction) at the 52 level of significance, a first order model was used, according to: Y = Bo + lel + 32x2 + sllexz + e (2) To characterize the main Mn fractions and soil properties 2 Development of a DTPA micronutrient soil test. Agron. Abstr., p.84. determining the Mn extracted by each solution, models were developed at selected time-ratio combinations relating extractable Mn to the following variables: pH (X1), organic C (X2), chelated Mn (X3), Mn oxides (X4), total Mn (X5), milliequivalent ratio of Ca+Mg/K (X6) and free CaCO3 (X7). A modification of the stepwise regression procedure (10) was used. This system3 allows all the variables to be in the model at the begining of the regression. Variables are deleted according to a predetermined level of significance, but deleted variables can be added again to the model based also on a predetermined level of significance. The following model was used: 7 Y = so + ingixi + e (3) where the response variable (Y) was extracted Mn. A 52 level of significance was used to delete or add variables. Variable X7 was used as a qualitative variable with a value of 1 for the presence and a value of 0 for the absence of CaCO . The data of all 12 soils were used for 3 0.1N HCl and HBPO4 and from 11 soils for DTPA and 1N NH4 Mn was not allowed to enter the model as an independent variable when OAc. Chelated developing the model for DTPA extractable Mn. Results and Discussion Results in Table 2 show the ranges of Mn concentrations (soil 3P°4 OAc (pH 7) the low values were obtained at a ratio of 5 with basis) extracted by the four extractants. For 0.1N HCl, 0.1N H and 1N NBA 10 min of shaking, while the higher ones occurred at a 25-120 min ratio-time combination. Results for DTPA varied depending on the soil 3Michigan State University Stat System. 1974. Part 12, LSSTEP Program. 10 considered, some yielding the lowest results at different ratio-time combinations than others. These differences were minimal in most cases. The left column for DTPA (Table 2) corresponds to a ratio of 5 with 10 min of shaking while the right column is for the same ratio with 120 min of shaking. Table 2. Ranges in extractable Mn by four procedures as a result of varying solution:soil ratios and shaking times. Soil No. 0.1N HCl 0.1N H P04 0.005M DTPA 1N NHAOAc PPm# 1 32.6 - 52.7 11.0 - 50.0 1.6 - 2.4 0.52 - 2.8 2 13.0 - 36.8 4.4 - 20.3 1.4 - 2.1 0.41 - 1.0 3 20.2 - 44.6 6.8 - 26.3 8.2 - 9.8 4.7 - 7.3 4 28.4 - 59.1 5.8 - 31.3 5.4 - 7.4 2.4 - 5.8 5 3.3 - 10.1 1.9 - 6.2 -- -- -- -- 6 6.3 - 20.5 1.7 - 9.9 2.6 - 2.8 1.1 - 2.1 7 36.8 - 101 25.5 — 66.0 9.0 - 11.9 6.4 - 9.9 8 72.8 - 181 34.9 - 106 31.6 - 42.5 17.2 - 26.9 9 65.0 - 131 39.7 - 86.4 31.6 - 40.2 21.2 - 31.6 10 27.3 - 65.4 12.3 - 40.5 10.1 - 12.6 6.0 - 9.5 11 4.4 - 174 0.51 - 17.5 6.2 - 15.7 2.1 - 6.5 12 5.2 - 12.6 2.2 - 11.0 0.71 - 1.0 0.42 - 0.71 #Minimum values at a 5-10 min ratio time combination; maximum values at 25-120 min, except for DTPA whose maximum was at 5-120 min. 11 The complete set of results was summarized in the form of regression equations (lst. and 2nd. order models, Eq. 1 and 2) and are shown in Tables 3 to 6. In these tables the equations labelled as Average describe the response surfaces shown in Fig. l and were obtained by fitting the data for all soils with the 2nd. order model (Eq. 1). Except for soil 2 when extracted with 1N NH40Ac, these models were useful in explaining variations in extracted Mn at the 12 level of significance. Some equations, however, failed to explain a sizeable part of the variation in the data, as shown by R2 values smaller than 85%. This fact does not invalidate the trends considered significant by the regression but would cause error if the original data were to be reconstructed by replacing the X1 and X2 variables with their corresponding values. By determining which regression coefficients are significant for a particular extractant and soil, the range in extractable um can be assigned to the linear and/or quadratic effects of time and ratio and/or to the interaction between both variables. The results in Table 2 show that the two acids were very effective in extracting Mn from most soils. A comparison between Fig. 1A and 13 indicates that, on the average, 0.1N HCl extracted twice the quantities of Mn extracted by 0.1N H3P04. These surfaces also show that going from a 5-10 min to a 25—120 min ratio-time combination produced a 3-fold increase in 0.1N HCl extractable Mn. In the case of 0.1N H3P04 it was, on the average, a 4-fold increase which indicates that Mn extracted by this procedure is more dependent upon the time and ratio selected. Further on in this study, it will be shown that solubilization of Mn oxides plays an important role as a source of Mn extracted by these 12 .%Ho>wuoounou .Ho>oa NH one an oau um unmuamunwfim as .0 «« 0.00 mun.o mo.m no.0: ¢~.ol as 0m.0 n.0m owmuo>< «« N.om onwo.o Nm.H eoam.ou Hw.e| «« om.H Hm.n NH «0 o.wm «a «w.e II II «« o.om wN.~I mm.oHI 00 «« w.0m wmao.o ommo.on mm.~u om.0 «a ww.o 0.00 ea «0 0.00 000.0- «A 0.00 00.0- 0.00- 0 00.0 0.00 0 «« m.mm omm.o 0.00 om.0l 0.00: « w.~H o.mn m «a m.mm unno.o w0.~0 mw.H| 0.0m: « 0N.0 m.o< 0 «a m.0m .00 o0m.o II II oa~.c 000.0 nm.o o «a «.00 a «00.0 II II om0.c I ooa.o m~.m m «a c.~m emu.o « no.0: mm.0: « “.mm « om.m m0.a 0 «a H.0m omm.o in in m~.0 « NN.H m.H~ m «a a.w¢ « 00m.o 000.on as hm.au mm.0 «a o0.n wo.m m «a ~.mw mom.o nmm.o «« n0.mt mum.o I as oo.m c.o~ a N 000000 000 000000 000 000000 000 00000 00 00000 00 on .oz a mucouuwmmooo unannouwom 000m .000 000 000 000000000 00 000000000 .000 20.0 00 oouomuuxo a: mo mofiuwucmsu 050 no Auxv 00000 0000060009000 was AHNV wcfixmnm Ho 0800 «0 pummmm .m manna 13 .mau>auuoauuu .H«>~H NH can an «nu ”a unuoamaamfim *« .« «« m.m¢ .« m~.q «. no.» I «o-.ou «« ¢~.~ «« mm.” omc.o I omaum>< «« 0.50 mNN.0 «« Nm.~ I « «n.0I ««mom.0 «« ma.a mn.m I NH «« 0.00 «« 00.m « mn.a Hmm.0I mma.0I Hm.0 000.0 Ha «« H.mm «« 00.m «« 00.n I H0.0I «« HH.~ « mm.H H0c.0 0H «« 0.00 «« ¢.HH «« n.0HI «« 0H.NI «« nm.m «« 50.0 n.0H m «« 0.00 «« 0.0H «« n.mHI «0 00.HI *« 00.m *« H0.N «~.0 w «« ~.00 05.H «« n.0aI 00.HI «« «0.0 « m0.e 00.~ n «« H.0m «« 00.H «« H0.H I 000.0I «a0w0.0 00H.0 00.H I 0 *a 0.00 Hem.0 « om.~ I NNn.0I «t000.0 0n0.0 00.N I m «« H.00 «{ m~.m «0 00.0 I 0-.0I «« 0n.~ 00~.0 Hm.m I 0 *a 0.0m «« Hm.c «« 0~.0 I NNN.0I «« ~0.H 0H~.0 mem.0 I m «« n.0m om.a «« 0m.¢ I 050.0I «« 00.H * 0H.H no.0 I N «« H.50 «« 50.0 «« ~.HHI 00.HI «« 0H.0 00.H H.0aI H N Amoaxv «Hp ANOHxV mm; Amoaxv Has «a Aofixv an on .02 m muamaoammmoo aowmmmuwmm Haom .AHV aofiuusum ou magnuooua .eom m za.o mp wouomuuxm a: mo mmaufiuamsv ecu co ANNV oaumu Hfiomucowusaom 00w AHNV wafixmnm mo mafia mo uomwmm .0 manna 14 two procedures. For a fixed ratio, responses to time of shaking may indicate a solubilization process that should tend to slow down as the Mn activity in the extracting solution increases. This is confirmed by the shape of the response surfaces in Fig. 1A and 13 and by the consistent negative b coefficient for all soils when using these two 11 extractants. The fact that only three soils showed this coefficient to be significant suggests that longer shaking periods should have been used with both extractants. According to Sorensen, et a1., (12), if the increase in Nb activity in the extracting solution is the limiting step in the extraction of more Mb at any fixed time, increases in ratio should produce a linear response in extractable Mh. Fig. 1A and Table 3 indicate that this may be the case for the extraction with 0.1N HCl since only two soils showed significant b22 at the 52 level of significance. Results from Table 4 and Fig. 1B clearly show that factors other than Mn activity in the extracting solution are limiting the extraction by 0.1N H3P04. It is worthwhile noticing the effect of time and ratio upon Mn extraction by both extractants from the organic soil (No. ll). It was the only soil where 0.1N HCl extracted more Mn than the sum of Mn oxides and chelated Mn (Table 1). Furthermore, 0.1N HCl extractable Mn never exceeded 252 of the total Mn content of each mineral soil, but accounted for 552 of this amount from the organic soil. Increasing the time of extraction with this extractant from 10 to 120 min at any ratio produced a 2 to 3-fold increase in Mn extracted. Increasing the ratio from 5 to 25 at any shaking time produced a 12 to 13-fold increase. A similar maximum amount of Mn was extracted from soil 8 (mineral) (Table 2), but in this case while time gave a 2.2 to 2.3-fold increase, ratio only 15 .‘h attuned (you) til tunnel ("I1 1’!- o! umm (Bill) 11. of shaking (In) an extracted (on) I. extracted (9") 1 1 10 so 00 ”a flu- “ INN»: (I10) ”- gt shout-l (Ila) Figure 1. Effect of shaking time and solution:soil ratio in the amounts of Mn extracted by: A. 0.1N HCl; B. 0.1N H P04; C. DTPA and D. 1N NH40Ac (pH 7). (Average for 12 soils . 16 yielded a 1.1 fold increase in extractable Mn. 0.1N HBPOA showed the same pattern with the organic soil as 0.1N HCl. In the best case (ratio of 25) there was a 2-fold increase in extractable Mn when increasing the shaking time from 10 to 120 min. Increasing the ratio from 5 to 25 at 10 and 120 min of shaking yielded 14 and 21-fold increases, respectively, in Mn extracted from this soil. This exponential effect of the ratio variable with both acids suggests that the total H+ activity per unit soil is the main driving force, probably by competing with Nb held by carboxyl groups of the organic matter (1). The pH of the 0.1N HCl extracts for all soils oscillated between 1.1 and 1.3, while those of 0.1N H PO oscillated between 2.2 and 2.4, 3 4 which is a lO-fold difference in H+ activity between both extractants. Since 0.1N HCl extracted 10 times as much Mn as 0.1N H3PO4 from the organic soil at both extremes of the range (Table 2), there is reason to believe that, at least for this soil (No. 11), Mn extraction is closely related to the H+ activity of the extracting solution. Differences between Mn extracted by both acids from the mineral soils do not show this degree of dependence on H+ activity. With the exception of soils 8, 9 and 11, ranges for DTPA extractable Mn were of 3 ppm.Mn (soil basis) or less (Table 2). The average response surface (Fig. 10) is somewhat misleading, particularly the effect of time of shaking at a ratio of 2 (it does not include soil 11) which was caused mainly by soils 8 and 9 alone. Therefore, the soil distribution about the 12.2 ppm of Mn average for the 2-120 min ratio-time combination (Fig. 16) is skewed, with seven soils lying below and only three soils above this average. Disregarding for a moment the results from soils 8, 9 and 11 (Table 2), a S-fold variation in ratio and a 12-fold 17 .oOHuoovo mHsu you vowsHonH uoa N OHumu HHomuoOHusHom I .mHo>Huoonomu .Ho>mH NH was an may on uaooHMstHm II .I II m.wa II ow.I I II mm.m I II «.mHI II H.eH II mm.I Im.s ImmuIII II o.om mHe.o II II I cm.HI IIoom.o Ins.o NH II o.mo II m.nHI oo.H I I n.0HI Is.