The soil samples were obtained from a relatively undisturbed field in Bukidnon, Central Mindanao, Philippines (coordinates: 7.8324 N, 124.8829 E). It is located at an altitude of 790 m above sea level. The soil belongs to the La Castellana clay series in the order of Alfisols in the Hapludalf’s great group. The climate belongs to type III according to the Corona climate classification. Soils were collected from the top soil layer to a depth of 10 cm, and brought to the Molecular Ecology and Physiology (MEP) Laboratory of the Premier Research Institute of Science and Mathematics (PRISM), Mindanao State University - Iligan Institute of Technology (MSU-IIT), Iligan, Philippines. Soil properties such as pH, OM, N, Phosphorus (P) and Potassium (K) were measured at the beginning of soil collection and at the end of the experiment (45th day). At the time of soil collection soil pH was determined in a 1:1 soil soil-water suspension. The OM content was measured using the Walkley-Black titration method, which is based on the oxidation of OM by a potassium dichromate-sulphuric acid mixture. To extract the available P in the soil, the Olsen method was used, while K was measured using the cold sulphuric acid method (Labajo & Pabiona, 2022).

The experiment was conducted in a screen house. A total of 20 plastic pots were prepared, consisting of four concentrations with five replicates for each treatment. Each plastic pot was filled with 5 kg of soil. Based on the chemical soil analysis of the Department of Agriculture of the CARAGA region, Philippines, the recommended amount of urea fertiliser in the field was 78.26 kg of urea per ha. Considering that 1 ha is equivalent to 2 x 10⁶ kg of soil, this recommended amount is approximately 196.65 mg of urea per 5 kg of soil or 39.33 mg/kg of soil. For the trial, urea with an NPK ratio of 46-0-0 was added to the soil pots at the following concentrations: 19.67 mg/kg soil (labelled as T1), which is 50% below the recommended value; 39.33 mg/kg soil (T2), which is the recommended value in this area based on the soil quality test; 78.66 mg/kg soil (T3), which is twice the recommended value and 0 mg/kg soil as a control. These amounts were applied according to the farmers’ usual schedule in two separate applications with the first 50% of the urea applied directly in solid form and mixed evenly into the soil, while the remaining urea was dissolved in water and sprayed evenly onto the soil 25 days after the first application. The solution was prepared by dissolving urea in tap water, resulting in mass concentrations of 4.66%, 8.9%, 17.8% and 0% for T1, T2, T3 and the control, respectively.

Immediately after soil sampling, 100 g of soil were taken to represent the original nematode communities and designated as day 0. While the rest of the homogenised soil was distributed among the pots, approximately 100 g of fresh soil were taken from each pot on day 15, 30 and 45. Following Martinez et al. (2019), nematodes were extracted using a modified tray method and picked using a copper needle under a stereomicroscope (Olympus, BX 41). Nematodes were mounted on permanent slides (Siddiqi, 1997) for identification to genus level based on morphological characteristics (Andrássy, 2005, 2007, 2009) under a binocular Olympus CX 22® microscope (400–1000x magnification). The nematodes were assigned as the “oloniser-persister” (cp) scale (Ettema & Bongers, 1993), and divided into trophic groups, namely bacterivores, fungivores, omnivores-predators, and plant parasites (Ferris & Bongers, 2009; Sieriebriennikov et al., 2014).

For the urea-free experiment, an independent samples t-test was used to measure the difference in soil physico-chemical properties and community descriptors (i.e., abundance, no. of genera, Shannon-Weiner Index (H’), Simpson’s Evenness Index and Maturity Index (MI)) between day 0 and day 45. Non-metric multidimensional scaling (nMDS) based on the log (x+1) transformation was also used to visualise trends in nematode community composition between day 0 and day 45. Moreover, significant differences in nematode genus composition between day 0 and day 45 was determined using multivariate permutation analysis of variance (PERMANOVA) in PRIMER+ with 999 permutations at p<0.05. SIMPER (Similarity Percentages) analysis was used to calculate the contribution of each species (%) to the dissimilarity between the two groups. To determine the effects of urea on nematode communities, a two-way ANOVA was used to compare the nematode community descriptors, between treatments over time using the Statistica ® software package version 7.0. Prior to ANOVA, data were subjected to meet the normality assumptions. p-values < 0.05 were considered significant.

