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Sequestration and storage of carbon (C) by agricultural soils has been cited as one potential part of the solution to soil degradation and global climate change. However, C sequestration in soils is a slow and dynamic process. The objective of this study was to evaluate the effects of crop rotation and N fertilizer management on soil organic C (SOC) levels at several points in time during 18 yr of a long-term study in the Western Corn Belt. Seven cropping systems (three monoculture, two 2-yr, and two 4-yr rotations) with three levels of N fertilizer were compared. Soil samples were taken in the spring in 1984, 1992, 1998, and 2002 to a depth of 30 cm in 0- to 7.5-, 7.5- to 15-, and 15- to 30-cm increments. No differences were obtained in SOC levels in 1984 at the beginning of the study. After 8 yr, rotation significantly increased SOC 449 kg ha-1 across all cropping systems. From 1992 to 2002, SOC levels in the 0- to 7.5-cm depth decreased by 516 kg ha-1 across all cropping systems. Soil organic C levels in the 7.5- to 15-cm depths in 1992 and 2002 demonstrated similar rotation effects to those in the surface 0- to 7.5-cm, being not significantly affected from 1984 to 1992 but being significantly decreased from 1992 to 2002 (568 kg SOC ha-1 across all cropping systems). Many of the SOC gains in the surface 30 cm measured during the first 8 yr of the study were lost during the next 10 yr in all but the 4-yr cropping systems after 18 yr. The loss of SOC in this latter period occurred when depth of tillage was increased by using a tandem disk with larger-diameter disks. These results demonstrate that more than one point-in-time measurement from long-term experiments is necessary to monitor SOC changes when several management variables, such as cropping system and N fertilizer, are being used. They also indicate that apparent small changes in cultural practices, such as in depth of tillage in this experiment, can significantly change SOC dynamics in the soil. Subtle changes in cultural practices (e.g., tillage depth) can have significant long-term results, but long-term experiments are required to quantify their impact under variable climatic conditions.