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SOIL SCIENCE degree in Dwagflnflm Major professor Date/QMMB 3 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. ‘ TO AVOID FINES return on or before date due. Ff DATE DUE , DATE DUE DATE DUE bLPI4I9fl . i KN MSU Is An Affirmative Action/Equal Opportunfly Institution cw WG-9- 1 THE EFFECT OF CROP ROTATION SEQUENCE ON ORGANIC CARBON AND NITROGEN LEVELS OF A CHARITY CLAY BY Richard Cleveland Zielke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of CrOp and Soil Science 1983 ABSTRACT THE EFFECT OF CROP ROTATION SEQUENCE ON ORGANIC CARBON AND NITROGEN LEVELS OF A CHARITY CLAY BY Richard Cleveland Zielke The objective of this work was to evaluate changes in soil organic C and N and their effect on N mineralization characteristics of six crap rotation sequences. The six rotation sequences consisted of combinations of corn (Zea mays L.), oats (Avena sativa L.), alfalfa (MedicaLo sativa L.), sugarbeets (Beta vulgaris L.) and navy beans (Phaseolus vulgaris L.). After 9 years of crOpping there were significant differences between sequences in organic C and N losses, mineralizable N and N uptake in a greenhouse study. These differences were attributed to differences in organic matter return rates. Losses ranged from 0.180 2 C (4030 kg C/ha) and 0.019 2 N (417 kg N/ha) for the navy bean-sugarbeet rotation, to 0.053 2 C (1200 kg C/ha) and 0.0066 2 N (148 kg N/ha) for the corn-corn-corn-sugarbeet rotation. Nitrogen mineralization estimates were linearly correlated with soil C and N levels but were not linearly related to sugarbeet yield parameters measured in the field or uptake of N measured in the greenhouse. DEDICATION To Betty ii ACKNOWLEDGMENTS ime author wishes to express sincere thanks to Dr. D. R. Christenson for his help in preparing this manuscript and for serving as the guidance committee chairman. Appreciation is extended to Drs. B. G. Ellis and D. KrauskOpf for their editorialauuommmu Ho>ofl do.o .mo.o I mzqao um woumaouuoo haucmo«MHanmse.a lessee Housman as. mast.3m amassed “wastrel Aoomfiv Sufism Anemav case Acumdv wfiucoxowz vow nmmmnumamwuwvwx Aooofiv mono use messes Aeemav umemo AONGHV mmMSBUHQM UQQ meme Anomav auuowauoo «sfimm.o «somc.o modem vumnouo «sew.o «ecm.o A.A uoaoofin Eonwuomv aonmuom «mom.o «soom.o wanna: annmuomlouwsm «smw.o amm.o mmmuwmmm «amom.o its: A.A wumumaoaw mwamuomav ammum uuwsouo «kmm.o eemm.o A.A maneuom‘aowaoqv ammuwo%m ««w~m.o ««~om.o A.n_mwmm.mmmV anoo Auv powwofimwwoo cowumaouuoo ooamuommm uanouwwc< uwnouo< Ahaoo mmouv mofioumw ucoam coaumnsuafi Nuanouowdm mam Hoanoumm .mmaonaoouw can ca 2 mo mxuums saws avenues mafiumoaoo mofivaum aw veafimuno muaoaowmwooo nowuuaouuoo mamawm .H wanna 14 log(Nmp-Nt) = log N - kt/2.303 (8) mp where Nt - cumulative amount of N mineralized at time t, and k = the mineralization rate constant. The first estimates of Nmp were obtained using the equation: l/Nt - 1/Nmp + b/t (9) where b - slape. Therefore Nmp could be obtained by finding the reciprocal of the intercept of the regression line obtained from a plot of l/Nt vs 1/t for each individual soil. Next, (Nmp'Nt) was calculated using the value of Nmp obtained in equation (9) and the N: observed. Plotting (Nmp’Nt) vs t on semilog paper produced a concave up curve if Nmp was smaller than, concave down curve if Nmp was larger than, or a straight line if Nmp was equal to the Nmp denoting best linear fit. The Nmp denoting best linear fit was finally obtained using an iterative process involving successive evaluation of (Nmp’Nt) vs t. The mineralization rate constant (k) was evaluated from equation (8) whereby the slope of a plot of log(Nmp-Nt) vs t is equal to k/2.303, and hence k - 2.303 x slope. Stanford concluded the pool of potentially mineralizable N was similar based on the observation that the rate constants observed for many different soils were statistically poolable. The average rate constant obtained for 39 diverse soils was 0.054 weeks-1. As can be seen, the mathematical method for estimating Nmp is tedious. The chemical determinations and extractions are equally laborious, however recent modifications by MacKay and Carefoot (1981) allow for incubation of the sample in a 0.2 um Falcon disposable filter unit which greatly simplifies the extraction and moisture equilibration processes. 15 Stanford et al. (1973b) conducted greenhouse experiments on soil N availability using labeled plant materials and concluded that Nmp may have use in predicting N mineralized under varying field and greenhouse conditions. These conclusions were also based on work that established moisture and temperature coefficients for Optimum N mineralization (Stanford et al., 1973a; Stanford and Epstein, 1974). Stanford et a1. (1974) attempted to develOp a short term (8 week) incubation method using the same procedure described above. Thus Nmp could be calculated using the following regression equation and the pooled rate constant (k = 0.054 wk’l @ 35 c): Nm . Nt/(l-lo-kt/2'3O3) (10) P Since their develOpment, the short and long term methods have received a considerable amount Of attention, primarily for purposes of establishing their validity under field and greenhouse conditions. Verstraete et a1. (1976) investigated the use of Nmp in predicting N mineralized as a function of crop, organic amendment, and liming variables. His conclusion was that Nmp was not useful in detecting differences in N mineralized due to the above factors but also added ”lack of sensitivity and/or accuracy to differentiate in a statistically significant way the impact Of the experimental variants examined is not surprising in view Of the relatively small differences in soil characteristics between the various variants”. Smith et al.(1977) reported that the correlation Of Nmp with estimates Of N mineralized under modified field conditions using a buried polyethylene bag technique was quite high (r - 0.92). Westerman and Crothers (1980) also used the buried polyethylene bag technique to determine N mineralized under field conditions. They reported that l6 correlation between predicted N uptake and measured N uptake by corn was highly significant (r -= 0.98) and further concluded that the buried bag technique was as useful as Nmp in predicting N mineralized in a single growing season or several growing seasons if crOpping practices do not change significantly. Hsieh et al. (1981) used the method Of Stanford (1972) to estimate the possible N pollution potential associated with sewage sludge decomposition in soil. Marion et al. (1981) used the Nmp method to predict N mineralized by Chaparall soils. He also reported that the method has potential in predicting N mineralization in terrestrial ecosystems. Keeney (1981) discussed the problems associated with estimating Nmp in forest ecosystems. Recently, the assumption that N mineralization follows first order kinetics has come under criticism. One such criticism is that a significant amount of soluble organic N is removed during leaching and thus is ignored in estimating Nmp if only mineral N is determined (Smith et al., 1980; Legg et al., 1971; Broadbent and Nakashima, 1971). Other criticisms exist ( Talpaz et al., 1981; Molina et al., 1980; Stanford et al., 1980), but they are difficult to verify because of the complex and Obscure mathematical principles involved. Probably the most important criticism is that most researchers air dry the soil before incubation, a process which tends to retard the soil biological activity for some time. It would seem that these estimates would be more meaningful if the soil used were maintained more closely to ”steady state” field conditions. The Obvious problem with this concept however is that heterogeneity Of subsamples (particularly in the fine textured soils) increases drastically with field moist soils. Still the method of Stanford and Smith (1972) has become quite popular primarily because of 17 the potential for its use in kinetic and quantitative (as Opposed to relative) measurement of N availability. Correlation studies of Nmp with uptake of N in greenhouse and field experiments are still neccesary to better establish the validity of Nmp. There are an enormous number Of prOposed chemical indices of soil N availability. These methods have been reviewed by Bremner (1965a), Jenkinson (1968), Danke and Vasey (1973), Cambell (1978) and Stanford (1982). The disadvantage of chemical methods is that they can in no way simulate the microbial considerations that are involved in N mineralization. Their use lies primarily in demonstrating a high degree of correlation with previously established methods or with uptake of N in the field or greenhouse. One method which meets these criteria is the autoclavable NH4+ method of Stanford and Demar (1970). Thus the following dicussion will be limited to the development of the autoclavable NH4+ method as an index of N availability. Stanford and Demar (1969) compared the autoclavable method with the anaerobic method of Waring and Bremner (1964) and Obtained a correlation coefficient r - 