~ II ~.~H H~.n III II m.mm mo.m I I~.~ I as.o I I.II I us.m aw.m on II m.ms II m.m~I I .s.oHI I o.HII II m.mm II ~.mH m.s~ a II 0.00 I ¢.0~I «0.0 I I 0.0mI II m.~m II n.5H m.s~ 0 II o.ao oe.s I II II Hm.s II mo.n I~.m I II o.sh II m~.~ II II II ms.mI on.o ss.~ I II II II II II li- II M II ~.wm «mao.o I o~.I I so.~ I I m.ms mw.H nI.I I II m.ss mm.m I I as.m I mm.s I I m.II I Hm.m Io.s m II m.Iw o~I.o II HI.m I no.0 I I mm.s I HI.H owa.o N II ~.Im II ~0.~ I II NH.m I Hm.N I II mm.0 II 0N.H HH.H H N onsxv Nan Amofixv «up Insane Hap Amoflxv «a ANOHIV an on .02 a N Hfiom muaoHonmooo oOHmmouwom .ANV saw AHV maofiumaum on waIsuoooa .«msn In oouomuuxm dz «0 mmHuHuawov msu no Amxv OHumH HHomI=0HusHow 0am AHNV wafixmnm mo oaHu mo uoowmm .m oHpmH 18 .hHo>Huooamou .Hu>oH «H can am one an unuofimaamfim II .I II «.00 m.0H II 00.0 00.n I 00.0 I II 05.H «H.m owouo>¢ II «.00 «0.0 I I 00.« H00.0 I 00.0 I h«H.0 000.0 «H II H.00 I «.00 000.0I 0.0HI 0H.0 II 00.0 H0.H HH II 0.0m 0.H«I II II I 0.0H H00.0 00.0 0H II «.00 >0.0 II «.00 00.5 II 0.00I 0n.H H.m« 0 II 0.00 0.«n II II II «.Hm 0H.H 0.0H 0 II «.00 m.««I I 00.0 m«.n I m«.H I II H0.« «0.0 5 II 0.H0 I m.m« II II II mm.« H00.0I «0.H 0 II II II II II II II 0 II n.00 II 0.00 II II 00.0 00H.0 00.« 0 II 0.00 0.00 I m0.m 00.0 I H.HHI 00.H 0«.n m I m.«0 «.mmI II II II «0.0 «00.0 000.0 « II 0.00 «0.0 II II I we.« H0.H 00H.0 H N Amoaxv «In Amosxv «Np Insane Han x~OHIV up ANOHIV In on .02 m « mucmHonmooo aOHmmouwom HHom .A«v was HHV 0:0Humsvm ou waHuuooow IAN mnv 040032 2H 00 wouomuuxm oz mo mmHuHuamsv oSu co A«xv OHumu HHomuaOHusHom 0am AHNV wonmnm mo mafia mo uuommm .0 anma 19 variation in shaking time accounted for an average 1.38 ppm variation in extractable Mn. Therefore, and for most practical purposes, results were relatively unaffected by the time and ratio variables in 8 out of 11 soils. The low R2 values of the equations for some soils (Table 5) were probably due to experimental error, since the quantities of Mn extracted between two consecutive ratios or shaking times differed in several cases by only 5 to 102. Results for 1N NHAOAc (pH 7) show that the minimum amounts of Mn extracted by this procedure were approximately half of those extracted by DTPA. This difference was reduced when considering the maximum valuesextracted by both procedures. With the exception of soils 8 and 11, the 1N NHAOAc extracted approximately 752 of the Mn extracted by DTPA and above 1002 in the case of soil No. l. The exponential type of response to increases in the ratio variable (Fig. 1D) is logical, since both effects, increased ratio and larger quantities of NH: per unit of soil,are contibuting additively in the Mn extraction. Mn Extractability and Correlation with Plant Mn Uptake In the previous section, the characterization of the release of Mn from soils, as affected by the shaking time and solution:soil ratio, has been discussed. This type of study is useful when the experimental conditions to maximize extractable Mn must be determined. For soil testing purposes, however, the interest lies in those conditions that give the highest correlation between extracted Mn by any procedure and plant uptake. For this purpose, the Mn extracted from.the 12 soils at every time-ratio combination by each extractant was correlated with the total Mn uptake by soybeans and sorghum grown in the greenhouse 20 in these same soils. The complete data analysis of these two greenhouse studies is given in Parts II and III of this study. Here, only the plant data of the check pots (three replicates per soil) were used. Equations (1) and (2) were used for the regression analysis, where the response variable (Y) was now the correlation between Mn uptake and extractable Mn at each time-ratio combination. Table 7 and Fig. 2 show the equations obtained and their graphical representation, respectively. When comparing the response surfaces of Fig. 2 with those of Fig. 1 the change in the direction of the abscissas should be noticed. Results show that the treatment combination thattmximized 0.1N HCl extractable Mn (Fig. 1A) minimized its correlation with plant Mn uptake by both crops (Figs. 2ArB). While in the previous section the interaction between time and ratio was, on the average, non-significant and positive (Table 3), it is now negative and significant (Table 7). Results for 0.1N HBPOA (Figs. ZC-D) were similar to those of 0.1N HCl except for a lesser degree of interaction between both variables, particularly with sorghum, and the quadratic effects being more important. The best time-ratio combination was the same for both extractants and crops: 120 min of shaking with a solution:soil ratio of 5. Response surfaces for 1N NHAOAc and DTPA are not shown since the overall regression for three out of the four equations was not significant even at the 152 level with either model. Reconstruction of the original data by substituting the experimental values for X1 and X2 into the equation of DTPA with sorghum, yields a maximum Y value of 0.75 and a minimum of 0.71. This variation was significantly explained Conch! Ion C 9 d U . g C b L 8 I I p P ' 4 I 0 gr, I— b— | d o ,1) I P P | ‘ I 0 60 "'" " | " 0.60 1' i _______ I... .. __-... . J \ I _. ‘\ - 0.50 I, 0 \ b \ (’0 7' \ 4 0‘, \ I- 4" \ l' ’ a l _1 ° J 120 so 10 II The of sheklnr (Ma) 21 Correlation Correlation Time of skating (min) I I I I d | 4 | ‘ I ' d I '- I I h- ' ‘ I. : _ i J \ \ b \\ ‘ \\ b - AL \. j A 120 60 m "I The of choking (sin) Figure 2. Effect of shaking time and solution:soil ratio in the correlation between 0.1N HCl extractable Mn and Mn uptake by A. Soybeans, B. Sorghum and 0.1N H P0 extractable Mn and Mn uptake by C. Soybeans and D. Sorg um. 22 .hHo>Huooomou .Ho>oH NH 0cm N0 onu um unmoHMchHm II .I o.mo II.HI HH.H ooe.o ww.~ I mam.OI sum.o asewuom s 040 :2 2H 0.50 00.HI «q«.0 000.0 I m0«.0 I mo.« 0««.0 womanhom II m.mn HHn.0I I mq.H q«.« II 00.0 I HH.0I 005.0 aonwuom 4090 ~.om HII.¢I I mm.H Im.N I H~.I I o0.II mmo.o mammnsom II o.om I~.HI II em.n -.m I II I.m~I o~.m NNm.o asewuom I m on m zH.0 II 0.00 II 00.0I I «m.« I 00.0 I II 0.HHI II 0.0H 000.0 monophom II H.00 II 0«.nI II II II 00.0 I II m.HH 0«n.0 answuom Hum zH.0 II 0.00 II «0.0I II II II 00.0 I II 0.0H «0«.0 moomomom N Amosxc NHn Asoaxv Nun “coaxv Hap Incaxv up AIoHIV In on m mono uomuomuuxm « muooHonmwoo oOHmmouwmm .A«V vow AHV mcoHuosom ou wcHouooom .oxouno a: oomHo one :2 oHoouoouuxo comauon cowumHouuoo onu oH A«xv OHumu HHomIGOHusHom 0am AHNV mconno mo oaHu mo uomwwm .n oHnMH 23 (1% level) by the model used. However, such a small response in the correlation between extractable Mn and plant Mn uptake has no practical applications. Therefore, it may be concluded that the selection of a time of shaking and solution:soil ratio for 1N NH OAc and DTPA 4 extractions is of little consequence for extractable Mn and its correlation with plant Mn uptake. In the case of 0.1N HCl and H3P04 large variations in the Mn extracted at different times and ratios allowed for the selection of a treatment combination that maximized the correlation with plant uptake, and that differed from the one that maximized extractable Mn. Extractable Mn and Soil Characteristics The average response surfaces of Fig. l and results for individual soils (Tables 3 to 6) show that the shaking times and solution:soil ratios used affected the Mn extractions in different ways, depending on the extractants. In an attempt to find and explanation for these responses, a stepwise regression procedure was used. Extractable Mn at four selected time-ratio combinations, namely: 5-10 min, 5-120 min, 25-10 min and 25-120 min, was related to seven soil characteristics (Eq. 3). The basic idea underlying the use of a stepwise procedure was that each extractants would relate to a different set of variables when changing the ratio-time combination. To prevent biasing the results due to the high value of organic C and bases ratio (Table 1) of the organic soil (No. 11), this soil was excluded from the analysis. Table 8 shows which soil characteristics explained most of the variability among soils in the Mn extracted by the four extractants at each ratio—time combination. 0f the seven variables used at the 24 .%H0>Huooamou .Hm>oH NH can um ozu um unmoamaawwm II .I IIm.om II II II IInmm.0 II 0H.HI 0~H mm : II~.mm II II II II~0<.0 II mm.HI 0H mm : II0.<0 II II II IIN~¢.0 II 0<.HI 0NH m I: IIm.m0 II II II IImnm.0 II 0I.HI 0H m u<0 mz zH IIm.mm IIH000.0 m~0.0I II II II mm.~ 0~H mm : IIH.mm IIH000.0 «m0.0I II II II mm.~ 0H mN : II0.0m IIH000.0 mm0.0I II II II <0.H 0~H m : II0.mm IIH000.0 mN0.0I II II II mm.