Crop–livestock integration enhanced soil aggregate-associated carbon and nitrogen, and phospholipid fatty acid
Aggregate size distributionAs hypothesized, the improved soil aggregation was observed under ICL, which is attributed to the presence of animals resulting in higher organic matter contents of total C and N fractions that can significantly enhance soil health over time32. Moreover, well-aggregated soils as observed under ICL ( > 4 mm) at site 1 and NE (2–4 mm) at site 2 have a greater potential of retaining their structure and may have higher macropores, which facilitate sustained root growth than soils with low aggregation such as under CNT (corn–soybean without grazing or CC) in this study. It also explains the significance of ICL systems with no-tillage and undisturbed grassland, where the formation of stable macroaggregates may occur as a result of incorporation of plant residues, stimulation of root exudates and increased biological activity. Furthermore, it was noticed that ICL system not only enhanced the macroaggregates but accentuated the presence of microaggregates due to persistent binding agents, which are critical in SOC protection against microbial decomposition. When integrating grazing livestock into crop rotation, soil aggregation is typically improved under moderate and controlled grazing than the high intensity grazing systems33. Compared to the long-term sites ( > 30 years), short-term site 4 did not result in discernible effects of grazing or CC on soil aggregation. However, within this short-term study, grazed pasture mix was able to enhance aggregation of 1–2 and 2–4 mm sized aggregates compared with oats, oats with CC, oats with CC and grazing. To observe the influence of CC and grazing on > 4 mm or 4 mm) under ICL at site 1 resulted in 1.3–1.5 times significantly higher SOC concentration than NE and CNT. The greater concentration of SOC and N in ICL and NE is attributed to the lack of soil disturbance, crop residue retention, and rhizodeposition, which reduces macroaggregate turnover rate14. At site 3, NE enhanced aggregate-associated C and N concentrations and performed significantly better than both ICL and CNT treatments. The higher C and N accrual in the NE than ICL and CNT, especially at site 3, can be due to massive root systems, long-term establishment and absence of cultivation, which contributes to enhanced soil quality, while reducing nutrient vulnerability to loss by oxidation18,36. For short-term study at site 4, insignificant differences in aggregate-associated SOC suggested that longer study period of at least > 5 years is required for SOC to respond to grazing and cover crop management. The higher total N under ICL and NE can also be due to the presence of legumes, and brassicas in CC, which are effective at recycling N and may have helped in scavenging N.An overall increase in C and N cycling under ICL and NE systems has been attributed to ingested pasture being converted into urine and manure. Under these systems, livestock catalyze nutrient cycling by breakdown of complex plant molecules, greater soil incorporation and decomposition of plant residues and soil organic matter, which can maintain or even improve soil fertility by production of organic acids such as fulvic and humic acids6,8,19. Moreover, grazing stimulates the carbohydrate exudation from grass roots, which is mostly composed of polysaccharides, a C-O alkyl source37. The enhanced C concentration under ICL and NE can also be associated with higher MWD. Integrated system cool-season pasture and winter CC had significantly higher total C and N than the non-integrated continuous corn in previous study6. The results from another integrated system study7 showed that soybean and oat-Italian ryegrass CC increased total C (1.16 Mg ha−1 yr−1) and N stocks (0.12 Mg ha−1 yr−1) under 7 year study period. It is previously reported that ICL system contains labile organic matter pools10,38, subsequently showing higher C stocks and greater root densities near soil-surface, which promotes aggregate-associated C stabilization18,39,40, higher infiltration rates, thus providing likely benefits to soil function linked to erosion control and soil water relations41.Soil microbial community compositionTotal bacterial biomass, AM fungi, and PLFA were enhanced under NE, which can be result of accumulation of organic residues and higher pasture root mass7,32, pasture being grazed can promote exudation of organic compounds by roots, serving as energy sources for microorganisms. The consistent increase in microbial population under NE can also be result of increased SOC and N, however, the same does not hold true for ICL system, where despite observing greater SOC and N, a significant decrease in the microbial population at site 2 was noticed. The enhanced total PLFA under NE system at site 2 is due to concomitant increase in AM fungi, gram (−), fungal/bacterial ratio, and total bacterial biomass compared to ICL. The fungal to bacterial ratio was reduced under ICL compared to NE at sites 1 and 2, pertaining to relatively low abundance of the fungal fatty acid 18:2ɷ6 in grazed system as compared to unmanaged grassland. This finding corroborates the notion that livestock-grazing systems contain bacterial-based