0.94. Smith and Stanford (1971) reported that the correlation between NHz,+ released upon autoclaving and N mineralized by the aerobic and anaerobic methods previously discussed were quite different (r - 0.70 and 0.92 respectively). Smith et al. (1977) reported significant correlation Of field estimates of Nmp with autoclavable NH4+ (r - 0.86). Stanford and Smith (1976) were the first to attempt tO estimate N from a chemical index Of N availability mp (autoclavable NH4+) for a large number of U.S. surface soils. Excepting a few calcareous western soils, Nmp could be estimated fairly accurately. The following equation is the pooled regression equation 18 for 275 diverse soils: Nmp - 4.1 N1 + 6.6 (11) where Ni - NH4T-N released upon autoclaving at 121 C for 16 hrs. The correlation between Nmp and Ni was high (r - 0.92) indicating that the method does indeed have potential for use in predicting Nmp. The reason for the lack Of fit for the western calcareous soils is unknown. More recently, Fox and Piekielek (1978) reported significant correlation.coefficients (r - 0.92) resulting between autoclavable NHz,+ and soil N supplying capacity ([total plant N - (.75 x amount of starter N added)]) under field conditions. The autoclavable NH4'+ method has been shown to be well correlated with all three biological methods previously mentioned. The significance of this correlation is that N1 is easier and cheaper to determine therefore lending itself to adoption by soil testing laboratories for routine use. MATERIALS AND METHODS General Overview The Objectives Of these investigations have been mentioned. In the spring of 1981 a soil sample consisting Of 20 probes per plot was collected from a long term rotation experiment and chemical analyses for organic C and N were conducted. These values were compared to values Obtained from corresponding 1972 soil samples. The soils at the field site are very uniform and it was assumed that any changes in organic C and N level resulted from differences in factors such as the amount of crop residue produced and its associated decomposability, therefore no ‘biomass estimates were Obtained. If the foregoing assumption is valid, it is reasonable to expect that differences in N availability will eventually result. Therefore N availability estimates were Obtained and correlated with sugarbeet parameters. Sugarbeets were chosen as an ”indicator crOp” because of their sensitivy to excess mineral N. Results Obtained the first year suggested that some significant differences existed and therefore warranted a more in-depth study Of the N status of these soils as affected by rotation sequence. In the fall of 1981 a soil sample consisting of 80 probes per plot was collected from plots that would be planted in sugarbeets the following spring. Chemical determinations were repeated and compared to values from corresponding plots sampled in 1972. Since sugarbeet parameters would not be available the second year, a detailed 19 20 investigation of the relationship between uptake of N in the greenhouse and estimates Of potentially available soil N (including Nmp) was undertaken. The following sections describe specific procedures that were used in these investigations. Field Experiment A field study was conducted at the Saginaw Valley Bean and Beet Research Farm in 1981 to determine the magnitude and direction of change of organic C and N levels Of the soil due to rotation sequences which differ in crOp residue return rates and to determine if these changes had a significant effect on yield and quality Of sugarbeets. The study was composed of rotation sequences which were selected from a larger, long term experiment that was initiated in 1972, and was arranged as a randomized complete block with six treatments and four replications. Each experimental unit measured 20.13 by 5.69 m, comprising a total area of 1.15 x 10'”2 ha. Sequences selected include combinations of corn (_Z_§_a_ mays L.), oats (Avena sativa L.), alfalfa (Medicago sativa L.), sugarbeets (Beta vulgris L.), and navy beans (Phaseolus vulgaris L.). The six treatments were as follows; (1) corn-sugarbeets (C-B); (2) navy beans-sugarbeets (Be-B); (3) corn-corn-corn-sugarbeets (C-C-C-B); (4) corn-corn-navy beans-sugarbeets (C-C-Be-B); (5) oats-navy beans-sugarbeets (O-Be-B); and (6) oats-alfalfa-navy beans-sugarbeets (O-A-Be-B). Tillage practices remained constant since 1972, and consisted of fall plowing and harrowing before planting in the spring using a spring tooth-spike tooth harrow combination. Fertilizer applied in a band below and to the side Of the seed in 1981 consisted Of 336 kg 18-46-0 21 plus 12 B and 3% Mn/ha. The navy bean-sugarbeet sequence was sidedressed with an additional 28 kg N/ha in June. Nitrogen fertilizer applied to plots sampled in the spring of 1981 since the initiation of the experiment can be found in Table 2. Potassium fertilization was unnecessary since soil test K levels were sufficient (500 kg exchangeable K/ha). Sugarbeets (variety US H20) were planted at a 71 cm row spacing on April 22 and were thinned to 20 cm between plants approximately five weeks after planting. Weeds were controlled by a preemergence application of 6.72 kg trichloro-acetic acid (TCA) and 4.48 kg 5-amino-4-chloro-2-pheny1-3(2H)-pyridazmone (Pyrimin)/ha. On October 24, the sugarbeets were tOpped with a beater/topper and mechanically harvested. Yields were calculated by weighing the beets harvested from two 20.13 m rows. Ten average sized sugarbeets were selected from each plot, mechanically sliced, the pulp hand squeezed and the resulting juice was sent to Michigan Sugar Company where determinations Of clear juice purity, recoverable sucrose and alpha-amino N were conducted according to the methods described by Dexter et al. (1967), Caruthers and Oldfield (1961), and Moore and Stein (1954), respectively. Sample Collection and Preparation All soils used in these investigations were obtained from the Saginaw Valley Bean and Beet Research Farm located in Swan Creek Township, Saginaw County, Michigan. There were three sampling dates; the summer Of 1972 when the larger study was initiated; the spring of 1981 from plots that were planted in sugarbeets in 1981 (designated Sp 1981); and the fall of 1981 from plots that were planted in sugarbeets in 1982 (designated F 1981). All soils were sampled to a depth of 20 cm Table 2. CrOps grown and N applied to crop rotation sequences since 22 19721. C-B Be-B C-C-C-B C-C-Be-B O-Be-B O-A-Be-B Year CrOp N CrOp N Crop, N Crop N Crop N Crop N kg/ha kg/ha kg/ha kg/ha kg/ha kg/ha 1972 C 150 Be 30 C 150 Be 30 B 60 Be 30 1973 B 50 B 84 B 50 B 50 O 56 B 50 1974 C 224 Be 28 C 224 C 224 Be 28 0 56 1975 B 50 B 84 C 168 C 140 B 50 A 0 1976 C 224 Be 28 C 106 Be 28 0 56 Be 28 1977 B 56 B 84 B 56 B 56 Be 28 B 56 1978 C 224 Be 28 C 168 C 168 B 56 O 56 1979 B 56 B 84 C 168 C 168 O 56 A 0 1980 C 224 Be 28 C 168 Be 28 Be 28 Be 28 1981 B 56 B 84 B 56 B 56 B 56 B 56 lEach crop in each sequence is grown each year. The above table corresponds to research plots that were sampled in the spring of 1981. C = Corn, Be - Navy beans, B - Sugarbeets, O - Oats, A - Alfalfa 23 and consisted of 20 probes per plot with the exception of the fall 1981 sampling which consisted of 80 probes per plot. Samples collected in the field were air dried for 72 hours, ground in a hammer mill until the entire sample passed a 20 mesh sieve and stored in airtight containers until chemical analysis and laboratory experiments could be conducted. Soils used for total N and dry combustion C determinations were subsampled and ground in a Spex Mixer/Mill until they passed a 100 mesh sieve. General Chemical andLMineralogical Properties The soil used in this study is tentatively classified as Aeric, Haplaquept, fine, illitic (calcareous) mesic (Charity clay)l, with 152 sand, 39.3% silt and 45.72 clay. Textural analysis was determined by a ‘modified hydrometer method after reducing the soluble salt content with acidified sodium acetate followed by removal of easily oxidizable organic matter with hydrogen peroxide (Day, 1965). Preparation and X-ray diffraction Of an oriented aggregate was conducted according to methods described by Kunze (1965) and Whittig (1965) after each Of three consecutive treatments; glyceration and Mg saturation; saturation with 1 .N KCl followed by air drying; and heating the K saturated sample to 500 C. The clay fraction was found to contain a large amount Of vermiculite and smaller amounts of montmorillonite, chlorite, illite, kaolinite and quartz. The relative abundance Of each of the various minerals (particularly vermiculite), was determined from information on the diff_r_actogram__as well as from differences in estimates of the cation 1 Personal communication, Dr. E.P. Whiteside, Professor Emeritus, Crop and Soil Science Department, Michigan State University. 