~ 0H m mm up :2 oHnmuowuuxo mo moaufiuamav onu ou ouwawu umcu mucuomw Hfiom can: .0 manna 25 begining of the stepwise procedure (Eq. 3) only one or two out of four remained in each model. These variables were significant at the 5% or 1% level except for the linear term of total Mn in the DTPA equations; this particular case will be discussed later on. The two principal variables for 0.1N HCl were chelated Mn and Mn oxides at short and long shaking periods respectively. Although significant, pH is only contributing 5 to 6% of the R2 value in the two equations in which it was included. These two main variables may explain why soils 5 and 6 (Table 3) did not show a significant simple effect for the time variable, since both had the lowest amounts of chelated Mn and Mn oxides (Table 1). The four equations for this extractant also show that the Mn sources are the same when changing ratios at fixed shaking times, which is probably the reason why most soils did not respond to changes in ratio. Since 0.1N HCl is a powerful extracting agent for Mn (Table 2), most of the Mn from any one source should be extracted, within limits, independently of the ratio used. The limits are set by the increase in the intercept when increasing the ratio at long shaking times, while the slopes of both equations remain relatively constant (Table 8). Results for 0.1N H3P04 show extractable Mn mainly as a function of the Mn oxides content at all ratio-time combinations. The strong effect of the interaction between time and ratio with most soils (Table 4) can now be realized by observing the changes in slope when changing one variable at a fixed level of the other one. Extractable Mn was higher when any one variable was increased at the high level of the other. Due to the small number of points defining each equation, the number of independent variables was kept to a minimum, making no allowance for interactions or quadratic effects. Total Mn in soil was the only 26 variable that showed a significant relationship with DTPA extractable Mn, at all treatment combinations. A plot of these results showed a strong non-linear relation between both variables. After the 2nd order term was included in the equation, the linear term lost all its significance and explains 12 or less of the R2 values for those equations. It was left in the equations to maintain all the terms of the polynomial. Since total Mn comprises several chemical species of very different characteristics, no explanation can be offered for the soils behavior upon changes in the time and ratio variables. Results for IR NH OAc (pH 7) extractable Mn show this variable as a 4 function of chelated Mn at all ratio-time combinations. Chelated Mn was defined as the Mn extracted from finely ground soils by two successive extractiomwith 0.005M DTPA. This close relationship and similar maximum amounts of Mn extracted by DTPA and 1N NH OAc (Table 2) suggest 4 that part of the Mn extracted by DTPA is in exchangeable positions. Smaller amounts of Mn extracted from most soils by the IN NHAOAc seem to be related to ratios and extractants concentrations. This tended to be confirmed by positive quadratic effects of the ratio (bzz) variable upon 1N NHAOAc and negative ones for DTPA for all soils. Figures 10 and 1D show these effects very clearly at long shaking times. Summary and Conclusions The effect of 12 shaking time-solution:soil ratio combinations on the quantities of Mn extracted by 0.1N HCl, 0.1N H3P04, 0.005M DTPA and 1N NH40Ac (pH 7) from 12 soils was studied. Effects of these variables upon extractable Mn were studied by multiple regression analysis. Extractable Mn from all soils at each time-ratio combination was then 27 correlated with yield data from two greenhouse studies using these same soils. Hence, the effect of both variables in the degree of correlation between extractable Mn and Mn uptake by soybeans and sorghum was obtained. To study which soil Mn fractions and soil characteristics were important in determining the quantities of Mn extracted, a stepwise regression procedure was used. Results can be summarized as follows: 1) The time-ratio combination that yielded the largest quantities of extractable Mn by all extractants (average for 12 soils) was a solution:soil ratio of 25:1 (volume:air dry weight) and a shaking time of 120 min. 2) For 0.1N HCl and 0.1N H P04, a ratio of 5:1 with 120 min of 3 extraction gave the highest correlation between extractable Mn and plant Mn uptake by both crops. For most practical purposes, the correlation of 1N NHAOAc and DTPA extractable Mn with plant Mn uptake was unaffected by the time-ratio combination used. 3) With 10 min of shaking, chelated Mn was the main variable relating to 0.1N HCl extractable Mn; at 120 min it was Mn oxides. These variables were selected at both ratios, 5 and 25. The other three extractants correlated with a single variable at all four time-ratio combinations: 0.1N H OAc with chelated Mn and P0 with Mn oxides, 1N NH 3 DTPA with (total Mn)2. 4 4 4) 0f the four extractants used, 0.1N H P04 was the one giVing 3 the highest correlation with plant uptake, at a ratio-time combination 5) For soil testing purposes, the use of 0.1N H3PO4 with a ratio- time combination of 5-60 min is recommended for mineral soils. 10. ll. 12. 13. Literature Cited Basu, A.N., D.C. Mukherjee and S.K.Mukherjee. 1964. Interaction between humic acid fraction of soil and trace element cations. J. Indian Soc. Soil Sci. 12:311-318. Boken, E. 1958. Investigations on determination of the available manganese content in soils. P1. Soil 9:269-285. Browman, M.G., G. Chesters and H.B.Pionke. 1969. Evaluation of tests for predicting the availability of soil manganese to plants. J. Agri. Sci. 72:335-340. Bundy, L.B. and J.M. Bremner. 1972. A simple titrimetric method for determination of inorganic carbon in soils. Soil Sci. Soc. Am. Proc. 36:273-275. Chao, T.T. 1972. Selective dissolution of manganese oxides from soils and sediments with acidified hydroxylamine hydrocloride. Soil Sci. Soc. Am. Proc. 36:764-768. Chapman, H.D. 1965. Cation-exchange capacity. .12 C.A. Black, et al., (ed.) Methods of soil analysis. Agronomy 9:891-900. Am. Soc. of Agron., Madison, Wis. Cox, P.R. and E.J. Kamprath. 1972. Micronutrient soil tests, p. 289-317. IB.J°J- Mortvedt, et al., (ed.) Micronutrients in Agriculture. Soil Sci. Soc. Am., Madison, Wis. Day, P.R. 1965. Particle fractionation and particle-size analysis. lg_C.A. Black, et al., (ed.) Agronomy 9:545-566. Am. Soc. of Agron., Madison, Wis. Dolar, 8.6. and D.R. Keeney. 1971 Availability of Cu, Zn and Mn in soils. I.- Influence of soil pH, organic matter and extractable phosphorus. J. Sci. Fd. Agric. 22:273-278. Draper, N.R. and H. Smith. 1966. Applied Regression Analysis. John Wiley and Sons, New York. Hoff, D.J. and H.J. Mederski. 1958. The chemical estimation of plant available soil manganese. Soil Sci. Soc. Am. Proc. 22:129-132. Sorensen, R.C., D.D. Oelsligle and D. Knudsen. 1971. Extraction of Zn, Fe, and Mn from soil with 0.1N HCl as affected by soil properties, solution:soil ratio and length of extraction period. Soil Sci. 111:352-359. Viets, F.G. and W.L. Lindsay. 1973. Testing soil for Zn, Cu, Mn and Fe. p. 153-172. In_L.M.‘Walsh and J.D. Beaton (ed.) Soil testing and plant analysis. Soil Sci. Soc. Am., Madison, Wis. 28 PART II STUDIES IN SOIL MANGANESE 2. EXTRACTABLE MANGANESE AND PLANT UPTAKE 29 Introduction Considerable effort has been made in the development of tests to estimate the availability of Mn in soils. This element occurs in several oxidation states and is associated with different soil fractions, namely, several types of Mn oxides, Mn associated with the organic matter, Mn related to the inorganic colloid and Mn in soil solution. A cycle for this nutrient in soils has been proposed by Ghanem, et al., (10). The nature of this cycle has justified the use of different types of extracting solutions in an attempt to characterize the fractions which are in close equilibrium with the Mn in solution and, therefore, responsible for the replenishment of Mn extracted by a crap throughout the growing season. Reviews on this subject have been published (6, 24). Several authors (12, 11, 3, 15) have found 0.1N H3P04, 1.5 and 3M NH4H2PO4 extractable Mn to correlate well with Mn uptake by plants. Manganese extracted by 1N NHAOAc has also been used with some success (1, 20) and correlated better than 0.1N HBPO with plant uptake when the 4 soil pH factor was included in the prediction equation (3). DTPA has been proposed as a single test for available Cu, Zn, Fe and Mn in soils (Lindsay and Norvell, 1969)1; however a study by Dolar, et al., (8) showed that DTPA was not the best extractant for this purpose in soils of Wisconsin and they propose, instead, the use of an extraction with 1N NH4OAc-O.01M EDTA. Correlation of DTPA extractable Mn with Mn concentrations in wheat and soybeans was shown to depend on the pH of soil (20). There have been several reports on the increased availability of Mn 1 Development of a DTPA micronutrient soil test. Agron. Abstr., p.84. 30 31 after steam treatment of the soils (2, 21, 22), in some cases developing problems of Mn toxicity in plants. Tests performed in this laboratory (unpublished data) subjecting soil samples to a steam pretreatment have shown that the quantities of Mn extracted correlated well with plant Mn uptake. In recent years, the incorporation of soil characteristics to the prediction equations of soil tests have been successffully used (3, 8, 6, 18). Organic matter and pH have been the variables most used. In the investigation described herein, several soil tests for Mn are compared, including steam sterilization followed by 1N NH40Ac extraction. Furthermore, the possiblity of including certain soil characteristics in the prediction equation of these soils tests is also explored. Materials and Methods Soils Surface soil samples of 12 different soil types from Michigan and Indiana were used. The main soil characteristics have been reported previously (Part I). Methods Greenhouse experiment: The soil samples were air dried, screened through a one cm sieve and placed in one gallon cans lined with polyethylene bags - 3.5 kg/pot for mineral soils and 1.6 kg/pot for the organic soil (air dry weight). All soils received a basic application of 50 ppm of K and P and 10 ppm of Zn. The mineral soils received the following treatments: 8) basic fertilizer application only; b) 10 ppm of Mn as 32 MnSO4 (reagent grade) and c) 20 ppm of Mn as MnSO4 (reagent grade). The organic 8011 received 22 and 44 ppm of Mn and 110 ppm of P and K and 22 ppm of Zn. A randomized complete block design with three replications was used. After mixing the fertilizer with the soil, these were incubated for 2 weeks at 80% field capacity and then planted with 10 seeds of soybean (Glycine max, var. Hark). After emergence the plants were thinned to 3 per pot and supplemental fluorescent light was initiated to give a day length of 14 hours. Each pot was weighed daily and brought to field capacity with deionized water. To prevent uneven root distribution, N fertilizer was not added as a basic application, but rather applied when necessary, at a rate of 30 ppm of N per application. The tops were harvested from each pot when at least two plants were at blooming stage, placed in paper bags and dried at 60 °C, weighed and ground in a Wiley mill to pass a 40-mesh screen. After harvest soil samples were collected from each pot using a soil sampling probe, air dried and ground to pass a 2 mm sieve. Plant analysis: Total Mn in plant tissue was analyzed by dry ashing according to the procedure described by White (25) and Mn determined by atomic absorption spectrophotometry. Sbil Analysis: A solution:soil ratio (volume:air dry weight) of 5 (20:4) was used for all the extractions. The lengths of extraction were 30 min for DTPA and 1N NHAOAC (pH 7), and 60 min for 0.1N HCl, 0.1N H3P04 and 1.5MINH4H2P04. For the proposed procedure 4 g of soil were subjected to 30 min of steam heat at 15 psi and then extracted with 1N NHaoAc as indicated above. Solution:soil ratios and lengths of extraction were selected according to results obtained in Part I. In all cases Mn was analyzed in the filtrates by atomic absorption spectrophotometry. 