The deterioration of soil biological health is the most important aspect associated with the sustainability of cereal-based food production systems. The application of organic nutrient sources is widely accepted and recommended for sustaining crop productivity and preserving soil fertility. Therefore, a study was carried out to assess the effects of different levels of farmyard manure (FYM10: 10 t ha−1, FYM20: 20 t ha−1, FYM30: 30 t ha−1), including inorganic fertilizer (NPK) on the soil and the biological properties of five high-yielding wheat cultivars (HD 2967, DPW 621-50, PBW 550, and WH 1105) over a three-year period (2014–2015 to 2016–2017). The results showed that the application of NPK produced significantly higher yields compared to different levels of FYM and the control during all the study years. The continuous addition of a higher rate of FYM at 30 t ha−1 was found to be beneficial in terms of enhancing crop yield gain, thereby bridging the yield gap to only 7.2% in the third year; the gap was 69.1% in the first year with NPK application. The microbial population and microbial biomass carbon were significantly higher in the FYM treatments compared to the NPK treatment. The activities of different soil enzymes were observed to be significantly maximum in the FYM30 treatment. Similarly, the addition of FYM significantly improved the soil respiration and microbial activity over the NPK and control treatments. Based on the principal component analysis, fluorescein diacetate, bacteria, fungi, and actinomycetes were observed as sensitive biological parameters for the assessing of soil biological health. The soil biological index (SBI) determined with the sensitive parameters was in the decreasing order of FYM30 (0.70), FYM20 (0.61), FYM10 (0.55), NPK (0.18), and control (0.15). Considering both the SBI and the sustainability yield index together, the performance of WH 1105 was found to be better compared to the rest of the wheat cultivars. Our results conclude that the application of FYM in the long run increases the crop yield (24.3 to 38.9%) and improves the soil biological process, leading to the improved biological index of the soil.
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