24 exchange capacity (CEO) Of the purified clay sample Obtained in Ca/Mg and K/NH4 matrices. The CEC of the soil was estimated to be 290 cmmol (p+)/kg soil and was determined by saturating 2 g soil with neutral NH4OAc followed by three washings and centrifugations to remove excess NH4+ in solution. Ammonium on the CEC was determined by alkaline steam distillation into H3B03 and titration with standardized H2804.2 Soil pH was 7.9 and was measured on a 1:1, soil:water suspension using a glass electrode. Greenhouse Experiment A greenhouse experiment using soils samples collected in the fall of 1981 was conducted to investigate the relationship between estimates of potentially available soil N and uptake Of N at three levels Of N fertility, and to determine if future sugarbeet yields would be affected by culturally induced soil N changes. The experimental design consisted Of a 3 x 6 factorial (arranged as a ramdomized complete block) with three levels Of N fertility (0, 37.5 and 75 ppm N), six rotation sequences, and four replications, giving a total Of 72 separate experimental units. Three consecutive crops were grown; oats (var. Garry), corn (var. Pioneer 3780) and oats, respectively. Before each growth period, each block was rerandomized and placed under high intensity discharge lights in the greenhouse. Daytime temperatures ranged from 20 to 29 C over the entire experiment. Night temperatures were constant at 20 C and the photOperiOd was 16 hours per day- Moisture was periodically adjusted to 20 2 (w/w) by gravimetric means. 2 Unpublished mimeo, D.D. Warncke, Crop and Soil Science Department, Michigan State University. 25 Initially, the potting and fertilization procedure was as follows. Twelve hundred fifty g air dried soil (20 mesh) was deposited in tared pots with plastic liners. In order to ensure that N was the only growth limiting nutrient, each pot received applications of 63 and 50 ppm K and P respectively by the addition of an aqueous solution of KH2P04. The moisture content was adjusted and the pots were allowed to stand unplanted for three days. At the end of this period the moist soil was screened through a 4 mesh sieve, carefully mixed and returned to the pot for planting. One week after planting of the first crop, each pot received a predetermined amount of N fertilizer as an aqueous solution Of C8(N03)2 ' 4 H20. NO additional N, P, or K fertilizer was applied in the second or third crOpping periods. When oats were grown, thirty seeds were planted to a depth Of 1 cm and thinned to 25 plants per pot upon emergence. Five corn seeds were planted and thinned to 3 plants per pot upon emergence. At the end Of five weeks the above ground portion was harvested by clipping at the soil surface. The moist soil was then sieved through a 4 mesh screen and 50 g were removed for chemical analysis. All soils sampled in the greenhouse experiment were rapidly air dried, ground in a mortar and pestle to pass a 20 mesh sieve, carefully mixed, extracted in a 10:1 v/w ratio of 2 _N_ KCl and analyzed for N03‘ and NH4+—N by the automated methods discussed in the section on laboratory analyses. Results obtained were reported as ppm N03” and NH4T-N in air dry 8011. All plant samples were dried in an oven at 60 C for 48 hours, after which time dry weights were obtained and the samples were ground to pass a 40 mesh sieve in a Wiley Mill. Total N in the plant tissue 26 was determined by the micro-Kjeldahl method. Laboratory Analyses and Experiments Organic carbon determinations Organic C content of the soil was estimated by two methods; Walkley-Black wet combustion with chromic acid (Walkley and Black, 1934; Walkley, 1935, 1947), and dry combustion using a Leco 70-second C analyzer (Tabatabai and Bremner, 1970). The dry combustion method was used only on the 1981 spring and corresponding 1972 samples. In the Walkley~Black method, 10 ml 1 N_K2Cr207 was added to 1.5 g soil (20 mesh) in a 500 ml Erlenmeyer flask followed by the addition of 20 ml 36 1! H2804. The sample was allowed to digest and cool for 30 minutes. To this mixture, 170 ml distilled water and 3 drops of Ferroin (1,10-phenantroline ferrous sulfate) were added, and the sample was titrated to a maroon endpoint using 0.5 N Fe(NH4)2(SO4)2. Results Of duplicate determinations are reported as Z organic C in air dry soil (2 moisture - 4.60 w/w). Estimates of organic C tw'the dry combustion method were Obtained by weighing 100 mg soil (100 mesh) into a combustion crucible and adding three 1.1nl aliquots Of 5% H2803 to destroy carbonate C (Allison, 1965). The sample was allowed to stand overnight after which time the crucible was dried in an evacuated desiccator containing NaOH pellets. The sample was then analyzed for C by combusting in a Leco 70-second C analyzer. The mechanism for this process is described in detail by Belo (1970) and Tabatabai and Bremner (1970), but basically consists Of two steps: first the sample is combusted in a high temperature, high 27 frequency induction furnace, and secondly, the amount of C02 evolved is detected by a thermocouple which allows the C content of the sample to be read directly from the instrument. Results Obtained for duplicate determinations of each sample are reported as Z organic C in air dry 30110 Total nitrogen determinations Total N content (not including NO3‘) of the soil was determined by the micro-Kjeldahl method described by Brenmer (1965b). For soils, two ml distilled water was added to 400 mg soil (100 mesh) in a 100 ml Kjeldahl flask and allowed to stand for 30 minutes. Each flask received 1.1 g catalyst mixture (100:10:l ratio Of K2804 : CuSOz, : Se, respectively), 3 ml 36 N H2801, and was digested for 3 hours. After cooling, each sample was diluted with 20 ml distilled water. Ammonium liberated by alkaline steam distillation was collected in H3803 and total N was determined by titration with standardized H2804. Results obtained for duplicate determinations of each sample are reported as Z N in air dry soil. For plant samples, 100 mg plant tissue (40 mesh) were digested using 2 ml distilled water, 2 ml 36 N H2804 and 1.1 g catalyst mixture. Results are reported as Z N in oven dry plant tissue. Nitrate and ammonium determinations Nitrate and NH4+-N in the soil were determined by a modified automated colorimetric Cd reduction method and automated alkaline phenate method, reqpectively (Technicon industrial methods 1973a and 1973b). Duplicate 5 g soil samples were extracted with 50 ml 2 N KCl on a reciprocating shaker for 1 hour. The samples were then filtered and 28 analyzed immediately or stored at -4 C until the analysis could be conducted. Simultaneous determination of N03‘ and N02‘-N and NH4+rN was accomplished using a Technicon Autoanalyzer System II. Values Obtained are reported as ppm N03- and NH4+-N in air dry soil (NOZ' was considered to be negligible and therefore was contained in the N03“ fraction). Potentially available soil nitrogen Three methods Of estimating potentially available N were investigated. These involved; (1) aerobic incubation (Bremner, 19650; (2) N released by autoclaving in 0.01lM.CaC12 (Stanford and.Demar, 1969); and (3) N mineralized in short term incubations at 35 C (Stanford, Carter and Smith, 1974). Due to the effect Of long term storage on the mineralization of N by soil, estimates were Obtained only for samples collected in 1981. In the aerobic method, 10 g air dry soil (20 mesh) was mixed with 30 g acid washed quartz sand (30-60 mesh), moistened with 6 ml distilled water and allowed to incubate in 8 ounce bottles at 30 C for 14 days. Water loss was prevented by covering each vessel with polyethylene plastic and aerobic conditions were maintained by puncturing three small pin holes in each top. Mineralized nitrogen (NO3' and Nnghnn was extracted by shaking for 1 hour with 100 ml 2 _N KCl. Mineral N in the filtered extracts was determined by the automated Cd reduction and alkaline phenate methods Previously mentioned. Estimates of mineralizable N were Obtained by subtracting the amount of N03" and NH4+¥N contained in an unincubated 3:1, sand:soil mixture from mineral N contained in the extracts of incubated samples. Values Obtained for triplicate determinations are reported as ppm NO3"and NH4+-N 29 mineralized. Nitrogen released by autoclaving in 0.01 1;! CaC12 was determined in the following manner. Twanty-five ml 0.01 M CaC12 was added to 10 g air dry soil (20 mesh) in a 50 ml plastic centrifuge tube. Each tube was covered with Al foil and placed in an autoclave for 16 hours at 121 C. Each sample was centrifuged and resuspended in two more 25 ml aliquots Of CaClz and the extracts were combined and adjusted to 100 ml in a volumetric flask. Distillable N in the filtrate was determined by micro-Kjeldahl alkaline distillation whereby 10 ml 0.1 _N NaOH was added to 30 ml extract in a 100 ml Kjeldahl flask and NH3-N was distilled into H3B03 and titrated with standardized H2804. Estimates Of N released by autoclaving were obtained by subtracting NH4+-N contained in a non-autoclaved sample. Results Obtained for duplicate determinations Of each sample are reported as ppm NH4T-N released by autoclaving at 121 C for 16 hours. Nitrogen released in short term incubations was determined by a method similar