There were no differences in the number of nematodes, their biomass, or any of the ecological indices measured between cropping cycles, but the basal index (BI), an indicator of depleted soil food webs (Du Preez et al., 2022), and the enrichment footprint (EF), an indicator of organic enrichment and soil fertility (Du Preez et al., 2022), varied across cropping cycles. The BI was lower in 2023-2024 (32.25 ± 1.58) than in the 2022–2023 cropping cycle (36.23 ± 1.10) (p<0.05) and the EF was lower in 2022-2023 (37.18 ± 4.14) than in the 2023-2024 (48.428 ± 5.78) cycle (p<0.05), indicating a general nutrient enrichment in soils from 2022 to 2024.

Table 3 illustrates the variation of different nematode based indices across various sampling times, without considering the effects of the different background fertilization treatments. Overall, total nematode abundance and biomass were maintained or reduced from P0 to Pi but increased from Pi to Pf (p<0.05).

The maturity indices (MI, MI 2-5, ∑MI), indicators of soil food web succession (Du Preez et al., 2022), were maintained from P0 to Pi, then reduced from Pi to Pf (p<0.05) ( Table 3 ). Conversely, there was an increase in the plant parasitic index (PPI), a maturity index applied to the plant-parasitic assemblage, from P0 to Pf ( Table 3 ).

The channel index (CI), an indicator of fungal-mediated decomposition channel (Du Preez et al., 2022), the basal index (BI), and the structure index (SI), indicators of soil food web complexity (Du Preez et al., 2022), were slightly increased or maintained from P0 to Pi, but reduced from Pi to Pf (p<0.05) ( Table 3 ). Conversely, the enrichment index (EI) was reduced from P0 to Pi and then increased from Pi to Pf (p<0.05) ( Table 3 ).

Finally, our results on nematode metabolic footprints, indicators of the magnitude of the function developed by different nematode functional guilds (Du Preez et al., 2022), showed that the composite footprint (CF), which included the whole community, the enrichment footprint, which accounts for bacterial-feeding enrichment indicators (EF), the bacterivore footprint (BF), which includes all bacterivores, and herbivore footprints (HF), which considers all plant parasites, were maintained from P0 to Pi, but increased from Pi to Pf (p<0.05) ( Table 3 ). The structure footprint (SF) and omnivore footprint (OF), which look at nematodes in all high trophic groups and omnivores, did not change from P0 to Pi but decreased from Pi to Pf (p<0.05) ( Table 3 ). We found no differences in the fungivore footprint (FF) at any sampling time.

When comparing the effects of the different background fertilization treatments at the three sampling times during the crop cycle, the increase in nematode biomass from Pi to Pf was significant (p<0.05) only in the following organic amendment treatments: pelletized chicken manure (Pi: 7.93 ± 1.47 vs. Pf: 34.49 ± 8.64 µg per 250 cm³ of soil), fresh cow manure (Pi: 8.29 ± 0.52 vs. Pf: 35.51 ± 7.13 µg per 250 cm³ of soil), and composted cow manure (Pi: 6.91 ± 1.00 vs. Pf: 23.95 ± 3.10 µg per 250 cm³ of soil) ( Figure 1 ).

The maturity indices decreased from Pi to Pf in the pelletized chicken manure, with MI showing values of 2.36 ± 0.06 for Pi and 1.72 ± 0.08 for Pf ( Figure 2A ), and ∑MI showing 2.70 ± 0.02 for Pi and 2.25 ± 0.06 for Pf ( Figure 2B ). MI 2–5 decreased from P0 to Pi in both the fast-release (P0: 2.36 ± 0.02 vs. Pi: 2.27 ± 0.03) and slow-release (P0: 2.39 ± 0.03 vs. Pi: 2.29 ± 0.05) inorganic fertilizers ( Figure 2C ).

All background fertilization treatments showed a trend to reduce CI from Pi to Pf, but these reductions were statistically significant (p<0.05) only in the slow-release inorganic fertilizer (Pi: 57.76 ± 6.85 vs. Pf: 10.76 ± 1.86), fresh chicken manure (Pi: 60.89 ± 9.90 vs. Pf: 6.40 ± 1.36), and fresh cow manure (Pi: 63.08 ± 5.63 vs. Pf: 10.70 ± 2.62) ( Figure 3A ). The increase in the EI from Pi to Pf was significant (p<0.05) only in chicken manure treatments, fresh (Pi: 39.68 ± 4.34 vs. Pf: 70.46 ± 4.36) or pelletized (Pi: 38.19 ± 7.60 vs. Pf: 72.88 ± 4.48) ( Figure 3B ). The SI was reduced significantly (p<0.05) from P0 to Pf in both inorganic fertilizers, fast- (P0: 47.11 ± 1.17 vs. Pf: 9.25 ± 3.43) and slow-release (P0: 49.11 ± 2.54 vs. Pf: 8.05 ± 2.84) ( Figure 3C ).