33 Methods used to determine other soil characteristics mentioned in this study were given previously (Part I). Results and Discussion Results for total Mn uptake by soybeans are given in Table 1, together with the soil Mn extracted by each method. Extraction with 0.1N HCl gave the largest quantities of extractable Mn with the exception of the organic soil (No. 11) where steam/NH4OAc and 1.5M NH4H2PO4 were stronger extractants. Although Boyd (2) proposed that organic matter was the source of the Mn released by the steam treatment mineral 801187, 8, 9 and 10 released larger amounts of Mn than the organic soil itself. Correlation and Regression Analysis Evaluation of the soil tests and soil factors as predictorsof Mn uptake by soybeans was done using multiple regression analysis according to the following model: Y = so -I- 81x1 + 81x1 + 82x2 + e (1) where X1 (X8...Xf) represents the Mn extracted by the different soil tests, X1 2 the variable response (Y) is total Mn uptake. The results of this = pH, X - Ca+Mg/K (bases ratio using meq/100 g of soil) and analysis are shown in Table 2. An indication of the degree of fit gained by the inclusion of X1 and X2 into the model is given by comparing the square of the simple correlation coefficient for any extractant with the R2 of the complete model. Soil reaction is known to relate negatively with soluble Mn (14) and inclussion of this variable in prediction equations has been proved 4H2P°4 Steam/ NH40Ac Extractable MnT NHAOAc ppm (soil basis) H3PO4 34 DTPA HCl 45.1 51.7 57.5 uptakei 117 155 145 Mn uptake by soybeans and soil extractable Mn by each method. ug/Pot added PPm 0 10 20 Mn Table 1. Soil No. 485 330 841... 313 825 756 522 231 161 577 661 .0. .0. 0.. .0. 0.. .0. .0. 0.. .0. 0.. 0.. 914 937 195 258 480 297 146 924 591 927 471 11 11 112 l 444 555 455 112 233 1 496 404 635 011 428 935 406 2 496 713 671825 580 124 368 631 903 44.8 058 239 258 1 4 55 22 2 160 667 559 269 751 639 796 521.. 615 929 498 00. 000 no. no. on. 0.0 000 O. to. no. one 791 11.19 125 655 782 536 338 644 927 880 501 O O C C O O O C . . C C O O O C O O 6 O 703 589 588 260 135 569 02 ll 1 333 333 3 111 234 772 272 212 044 090941508 459 637 0.. 0.. .0. .0. .0. .0. .0. O .0. 0.. .0. 234 470 246 001 134 789 002 823 699 890 012 1 222 11. 572 439 076 815 671 860 614 883 210 580 I 0.. O .0. O O. 0.. 0.. .0. .0. 749 982 818 595 108 536 235 977 071 891087 4 31 31141 81 0 134 965 263 745 700 250 266 321 108 964 070 140 700 790 45 591 094 303 690 314 582 805 112 11 l l 434 233 334 222 1 11 000 000 000 000 000 000 000 000 000 000000 12 12 12 12 12 12 12 12 12 12 12 2 3 4 5 6 7 8 9 o l 2 1 1 1 T Each result average of three replicates. 35 successful when using lN NH4OAc (3), double acid (6) and other Mn extractants (8, 18). It is also reasonable to think that Ca2+ and to a lesser degree Mg2+ are the main divalent ion competitors of Mn2+ for positions on the exchange complex. Thus, including a factor accounting for this relationship seems justified -- the significant negative simple correlation of X2 with plant uptake confirms this. The advantage of using these variables instead of others that may correlate even better with plant uptake (Part I) is that they are determined routinely in soil testing laboratories and therefore no extra analyses are necessary. The list of the extractants in order of decreasing simple correlation with plant uptake is: H P0 > steam/NHAOAC > NH H P0 > 3 4 4 2 4 HC1 > NH4OAc > DTPA. When using the R2 for the complete equations, the order becomes: H P0 > steam/NH OAc = NH H P0 > HCl > NH OAc = DTPA. 3 4 4 4 2 4 4 To study the relative contribution of X and X2 in each model, 1 the R2 delete value for each variable was included. This value gives the percentage of the variance that would be accounted for by the remaining variables, with that particular variable not being included in the equation. In this context, deletion of the X variable (soil 1 test) from any equation, would leave pH and bases ratio explaining more than 402 of the variation in the Mn uptake data. Bases ratio was more important than pH in explaining the variation of the data in the prediction equations of NHAHZPOA’ steam/NHaoAc, NH40Ac and DTPA -- 152 in the case of this last extractant. For HCl and H P04, pH was more 3 important than bases ratio. Bases ratio was not significant when included in the prediction equation of 0.1N H P04, which is shown by the 3 magnitude of its R2 delete value. The only extractant that alone could explain most of the variation in mm uptake by soybeans was 0.1N H3P04 36 Table 2. Relationship of Mn uptake by soybeans (Y) with several soil Mn tests, pH and bases ratio, according to Equation (1). Soil Test Correlation Coefficients R2 R2 Simple Partial Delete 2 Z 1.5M NH4H2P04 (Xa) 0.796** 0.805** 40.2 pH (X1) -0.347** -0.526** 70.9 Bases ratio (X2) -0.458** —0.588** 67.8 Regression Eq. Y = 637 + 4.79Xd - 77.7X1 - 0.99X2 78.9** Steam/1N NHaoAc (Xb) 0.840** 0.806** 40.2 pH (X1) -0.347** -0.281** 77.2 Bases ratio (X2) -0.458** -0.529** 70.9 Regression Eq. Y = 389 + 4.88Xb - 39.5X1 - 0.87X2 79.0** IN NH40Ac (XC) 0.747** 0.643** 40.2 pH (X1) -0.347** -0.273** 62.1 Bases ratio (X2) -0.458** -0.444** 56.3 Regression Eq. Y = 485 + 19.2Xé - 50.1X1 - 0.91X2 64.9** DTPA (Xd) 0.690** 0.634** 40.2 pH (X1) -0.347** -0.337** 59.7 Bases ratio (X2) -0.458** —0.547** 48.9 Regression Eq. Y = 581 + 9.95Xd - 61.9X1 - 1.15X2 64.2** 0.1N HCl (Xe) 0.749** 0.692** 40.2 pH (X1) -0.347** -0.534** 56.4 Bases ratio (X2) -0.548** -0.244** 66.8 Regression Eq. Y = 710 + 2.92Xé - 95.0X1 - 0.48X2 68.8** 0.1N H3PO4 (Xf) 0.919** 0.901** 40.2 pH (X1) -0.347** -0.482** 85.2 Bases ratio (X2) -0.458** 0.055 88.6 Regression Eq. Y = 388 + 8.29X - 52.2X 88.6** f' 1 ** Significant at the 1% level. 37 Relationship Between Mn Uptake and Soil Properties In an attempt to establish the soil Mn fractions and soil properties most important in explaining the variations of the Y variable, a stepwise regression technique was usedzaccording to the following model: 7 Y= so + iglaixi + e (2) where the variable response (Y) was Mn uptake and the seven independent variables were: pH, Ca+Mg/K, CaCO , organic C, chelated Mn, Mn oxides and 3 total Mn. A 12 level of significance was selected for the deletion and addition of variables. The list of variables left in the model are shown in Table 3. Table 3. Relationship between Mn uptake by soybeans and several soil characteristics. 2 Simple Partial R 2 8011 Properties Correlation Correlation Delete R Z Z pH (X1) -0.347 ** -0.400 ** 78.2 Mn oxides (X2) 0.815 ** 0.715 ** 62.6 Bases ratio (X3) -0.458 ** -0.451 ** 77.1 Total Mn (X4) 0.690 ** -0.522 ** 74.9 Regression Eq. Y = 543 - 53.1X1 + 2.26X2 - 0.677X3 - 0.655X4 81.7** ** Significant at the 11 level. By the magnitude of the R2 delete values, pH is the parameter that explain the least variation in the uptake data, in the presence of the other variables, while Mn oxides is the parameter explaining most of 2 Michigan State University Stat System. 1971. LSSTEP Program, Chap.12. 38 the variation. Furthermore, a regression equation with Mn oxides as the only variable would account for a greater percentage of the variability of Y (66.42) than the extraction.with 1.5M NH4H2P04, 1N NH4OAc and 0.1N HCl as single determination, and than 1N NH OAc and DTPA when 4 including pH and bases ratio in the prediction equation. The strong correlation of plant uptake with Mn oxides and the fact that chelated Mn did not appear in the model as a significant variable is an indication as to why DTPA extractable Mn was not useful as a predictor of Mn uptake. This conclusion would only hold for the mineral soils studied since a recent study by Randall, et al., (18) using 20 organic soils showed DTPA to be a good predictor of Mn uptake in this type of soil. The high correlation of plant uptake with Mn oxides is in good agreement with results of other researches. According to Passioura and Leeper (17) Mn oxides in soils are not necessarily crystalline or concentrated in a few course nodules. Ross, et al.,(l9) reported that the soil Mn compounds that they studied were largely amorphous to X-rays due to poor crystallinity, fineness of both. Therefore high specific surfaces coupled with low degree of crystallinity may render part of this fraction available to plants through action of root exudates and modification of the pH-redox system of the root envirmnent produced by the release of H+ (26). Availability of higher oxides of Mn to oats has been reported (13, 16). Results by Randall, et al., (18) show hydroxylamine hydrochloride among the three best extractants out of 18 in predicting plant Mn uptake and this was the same extractant used (5) in defining the Mn oxides fraction, although the acid concentrations differed. Simple correlation coefficients among extractants and several soil 39 properties are shown in Table 4. Although different procedures were used to define chelated Mn (Part I) and DTPA extractable Mn, 3 close relationship between both variables should be expected since the same extractant was used in both cases (Table 4). 