In the urea-free experiment, the physicochemical properties of the soil were compared between the beginning (day 0) and the end (day 45) of the experiment. The results showed that the pH decreased significantly (p<0.05), while K increased significantly after 45 days (p<0.5). Other soil properties, such as OM, P and N, showed no significant changes. Nematode density (individuals/100g soil) and number of genera decreased significantly (both p<0.05) by 28% and 35%, respectively, at the end of the experiment. As shown in the nMDS, there was no overlap in nematode genera between day 0 and day 45, indicating distinct nematode assemblages at the start and end of the experiment (Fig. 1). In the same period of time, there was a significant decline in the H’ and the Simpson’s Eveness Index (both p<0.05), as well as the MI (Table 1). Among trophic groups, herbivores (32.4%) dominated the nematode assemblages on day 0, followed by bacterivores (25.2%), fungivores (11.9%), predators (16.3%) and omnivores (14.1%). A shift in the nematode communities was then observed on day 45, with the proportion of herbivores (7.3%), bacterivores (63.9%), fungivores (11.4%), predators (6.4%) and omnivores (11%) (Fig. 2).

The percentage dissimilarity between day 0 and day 45 was 49.93%, which can be attributed to the decrease of herbivores (Rotylenchus, Rotylenchulus and Belondira), predators (Mylonchulus, Iotonchus, Oxydirus and Eudorylaimus), bacterivores (Cephalobus) and omnivores (Crassolabium and Drepanodorylaimus). On the other hand, several genera also increased towards the end of the experiment, such as Rhabditis (bacterivore), Filenchus (fungivore) and Mesodorylaimus(omnivore) (Table 2).

On day 15, the nematode abundance in T2 (39.33 mg/kg urea) was significantly higher than the other treatments (p<0.05) (Fig. 3A), while there was no significant difference (p>0,05) among the treatments on days 30 and 45. There was also no significant difference (p>0.05) in the number of genera (Fig. 3B), Shannon-Weiner Index (H’) (Fig. 3C), Simpson’s Evenness Index (Fig. 3D) and Maturity Index (MI) (Fig. 3E) between the treatments.

Field studies capture the complexity and variability of natural ecosystems, including interactions among multiple species and environmental factors. However, with such environmental variability, attributing observed effects caused by specific stressors can be challenging. On the other hand, microcosms offer a higher level of control over experimental conditions, allowing researchers to isolate and manipulate specific variables. By controlling specific factors, microcosm studies help in understanding the mechanisms underlying ecological processes. Thus, in this experiment, a microcosm approach was used to investigate the effects of urea on nematode communities.

The results showed a 28% reduction in abundance and 35% in the number of genera. These findings were consistent with previous studies involving nematodes in a terrestrial microcosm (Martikainen et al., 1998; Martinez et al., 2019). Although changes in physical and chemical properties have been shown to directly influence nematode abundance and diversity (Nisa et al., 2021), the decline in abundance and genera, particularly herbivores, was influenced by the removal of plants (Martinez et al., 2019; Ewald et al., 2020). Aside from providing nourishment to plant-feeding nematodes, plants can also alter soil conditions that can affect nematodes or can attract other organisms that serve as a food source of nematodes. Physical disturbance can also alter the nematode genera (Lazrova et al., 2021). A decline in abundance was observed among cp 4 and 5 genera, such as Mylonchulus, Iotonchus and Oxydirus (all predators) and Athernema, Crassolabium, Eudorylaimus, Drepanodorylaimus, Laimydorous and Paractinolaimus (all omnivores), suggesting that in addition to their high susceptibility to environmental disturbance (Sun et al., 2024), predators and omnivores may also be sensitive to low food availability (Martinez et al., 2019; Karuri, 2023). The absence of a plant host may have caused the trophic groups to shift from herbivores to bacterivores after 45 days.