The EF increased at Pf in all treatments except fresh or composted cow manure ( Figure 4A ). Fast- (P0: 195.12 ± 38.34 vs. Pf: 21.17 ± 13.74) and slow-release inorganic fertilizers (P0: 125.41 ± 14.71 vs. Pf: 19.69 ± 8.70) reduced the SF from P0 to Pf ( Figure 4B ).

Herbivore footprints increased from Pi to Pf in all organic amendments, but not in inorganic fertilizers ( Figure 5A ). The omnivore footprint decreased from P0 to Pi in the fast-release inorganic fertilizer (P0: 194.43 ± 38.19 vs. Pf: 20.40 ± 13.84) ( Figure 5B ).

Our study demonstrates the potential of organic amendments to partially suppress RKN-disease in cucumber. At the pre-treatment stage (P0), RKN abundances were uniformly distributed across the field plots. At the transplanting stage (Pi), five days after the background fertilization treatments, RKN soil abundances (83 ± 27 J2 per 250 cm³ of soil) exceeded the tolerance limits estimated for the C. sativus-M. incognita pathosystem (0.13-0.25 J2 per 250 cm³ of soil) (Giné et al., 2014; Talavera-Rubia et al., 2022), and hence some yield losses were expected. Average cucumber yields in our field trials ranged from 5.45 to 8.26 kg per plant. The fast-release inorganic fertilization treatment, the standard for intensive horticultural production in the area, gave the lowest yield value. In comparison with the fresh chicken manure treatment, the fast-release N inorganic fertilization treatment was 44% less productive.

Previous research found that the application of chicken manure to soil at high dosages suppressed RKN-disease in numerous crops worldwide by releasing compounds like ammonia, organic acids, and phenolics during decomposition, which are toxic to nematodes (Peiris et al., 2020). The reported quantity of organic amendments required for effective disease control (20000–50000 kg/ha) (Melero-Vara et al., 2012) is often not affordable in many cropping systems, prompting the exploration of various approaches to optimize their effects. Our research demonstrates that when RKN soil inocula is not very high (<102 ± 9 J2 per 250 cm³ soil), even low dosages of organic amendments (400–1200 kg/ha) can achieve effective and profitable control of the RKN disease in highly susceptible crops such as cucumber and likely in less susceptible crops like tomato or pepper. Effectiveness of low dosages of organic amendments when RKN soil inoculum is higher or in spring-summer cropping cycles, when RKN activity is higher and damage threshold lower, still needs to be investigated.

The mechanisms of RKN-disease suppression by organic amendments can be direct, involving the release of antibiotics, toxic metabolites, or volatile organic compounds (VOCs) that are harmful to nematodes, or indirect, by enhancing populations of saprophytic or antagonist organisms in soil, boosting plant resistance to nematodes, or improving plant nutritional status, thereby increasing plant vigor to overcome the impacts of RKN (Melero-Vara et al., 2012; Rosskopf et al., 2020). Organic amendments with a higher N content (a lower C:N ratio) and easily oxidizable C have been found to produce higher concentrations of organic acids, thereby enhancing soil disinfestation efficacy (Liu et al., 2016). Nevertheless, organic amendments with high N concentration may lead to an undesired flash release of nutrients. Liu et al. (2016) highlighted the need to choose the right organic amendments and application rates that can control soilborne diseases without causing harm to the environment. In our study, the most effective organic amendments for reducing RKN abundances were those with the lowest C:N ratio; specifically, fresh and pelletized chicken manure resulted in RKN mortalities of 47.37 ± 2.02 and 37.31 ± 1.98, respectively, which aligns with previous reports indicating that higher N content improves pathogen suppressiveness (Liu et al., 2016). Additionally, chicken or chicken manure enhances soil structure, increases microbial activity, and boosts enzyme functions such as phosphatases and urease, which are essential for nutrient cycling and plant resilience. It also improves cucumber plant performance in soil infested with RKN, further explaining the higher cucumber yield observed in the fresh chicken manure treatment (Mardani et al., 2024).

The Seinhorst models indicate that for the C. sativus–M. incognita pathosystem, if a nematicidal treatment costs between 50 and 100 €/ha, the RKN abundances at which the cost of treatment equals the loss in crop value (economic threshold) will be around 5–10 J2 per 250 cm³ of soil (Talavera-Rubia et al., 2022). If RKN abundance is higher than the economic threshold, using nematicidal treatments makes financial sense because the extra money earned from reducing yield losses will be greater than the cost of the treatment. Therefore, since the efficacies of organic amendments in reducing RKN abundances in soil ranged from 25 to 47%, their use would be profitable for RKN abundances over P0: 7–15 J2 per 250 cm³ of soil. Organic amendments were better at killing nematodes than inorganic fertilizers (10–12%), but not as good as most chemical nematicides allowed in the European Union (40–64%); however, organic amendments are usually free or inexpensive if obtained locally, and even when considering transportation and application costs, they were cheaper (45–100 €/ha) than the cost of approved chemical nematicide treatments in the area (170–849 €/ha) (Talavera-Rubia et al., 2022).