to the aerobic incubation method. Triplicate 20 g soil samples (20 mesh) were mixed with 20 g acid washed quartz sand (30-60 mesh) and placed in Nalgene disposable filter membrane units (model # 120-0020) each containing a 0.20 um Millipore filter. A piece Of filter paper was placed on the surface of each vessel to prevent particle separation during leaching. Samples were leached with 4-10 ml aliquots of 0.01 M CaClz, and the filtrate saved for NO3" and NH4+-N analysis. Twenty-five ml of a minus N nutrient solution (0.002 _M CaSO4 ° 21120; 0.002 M MgSO4; 0.005 M Ca(H2PO4)2 ' H20; and 0.0025 _M_ K2804) was then added to each sample. The samples were placed on a vacumm manifold and allowed to equilibrate under 60 cm suction for 12 hours. The resulting 30 filtrate was combined with the previously collected filtrate and equilibrationiweights were Obtained for each tared vessel to ensure uniform moisture content. Each vessel was covered with polyethylene plastic and placed in the incubation chamber at 35 C. The system remained aerobic because of diffusion Of 02 through the bottom of the filter unit. The leaching and equilibration processes were repeated for each successive incubation period. Successive incubation periods consisted Of 1, 1, 1, 2 and 3 weeks respectively, corresponding to cumulative incubation periods Of 1, 2, 3, 5 and 8 weeks respectively. Nitrate and NH4T-N contained in the filtrate were determined by the automated Cd reduction and alkaline phenate methods previously cited. Values Obtained are reported as ppm N03" and NH4+-N mineralized in the soil. Statistical analyses Statistical methods described by Snedecor (1978) and Steel and Torrie (1980) were used for pertinent statistical analyses. RESULTS AND DISCUSSION Comparison Of Organic Carbon Levels by Two Methods Before a major investigation Of the magnitude and direction of change of organic C and N levels of soils could be conducted it was necessary to decide upon an apprOpriate analytical method. The criteria for selection was reproducibility of results (i.e. duplicate determinations were required to have a relative error of less than 5%). Organic N in soil is readily determined by the micro-Kjeldahl method. However, organic C is more difficult to measure. This problem is compounded in these investigations because of the presence of free carbonates in the soil. Table 3 contains a comparison of organic C levels of the Sp 1981 and corresponding 1972 soil samples Obtained by two methods. Both methods indicate that organic C levels have declined over the cropping period. Furthermore, organic C levels between rotation sequences upon initiation Of the experiment were not significantly different, whereas after nine years of crOpping, significant differences have resulted as measured by both methods. In general, the dry combustion results are slightly lower than the Walkley-Black estimates. This Observation suggests that the correction factor used in calculating X C via the Walkley-Black method may be too large or that pretreatment with dilute H2803 to destroy carbonate-C in the dry combustion method may oxidize organic C as well. The latter explanation is more feasible owing to the 31 32 Table 3. Comparison Of organic carbon levels of soils sampled in the spring of 1981 and their corresponding 1972 levels as measured by two different methods.1 Walkley-Black Dry Combustion Sequencez 1972 1981 72-81 1972 1981 72-81 z c C-B 1.67a3 1.55a 0.128ab 1.46s 1.38ab 0.078s Be-B 1.65a 1.47b 0.175b 1.49. 1.34b 0.143s C-C-C-B 1.68a 1.63c 0.0533 1.56s 1.50c 0.060s C-C-Be-B 1.66a 1.58ac 0.083ab 1.62a 1.55c 0.068a O-Be-B 1.63a 1.46b 0.173b 1.59s 1.46ac 0.128a O-A-Be-B 1.66s 1.48b 0.180b 1.62a 1.50c 0.118a cv 4.962 2.272 46.042 6.612 4.47% 79.672 1Means of 4 replications, duplicate determinations. 2C - Corn, Be - Navy bean, B . Sugarbeet, O . Oat, A = Alfalfa 3Means followed by same letter within a column are not significantly different, alpha - 0.05 (Duncans new multiple range test). 33 large number of samples that had to be rerun in order to Obtain a relative error of less than 52. Conversely, the Walkley-Black method had a very low relative error test failure rate. A closer look at the organic C levels in 1972 as estimated by both methods will show that results Obtained by the dry combustion method are more highly variable than the Walkley-Black estimates. Consequently, the Walkley-Black method was employed in these investigations. The dry combustion method would be extremely useful for determining organic C in acid soils where free carbonates are not present and hence do not result in positive errors or variation due to sample pretreatment. Trends in Culturally Induced Soil Carbon and Nitrogen Changes Gains and losses of organic C and N in soil are considered to be first order processes. The major Objective in this study was to first identify the magnitude and direction of change Of these organic matter constituents. Verification that these changes proceed via first order kinetics using equations derived by Salter and Green (1933), Jenny (1941), Bartholomew and Kirkham (1960), and Russell (1976) is not possible since the data base is only nine years Old and is comprised of two measurements. Thus the discussion Of C and N changes is relative to initial organic C and N levels without regard to quantitative kinetics. Each crop in each sequence is grown every year. Table 2 shows the crops grown and N rates from 1972 to 1981 for the experimental units sampled Sp. 1981. A similar table could be constructed for the plots sampled F 1981. Distinction between date of sampling, size of sample Obtained and experimental unit represented is critical, since these samples appear to represent significantly different pOpulations (their 34 variances are not homogeneous for all soil parameters estimated). Results of these investigations are contained in Table 4 (Sp 1981 sampling) and Table 5 (F 1981 sampling). In both cases, C and N levels Observed are compared to levels Observed for corresponding 1972 samples. Table 4 (Sp 1981) shows that organic C levels in 1972 ranged from 1.63 to 1.67 2 C (36500 and 37400 kg C/ha, respectively). Total N levels ranged from 0.192 to 0.197 Z N (4300 and 4410 kg N/ha, respectively). The lack of significance in both cases verifies the assumption of homogeneity of experimental units upon initiation of the experiment. Organic C and N levels observed in Sp 1981 are significantly different. A useful quantity in assessing the effect Of rotation sequence upon these levels is Obtained by difference (1972 minus 1981 levels). In all cases, C and N levels of Sp 1981 samples have declined after nine years Of cropping. Specific organic C changes range from 0.053 2 for the C-C-C-B rotation to 0.180 Z for the O-A-Be-B rotation, corresponding to losses of 1180 and 4030 kg C/ha, repectively. The change in C for the C-C-C-B rotation was significantly lower than the O-Be-B, Be-B and O-A-Be-B rotations, but not significantly different from the C-C-Be-B and C-B rotations. Organic N losses ranged from 0.0066 2 for the C-C-C-B rotation to 0.0186 2 for the Be-B rotation (148 and 417 kg N/ha, respectively). The change in N for the C-C-C-B rotation was significantly lower than the O-Be-B, Be-B and O-A-Be-B rotations, but again not different from the other rotations containing corn. The change in N for the C-C-Be-B rotation was significantly less than the change for the Be-B rotation, but not different from the O-Be-B rotation. 35 .Aumuu awash oaawuana sun mononanv mo.o I ocean .ucouOMMHo hauomowmwomfim no: one naoaoo w segues uOquH mama onu he voSOHHOm mOOoZm mmaoma< I < .uoo I o .uoonuwwam I m .omon h>mz I on .ouoo I om .moowuunaauOuov oumouaaov .maowuoowaoou 0 mo mucosa Nao.~ Noe.~ Nn~.ms Nae.~ Nn~.s Noo.oe non.~ Noa.e >0 mm~.m aem.m momflo.o unmaa.o m~m~.o pom~.o nwa.~ moo.a mummnOH 2 can u Ofiommuo no mooosvom aOHuwuou mo uoommu may .¢ manna 36 .Aumou Owouu Oamuuaaa son momoosav no.0 I mamas .uoouommwo mauomOfimaowfim uo: one caoaou a canoe: uOquH meow Ono an mascaaom mowozm wmaoma< I < .uoo I o .uoonumwnm I m .omom m>mz I on .nuoo I om moOfiumofiauouOv OuoOHHmsv .ooowumOHHmou a mo memo:H Nma.m Nam.a Nmo.mN NwN.n Neo.n NoN.on Nom.e Nma.s so mmo.m mmN.m amomoo.o mmNN.o mmNe.o ammo.o emm.a aeo.~ mummuero maa.a mmH.a uaomao.o «Noa.o mmm~.o amnN.o mmm.~ mw0._ mummuo mm~.o aeo.a oaemao.o mmNH.o amma.o ammo.o aeo.~ eeN._ mnemuuuo m-.m m-.m aaeNoo.o mmNN.o amma.o mmoo.o moo.~ moo.~ muououo mam.m mos.a omeno.o swea.o mme.o noo~.o mom.a «SN.N muse m~o.m mm~.a momoo.ou mwNH.o anN~.o m085 a~m.~ meao.