1N NH OAc also relates l, closely to chelated Mn and, therefore, both DTPA and 1N NH OAc 4 extractable Mn show a high correlation with each other. The high correlation between Mn extracted by steam/NHAOAC and 1.5M NH4H2P04 can be explained by the fact that both are closely correlated to the Mn oxide fraction. The relationship between Mn oxides and steam/NH OAc extractable Mn indicates that this fraction may be more I. labile to the steam treatment than proposed by Boyd (2). Growth Response The results of the Mn uptake by the soybeans were shown in Table l. The variances for the combined experiment were not homogeneous and a logarithmic transformation was necessary for the analysis of variance. The effect of the fertilizer application on each soil was studied by using orthogonal polynomials, partitioning the two degrees of freedom of the fertilizer treatment into a linear and a non—linear response (23). The results of this analysis are shown in Table 5. Based on the Mn deficiency symptoms observed in the soybeans growing in soils 3 and 4 a more significant response to the fertilizer application was expected. However, these two soils had more than 40% clay and there were delays in the germination and emergence of the soybeans plants; probably problems related to root growth and distribution did not allow for an efficient use of the applied fertilizer. No explanation can be offered for the significant response at the 52 level of soil 8, since this soil 40 .Hm>ma NH we“ um acmowmaoowm n~.0 omnu nooHMH moSHo> «« 00.H a: kuoe oo.0 00.H movaxo a: oo.0 no.0 00.H a: omuwaono an.0: qn.0: NH.0: 00.H owumu momoo mq.ou oo.0- os.o: mo.c- oo.H moomo oo.o: mo.o- No.o- ~q.o NH.o oo.H o oocmmuo nn.0: oo.0: oo.0: oH.0: no.0 nn.0: 00.H :0 em.o Nm.o hm.o s~.o- om.o- Ho.o- mH.o- oo.H scammqmz oo.0 no.0 no.0 nN.0: no.0: n0.0: nn.0: no.0 00.H o¢0oo on :2 oHnmuomuuxo oaoam muoofiofimwooo coauMHouuoo oHQaHm .MIMMNMW Table 5. Response in Mn uptake by soybeans to Mn fertilization as MnSO4. igglicziigz Logarithm of Mn uptake (pg/pot) PPm Soils l 2 3 4 5 6 0 2.071 2.047 1.900 1.890 0.862 1.759 10 2.192 2.155 2.026 1.980 1.644 1.955 20 2.161 2.310 2.019 2.013 1.742 2.042 Sum of Sq. Linear effect 0.012 0.104** 0.022+ 0.023+ 1.159** 0.120** Quadr. effect 0.011 0.001 0.009 0.002 0.235** 0.006 7 8 9 10 11 12 0 2.605 2.366 2.560 2.363 1.777 1.903 10 2.597 2.486 2.594 2.323 1.935 2.030 20 2.643 2.526 2.603 2.394 2.092 2.176 Sum of Sq. Linear effect 0.002 0.038* 0.003 0.001 O.194** 0.112** Quadr. effect 0.001 0.003 0.001 0.006 0.001 0.001 T, * and ** Significant at the 10%, 5% and 1% level, respectively. .Ho>oa N0H any um coauMNfiHauuom :2 cu oncoemou unmowmwawwm no.0 muomoawfiaowm won a. .Ov .ouavoooua oomHQZImumo man on a: oHnmuomuuxo A5 «chumemz Entn 0cm o<0qmz zH\aoouo now can AoH Hoofiuwuo osu mo oofluoafiauouon .H ouowfim 42 Aamov oouomuuxo oz Aaanv wouumuuxo oz 0n 0e co co 0H 0c 0n 0N 0H . . . . q . . _ a . _ q . . _ _ q . . c N e . . ”m x =2 zm H .+.x .31 «ammo 56 ”0.0 o L 03 oz zQawoum 3.0 10~ 10w 10¢ :0: .+ 0 +0 I 0.. +0 0 O + o 0 10o :00 +0 1 o I. x. 0 0o 0 0o x 1 1 x o o o x O < o L00H :00H asuodsax p131; z 43 showed large amounts of extractable Mn by all the procedures used. The determination of the critical level of Mn for the three best extractants was done by the method proposed by Cate and Nelson (4). These levels were about 12 ppm of Mn for 0.1N H3P04 and 14 ppm of Mn for steam/NHAOAc and 1.5MZNH4H2PO4 (Fig. 1). None of these extractants were able to separate soil 8 as a deficient one and as said before, no explanation can be offered for this behavior other than caused by experimental error. Except for this soil, 0.1N H3P04 was able to separate the rest of them into deficient and nonedeficient, including the organic soil (482 of maximum yield), while the other two extractants did not place this last soil into the deficient group. Furthermore, the steam/NH OAc extraction underestimated the available Mn of soil 4 l (81.22 yield) while the other two procedures did not. Results from Table 5 show that only one soil of those that responded significantly to the fertilizer treatment had a significant quadratic effect. This would indicate that a higher maximum rate of Mn application should have been used. Summary and Conclusions A greenhouse experiment with 12 soils from.Michigan and Indiana was used to evaluate six extractants in their ability to predict Mn uptake by soybeans. A randomized complete block design experiment with three replicates and three rates of Mn fertilization (as MnSOA) namely, 0, 10, and 20 ppm of Mn for the mineral soils and 22 and 44 ppm.of Mn for the organic soil was conducted. Soybeans tops were harvested at bloom stage, dry weight recorded and the tops analyzed for Mn. All statistical analyses were based on total Mn uptake data. 8011 samples were taken 44 from each pot with the aid of a probe and extracted for Mn by the following extractants: 0.1N HCl, 0.1N H3P04, 1N NHAOAC (pH 7), 1.5M NH4H2PO4 and steam/1N NH40Ac. Correlation and regression techniques were used to evaluate the behavior of each 0.005M DTPA (pH 7.3), extractant to study the relationships between Mn uptake and soil properties and to observe interrelations among extractants and soil properties. The results can be summarized as follows: 1) The listing of extractants in decreasing order of correlation with Mn uptake by soybeans was: H3P04 > steam/NHAOAC > NH4H2P04 > HCl > NHAOAC > DTPA. 2) If pH and Ca+Mg/K were included in the prediction equation, the order was as follows: H3P04 > steam/NH40Ac = NH4H2P04 > HCl > NH40Ac = DTPA. 3) Bases ratio was more important than pH in the prediction equations of 1.5M NH H P0 DTPA, 1N NH OAc and steam/1N NH OAc. The 4 2 4’ 4 4 reverse was true for the 0.1N HCl prediction equation. The prediction equation of 0.1N HBPO4 showed almost no improvement when bases ratio and pH were included. 4) The Mn oxides fraction of the soils explained 66.4% of the variation of the Mn uptake data and inclusion of pH, bases ratio and total Mn in the equation raised this value to 81.7%. 5) A correlation coefficient of 0.97 was obtained between DTPA and 1N NH OAc extractable Mn, and of 0.95 between steam/NHAOAc and 1.5M 4 NH4H2P04. correlated with the chelated Mn fraction of the soils, while the second Mn extracted by the first pair of extractants was highly pair correlated very closely with the Mn oxides fraction. 45 7) The critical levels of extractable Mn in soils were 12 ppm for 0.1N H P0 3 4 and 14 ppm of Mn for steam/1N NH OAc and 1.5M NH H P0 . 4 4 2 4 10. 11. 12. 13. Literature Cited Boken, E. 1958. Investigations on determination of the available manganese content in soils. P1. Soil 9:269-285. Boyd, H.W. 1971. Manganese toxicity to peanuts in autoclaved soils. Pl. Soil 34:133-144. Browman, M.G., G. Chesters and H.B. Pionke. 1969. Evaluation of tests for predicting the availability of soil manganese to plants. J. Agri. Sci. 72:335-340. Cate, R.B. and L.A. Nelson. 1965. A rapid method for correlation of soil test analysis with plant response data. Inter. Soil Testing Series Tech. Bull. 1. North Carolina State Univ. Agr. Exp. Sta., Raleigh. . Chao, T.T. 1972. Selective dissolution of manganese oxides from soils and sediments with acidified hydroxylamine hydrochloride. Soil Sci. Soc. Am. Proc. 36:764-768. Cox, P.R. 1968. Development of a yield response prediction and manganese soils test interpretation for soybeans. Agronomy J. 60:521-524. Cox, P.R. and E.J. Kamprath. 1972. Micronutrient soil tests. p.289-3l7. .Ig_J.J. Mortvedt, et al., (ed.) Micronutrients in agriculture. Soil Sci. Soc. of Am., Madison, Wis. Dolar, S.G. and D.R. Keeney. 1971. Availability of Cu, Zn and Mn in soils. I.- Influence of soil pH, organic matter and extractable phosphorus. J. Sci. Fd. Agric. 22:273-278. Dolar, S.G., D.R. Keeney and L.M. welsh. 1971. Availability of Cu, Zn and Mn in soils. III.- Predictability of plant uptake. J. Sci. Fd. Agric. 22:282-286. Ghanem, 1., M.M. El—Gabaly, MLN. Hassan and V. Tadros. 1971. Effect of organic materials addition on transformation of added manganese dioxide to alkali calcareous soils. Pl. Soil 34:653- 651. Hammes”J.K. and K.C. Berger. 1960. Chemical extraction and crop removal of manganese from air dried and moist soils. Soil Sci. Soc Am. Proc. 24:361-364. Hoff, D.J. and H.J. Mederski. 1958. The chemical estimation of plant available soil manganese. Soil Sci. Soc. Am. Proc. 22:129-132. Jones, L.H.P. and G.W. Leeper. 1951. Available manganese oxides in neutral and alkaline soils. P1. 8011 3:154-159. 46 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 47 Lindsay, W.L. 1972. Inorganic phase equilibria of micronutrients in soils. p. 41-57. .12; J.J. Mortvedt, et al., (ed.) Micronutrients in agriculture. Soil Sci. Soc. Am., Madison, Wis. Pailoor, G., J.C. Shickluna and K. Lawton. 1970. Manganese availability in several Michigan soils. Michigan State Univ. Agr. Exp. Sta. Res. Rep. 97. Page, E.R. 1962. Studies in soil and plant manganese. III.- The availability of higher oxides of manganese to oats. P1. Soil 17:99-108. Passioura, J.B. and G.W. Leeper. 1963. Available manganese and the X hypothesis. Agrochimica 8:81-89. Randall, G.W., E.R. Schulte and R.B. Corey. 1976. Correlation of plant manganese with extractable soil manganese and soil factors. Soil Sci. Soc. Am. J. 40:282-287. Ross, S.J.,Jr., D.P. Franzmeier and 0.8. Roth. 1976. Mineralogy and chemistry of manganese oxides in some Indiana soils. Soil Sci. Soc. Am. J. 40:137-143. Shuman, L.M. and 0.E. Anderson. 1974. Evaluation of six extractants for their ability to predict manganese concentrations in wheat and soybeans. Soil Sci. Soc. Am. Proc. 38:788-791. Singh, M. and A.N. Pathak. 1970. Effect of heating and steam steriliztion on soil manganese. P1. Soil 33:244—248. Sonneveld, C. and S. Voogt. 1973. The effects of soil sterilization with steam-air mixture on the development of some glasshouse crops. P1. Soil 38:415-423. Steel, R.G. and J.H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Co., New York. Viets, F.G. and W.L. Lindsay. 1973. Testing soil for Zn, Cu, Mn, and Fe. p.153-172. .I§_ L.M; walsh and J.D. Beaton (ed.) Soil testing and plant analysis. Soil Sci. Soc. Am., Madison, Wis. White, R.P. 1969. Hydroxylamine hydrochloride as a reducing agent for atomic absorption determination of manganese in dry ashed plant tissue. Soil Sci. Soc. Am. Proc. 33:478-479. Wilkinson, H.F. 1972. Movement of micronutrients to plant roots. p. 139-169. .13 J.J. Mortvedt, et al., (ed.) Micronutrients in agriculture. Soil Sci. Soc. Am., Madison, Wis. PART III STUDIES IN SOIL MANGANESE 3. MANGANESE LABILE POOL AND PLANT UPTAKE 48 Introduction Extensive use of radioisotopes has been done in soil fertility studies. Most of these studies have concentrated in the macronutrients in soils, particularly P (4, 2, 9), although studies in S, Ca and N (6) have also been done. By comparison, micronutrients have received much 54Mn is particularly suited for studies less attention. The use of about the chemistry and plant nutrition of Mn, since it is a high energy gamma emitter with a long half-life. This radioisotope has been used to study Mn distribution in plants (13), its interaction with other micronutrients (20), the fate of Mn applied to soil as MnSO4 (l6), and its interaction with clays (3). The determination of the Mn labile pool of several soils was done by Lamm (8) who determined isotOpically exchangeable Mn by laboratory procedures, by E value (18) and with the use of plants by calculating the L values (9) and by A value (7). More recently (11, 12) DTPA.was used as the equilibrating solution in the determination of isotopically exchangeable Mn, Fe, Zn and Cu. All the theoretical considerations concerning the use of these values and comparisonsamong them have been covered extensively (10, 6, 5). The objectives of this study were to determine the E and L-values of various soils, to assess their effectivenes as predictors of plant Mn uptake by comparison to other more commonly used Mn extraction methods, and to study the residual effect of Mn fertilization as MnSOa. 