decomposition channels and are mostly dominated by gram (+) bacteria and that the fungal population is comparatively more important in decomposer food-webs of native grasslands. These results coincide with previous studies42,43. Moreover, the increase in fungal to bacterial ratios under NE system in contrast to ICL at sites 1 and 2 can relate to modifications in soil health with C sequestration, as fungal populations incline towards higher C assimilation proficiencies and greater storage of metabolized C than bacterial populations9,44. The grazing intensity also plays a significant role in bacterial and fungal presence. It is previously reported that high grazing intensity had greater bacterial PLFA concentration than the low grazing counterparts in grassland systems45. It is considered that under heavily grazed sites in grasslands, bacteria-based energy channels of decomposition dominate other microbial communities, while fungi can successfully enable decomposition in both slightly grazed and non-grazed systems43. Grazed pasture mix at short-term study site 4 showed 12–21% higher total PLFA than the oats, oats with CC, oats with CC + grazing systems. It is also possible that this increased total PLFA at site 4 under grazed pasture mix contributed to enhancing the 1–2 and 2–4 mm sized aggregates compared to other treatments. It indicated that though physicochemical properties can take longer ( > 8–15 years) in significantly responding to changes in management systems, soil microbial community and structure may show a rapid response (~ 3 years), thus it can be used as an early indicator while assessing the variations in soil health18,46.Overall, NE exemplified the undisturbed grazed mixture with a greater microbial population at sites 1, 2, and 3, when compared to other agricultural systems. Our findings coincide with previous studies where pasture systems performed better than the agricultural soil, in terms of, showing greater microbial biomass and fatty acid signatures related to bacterial and fungal populations, which is mostly attributed to greater surface coverage and absence of tillage practices in pasture systems9,47,48. Lower soil microbial communities under ICL system than native Cerrado pasture have been found previously because of reduced soil porosity and macropore continuity resulting in restricted gas diffusion and water movement18.Although the AM fungi abundance was not significant for sites 3 and 4, and significantly lower for ICL system than NE at sites 1 and 2, it should be taken into consideration that FAME analysis cannot reflect species-level changes for fungi and/or bacteria and the variations in microbial community structure for ICL system can be due to changes in abundance and distribution among microbial groups. For example, in a previous study9, while increased bacterial population was observed for continuous cotton compared to the ICL system, however, pyrosequencing for bacterial diversity assessment demonstrated disparities between both systems, where greater Proteobacteria was seen under ICL system than continuous cotton. Numerous factors such as degree of disturbance, pH level, bulk density, porosity, soil water content, C and N distribution, and residue positioning regulate the amount of bacterial and fungal biomass in agroecosystems18,49. Arbuscular mycorrhizal fungi are responsible for formation of macroaggregates ( > 0.25 mm) by producing a glycoprotein called glomalin, which is present abundantly in natural and agricultural systems. However, increased grazing intensity, use of excess fertilizers and fungicides can directly or indirectly reduce mycorrhizal population by influencing soil organisms accountable for converting soil organic matter into plant nutrients38. Animals may also cause moderate soil compaction affecting the fungal biodiversity and soil pore space6,38.Relationship among measured soil propertiesBased on PCA, it is derived that integrated crop–livestock and natural ecosystem of native grassland can provide substrate for the microbial composition and enhance aggregate-associated C and N fractions. A positive correlation between SOC and microbial communities suggested the inclination of microbes to affect SOC and N turnover and vice-versa through interaction with crop–livestock grazing, vegetation, and soil properties. Fungi exhibited insignificant responses to changes in soil pH and bulk density than bacteria because chitinous cell walls make fungi more resistant and resilient to variations in soil conditions50,51. A reduction in gram (−) bacteria may indicate the presence of stressed soil conditions due to pH and increased bulk density, which has previously been observed in other studies52,53. A significant negative correlation between bulk density and SOC, N, gram (−), actinomycetes, total bacteria, and total PLFA indicated that the microorganisms influenced the soil compaction and related SOC and N. Moreover, positive correlation between SOC and microbial composition suggested that microbes can influence C sequestration in the soil via a shift in community structure. Microbial composition is influenced by soil C, whereas N is a critical biogenic element that improves microbial growth and their ability to utilize soil C54. More