~ era 2 N o N Heme NNAN ”QINN amen NNAN HwINN Lesa «NAN Necessamm cause z\o ammonuez Hence 0 xumNmnaoNaNms ~.wOquOuO mo memo» m nouns meu wo Hana ecu aw endgame mHHom no mHO>OH 2 was 0 Ofiammuo :O moaosoom ooquuou mo uoomwo any .n nanny 37 In general, where losses of C were high, N losses were high. For Sp 1981 samples, it was Observed that rotations containing corn tended to result in higher levels Of 8011 C and N, while rotations containing combinations of a legume and oats resulted in lower C and N levels. Salter and Green (1933) observed the Opposite although this anomally may be due to the fact that substantial use of N fertilizer did not occur until 1950 and alfalfa is grown only one season. Furthermore, alfalfa has been grown twice since the initiation Of the experiment and apparently the beneficial effects on soil organic C and N are not observable under these management practices in the time interval investigated. For the system studied, it is reasonable to assume that rotations containing 50 Z corn return a larger amount of crop residue than the Be-B and O-Be-B rotations, and therefore result in higher C and N levels. Table 5 (F 1981) shows that organic C and N levels in 1972 were not significantly different between sequences. The range of these values is slightly wider and CV's are slightly larger than those observed in Table 4. Furthermore, organic C and N levels observed in F 1981 were not significantly different, however the differences between the C and N levels in 1972 and F 1981 were significant. Losses of C ranged from 0.055 2 for the O-A-Be-B rotation to 0.200 2 for the Be-B rotation (1230 and 4000 kg C/ha, respectively), while losses Of N ranged from -0.0030 Z for the C-B rotation to 0.0198 2 for the Be-B rotation (-67 and 444 kg N/ha respectively). Changes in C for the C-C-C-B, C-C-Be-B, C-B and O-A-Be-B rotations were all significantly lower than changes for the O-Be-B and Be-B rotations. With respect to N changes, the C-B rotation was significantly lower than the C-C-Be-B, O-Be-B and 38 Be-B rotations, while the N changes for the O-A-Be-B and C-C-C-B rotations were significantly lower than the Be-B rotation. The increase in N Observed for the C-B rotation is inconsistent with results Obtained on Sp 1981 samples, as is the higher level of C and N Of the O-A-Be-B plots, although this Observation for a rotation containing alfalfa is more in line with results Obtained in similar long term cropping experiments. These inconsistencies may be due to the fact that a larger sample (80 probes per plot) was collected in the fall, and that these measurements were made on a different set of experimental units. Regardless, the trend toward higher C and N levels of sequences containing 502 corn is evident in both the spring and fall sampling. The lower values of C and N Obtained for the Be-B and O-Be-B rotations parallel observations made on Sp 1981 samples, again suggesting that these rotations return a smaller amount of crop residue to the soil. The C and N status of the O-A-Be-B and C-B rotations is less definitive since these rotations behaved differently the second year. Also noteworthy is the fact that C/N ratios calculated for Sp 1981 samples and corresponding 1972 samples are lower than those calculated for-1? 1981 and corresponding 1972 samples despite the fact that all the 1972 samples were collected at the same time. The lack of significance of the 1972 C/N ratios Of both sampling dates supports the hypothesis that experimental units were homogenous. The significant difference between the Sp 1981 C/N ratio for the C-C-C-B rotation and the other rotations is not suprising due to the larger differences in its components. Estimates of Potentially Mineralizable Nitrogen Table 6 contains estimates of potentially mineralizable N for both sampling dates. The Sp 1981 autoclavable mineralizable N estimates (AMN) were not significantly different, however they follow a pattern similar to the Bremner mineralizable N estimates (BMN) for both sampling dates. The Sp 1981 BMN estimates were significantly different. The C-C-Be-B rotation provided the largest estimate and was significantly different from the O-A-Be-B, C-B, O-Be-B and Be-B estimates, but not different than the C-C-C-B value. The C-C-C-B, O-A-Be-B, C-B and O-Be-B rotations all behaved similarly, however C-C-C-B and O-A-Be-B estimates were significantly higher than the Be-B rotation. The C-B, O-Be-B and Be-B estimates were not significantly different from each other. The F 1981 ANN estimates indicate that the C-B rotation is significantly lower in potentially mineralizable N than all the other rotations. This is surprising Table 5 indicates that the level of soil N increased over the nine year crOpping period whereas all the other levels decreased. The F 1981 BMN estimates paralleled Sp 1981 estimates, although the N levels and change in N levels did not parallel Sp 1981 observations. The C-C-C-B rotation is significantly higher in potentially mineralizable N than the O-Be-B and Be-B rotations, but not different from the other rotations. Furthermore, the C-C-Be-B rotation is significantly higher than the O-Be-B rotation but not different from the other rotations. The O-A-Be-B, C-B, Be-B and O-Be-B estimates were not significantly different from each other. 39 40 Table 6. The effect of rotation sequence on estimates of potentially mineralizable soil N for both sampling dates.1 Sp 1981 F 1981 Total N (NO3' + NOZT + NHQT) mineralized Sequence2 BMN3 AMN4 BMN AMN _NEQAS NmnHE. PPm C-B 30.5ab7 29.9a 27.1abc 21.3a 2.07a 6.30a Be-B 29.0b 29.1a 26.4bc 25.3b 1.38b 5.14s C-C-C-B 33.4ac 32.3a 30.6a 24.8b 1.82a 5.98a C-C-Be-B 35.3c 31.4a 29.9ab 27.3b 2.08a 6.83a O-Be-B 30.2ab 27.2a 25.9c 26.3b 1.81a 4.96a O-A-Be-B 31.6ab 30.2a 28.8abc 26.6b 1.93a 6.20a CV 6.892 7.002 8.02% 8.79% 10.65% 16.71% 1Means of 4 replications, BMN-triplicate determinations, AMN-duplicate determinations. 2C I Corn, Be I Navy bean, B I Sugarbeet, O = Oat, A I Alfalfa 3BMN I Bremner (1965) aerobic incubation method. 4AMN I Stanford and DeMar (1969) autoclavable method. SNmpA I Stanford et al. (1974) short term incubation method, adjusted. 6NmpU I Stanford et al. (1974) short term incubation method, unadjusted. 7Means followed by the same letter within a column are not significantly different, alpha I 0.05 (Duncans new multiple range test). 41 There are several.observations from Table 6 which are noteworthy when these values are compared to each other and to corresponding C and N levels of the soil for the different sampling dates. For both sampling dates BMN estimates are slightly higher than AMN estimates. This difference is not surprising since N mineralization in the BMN method proceeds via biochemical mechanisms. Secondly, the AMN and BMN estimates for the F 1981 samples are lower than the Sp 1981 estimates. This is thought to result from a higher concentration of mineralizable substrate in the spring samples because crop residues were fall incorporated. Fall samples were Obtained before residue incorporation, Biological activity is higher during the growing season, and hence one would expect available and potentially available N to be depleted at the lend of the season. The mineral N content of the soil is also lower for the fall samples (Table 7). Little statistical inference is implied In! the significant differences between sequences for these quantities since mineral N is a highly variable soil parameter, however the higher value of N03” for the F 1981 C-B sample may be attributed to a higher N fertilizer rate for that sequence (Table 2). If one excludes the F 1981 AMN and Nmp values in Table 6, ranking of the remaining means suggests that the chemical method may be as useful as the biological method, and more importantly, N mineralization as measured by the BMN method is affected by rotation sequence to the same degree on the separate experimental units. This ranking Operation results in the following ordering: C-C-C-B I C-C-Be-B > O-A-Be-B I C-B > Be-B I O-Be-B. The reason F 1981 AMN estimates deviate from this pattern is unexplained, however the AMN estimates of mineralizable N at the end of the growing season for rotations containing legumes appear to 42 Table 7. Mineral N concentrations Observed in the spring and fall soil samples in relation to rotation sequence. Sp 1981 F 1981 Sequence2 NO3' NH4+ N03“ NH4+ ppm N C-B 5.03a3 3.30a 5.43a 2.60a Be-B 5.63a 2.90a 2.91cd 2.63s C-C-C-B 5.22a 3.37a 3.73b 2.76a C-C-Be-B 6.54a 2.76a 3.22bc 2.88s O-Be-B 5.57a 3.05a 2.36d 2.51s CV 11.83% 14.50% 13.802 21.02% 1Means of 4 replications, duplicate determinations. 2c - Corn, Be - Navy bean, B - Sugarbeet, o - Oats, A . Alfalfa 3Means followed by the same letter within a column are not significantly different, alpha I 0.05 (Duncans new multiple range test). 