49 Materials and Methods Greenhouse experiment: The control and 20 ppm Mn treatment of the same soils used in a previous experiment (Part II) were used in this study. The soils were removed from their cans, clods were broken up and most of the root residues removed. Pots containing mineral soils were reduced to 3 kg/pot and those with the organic soil to 1.5 kg/pot. No additional Mn fertilizer was used, the rates being, therefore, 0 and 20 ppm Mn (as unso4) for the mineral soils and 44 ppm of Mn for the organic soil. A split-plot design was used for this experiment, with the Mn rates being layed out as randomized complete blocks with three replicates and harvests being the split-plot. Carrier free 54Mn was added to each pot mixing 10 m1 of an aqueous solution containing an activity of 0.2 uCi/ml with the soil on a heavy sheet of paper. Pots were taken to field capacity with water containing an equivalent of 33 ppm of N and 44 ppm of P (soil basis) and were allowed to incubate for two weeks. On April 26, they were sown with a Trudan No.2 sudangrass hybrid (Sorghum vulgare sudanense L.). After emergence pots were thinned to seven plants/pot and the use of supplemental fluorescent light was initiated. All the pots were taken twice daily to field capacity with an automatic irrigation system designed and constructed for this experiment. On May 21 the plant tops were cut 10 cm from the soil surface, placed in paper bags and dried at 60 °C. The dried tissue was weighed and then ground in a Wiley mill to pass a 40-mesh screen. On May 29 all the soils were fertilized with 50 ppm of K and N. The 2nd and 3rd harvests were done in June 4 and June 28, respectively, following the same procedure as outlined above. 50 51 Plant analysis: Duplicate l g samples of the ground plant tissue (except for the few cases where insufficient material was available) were counted for 54Mn activity in a well-type sodium iodide crystal scintillation detector (Packard Auto-Gamma Spectrometer). One milliliter of the 54Mn fertilizer solution was evaporated to dryness in a counting tube and counted with each batch of plant samples. Correction for radioactive decay was not necessary since the specific activity in plant tissue was compared with specific activity of the fertilizer counted the same day. The same plant samples were then analyzed for total Mn by dry ashing, according to the procedure of White (21). Sbil analysis: The isotopic exchange study was carried out in the soil samples collected from the pots after the soybean harvest(Part II). Four-gram air dried samples were allowed to equilibrate for 72 hours (11, 12) in a rotary shaker with 20 m1 of a 0.1N H3PO4 solution containing 0.015 uCi/ml. After this period the suspensions were filtered through Whatman No. 42 filter paper. A one milliliter aliquot was evaporated to dryness in counting tubes and 54nn activity measured in the same counter used for the plant samples. The same procedure was followed with three 20 m1 blanks of the extracting solution. The Mn extracted from the soil was measured in the remaining filtrate by atomic absorption spectrophotometry. Other soil data: The main characteristics of the soils used in this study and the results of extractable Mn by several soil tests to which reference is made in this study are given in Parts I and II. Calculations: L and E values were calculated according to the following formula: 52 L or E - B (3f - 1) where L or E = IsotOpically exchangeable Mn 54 B 8 Amounts of Mn added (0.000333 ppm soil basis) Specific activity of 54Mn solution added (Appendix III) sf 8 Specific activity of plant for L value, or specific activity of the 0.1N H P0 3 4 solution after 72 hs of shaking for E value (Appendix III). In all cases specific activity refers to counts per minute divided by the micrograms of Mn in the plant or solution aliquots. Results and Discussion Sudangrass M uptake data an L values as determined by isotopic dilution techniques (10) are shown in Table 1. No correction for seedborne Mn was done (19) since its effect upon successive harvests should be negligible. Allowance for 55M1: and 5[‘Mn removed in early cuttings (14) produced only minor changes in the L values of the 2nd and 3rd harvest. Results were analyzed according to a split-plot design; factor A was the fertilizer treatment for each soil and factor B was harvests. The variances for the combined experiment were not homogeneous and a logarithmic transformation of all the data was necessary for the analysis. Comparisons between means of different fertilizer treatments were done with the Tukey test, the same test was used to study the effect of harvest upon L values and Mn uptake. Critical values for these tests are not shown in the tables since they would only apply to the transformed data which were not included. The L values increased significantly from the first to the second 53 Table 1. Manganese labile pool (L-values) and Mn uptake by plants. 5 5 $011 Mn Harvest Harvest dd d 1 2 3 1 2 3 NO’ 3 e L-valucsi Mn uptake? ppm "Is/1003 ug/pot 0 1.653 2.67 b 2.64 b 71.7ab 67.93 97.6 b 1 ** *a ** ** t * 20 3.543 3.983b 4.70 bc 1263b 1043 153 b 0 1.463 2.12 b 2.77 c 1583 2193 323 b 2 ** ** 20 2.213 3.14 b 3:37 b 1733 2003 2483 0 0.8833 1.11 b 1.37 b 1733 1923 289 b 3 *3 ** ** + 1 20 1.573 2.17 b 2.77 c 1843 2573 409 b O 1.593 1.783 1.713 40.83b 39.03 51.2 b 4 ** *t ** t 20 2.433 3.51 b 2.6lab 48.83 52.13 60.03 0 0.2273 0.360 b 0.377 b 36.03 43.33 64.1 b 5 ** *t ** *t it ** 20 1.533 2.21 b 1.923b 113a 1193 1883 0 0.3503 0.4073b 0.610 c 1033 96.73 180 b 6 ** *3 ** *a ** + 20 1.323 1.503 1.633 1703 1573 245 b 7 0 5.053 7.23 b 8.36 b 2363 479 b 774 c * 20 6.993 7.933b 9.15 b 2513 388 b 769 c 8 0 8.073 9.743b 11.8 b 1873 365 b 641 c 20 9.263 11.9 b 14.3 b 2113 2693 633 b 9 O 7.153 7.543b 11.3 c 3083 551 b 1216 c 20 7.903 8.933b 10.6 b 3543 614 b 1163 c 10 0 2.693 3.37 b 4.21 b 2283 429 b 722 c * * 20 3.613 4.64 b 5.24 b 2163 440 b 777 c 11 0 1.963 3.20 b 3.78 b 80.63 98.23 194 b t at. 2.603 4.0211. 4.04 b 78.43 1321:. 153 b 12 0 0.5103 0.723 b 0.887 b 2053 2173 309 b ** *3 3* t f *t 20 1.583 1.973b 2.37 b 3173 3063 493 b * ,** Difference between means of fertilizer treatments significant at the 10%, 5% and 1% level, respectively, by Tukey test. 5 Any two means with the same letter in the same row do not differ significantly from each other (P<.05) by Tukey test. 1 Each value, average of three replicates. 54 harvest in most cases (Table 1). This may be due to lack of isotOpic equilibrium (l4, 8, 9) and/or to increases in the size of the root systems from one harvest to the next. The fact that the L values tend to stabilize, in most cases, from the 2nd to the 3rd harvest gives some basis to either explanation. Results for Mn uptake show the opposite trend. Except for the most fertile soils (No. 7, 8, 9 and 10), there were no significant increases in Mn uptake between the lat and 2nd harvests. Between the 2nd and 3rd, however, most soils showed a significant increase in Mn uptake, probably due to the N and K fertilization just before the 2nd harvest. Although not shown in the tables, dry matter almost doubled from the 2nd to the 3rd harvest while Mn concentration dropped in most cases due to a dilution effect, in this same period. The fact that the Mn uptake increased between the last two harvests while the L values did not is in good agreement with the theoretical considerations proposed by other authors (6, 9). According to these authors, enviromental changes affecting growth but not the quantities of available nutrient should not affect the L values. The study of the residual effect of the Mn applied in a previous experiment (Part II) using the L values results can be explained in the following way: in the unfertilized soils, the relative proportion of 54Mn to 55Mn uptake by the plants should favor the uptake of more saMh, particularly in the Mn deficient soils, hence yielding low L values. In the fertilized soils, the proportion of 5('Mn in the plants should be lower due to a greater isotopic dilution, thus giving higher L values. It is reasonable to expect that these changes in the relative uptake of the two Mn forms is going to be significantly larger in the soils chficientin.Mn that were fertilized, if this added Mn was still in 55 relatively available forms. Six Mn deficient soils showed significant differences for all harvests between the L values of the unfertilized and fertilized pots (Table 1). Considering that each pot sustained seven sudangrass plants it can be concluded that the applied Mn had a good residual effect. Similar results were found by other researchers (16). The L values of soils 2 and 11 were significantly different in the first two harvests, in spite of both being Mn deficient (Part II), although this significance was lost in the third harvest. No explanation can be offered for the significant differences in L values of soils 7 and 10 since both have high quantities of extractable Mn (Part II). In order to successfully use the L value concept with this particular approach, significant differences in L values between fertilized and unfertilized soils should relate closely to significant differences in Mn uptake. Results for individual harvests (Table 1) show this relationship to be reasonably good. In evaluating these results it must be considered that with a high number of plants/pot some nutritional unbalances may have occurred even though no visual symptoms were present. It is probable that this was the reason for a lack of response in the Mn uptake from soil 2. The average L values and Mn uptake data for the three harvests are shown in Table 2. Results for the isotopically exchangeable Mn (E value) are also shown in this table. LOpez and Graham (11, 12) used this technique to measure the Mn E values of several soils employing DTPA as the equilibrating solution. Prior studies (Parts I and II, 15) showed 0.1N HBPO4 to correlate better than DTPA with plant uptake; therefore, this acid was used as the equilibrating solution in this 56 Table 2. Effect of soil type and residual Mn fertilization upon measured parameters. Radio- E-values/ L—valueg/ 5:11 :3" d Liverlage§ “fiverigle: E-values activity total total 0. a e vs ues up 3 e in solution Mn Mn ppm mg/IOOg vg/pot mg/lOOg Z Z Z 0 2.32 79.1 5.30 81.4 26.4 11.5 1 ** *t ** 20 4.08 128 6.85 7.2 -- -- 2 0 2.11 233 4.91 93.1 39.0 16.7 *3 ** 20 2.91 207 6.71 93.5 -- -- 0 1.12 218 4.75 73.5 14.4 3.4 3 ** 1 ts 20 2.17 284 6.68 73.7 -- -- 0 1.69 43.7 4.88 57.0 13.8 4.8 4 3* 1 ** 20 2.85 53.6 7.57 59.0 -- -- 0 0.321 47.8 0.941 97.5 6.9 2.4 5 3* ** ** 20 1.89 140 3.17 98.6 -- —- 6 0 0.456 127 1.75 77.6 11.6 3.0 ** *t ** 20 1.49 191 4.42 76.9 -- -- 7 O 6.88 496 40.0 62.2 96.9 16.7 20 8.02 470 41.8 61.9 -- -- 8 0 9.87 398 47.7 53.8 72.8 15.1 * 20 11.8 371 49.8 53.7 -- -- 9 0 8.66 692 45.6 57.4 81.7 15.5 20 9.15 710 46.8+ 57.3 -- ~- 10 0 3.42 459 21.0 56.5 50.8 8.3 * 20 4.49 478 23.0 56.7 -- -— 0 2.98 124 25.4 12.8 81.9 9.6 11 + * 20 3.55 121 35.5 12.5 -- -— 0 0.707 244 2.87 59.2 29.8 7.3 12 ** *t ** 20 1.97 372 6.72 59.5 -- -- +,*,** Differences between means of fertilizer treatments significant at the 10%, 5% and 1% level, respectively, by L.S.D. test. 5 Average of three harvests. 