Sulfur nutrition is a crucial part of proper crop growth. In this study, we investigated the influence of organic fertilizers in a long-term field experiment (23 years) with continuous maize monoculture. We focused on: (a) changes in the soil sulfur fraction pools, (b) the balance of total sulfur inputs and outputs, and (c) sulfur uptake by maize. The following treatments were selected: unfertilized control (Control), urea and ammonium nitrate (UAN), UAN and wheat straw (UAN + St), sewage sludge (SS), farmyard manure (FYM), and slurry (Slurry). Using sequential extraction, we determined water-soluble (SW—in water), adsorbed (Sads—in 0.032 mol L−1 NaH2PO4), and available (Sav = SW + Sads) sulfur content. Microwave-assisted digestion in an Aqua regia solution was used to measure the pseudo-total sulfur content (Spt). Organic-bound sulfur (Sorg) was calculated as a difference between Spt and Sav. We found that average biomass yields responded to a uniform 120 kg N ha−1 year−1 dose, rather than the dose of S in fertilizers, with an increase over the Control by 34–49%. The effect of an additional 33.5 kg N ha−1 year−1 on UAN + St treatment was not significant. Average sulfur uptake responded to increased yields (69-121% higher than Control), rather than the sulfur application, with the exception of SS, where the dose of sulfur was high enough to cause an additional uptake. In the topsoil, we discovered a significant decrease over time (from 1997 to 2019) in water-soluble (SW), adsorbed (Sads), available (Sav), and pseudo-total (Spt) fractions on all treatments to 29, 59, 37, and 82% of their initial values, respectively. For all treatments, the proportion of Sorg in Spt increased over time, which was caused by the decrease in mineral fractions (SW, Sads, Sav). The absolute content of Sorg decreased over time for all treatments except SS and FYM to about 85% of the initial value. Using the simple balancing method, we calculated that UAN + St, SS, FYM, and Slurry treatments annually lost 8.04, 66.1, 21.4, and 26.8 kg of S ha−1, respectively. This loss was attributed to the decrease in atmospheric depositions, as well as the release of sulfur from soil organic matter (for UAN + St and Slurry treatments) and a high proportion of easily mineralizable and inorganic sulfur from the SS treatment. Generally, the FYM fertilizer provided the highest potential for maintaining soil Spt status.
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