43 be higher than rotations containing only corn and sugarbeets. This anomally may arise from differences in the biochemical as Opposed to chemical stability of the mineralizable substrate, and hence may be a reflection Of the chemical nature of organic matter present. Another characteristic of the data in Table 6 is the large difference between estimates of N mineralized by the short term incubation method of Stanford et. al. (1974), and those obtained by other methods. Owing to the fact that these incubations were carried out for 8 weeks at 35 C, and that the BMN method was carried out for only two weeks at 30 C, the results obtained are indeed unusual. Because of this the N mineralization potential as defined by Stanford and Smith (1972) could not be calculated. During the experiment, it was thought that N mineralized during the short term incubation was being lost through ammonia volatilization or biological denitrification. A monitoring of N lost by these processes for a few randomly selected samples indicated that little or no gaseous evolution occured. Nitrogen immobilization was possibly occurring due to the presence of carbonaceous filter paper which was placed in each vessel to prevent erosion and particle separation during the leaching process. The presence of visible microbial colonies residing on the filter paper in each vessel may be further evidence of immobilization due to an artificially high C/N ratio. Regardless of the cause of immobilization, the inhibitory effect seems to be quantitative since a similar ranking of the means reveals that although the values are much lower, they follow a pattern similar to the other estimates of N availability (again excepting the F 1981 AMN estimates). Previous crOp residue which is easily decomposed may influence the 44 degree of N mineralization, hence adjusted and unadjusted values of Nmp are contained in Table 6. Adjusted means that N mineralized during the first week of incubation was subtracted from the cumulative N mineralized at the end of 9 weeks. Unadjusted values follow the trends observed for adjusted values although there are no significant treatment differences. It is important to note that the adjusted estimate for the Be-B rotation is significantly lower than the other rotations, a fact which is consistent with estimates obtained by the other methods. The effect of crop rotation sequence on estimates of potentially mineralizable N is best illustrated using simple correlation. Tables 8 and 9 contain simple correlation coefficients obtained by correlation between organic C and N, AMN, BMN, and estimates of short term mineralizable N for the separate sampling dates. The Sp 1981 correlations (Table 8) will be considered first. Highly significant linear relationships are shown to exist between Walkley-Black C (WBC) and estimates of mineralizable N by the AMN and BMN methods (r I 0.89 and 0.79, respectively). Similar relationships exist between total N (TN) and AMN and BMN (r I 0.87 and 0.80 respectively). This significant linear relationship suggests that the degree of mineralization is proportional to the amount of mineralizable substrate present in the soil, and substantiates the assumption that N mineralization (under controlled conditions) proceeds via first order kinetics. Further evidence exists in the significant relationships observed between the change in C and N (DZC and DZN, respectively) and AMN and BMN (Table 8). The correlation coefficients in this case are negative and can be interpreted to mean that large losses of organic C and N will result in smaller estimates of mineralizable N. One must be .haoouuuoaou Ho>o~ No.0 .no.o I undue uo voumdouuoo hauoaouwuom«m«¢.e ‘45 oo.a ma.o an.o an.o a~.o. na.o aa.o ma.o o_.cu ma.o nN.o ao.o Ammav as sea aqua. .aoaoa « e s as oo.N NN.o NN.o no.o. aN.o aa.o nN.o Nn.oa m~.on oN.oI Nn.on Namuo aoa aoa aqua. .aoaam I «I «I oo.~ aa.o ao.o oa.o N~.o o~.c Nn.on Na.ou _n.on an.ou NzNav z N .aNua {i ii 8 i : oo.N nc.o a~.o «N.o an.o an.or na.on on.on oa.on NONaO o N «name as s e «I oo.N na.ou an.on No.ou ao.o a~.o cN.o -.c Az<uouno auOuOEONoo vane» uoonuowoo mo kahuna oowudouuou camawm .m manna 46 .hHO>HuOOmON HO>OH No.c .mo.o I sends us moumaouuoo haucmoamwowwm . «a « oo.N. ma.o , aN.o.. aa.a Na.or .a~.o, om.o ma.cu NN.o . No.o Nev aaaaafieaaa aaz fi * oo.a a~.ou mo.o am.ou a~.o- mo.o aa.ou am.o- mm.or Nev eaaaafiea aaz oo.a Na.o NN.o aa.o m~.o No.0 wa.o ma.o Amanda «Nana: aaaaaaaaau k i a. oo.a aa.ou m~.o No.o N_.ou N~.o aa.o Amozpv m aoaa .2 No «Nana: i oo.a ao.o mo.o am.o ma.o Na.o a~uzav N aoaa .z no manna: oo.N N~.o mo.ou Na.o Na.o Naozav N aoaa .2 No manna: I « oo.~ mo.o o~.o m~.o Azzmv z .eNa awesome co; afio :6 2:3 2 ozgeaooufi. «a «a oo.~ mm.o Azav z N Hence «I ao.N Aumzv o N sumamnaaaxaaz a < mane maze. Noza #02:}! 2:: zz< 29 on: . aaaaaaama .ooaoamm Haom uwmq Add“ man no moauuoqouq Hwom vouooaom cues omsonoOONw Ono ow oo>uomno mm HO>OH z ouou men no 2 mo Oxmuo: mo kahuna coauoaouuoo mamaam .a wanna 47 careful with this interpretation since the magnitude of these correlation coefficients are not as large as those previously discussed. A significant correlation between the AMN and BMN methods (r = 0.61) was also obtained. Table 9 contains similar correlation coefficients for the F 1981 sampling, however DZC and DZN coefficients are not included since they were not significant. Here, AMN is significantly correlated with WBC and TN (r I 0.75 and 0.79, respectively) whereas BMN is not significantly correlated (r I 0.23 and 0.26, respectively). As expected from the previous Observation, the relationship between BMN and AMN is poor (r I 0.03). Similarly, the adjusted and unadjusted Nmp estimates are poorly correlated with WBC, TN and BMN. The negative correlation between the adjusted estimates and AMN although small, is unusual. In conclusion, the relative magnitude and uniformity of results Obtained for the Sp 1981 estimates of mineralizable N suggest that it is best to collect soil samples for this purpose in the spring instead of the fall. This is supported by a similar observation for organic C and N levels of the soil. Furthermore, a first approximation of N mineralization appears to follow first order kinetics. Therefore a rotation which returns a larger amount of crop residue can supply a larger amount of mineralizable N to a growing crop, and is consistent with the ”feedback effect" (increasing yields increase crOp residue production and soil C and N levels) described by Russell (1976) and results Obtained by Larson et a1. (1972). For the system studied, rotations containing 50 Z corn have the potential to mineralize more N than the Be-B and O-Be-B rotations. The C-B and O-A-Be-B rotations are intermediate in their N supplying capacity. This pattern is similar to 48 that observed for changes in organic C and N levels of the soil. Effect of Crop Rotation Sequence on Sugarbeet Yield Parameters The relationship between culturally induced soil N changes and estimates of mineralizable N has been established in this and other similar studies. In general, N mineralization proceeds via first order kinetics and the rate of mineralization is therefore proportional to the amount of mineralizable substrate. Similarly, the detrimental effects of excess mineral N on the yield and quality of sugarbeets are well documented. Table 10 contains the results Of a field study which was conducted in 1981 to determine if sugarbeet yield parameters were significantly affected by culturally induced soil N changes. With respect to Z sucrose (Z S) and recoverable sugar per metric ton of beets (RST), only the C-C-Be-B rotation produced significant differences. Yield, clear juice purity (CJP), recoverable sugar per ha (RSH) and alpha amino N (AAN) were not significantly different. Table 8 contains simple correlation coefficients obtained from correlations between sugarbeet yield parameters and selected soil properties. Most importantly, there is no significant correlation between estimates of mineralizable N and yield parameters. There are some significant linear relationships between the yield parameters themselves. For example, yield is highly related to RSH (r I 0.93). Percent sucrose in the beet juice was positively correlated with CJP, RST, and RSH (r I 0.67, 0.99, and 0.46, respectively), and negatively correlated with AAN (r I -0.55). Clear juice purity was negatively correlated with AAN (r I -0.83) and positively correlated with RST and RSH (r I 0.79 and 0.43, respectively). Alpha amino N was negatively 49 Table 10. The effect of crOp rotation sequence on sugarbeet yield, Z sucrose (S), clear juice purity (CJP), recoverable sugar, and alpha-amino N (AAN) concentrations in sugarbeet juice obtained from sugarbeets produced on soils sampled in the spring of 1981.1 Sequence‘ Yield S CJP Recoverable S AAN Mg/ha kg/Mg Mg/ha meq/lOOg C-B 61.58a3 16.7a 96.2a 145.6a 10.28a 7.68s Be-B 60.91a 16.1a 95.7a 138.6a 9.77a 9.93a C-C-Be-B 57.57a 15.2b 95.0a 129.0b 8.76s 11.3a O-Be-B 59.31a 16.3a 95.7a 141.0a 9.67a 8.43a CV 8.62Z 2.49Z 0.62Z 3.20Z _ 9.68Z 22.142 1Means of 4 replications, duplicate determinations. 2C I Corn, Be I Navy bean, B I Sugarbeet, O I Oat, A I Alfalfa 3Means followed by the same letter within a column are not significantly different, alpha I 0.05 (Duncans new multiple range test). 