57 study. Except for soils 8 and 9, there is good agreement between L and E values in their significant response to the residual effect of the Mn fertilizer and also with Mn uptake (Table 2). In this table, the column indicating the radioactivity remaining in solution shows the extent of the isotopic dilution of the tracer with the Mn in solution and in the solid phase in the determination of the E value with 0.1N H3P04. will give the actual quantities of Mn extracted. In two of the sandy Multiplying these results by their corresponding E values soils (No. 2 and 5) almost all the labile pool was in solution. The opposite is true for the organic soil (No. 11). This is the only soil where the E value seems to have overestimated the Mn labile pool. With 310 ppm of total Mn (Part I), the 0.1N H3P04 at equilibrium extracted only 32.5 ppm of Mn (soil basis). The isotopic dilution indicates, however, that the equilibrium was established with 82% of the total Mn, or 254 ppm of Mn (E value). If this were true, Mn availability in organic soils should be adequate when, in fact, the opposite is true (17). Therefore, for this particular soil, the L value offers a more realistic result in terms of Mn availability to plants, than the E value as measured by 0.1N H3P04. When evaluating the L and E value results, it should be kept in mind that the E value determination was done in samples taken after the soybean harvest (Part II) but prior to the sowing of the sudangrass. This results, therefore, give an indication of what the residual Mn was after the first crop. The L values, instead, give an instantaneous picture of the equilibrium situation in the soil from harvest to harvest. Although L and E values are conceptually equivalent (10), E values were much higher than L values (Table 2). This is because both 58 isotopic exchanges took place in two completely different enviroments, one was the soil solution and the other the 0.1N H3P04 solution. By giving the L and E values as a percentage of total Mn in the unfertilized soils (Table 2), the relative strength of the soil solution-plant system and the 0.1N H3P04 system can be effectively compared. Results for the E values as a percentage of total Mn clearly show the big differences that exist among soils in the type of Mn compounds present. The correlation coefficients of E and L values with sudangrass Mn uptake are given in Table 3. Extractable Mn by_six other extractants was reported previously (Part 11); here only its correlation with plant uptake is reported. Allextractants show' an increase in the correlation between both variables in the succesive harvests probably due to a better samplirg of the soil volume by the roots. Table 3. Simple correlation coefficients between Mn uptake by sudangrass and several soil tests for Mn.** Soil test 1 HarZests 3 32:52:: Average R2 Z 0.1N HCl 0.457 0.563 0.639 0.608 37.0 DTPA 0.482 0.625 0.715 0.674 45.4 0.1N H3PO4 0.641 0.749 0.815 0.794 63.0 1N NHAOAc 0.587 0.713 0.808 0.771 59.4 Steam/NHAOAc 0.564 0.732 0.808 0.773 59.8 1.5M NH482P04 0.416 0.576 0.656 0.615 37.8 E value 0.488 0.675 0.746 0.708 50.1 A value 0.542 -- -- -- - A value -- 0.616 -- -- -- A value -- -- 0.762 -- -- Avge. A value -- -- -- 0.703 49.4 ** All correlation coefficients significant at the 1% level. 59 The list of the different tests in order of decreasing simple correlation with the Mn uptake for the average of the three harvests is: H3P04 HCl. 0Ac > E value > A value > DTPA > NH H P0 > > steam/NHaoAc > NH4 4 2 4 Inclusion of pH and Ca+MglK ratio in the prediction equation of each soil test increased greatly the usefulness of all of them, as shown by the R2 values in Table 4. In fact, for any soil test, the R2 delete value indicates that 52.7% of the variation in the uptake data is explained by pH and bases ratio alone. This is a larger percentage than that reported for soybeans (Part II). The R2 delete for any variable indicates which percentage of the variation would be explained by the remaining variables if that particular variable were deleted from the model. In this context, pH is more important than the soil test in the prediction equation of 0.1N HCl, DTPA , A value and 1.5M‘NH4H2PO4 and more important than bases ratio in all the equations. This last variable contributes to explain between 5 and 13% of the variation in the different equations with the exception of those for 0.1N HCl and 0.1N H3P04. The lack of significance of this variable in the prediction equation of 0.1N H P0 was also reported previously, 3 4 (Part II). The relationships among E and L values with other soil tests and soil characteristics are given in Table 5. Both values (E and L) are highly correlated with each other; this indicates that 0.1N H3P04 can be used as the equilibrating solution for determining E values in mineral soils. Mn oxides, steam/NHAOAc and 1.5M’NH4H2P04 are the soil characteristic and soil tests that correlate best with both, E and L values. Other interrelationships not included in this table were given 60 .Ho>oH NH one up uomuumwowam «e H c.33 *«omn.o- 3mm.o- Huey oHuaHHmmmmm *«3.3H me.H HxamH . namem.o mmmH u 3.33 ssmoo.cu mom.ou H so on H.Hm ssmme.o moH.o Hexv mosHmpum N H w H.oH ««o~3.o: 3mm.ou H «xv OHHuu manna ««H.HH HOH.H . HHHN . xaH.m «HoH . ~.om «thH.o- mon.o- HHHV ma H.Hm «tooH.o moH.o H mxv H. mw>om Sofia Amumo>umn mounuv oxouoo oz moouo>o onu mo ofinmoowumaom .q canny Table 5. Simple correlation coefficients 61 and soil characteristics. among several soil tests Soil tests and Average soil properties L value E value 0.1N HCl 0.891** 0.7l6** DTPA 0.880** 0.882** 0.1N H3P04 0.894** 0.797** 1N NH4OAc 0.864** 0.840** Steam/NHAOAC 0.934** 0.973** 1.5M NH4H2P04 0.938** 0.953** pH -0.152 -0.293** Organic C -0.104 0.024 CaCO3 -0.387** -0.402** Bases ratio -O.356** -0.l67 Chelated Mn 0.895** 0.902** Mn oxides 0.939** 0.944** Total Mn 0.860** 0.862** E value 0.922** -- ** Significant at the 12 level. previously (Part II). The determination of the critical L and E values below which response to Mn fertilization cambe expected were obtained by the method pr0posed by Cate and Nelson (1) and are shown in Figures 1A and 13. These critical values are about 3.2 mg Mn/lOO g for the L value and 7.0 mg/100 g for the R value determined with 0.1N H P0 Lack of 3 4' response in the Mn uptake from soil 2 misplaces it, although it has a low L value. As said before, this lack of response may have been caused by some factor other than Mn, particularly since soybeans growing in this soil showed a significant increase in Mn uptake upon fertilization 62 .wusvmooua comHmznuuau may hp a: no usHm>uw vcm opaw>ua now Ho>uH HmUHuHuo 0:» mo coHumcHEumuuo w oOHch as on as on ON CA _ _ H _ _ _ _ mmcoqwmu ucmuwuchHm v? omcoawuu unonuHCmHmnco: "x r+ 0N ow ow ow 00H ONH .uan>nw .m vcm osHm>aH .H musme w oeHH=z m: 0H m o c 4. _ _ _ _ _ _ I,o~ uncommon uchHWchHm x. +1 omconwou ucnoHuchHmuaoc ”x L 3 J < n1 00 +;. J +1+ ow x .1 ooa x x J IlomH asuodsal PI 0'! A z 63 (Part II). Soil 11 is also misplaced due to its lack of response. Here, probably the organic matter transformed the Mn into unavailable forms, since again, soybean responded to Mn fertilization in the prior study. The comments mado above apply to the E value figure of soil 2. Results for the organic soil (No. 11) were certainly overestimated by this procedure, and it would not be of application to organic soils when using 0.1N HBPO4 as the equilibrating solution. The critical level for 0.1N H3PO4 extractable Mn as determined by the Cate and Nelson procedure was 14 ppm of Mn (soil basis). This was the best predictor of Mn uptake by sudangrass whether used alone or with the inclusion of pH in its prediction equation. Summary and Conclusions To study the residual effect of Mn fertilization, the soils with 0 and 20 ppm of applied Mn (as MnSO4) that had been previously cropped with soybeans were sown to sudangrass. Prior to the sowing, soil samples were taken from each pot and carrier free 54Mn was mixed with the remaining soil. Isotopically exchangeable Mn (L value) was calculated by measuring the 54Mn in the plants at each of 3 harvests. E values were obtained by equilibrating soil samples with 0.1N H3PO4 containing a known amount of saMh. Soil extractable Mn by six extractants was reported in Part II. The results were used in the correlation with Mn uptake by the sudangrass together with the L and E values. Results can be summarized as follows: 1) It was shown that Mn applied as unsoé has a good residual effect, since three successive harvests of sudangrass differed significantly in their Mn uptake between fertilized and unfertilized pots. This happened in six out of eight soils known to be deficient in this nutrient. 2) The L and E values were useful parameters in predicting Mn uptake response due to the residual effect of Mn fertilization. The exception was for E value with the organic soil, in which case the Mn labile pool was greatly overestimated. 3) The listing of soil tests in decreasing order of correlation with the average Mn uptake (3 harvests) by sudangrass was: H P0 > 3 4 steam/NHaoAc > NHAOAc > E value > A value > DTPA > NHAHZPO4 > HCl~ 4) Inclusion of pH and bases ratio in the prediction equation of 64 65 the soil tests improved markedly the R2 values of these equations. The prediction equation of 0.1N H3PO4 accounted for 84.52 of the variation in the Mn uptake data. 5) The critical levels of Mn in soil as determined by L value, E value and 0.1N HBPO4 were 32, 70 and 14 ppm of Mn respectively. 10. 11. 12. 13. 