Our results show that both organic amendments and N fertilizers exert a significant effect on the soil nematode community. Soil nematodes exhibit increased abundances and total biomass in response to N-fertilization, mainly due to increased abundances of nematodes in lower trophic levels (Okada and Harada, 2007; Liu et al., 2016), which, in turn, promotes N mineralization and increases mineral N contents in the soil (Buchan et al., 2013). However, significant increases in nematode abundances were only noted in our trials at harvest, following cucumber cultivation cycles. Even though N fertilizers might have toxic effects on soil organisms in the short term (Sachdeva et al., 2025), no negative effects of organic or inorganic fertilization on total nematode abundances were detected five days after background fertilization treatments, in agreement with previous studies (Martinez et al., 2021). Nonetheless, a positive effect of fertilization on total nematode abundance was noted at harvest, after one crop cycle of cucumber, due to the initial low resource availability in the sandy-silty soil, the fertigation applied during the crop cycle, and the presence and activity of roots.

Maturity indices values (MI, MI 2-5, ∑MI) indicated significant soil disturbance with low food web structure at P0 and Pi, probably due to tillage and nutrient enrichment (Puissant et al., 2021), which further declined by Pf due to harmful effects of fertilizers in high soil food web levels and the promotion of lower guilds (Liu et al., 2016) from the enrichment caused by fertigation. Conversely, the plant parasitic nematode index (PPI) slightly increased post-cropping at Pf, driven by the increase in RKN nematode abundances at the end of the crop. Fresh and pelletized chicken manure reduced MI and ∑MI indices while increasing compound, enrichment, and herbivore metabolic footprints from Pi to Pf. In contrast, fast-release inorganic fertilizer reduced the structure index (SI) and MI2–5 from P0 to Pf. Therefore, the use of organic amendments or slow-release inorganic fertilizers as background fertilization maintained a more complex and structured soil food web than under a fast-release inorganic fertilizer treatment.

Before application of fertilization treatments at P0, soil food web indices (BI, CI, EI, and SI) exhibited low to intermediate values, indicative of moderate nutrient enrichment and a food web of low-intermediate complexity, where organic matter decomposition is predominantly carried out by bacteria, which is typical of agricultural soils with high nutrient resources (Ugarte et al., 2013). Organic amendments increased BI and CI at transplanting (Pi) due to the increase in generalist nematodes adapted to perturbation, but these indices were reduced across all treatments at harvest (Pf), while the EI showed an opposite pattern, decreasing from P0 to Pi and greatly increasing at Pf as a response to the mineralization of organic matter that boosts microbial communities, which, in turn, enhance enrichment-opportunistic nematodes (Leroy et al., 2009). The SI, an indicator of soil food web complexity, remained at intermediate values between P0 and Pi but was reduced at Pf, as commonly found in response to soil perturbation associated with agricultural management (Ferris et al., 2001). These findings indicate that although adding organic materials improved the soil food web’s complexity when plants were transplanted, this improvement decreased by the time of harvest, pointing to a change towards simpler soil food webs where bacteria break down organic matter because of ongoing nutrient supply from fertigation.

Metabolic footprints, which indicate nematode contribution to ecosystem services, showed no differences between P0 and Pi, suggesting that background fertilization treatments did not affect soil food web functioning in the 5-day period after application. However, after a crop cycle, compound, enrichment, bacterivore, and herbivore metabolic footprints increased at Pf, reflecting higher nematode abundances, biomass, and contribution to soil food web carbon flux through the bacterivore and herbivore channels at the end of the cropping cycle. Conversely, structure, fungivore, and omnivore metabolic footprints decreased at Pf, indicating reduced contribution of nematodes to the fungi-mediated organic matter decomposition and low participation in ecosystem services derived from soil food web complexity, e.g. pest control (Sánchez-Moreno and Ferris, 2007; Timper et al., 2012).

Our findings indicate that when more carbon (C) is added through fertilizers, the total number of nematodes increases. However, using more nitrogen (N) from fertilizers reduces the maturity index (MI) and the number of omnivore-predator nematodes, which means that high nitrogen levels make the nematode community less complex, as noted before (Sánchez-Moreno and Ferris, 2007). Furthermore, organic amendments differ in their impact on soil nematodes, as those with C-rich crop residues supported larger free-living nematode populations and promoted structure and enrichment index (EI), whereas N-rich animal manure was more effective in controlling plant-feeding nematodes (Liu et al., 2016).