50 correlated with RST (r I -0.48) while the relationship between RST and RSH was positively correlated (r I 0.48). These relationships are in agreement with the findings Of Hills and Ulrich (197l),IfiJJs et al. (1978), Carter and Traveler (1981) and numerous others. One must conclude that culturally induced soil N changes are not substantial enough to significantly influence sugarbeet yield parameters in the field. The significant differences arising in the C-C-Be-B rotation are not likely due to differences in mineralizable N since correlation between yield parameters and mineralizable N indexes were not significant. It is important to note that CJP which is a moderate indicator of sucrose recoverability, is negatively correlated with WBC and TN (r I -0.42 and -O.37, respectively). Although the first correlation is small and the latter is not significant, it establishes a trend and provides evidence indicating that in time sugar yields may decline. This trend will be further considered in the next section. Effect of Crop Rotation Sequence on Uptake of Nitrogen in the Greenhouse Results of the field study clearly established that sugarbeet yield parameters were little affected by culturally induced soil N changes, yet some significant differences in potentially mineralizable N appear to exist. A greenhouse experiment was conducted to determine if uptake of N was linearly related to the N availability indices. Three consecutive crOps were produced (oats, corn and oats, respectively), and yield, Z N in tOps, uptake and mineral N remaining in soil after cropping were determined. Results obtained are reported in Table 11. A complete statistical interpretation of Table 11 is not presented because uptake is the best indictor of N availability due to the inherent 51 uwaouu< I < .uuo I o .uoonuawam I n .coon h>uz I on .nuoo I UN .moouuooasNOOOO oueoaaanv .ooowuoounaou noon mo manure o~.~ m.mn m.mw and @.NN n.¢< c.no me.~ n~.m ~N.¢ nN~.~ n~e.~ oon.~ o.n~ w~.o no.~ ~.om o.no No.» n.- m.mo no.~ ~n.~ w~.e cow.o omn.~ won.~ m.hn o~.o -.N ~m.~ ¢.~¢ ~n.m oo.m m.n~ Nu." no.~ «N.~ «on.o waw.o nmo.~ o nromr<|o mm.~ n.cm ~.mm emu m.o~ «.mn o.no o~.~ ou.n nm.c cwo.~ m-.~ mme.~ o.nn nm.o nu.u o.~n 0.9m do.” m.w~ ~.eo e~.~ n~.~ o~.e mdn.o ocn.~ nem.~ n.5m ow.o va.— an.N n.5n «5.x cw.“ n.o~ m~.~ No.0 oo.~ cc~.o new.o muo.~ o mromlo om.~ c.0m —.~m and ~.on N.nn m.o~ ~¢.~ m~.n w~.e nnu.q no~.~ mac." o.nm m~.c m~.« n.~n n.wm so.a c.o~ «.mo oc.~ en.~ mm.c «no.0 mnn.~ mmn.~ n.nm o~.c Ne.N ~e.~ w.~e eh.w NN.n w.n~. _~.~ no.0 o~.~ nah.o nmu.o om~.~ o muonrolo wn.~ c.am ~.~o sna o.—m c.0n ~.wo -.N n~.n en.¢ ~n~.~ Qua.“ own.~ o.nn -.o «N.N «.mN s.ma ¢.o~ n.o~ o.o~ Nu.“ ~e.~ am.q mcm.o mmm.~ muo.~ n.5n m~.c on.~ mm.~ ~.m¢ so.a co.n n.n~ h~.~ do.— ~m.~ mam.o wm~.c mmo.~ o aloloro ¢~.~ c.wN N.oc “nu ~.¢~ n.ne e.ao o~.~ on.n c~.e m-.~ ww~.~ nno.~ c.ms c~.o ~o.~ N.~N w.nm -.o a.o~ ~.mo mo.— _~.~ n~.¢ mm~.o nnn.~ moo.~ n.~n nu.o ao.~ mn.~ n.wn oo.o cu.~ ~.NN om.~ cm.c n~.~ o~s.o mow.o neo.u 0 atom nn.~ ~.~c ~.oa m- o.nn ~.a~ u.OH z «mucosoom «woo ad 2 House“: 2 «O sauna: umou ea 2 case» «.0mnozoooum sea a“ ovowuom unannouo o>uuaooocoo sauna Hanna Haou "won «How can aw woauacaou z Houoofia one assume 2 .OOONcoo z .vaoqh co HO>OH muunwuuou 2 one ooooovoo oouuouou mono mo uoomuo one .uu canoe 52 variability in yield, Z N and mineral N estimates obtained for the different crOpping periods, however some general observations are noteworthy. During the first cropping period, N deficiency symptoms (stunting and yellowing of the Older leaves) appeared in all pots at the 0 N level. Comparison of yield and Z N in tops indicates no differential response to applied N occurred in crOpping period 1 for the 37.5 and 75.0 ppm N levels. Near the end of the second cropping period, N deficiency was apparent at all N levels, though to a much lesser extent at the higher N level. All pots showed Obvious signs of N deficiency during the final crOpping period. As expected, yield, Z N in tops and uptake of N increased in response to applied N and‘decreased in succesive cropping periods due to N depletion. The decreasing trend in mineral N remaining in the soil after crOpping is further evidence Of N depletion. The quantitative discussion is limited to uptake of N (yield x Z N) and the ratio of uptake of N (uptake at the 75 ppm level divided by uptake at the 0 N level), which is a measure of response to applied N. Table 12 contains the approximate level of significance of the F test for uptake of N by the three consecutive crops. There were significant differences between sequences and between N levels in all three cropping periods, and there was a significant sequence x N interaction for crOp 2 and cumulative uptake values. Significant differences in response to applied N were Observed only for the cumulative uptake values (Table 13). Thus the discussion is limited to cumulative uptake. As expected, there was a significant difference in cumulative uptake due to N level (Table 14). There are also significant 53 Table 12. Approximate significance of uptake of N by three succesive crOps in the greenhouse at three levels of N fertility. approximate level of significance‘ Source cropl . crop2 crop3 cum. Replication 0.001 0.070NS 0.072NS 0.020 Sequence 0.015 0.001 0.028 0.001 Nitrogen 0.001 0.001 0.001 0.001 Sequence x Nitrogen 0.486NS 0.001 0.455NS 0.018 NS I F test was not statistically different at a probability level of 5Z. 54 Table 13. Approximate significance of the F test for the response to applied N by three consecutive crOps grown at three N fertility levels in the greenhouse on the fall 1981 soil samples.1 ‘Approximate level of significance Source crgp 1 crop 2 crop 3 cum. Replication .293NS .122NS .133NS .024 Sequence .145NS .267NS .725NS .025 1Response I uptake at 75 ppm level divided by uptake at 0 ppm level. NS I F test was not statistically different at a probability level of 5Z. 55 Table 14. The effect of rotation sequence on the cumulative response to applied N and cumulative uptake Of N by three consecutive crOps grown at three N fertility levels in the greenhouse on the fall 1981 soil samples.1 Cumulative N uptake Response2 Seqpence3 0 ppm N 37.5 ppm N 75 ppm N mg/culture c-B 43.2Aa4.5 92.58a 128Ca 2.98a Be-B 38.5Aab 93.88ab 137Cb 3.61bc C-C-C-B 43.2Aa 99.7Bc 137Cb 3.18ab C-C-Be-B 41.8Aab 98.3Bbc 138Cb 3.3Zabc O-Be-B 37.3Ab 90.6Ba 134Gb 3.64c O-A-Be-B 41.4Aab 95.6Babc 1380b 3.34abc 1Means of four replications, duplicate determinations. 2Response I uptake at 75 ppm N level divided by uptake at 0 ppm N level. 3C I Corn, Be I Navy bean, B I Sugarbeet, 0 I Oat, A I Alfalfa 4Means followed by the same letter (e.g. A) within a row (excluding the last column) are not significantly different, alpha I 0.05, (Duncans new multiple range test). 5Means followed by the same letter (e.g. a) within a column are not significantly different, alpha I 0.05, (Duncans new multiple range tCSt) o 56 differences between sequences at the individual N levels. Uptake at the 0 N level for the C-B and C-C-C-B rotations was significantly higher than the O-Be-B rotation but not different than the other rotations. The C-C-Be-B, O-A-Be-B, Be-B and O-Be-B rotations behaved similarly. Since uptake at the 0 N level is a reflection of the N supplying capacity Of the soil, the greater uptake by the C-B rotation is not surprising owing to the apparent increase in soil N over the nine year cropping period (Table 5). Soil N and mineralizable N estimates were lower for the Be-B and O-Be-B rotations for both years, and hence lower values Of uptake at the 0 N level were observed. Uptake at the 37.5 ppm level for the C-C-C-B rotation was significantly higher than the Be-B, C-B, and O-Be-B rotations, while uptake for the C-C-Be-B rotation was significantly higher than the C-B and O-Be-B rotations. Uptake by the remaining rotations was not significantly different. At the 75 ppm N level, C-C-Be-B, O-A-Be-B, C-C-C-B, Be-B, and O-Be-B rotations were all significantly higher in uptake than the C-B rotation. Uptake by the C-B rotation was highest at the 0 N level and lowest at the 75 ppm N level. A similar trend was observed for the C-C-C-B rotation. The significant sequence x N interaction appears to result from the presence Of corn and the absence of legumes in a rotation, and from a differential response of the corn containing rotations when corn was grown as a second crOp instead Of oats. Additixn: of N to the corn containing rotations has apparently inhibited N mineralization or conversely, stimulted the mineralization for the legume containing rotations to a greater extent. This suggests that there are qualitative differences in the degradability and type of humus 57 formed under these rotations. The ratio of uptake inversely parallels uptake of N at the 0 N level. The response to applied N was significantly lower for the C-B and C-C-C-B rotations than for the O-Be-B rotation. This is most likely due to the sequence x N interaction. The C-B response was also significantly lower than the Be-B response. The response of the remaining rotations was not significantly different. Table 9 contains simple correlation coefficients obtained from correlations between greenhouse results and selected soil properties. There were no significant linear correlations between uptake at the 0 N level (cumulative or otherwise) and N availability indexes, and hence these results are not consistent with results reported in Table 1. Similarly, it will be noted that Sp 1981 correlations with the same soil parameters are higher. For