14. 15. Literature Cited Cate, R.B. and L.A. Nelson. 1965. A rapid method for correlation of soil test analysis with plant response data. Inter. Soil Testing Series Tech. Bull. 1. North Carolina State Univ. Agr. Exp. Sta., Raleigh. Ensminger, L.E. and R.W. Pearson. 1957. Uses of macronutrient isotopes in soil fertility research. p. 19-47. Atomic energy and agriculture. Epstein, E. and P.R. Stout. 1951. The micronutrient cation Fe, Mn, Zn and Cu: their uptake by plants from the adsorbed state. Fried, M. 1957. Measurement of plant nutrient supply of soils by radioactive isotopes. p. 1-17. Atomic energy and agriculture. Fried, M. 1964. "E", "L", and "A" values. Proc. 8th. Intern. Congr. Soil Sci., Bucharest, Rumania. Fried, M. and H. Broeshart. 1967. The soil-plant system in relation to inorganic nutrition. Academic Press, New York. Fried, M. and L.A. Dean. 1952. A concept concerning the measurement of available soil nutrient. Soil Sci. 73:263-271. Lamm, C.G. 1960. Some investigations of the chemistry and plant uptake of manganese in soils by use of radioactive . Proc. 7th. Intern. Congr. Soil Sci., Madison, Wis., USA. Larsen, 5 1952. The use of P32 in studies on the uptake of phosphorus by plants. Pl. Soil 4:1-10. Larsen, 8 1967. Soil phosphorus. Adv. Agron. 19:151-208. Lopez, P.L. and E.R. Graham. 1970. Isotopic exchange studies of micronutrients in soils. Soil Sci. 110:24-30. Lopez, P.L. and E.R. Graham. 1972. Labile pool and plant uptake of micronutrients: 1. Determination of labile pool of Mn, Fe, Zn, Co and Cu in deficient soils by isotopic exchange. Soil Sci. 114:295-299. Millikan, C.R. 1951. Radio-autographs of manganese in plants. Austr. J. Bio. Sci. 4:28-41. Probert, M.E. 1972. The dependence of isotopically exchangeable phosphate (L-value) on phosphate uptake. P1.Soil 36:141-148. Randall, G.W., E.E.Schulte and R.B.Corey. 1976. Correlation of plant manganese with extractable soil manganeseand soil factors. Soil Sci. Soc. Am. Proc. 40:282-287. 66 16. 17. 18. 19. 20. 21. 67 Romney, E.M. and S.J. Toth. 1954. Plant and soil studies with radioactive manganese. Soil Sci. 77:107-117. Rumpell, J., A. Kozadiewicz, B. Ellis, G. Lessman and J. Davis. 1967. Field and laboratory studies with manganese fertilization of soybeans and onion. Michigan State Univ. Agr. Exp. Sta. Quaterly Bull. 50:4-11. Russell R.S., J.B. Rickson and S.N. Adams. 1954. Isotopic equilibria between phosphates in soil and their significance in the assessment of fertility by tracer methods. J. Soil Sci. 5:85-105. Russell, R.S., E.W. Russell and P.G. Marais. 1957. Factors affecting the ability of plants to absorb phosphate from soil. I.- The relationship between labile phosphate and adsorption. Toth, S.J. and E.M. Romney. 1954. Manganese studies with some New Jersey soils. Soil Sci. 78:295-303. White, R.P. 1969. Hydroxylamine hydrochloride as a reducing agent for atomic absorption determinations of manganese in dry-ashed plant tissue. Soil Sci. Soc. Am. Proc. 33:478-479. APPENDIX 68 Table II-l. Soybean yield and Mn content as affected by soil type and Mn fertilizer. Yield Mn Content Soil Mn Replication Replication No. added 1 2 3 1 2 3 ---------g/pot ————— --------ppm-------- 1 0 4.90 4.68 4.48 24.3 25.5 25.6 10 4.68 4.92 4.70 31.5 31.6 34.8 20 5.00 4.15 5.28 35.1 29.1 27.2 2 0 5.60 5.00 4.12 26.4 20.9 21.7 10 4.85 4.65 5.05 25.7 36.4 27.3 20 5.00 4.98 7.35 41.6 27.7 40.4 3 0 7.10 5.90 6.45 14.8 12.7 9.83 10 8.80 5.25 5.35 16.8 16.6 17.3 20 8.90 7.50 4.70 19.2 11.7 16.1 4 0 6.35 6.70 6.75 12.3 11.2 11.7 10 6.62 8.00 8.15 12.4 14.0 11.6 20 7.90 6.78 7.42 14.3 14.2 13.5 5 0 0.620 0.751 0.583 11.6 12.7 9.76 10 1.80 1.55 1.25 33.5 27.3 26.8 20 1.22 1.48 1.50 38.4 42.6 37.8 6 0 6.45 4.80 5.35 9.47 11.4 10.6 10 7.65 4.73 7.55 12.1 15.9 13.9 20 5.80 5.28 6.50 25.3 14.3 18.6 7 0 6.40 6.08 6.25 58.0 63.4 72.9 10 7.50 6.25 6.62 57.5 59.2 58.5 20 7.35 7.40 6.15 60.3 63.1 66.9 8 0 7.60 4.70 5.60 42.7 42.8 34.3 10 7.05 6.52 7.45 45.1 43.9 42.4 20 7.00 7.00 7.50 50.0 51.8 39.8 9 0 9.27 8.88 9.90 39.9 40.2 36.6 10 9.75 7.70 7.40 42.8 49.2 51.6 20 10.3 7.72 8.75 42.1 45.6 48.4 10 0 8.38 8.35 8.80 19.9 37.0 27.1 10 10.9 4.70 7.80 29.9 23.8 32.8 20 8.10 6.95 8.68 25.8 31.9 37.9 11 0 7.20 8.40 5.82 8.08 8.30 9.05 10 8.32 7.38 6.80 13.5 11.0 10.3 20 9.60 8.83 7.05 14.3 15.7 14.1 12 0 3.50 3.55 3.70 17.2 23.8 27.2 10 3.85 3.32 3.58 29.8 31.5 28.7 20 4.00 3.70 4.10 36.6 44.8 33.9 69 Table 111-1. Sudangrass yield and Mn content as affected by soil type and Mn fertilizer (lst harvest). Yield Mn Content Soil Mn Replication Replication No. added 1 2 3 1 2 3 ------- 3/pot------ rpm 1 0 2.40 2.60 2.60 23.5 33.2 27.8 20 2.35 3.12 3.02 42.9 41.8 48.2 2 0 4.08 3.88 3.10 39.2 44.0 46.5 20 3.25 3.72 2.82 53.5 54.4 50.6 3 0 4.60 4.50 3.10 35.3 55.1 35.3 20 4.88 4.86 4.60 44.5 33.3 37.7 4 0 2.68 3.75 4.40 10.0 12.7 10.9 20 4.58 2.95 4.82 10.0 12.5 13.2 5 0 1.82 3.68 2.42 13.8 11.4 16.9 20 2.55 4.38 1.90 35.7 35.0 49.8 6 0 4.10 3.22 3.52 27.0 27.5 31.4 20 4.80 3.85 5.05 29.0 40.8 42.2 7 0 2.12 3.00 2.92 92.1 93.7 79.9 20 2.15 2.82 2.70 93.9 100 99.9 8 0 4.78 3.32 2.70 54.0 46.2 55.5 20 3.95 3.90 3.15 49.9 70.9 50.3 9 0 3.68 4.10 2.82 90.6 83.7 87.6 20 4.05 5.22 3.75 85.8 72.0 90.5 10 0 4.05 3.75 3.52 58.2 72.8 49.5 20 3.40 4.05 3.55 65.3 57.2 55.0 11 0 3.88 3.65 4.42 30.5 19.8 11.6 20 4.92 4.85 4.85 11.9 12.6 23.8 12 0 3.57 3.80 3.52 58.2 57.7 53.4 20 4.55 2.80 4.50 80.6 77.3 81.8 70 Table III-2. Sudangrass yield and Mn content as affected by soil type and Mn fertilizer (2nd harvest). Yield Mn Content Soil Mn Replication Replication No. added 1 2 3 1 2 3 -----g/pot---- ———————— ppm 1 0 3.40 3.10 3.35 21.3 21.8 19.0 20 2.80 3.80 4.25 31.4 29.8 26.4 2 0 4.50 4.65 3.40 52.8 51.2 53.6 20 2.85 4.15 4.30 49.6 56.3 52.5 3 0 3.65 3.45 3.85 64.5 53.5 44.9 20 4.10 3.26 4.55 67.6 65.3 61.7 4 0 2.85 6.15 4.00 7.0 8.5 11.2 20 3.68 2.50 3.88 17.7 17.8 12.0 5 0 1.72 2.60 2.10 18.4 20.1 21.9 20 2.28 2.35 2.05 56.9 53.3 49.4 6 0 3.30 3.60 4.10 26.9 31.1 21.8 20 4.00 3.60 4.20 40.6 39.4 39.5 7 0 2.82 3.90 4.10 158 141 108 20 3.05 3.75 3.78 105 106 119 8 0 5.62 5.38 5.49 71.3 65.2 62.7 20 4.18 2.52 4.65 77.8 89.2 55.3 9 0 4.50 4.40 4.75 139 130 96.4 20 3.58 4.68 5.82 111 142 134 10 0 4.60 4.10 4.85 102 95.7 87.9 20 4.65 4.45 5.05 96.9 86.0 96.1 11 0 6.50 6.40 5.22 17.0 17.9 13.3 20 4.80 6.65 7.40 25.7 23.3 15.7 12 0 2.15 2.32 2.30 94.6 89.6 104 20 3.55 2.15 2.20 121 100 124 71 Table III-3. Sudangrass yield and Mn content as affected by soil type and Mn fertilizer (3rd harvest). Yield Mn Content Soil Mn Replication Replication No. added 1 2 3 1 2 3 —-------g/pot ------ ---------ppm --------- l 0 6.60 4.60 7.28 18.0 18.7 12.1 20 5.90 6.72 8.70 29.3 25.3 18.5 2 0 7.30 7.00 7.82 45.8 45.9 39.9 20 5.92 4.85 7.25 32.6 45.0 45.7 3 0 4.92 6.58 7.95 60.2 46.7 33.2 20 6.25 9.28 8.42 76.2 41.8 43.3 4 0 5.00 5.40 9.25 9.0 9.5 6.2 20 6.80 3.75 10.9 7.7 12.8 7.3 5 0 3.85 4.50 6.08 12.5 14.5 13.0 20 4.58 6.70 5.45 35.6 33.8 31.7 6 0 5.45 6.20 7.98 28.5 30.7 24.3 20 6.90 7.25 6.70 36 0 35.6 34.1 7 0 7.12 7.00 7.55 108 114 99.6 20 5.25 7.42 8.20 134 113 93.3 8 0 7.98 9.52 9.38 59.9 78.8 74.1 20 6.12 8.50 9.35 93.8 84.3 65.2 9 0 12.3 9.82 9.38 119 125 102 20 8.30 10.8 10.2 131 109 122 10 0 6.00 8.20 8.18 118 91.3 86.4 20 8.45 8.70 9.82 96.3 89.7 75.0 11 0 10.3 9.65 9.20 26.8 14.8 17.8 20 7.58 8.25 7.35 20.7 21.8 16.6 12 0 4.02 5.20 4.72 68.9 62.0 69.4 20 5.38 4.40 5.78 98.1 95.3 92.2 72 mmNmoN mom «.0H mmmon wNo m.mN omoQNm oHoH H.mH o m mmnmoN mac w.aH mamon NNo N.oN oaomNm NHN o.Huom ucouaoo muH>Huom uaouaoo kuH>Huom uaouaoo om ASSN ax mm -oHvsH as mm .533 a: 32:. .oz .02 umo>umm ohm umo>umm ooN umo>umm umH oz .aom HHom .umm>um£ sumo um mmHaamm ammuwsmvsm mo mHmmHmsm qzqm pom :me mo muHsmom .eIHHH mHnoH 73 OONOON ONH OOH OOONNO OOH OOH OOOONO ONH O.nm ON O OHOOON OOH ONH NOONNO OOH OOH ONNNNO OHH 0.0N ON N OOOOON OHH OOH HOOONO OOH HHH OOOONO ONH 0.00 ON H mOOOON OHH NNH OOONNO NNH NOH ONOONO OOH 0.00 O O OONOON HOH OOH OOONNO OOH NOH NONONO OOH O.NO O N OONOON OOH OOH NOONNO ONH OOH OOOONO NOH O.Nm O H OH mOOOON 0.0m 0.0m HOOONO O.NO m.Om OOOONO 0.00 H.NO ON O OHOOON 0.00 OOH OOOONO 0.0N 0.0m ONOONO O.NO O.nO ON N ONOOON N.NN OOH OONONO 0.0N 0.0N ONNNNO 0.00 H.NO ON H OOOOON O.HN OOH OOOONO m.OO 0.0N ONOONO 0.00 0.00 O O OONOON 0.00 N.Om HOOONO 0.00 0.00 NONONO 0.00 0.00 O N OHOOON O.Nm 0.00 OOOONO N.OO m.ON OOOONO 0.00 m.NO O H OH OHOOON OOH OOH OOOONO OOH HHH ONOONO NOH OHH ON O ONOOON OHH ONH OONONO HOH 0.00 OOOONO ONH OHH ON N mOOOON NOH OOH OOONNO NOH NOH NONONO nOH H.OO ON H OOOOON ONH OHH NOONNO NOH m.OO NONONO OON m.OO O O ONOOON ONH OOH OOOONO OON OOH OOOONO OOH HHH O N OOOOON HOH OOH OOOONO ONN ONH ONNNNO ONN 0.00 O H NH NHOOON OOH N.OO OOOONO NHO 0.00 ONOONO ONO 0.00 ON O OONoON OON m.mO NOONNO ONN O0.00 OOOONO OOO H.HO ON N OHOOON NNN 0.00 OOOONO OHO N.NO OOOONO NON 0.00 ON H OONOON mom H.NO NOONNO OON O.HN ONOONO O.HO O O ONOOON OOO H.ON OONONO NOO H.NO ONOONO NOO O.NN O N OHOOON NNO 0.00 NOONNO Omm 0.0N ONNNNO ONN 0.0N O H HH OONOON OOH 0.00 mmmONO ONN O.mn ONOONO HOO H.NO ON O OOOOON OOH 0.00 HOOONO HON N.OO OOOONO HON 0.00 ON N OHOOON OHN 0.00 OOOONO OOO N.OO OOOONO ONO H.NO ON H Eco Emu O: Boo Boo O: Emu ago On OuH>Huum acoucoo muH>Huom acouaoo OuH>Huuo uoouaoo um uoHumm as mm uonmu e: um .OHuum a: woven .02 .oz umo>umm OuO umm>umm OaN umo>uom umH oz. .aom HHom umaaHuaoo .OuHHH oHpaH 74 ONOOON OOO 0.00 OOOONO ONO OOH OOOONO OOO 0.00 ON O OONOON OOO OOH HOOONO NOO 0.00 OOOONO OOO O.HN ON N OONOON ONO OHH OOOONO NOO NHH OOOONO OOO O.NO ON H OONOON OHO 0.00 OOOONO HOOH OOH OOOONO NOOH O.NO O O OHOOON OHO 0.00 OOOONO OOO H.OO OOOONO OOO 0.00 O N OHOOON OOOH O.NN OOOONO NOOH H.OO OOOONO ONON H.OO O H NH OOOOON 0.00 0.0N NOONNO 0.00 O.NH OOOONO 0.00 O.NN ON O ONOOON H.NO 0.0N OOONNO N.HN 0.0N ON N OOOOON 0.00 N.ON OOONNO 0.00 H.ON OOOONO 0.00 0.0H ON H OONOON N.OO H.ON HOOONO O.HO O.NH OOOOOO OHH N.HH O O OOOOON N.OO O.NH OOOONO 0.00 O.HN NONONO 0.00 O.NH O N OONOON H.OO 0.00 OOOONO 0.00 0.0H ONOONO OOH 0.00 O H OH NHOOON NOH 0.00 HOOONO OON N.OO NONONO ONN 0.00 ON O OOOOON HOH HHH HOOONO ONH N.ON NONONO OOH O.NO ON N OONOON NOH NHH NOONNO OON HHH ONOONO OOH O.HN ON H ONOOON HNN OOH OOONNO OOO 0.00 NONONO OON 0.00 O O OOOOON OON NHH HOOONO OOO 0.00 OOOONO NON N.ON O N OOOOON ONO ONH OONONO HOO OOH OOOONO ONN H.OO O H OH Baa ago On Baa Baa O: Baa ago On OuH>Huom ucmuaoo OuH>Huom uamuaoo OuH>Huom acouaoo mm IOHOOO a: «O IOHOQO oz mm IOHOMO oz Ouvcm .oz .02 umm>umm OuO umo>umm OON umo>umm HOH oz .Oom HHOO OmacHucoo .OIHHH mHnma MIC E «HIT/1711117111»:{1131191}ij RIES mgr/Nix)”