The research, conducted by Rafael Hernández Maqueda and Fernando Del Moral at the University of Almería, compared two adjacent greenhouses with similar soils and crop histories at the Palmerillas Experimental Station of the Cajamar Foundation. One greenhouse was managed conventionally, receiving inorganic fertilizers dissolved in the irrigation water—a practice known as fertigation that dominates Mediterranean greenhouse horticulture. The other followed an ecological intensification strategy: each year, the residues of the previous eggplant crop (Solanum melongena) were incorporated back into the soil together with organic amendments in the form of manure, while inorganic fertilizer use was deliberately restricted. In total, only about 40 percent of the nitrogen applied in the ecological system arrived in soluble mineral form, compared with 100 percent in the conventional greenhouse.

After six years of this contrasting treatment, the researchers sampled soil from the top 15 centimeters along randomly selected drip lines in each greenhouse, collecting nine samples per greenhouse in May 2021 at the end of the growing season. The samples were split between classical physicochemical analysis and molecular biology: aliquots frozen at minus 80 degrees Celsius yielded DNA for quantitative real-time PCR, which the team used to count copies of bacterial 16S rRNA genes, fungal ITS regions, and four genes central to denitrification—the microbial pathway that converts nitrate ultimately to nitrogen gas, and which can leak the potent greenhouse gas nitrous oxide (N2O) into the atmosphere if the process stalls midway.

The physical and chemical results were striking. Total organic carbon in the ecologically intensified soil reached 31.6 grams per kilogram, nearly 2.6 times the 12.2 grams per kilogram measured under conventional management—a proportional increase the authors note exceeds values typically reported in the literature, even though the levels still fall short of the thresholds recommended for optimal greenhouse horticulture. Water-soluble organic carbon, a rapidly cycling fraction that responds to management changes far faster than total carbon, more than tripled, rising from 0.23 to 0.68 grams per kilogram. Total nitrogen increased by roughly half, and the carbon-to-nitrogen ratio climbed from 5.98 to 9.1, a shift with important consequences for the microbial community structure.

Soil structure improved in parallel. The percentage of water-stable, resistant aggregates—the glues and scaffolds that hold soil particles together against erosion and compaction—rose from 21.3 to 33.2 percent, an increase of about 1.5-fold. Because aggregate stability is one of the most robust indicators of a soil’s capacity to resist degradation, this single number encapsulates much of what ecological intensification promises: a soil that behaves less like an inert growing medium and more like a living, self-organizing system. Soil pH, meanwhile, dropped by about 0.7 units, from 8.6 to 7.9, moving the alkaline greenhouse soil closer to the range in which most nutrients are most available to plants.

The biological data told an equally compelling story. Bacterial abundance, measured as 16S gene copies, remained statistically similar between the two systems. Fungal abundance, however, exploded: ITS copy numbers rose from 0.27 to 2.11 log10 copies per gram of dry soil, and the fungal-to-bacterial ratio increased roughly threefold, approaching but not quite reaching statistical significance. The researchers attribute this fungal boom to the lignified eggplant residues and mature compost incorporated under ecological intensification—substrates rich in complex carbon compounds that fungi, with their enzymatic apparatus for degrading recalcitrant material, exploit far more effectively than bacteria. Intensive mineral fertilization, by contrast, is well known to suppress fungal relative abundance and push communities toward bacterial dominance.

The denitrification genes provided perhaps the most nuanced findings. Both clades of the nosZ gene, which encodes nitrous oxide reductase—the only known enzyme capable of converting N2O back into inert atmospheric nitrogen—were significantly more abundant under ecological intensification. The nirK and nirS genes, which encode the nitrite reductases that initiate the nitrogen-producing steps of denitrification, also trended upward, though their increases did not reach significance. Critically, the ratio of N2O-consuming nos genes to N2O-producing nir genes exceeded 2 in both treatments and remained statistically similar between them, suggesting that neither system had an inherent advantage in its potential to suppress nitrous oxide emissions. The authors caution that this genetic proxy is imperfect: denitrification is only one of several microbial pathways that generate N2O, and gene abundance does not always translate into enzyme activity in the field.

Correlation analysis revealed the web of interactions underlying these patterns. Total organic carbon correlated positively with fungal ITS abundance (Rho = 0.700), and the carbon-to-nitrogen ratio likewise tracked fungal abundance. Soil pH showed consistent negative correlations with fungal ITS, nirK, and nosZ1 copy numbers, reinforcing recent findings that pH and organic carbon are among the dominant drivers of microbial community structure. Intriguingly, the four denitrification genes correlated positively with one another, hinting at tightly coupled denitrifier populations, while water-soluble organic carbon—despite its reputation as a microbial energy source—showed only weak links to the biodiversity indicators, suggesting it may not reliably reflect shifts in microbial abundance in these soils.

Then comes the caveat that gives the study its edge. Electrical conductivity, the standard proxy for salinization risk, was 7.4 times higher in the ecologically intensified greenhouse—4.52 versus 0.61 decisiemens per meter. Sodium quadrupled, potassium tripled, and magnesium rose 1.7-fold, while calcium actually declined by about a third, possibly because organic acids released from the decomposing residues promoted calcium leaching. The eggplant residues and compost derived from greenhouse crops are inherently rich in mineral salts, and under the low-rainfall, closed-canopy conditions of a Mediterranean greenhouse, those salts accumulate rather than wash away. The authors recommend rethinking amendment choices—avoiding high-salt biomass and exploring combinations of fresh and mature organic materials—to preserve the structural and biological gains without pushing salinity into dangerous territory, and to reduce reliance on salt leaching, which itself threatens groundwater quality.

The study’s design is deliberately exploratory—a single case study with two greenhouses rather than a replicated field trial—so its conclusions must be generalized with care. Yet its message resonates far beyond Almería. As European agriculture confronts the European Union’s Farm to Fork strategy and the global imperative to cut agrochemical inputs, ecological intensification offers a credible path toward soils that store more carbon, hold together better, and host richer microbial communities. What this six-year experiment makes clear is that the transition is not a simple substitution of organic for inorganic inputs but a systems problem: the composition of the amendments, the salt loads they carry, and the denitrifier communities they cultivate all interact in ways that only long-term, carefully monitored experiments can untangle. The next step, the authors argue, is to pair high-throughput sequencing with direct measurements of nitrous oxide fluxes and leachates, so that the genetic signals observed here can be translated into the climate and water outcomes that ultimately matter.

Subject of Research: Effects of ecological intensification on soil quality indicators and denitrification gene abundance in Mediterranean greenhouse soils

Article Title: Enhancing Greenhouse Soil Quality Through Ecological Intensification (EI). A Case Study

Article References: Hernández Maqueda, R., & Del Moral, F. (2026). Enhancing Greenhouse Soil Quality Through Ecological Intensification (EI). A Case Study. Spanish Journal of Soil Science, 15, Article 15184. https://doi.org/10.3389/sjss.2025.15184

Image Credits: AI Generated

DOI: 10.3389/sjss.2025.15184

Keywords: ecological intensification, soil quality, greenhouse horticulture, denitrification, nitrous oxide, soil organic carbon, salinization, fungal-to-bacterial ratio, qPCR, organic amendments, Almería, nitrogen cycling