the system under study, one mst therefore question the use and accuracy of N availability indexes in estimating mineralizable N in samples collected at the end of the growing season. Cumulative uptake and uptake during crop l at the zero N level were significantly correlated with WBC and TN (r I 0.47, 0.47, 0.48, and 0.48, repectively). The above observation and the significant linear relationship between AMN and BMN and their corresponding C and N levels (previously discussed) suggest that a nonlinear relationship may exist. The highly significant linear relationship between uptake by crop 1 and cumulative uptake (r I 0.94) at the 0 N level is further evidence that a discussion Of cumulative uptake is more useful than individual discussions of the results. It also tends to suggest that one extended cropping period is as useful as several consecutive crOpping periods. A significant correlation between cumulative uptake and uptake by crOp 3 58 at the 0 N level was also observed (r I 0.47). Uptake by crop three was negatively correlated with uptake by crOp 2 (r I -0.46). This relationship is believed to result from the fact that corn was grown during the second crOpping period, and corn containing rotations (C-B and C-C-C-B) behaved adversely. Larson (1972), Ketchenson and Beauchamp (1972) and Barber (1979) have all verified that corn and stover yields are reduced when corn follows corn (hence uptake of N may decline). SUMMARY hu>methods of determining organic C in the soil were investigated. The dry combustion results were highly variabLe owing to problems associated with the removal of carbonate C before combustion, therefore the WBC method was employed. Because complete oxidation is accomplished, the dry combustion method would be extremely useful for soils which do not contain carbonates. The same field experiment was conducted twice on separate experimental units. With respect to soil C and N levels, experimental units were shown to be statistically homogeneous upon initiation of the long term experiment. After 9 years of cropping to different rotation sequences, significant differences have resulted. Although there is a similarity of results, the variances of all soil parameters estimated were not homogeneous and hence results were not poolable between sampling dates. For the Sp 1981 samples, organic C losses ranged from 0.053Z for the C-C-C-B rotation to 0.1802 for the O-A-Be-B rotation (1180 and 4030 kg C/ha, respectively). Organic N losses ranged from 0.0066Z for the C-C-C-B rotation to 0.186Z for the Be-B rotation (148 and 417 kg N/ha, respectively). For the F 1981 samples, losses of C ranged from 0.055Z for the O-A-Be-B rotation to 0.200Z for the Be-B rotation (1230 and 4000 kg C/ha, respectively), while losses of N ranged from -0.0030Z for the C-B rotation to 0.0195Z for the Be-B rotation (-67 and 444 kg N/ha, respectively). 59 60 In general, losses of C paralleled losses of N. The trend toward higher C and N levels of sequences containing 50Z corn is evident in both sampling dates, as is the trend toward lower C and N levels of the O-Be-B and Be-B rotations. One can conclude that these significant differences have resulted from differences in the amount of organic matter returned to the soil. The next question which was asked is do these differences result in differences between estimates of potentially mineralizable N? Three methods were investigated, Bremner mineralizable N, autoclavable mineralizable N and N mineralization potential (BMN, AMN and Nmp,repectively). The Sp 1981 AMN and BMN and F 1981 BMN estimates closely paralleled one another, indicating that N mineralization under controlled conditions was affected by rotation sequence to the same degree. The F 1981 estimates of AMN did not follow this pattern, possibly because of qualitative differences in organic matter. The Nmp method provided anomalously low results, and thus was not very useful since the N mineralization potential as defined by Stanford (1972) could not be calculated. For both sampling dates the amount of N mineralized in the AMN and BMN methods was significantly and linearly correlated with Walkley-Black C and total N (WBC and TN). Thus one can conclude that the rate and amount of N mineralized in these processes was proportional to the amount of mineralizable substrate. In general, N mineralization estimates were high where soil C and N levels were high. For the system studied, rotations containing 50Z corn have the potential to mineralize more N than the Be-B and O-Be-B rotations. The C-B and O-A-Be-B rotations were intermediate in their N supplying potential. Smaller 61 correlation coefficients obtained for the F 1981 samples suggest that it is best to obtain soil samples for this purpose in the spring. Uniform results obtained for the Sp 1981 samples supports this statement and further suggests that a soil sample consisting of 20 probes per plot provides adequate statistical representation of the C and N and N mineralization status of the soil. The hypothesis that these differences can affect sugarbeet yield parameters in the field was investigated. Only the C-C-Be-B rotation produced significantly lower Z S and RST estimates. Since there was no significant linear correlation between estimates of mineralizable N and sugarbeet yield parameters, these differences did not appear to be related to differences in potentially mineralizable N. These results indicate that culturally induced soil N changes are not substantial enough to influence sugarbeet yield parameters at this time. In the greenhouse experiment there were significant differences between uptake for sequences and N levels in all three crOpping periods, as well as significant sequence x N interactions for the cumulative and crOp 2 results. The trend in cumulative N uptake at the 0 ppm N level closely paralleled C and N levels and a significant linear correlation between these values was found to exist. As the N level increased, cumulative uptake by the C-C-C-B and C-B rotations decreased. Thus a significant sequence x N interaction resulted possibly due to qualitative differences in organic matter formed under these rotations. Similarly, the relative response to N inversely paralleled trends in cumulative uptake of N at the 0 N level. There was no significant linear correlation between cumulative uptake at the 0 N level and N availability indexes as has been reported in the literature. This lack 62 of correlation cannot be explained. The highly significant relation between uptake by crop 1 at the 0 N level and cumulative uptake is evidence that one extended cropping period utilizing multiple clippings of a perennial, indeterminate (non N fixing) crop species may be as useful in vegetative tests for N uptake as several consecutive crOpping periods utilizing different crops. The former method can save a great deal of time and money. In conclusion, the greenhouse results indicate that significant differences in uptake have resulted, hence sugarbeet yield parameters may eventually be affected. All other factors being the same (eg. disease, insects, etc.), first order kinetics predict that the O-Be-B and Be-B rotations (being lower in organic C and N) will be the first rotations in which this effect will be observed. To prevent this effect from being manifested, new N fertilizer recommendations may eventually have to be formulated. 2. CONCLUSIONS Experimental.tnxits were found to be statistically homogeneous upon initiation of the experiment. After 9 years of crOpping in specific rotation sequences, significant differences in organic C and N have resulted. Because of the initial homogeneity, it is concluded that these differences have resulted from differences in the amount and nature of crop residue returned to the soil. Organic C and N losses have resulted in all cases but one, and in general organic C losses are paralleled by N losses. For tflue system studied, rotations containing corn resulted in smaller losses of C and N whereas O-Be-B and Be-B rotations resulted in larger losses of C and N. The amount of N mineralized in the AMN and BMN’nmthOds was significantly and linearly related to the initial concentration of mineralizable substrate (WBC and TN). 63 6. 64 Significant differences in the amount of potentially mineralizable N were shown to exist and in general were high where C and N levels were high and where losses were lowest. The significant differences between potentially mineralizabhebl estimates were not substantial enough to influence sugarbeet yield parameters at this time. Cumulative uptake in the greehouse at the 0 N level was not linearly related to estimates of potentially mineralizable N as has been observed in the literature. Significant differences in cumulative uptake between sequences were observed at all three N levels. The relative response to applied N indicates that the O-Be-B and Be-B rotations appear to benefit most from applied N while rotations containing corn produced the lowest response. 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