Nematodes are generally more abundant and diverse when the soil pH is close to neutral (approx. 6.5-7.5) (Li et al., 2023). Nisa et al. (2021) reported that acidic soils have a lower abundance of nematodes compared to alkaline soils. Soil pH can also indirectly affect nematode diversity by influencing the abundance and diversity of other soil organisms that serve as a food source for nematodes. When pH decreases, soil nematodes increase the secretion of epidermal collagen to strengthen the cuticle which protect its body from osmotic pressure (Cong et al., 2020). This suggests that nematodes can adapt to their environment and protect themselves from external stressors (e.g. osmotic pressure) and have the ability to strengthen their cuticle which can help them survive under adverse environmental conditions (Greiffer et al., 2022). The decrease in pH by 0.5 after 45 days, along with physical disturbance, could also probably explain the decrease or disappearance of nematodes with high cp values. The omnivores and predators, e.g. Laimydorous and Paractinolaimus, were no longer found at the end of the experiment, however, there was an increase in Rhabditis and Filenchus. The response of Rhabditis in this study contradicts the results of Nisa et al. (2021), who observed that bacterivorous (i.e. Rhabditis) nematodes are sensitive to decreasing pH. Other nematodes with similar cp values of 1 or 2 were found to be sensitive to changes in soil pH (Li et al., 2023), such as Cephalobus, whose abundance decreased on day 45. Cephalobus are known to feed on fungi and bacteria associated with plant roots, and their abundance can also be affected by the absence of plants. On the other hand, one genus (i.e. Ironus) with cp value of 4 was found at the end of the experiment but not recorded on day 0. This may have originated from an “egg bank” in the soil at the beginning of the experiment (Martinez et al., 2019).

The soil ecosystem can be disturbed by agricultural practices such as fertilisation (Ewald et al., 2022). Adding fertilizers might make the soil more acidic or alkaline, and disrupt the soil ecosystem by altering nutrient availability.

Previous studies have shown a negative correlation between high levels of nitrogen fertiliser and soil nematode abundance (Liang et al., 2020; Hu et al., 2022; Li et al., 2023; Wang et al., 2023a). As observed in all treatments (including the control) except T2 on day 15 (Fig. 3A), abundance decreased so this trend cannot be attributed to urea application alone. This may be caused by predation, resource competition or physical disturbance (Pothula et al., 2022).

On the other hand, previous studies have reported an increase in nematode abundance and/or species richness with nitrogen application (Li et al., 2020; Teshita et al., 2023). This was observed in T2 (39.33 mg/kg), where a significantly higher abundance was recorded than in the control. Nitrogen fertiliser can stimulate microbial activity in the soil, leading to an increase in bacterial abundance which can serve as food for bacterivorous nematodes (Ni et al., 2024). Hu et al. (2022) also reported that nitrogen input into the soil increases the abundance of nematodes. This was demonstrated in the present work in which an increase in the bacterivorous Rhabditis was observed.

The number of nematode genera and trophic groups tends to be affected by fertilisation (Hu et al., 2022). The effects of fertilisation on nematode genera and trophic groups can be complex and variable and depend on various factors, such as the type and amount of fertiliser applications, the soil type and the original composition of the nematode community. Bacterivorous nematodes tend to respond more to fertilisation than other trophic groups (Wang et al., 2023b) (Fig. 2). On the other hand, fertilisation can have a negative impact on fungal populations due to the increased growth of microbial groups, leading to a decline in the abundance of fungivorous nematodes (Zhang et al., 2024). Moreover, the availability of nutrients, especially nitrogen, can also play a role in the shift from herbivorous to bacterivorous nematodes (Wilschut et al., 2021). In contrast, bacterivorous nematodes can obtain nitrogen from the bacteria they consume, which may allow them to thrive in nutrient-poor environments (Mayrhofer et al., 2021; Shokoohi, 2024). The results on day 0 showed that herbivores dominated the trophic groups, but as the days progressed, the dominant trophic groups shifted to bacterivores. In addition, the percentage of fungivores did not vary greatly from day 0 to day 45 in all treatments, including the control.

The average dissimilarity was 50%, due to the significant decline and disappearance of certain herbivorous and predatory species with cp 4-5 (Rotylenchus, Iotonchus, Belondira, Nygolaimus and Mylonchulus). These genera are known to be sensitive to disturbance (Krashevska et al., 2019) and the experimental set-up could be a disturbance to these nematodes. It was also observed that the bacterivorous community (Rhabditis) increased steadily across all treatments, including the control, as Rhabditis is known for its tolerance to pollutants (Allouche et al., 2020). Ni et al. (2024) found that fertilised plots were associated with an increase in the abundance of bacterivorous and suppressed the omnivorous nematodes. It suggests that the addition of nutrients (including NPK) can influence the nematode community by increasing the abundance and diversity of their microbial food sources in the soil. However, in this study the concentrations of urea used had no effect on the structure of the nematode community. This could be due to the soil’s natural ability to store, immobilise and degrade pollutants (Zhang et al., 2019). In addition, since this is the first application of urea, the effects on the nematode community might not yet be noticeable. However, long-term and continuous application of urea over a longer period of time beyond the number of applications in this study, may alter the structure of the nematode community.