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Article

Impacts of Silage Biostimulants on Nematofauna in Banana Crop Soils: A Sustainable Alternative to Nematicides

by
Pedro E. Torres-Asuaje
1,
Ingrid Varela-Benavides
1,
Alba M. Cotes
2,
Fabián Echeverría-Beirute
1,
Fabio Blanco
3 and
Juan E. Palomares-Rius
4,*
1
Instituto Tecnológico de Costa Rica, San Carlos 223-21001, Costa Rica
2
Corporación Colombiana de Investigación Agropecuaria—AGROSAVIA, Km 14 Vía a Mosquera, Cundinamarca, Bogotá 250047, Colombia
3
Independent Researcher, Barva 40202, Costa Rica
4
Institute of Sustainable Agriculture (IAS), Higher Council for Scientific Research (CSIC), Apartado 4084, 14080 Córdoba, Spain
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(8), 1860; https://doi.org/10.3390/agronomy15081860
Submission received: 17 June 2025 / Revised: 28 July 2025 / Accepted: 28 July 2025 / Published: 31 July 2025
(This article belongs to the Special Issue Progress and Innovations in Biological Control of Plant-Parasitic Nematodes)
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Abstract

Radopholus similus, commonly known as the burrowing nematode, is one of the major pathogens affecting banana production. Currently, the control of this pathogen relies on chemicals, as no resistant varieties are available. However, new control methods, such the application of ensilage biostimulants (EBs) near the banana rhizosphere, have shown effectiveness. Nevertheless, the impact of this organic control method on soil nematodes and other microbial components remains unknown. This study evaluates the effects of EB application on the native nematofauna of banana. EBs altered the flow of carbon, nutrients, and energy in ways that influenced the abundance of fungivorous and bacterivorous taxa, while consistently reducing the number of plant-parasitic nematodes throughout the experimental period. Specifically, EB application in the soil increased the abundance of certain free-living nematodes, including Aphelenchus, Aphelenchoides, Cephalobidae, and Rhabditidae, while decreasing both the abundance and diversity of phytoparasitic nematodes. In contrast, Criconematidae, Hoplolaimidae, Meloidogyne, Tylenchidae, and R. similis were more abundant in the control and oxamyl-treated soils. EBs can play a crucial role in strategies aimed to improve soil resilience, fertility, and natural suppression, provided that more sustainable production practices are adopted.

1. Introduction

According to the Food and Agriculture Organization of the United Nations [1] (FAO, 2024), bananas are the most internationally consumed fruit. In 2024 alone, the global banana market exported 19.1 million tons of the fruit. Nevertheless, this crop is increasingly affected by phytosanitary issues that jeopardize its worldwide output. For instance, the root endoparasitic nematode Radopholus similis is considered the third most significant phytosanitary issue affecting banana crops [2], surpassed only by Fusarium wilt, caused by Fusarium oxysporum tropical race 4 [3], and Black Sigatoka, caused by Pseudocercospora fijiensis [4]. Additionally, R. similis is a destructive pathogen affecting citrus and black pepper, and it has been found on over 250 different plant species throughout the tropics and subtropics, with banana as its primary host [5]. Control of the pathogen is mainly based in the use of nematicides such as oxamyl [6,7], and plant resistance is not available in the major marketable cultivar, the Grand Naine, which is highly susceptible to this pathogen [8]. However, the use of chemicals is highly restricted in some markets, in which consumers demand low or no residues from phytochemicals, and they could have important effects on the environment and workers/consumers [9]. For this reason, new alternatives to the use of nematicides are needed. In this regard, Ref. [10] demonstrated that subjecting native microbiota, both from soil conductive and suppressive to Radopholus similis (the burrowing nematode), to an ensilage process resulted in a solid microbial matrix that effectively suppressed the endoparasitic nematode R. similis. Suppression of this pathogen was achieved by applying both ensiled biostimulants (EBs) near the rhizosphere area, both in seedlings and succession suckers in commercial plantations. Additionally, EBs increased the proportion of healthy root tissue despite the presence of the pathogen. However, the effect of this practice on the soil microbiota has not been evaluated in detail.
Free-living nematofauna can play a beneficial role in assessing the degree of soil disturbance in banana crops [11,12]. Soil nematodes are classified into five functional groups, bacterivores, fungivores, omnivores, predators, and herbivores [13], with R. similis among the latter. In addition to nematodes, springtails and mites also have advantages over other soil microorganisms as bioindicators because (i) they are one or two trophic levels higher in the food chain and serve as integrators of physical, chemical, and biological properties, facilitating food availability, and (ii) their generation time (from days to months) is longer than that of microorganisms (from hours to days), which gives more stable data over time [14,15]. Changes in nematofauna are correlated with soil conditions and are reflected in various indices, including maturity indices, basality indices, community structure indices, and abundance, diversity, and metabolic footprints [16]. Additionally, there are tools on the international computer network appropriate for analyzing these parameters, such as the Nematode Indicator Joint Analysis (NINJA) [17]. These parameters aid in diagnosing the impact of agricultural practices and other external factors on soil ecology, contributing to the development of strategies to mitigate their impact. Native nematofauna have been used as ecological indicators to evaluate the effects of agricultural practices on soil ecology in various crops, such as sugarcane [18], melon [19], banana [20], pineapple, and other ecosystems [21]. In this context, nematodes could provide valuable insights into ecological processes within the banana rhizosphere following the application of EBs. Specifically, their interactions with plant-parasitic and free-living nematodes, as well as the trophic groups they depend on (such as bacteria and fungi), could be monitored over time to evaluate the broader ecological impact of this successful organic technique beyond the control of R. similis.
In this context, the objective of this research was to evaluate, over different periods of time, the effect of the application of EBs derived from both conductive and suppressive soils of R. similis on the nematofauna of a banana-cultivated soil.

2. Materials and Methods

The experiment was conducted in duplicate in a greenhouse at the National Banana Corporation (CORBANA) La Rita, Limón, Costa Rica (10°15′54″ N and 83°46′26″ W). The first experiment was conducted between June and October 2021. The second experiment was conducted between October 2022 and February 2023.

2.1. Preparation of Ensiled Biostimulants (EB)

Both EBs were developed by adapting the methodology of [22]. To obtain 100 kg of dry matter (DM) of the EBs, 200 kg of fresh material were prepared. The formulation included conductive and suppressive soils (4.27 kg each, 70% DM) as sources of native microbiota, sugarcane molasses (10 kg, 75% DM) for microbial activation, rice semolina (92 kg, 90% DM) as a growth substrate, sawdust (Gmelina arborea Roxb., 31 kg, 70% DM), and fresh chopped grass (Axonopus compressus (Sw.) P. Beauv., 48 kg, 28% DM) to increase volume and enhance fermentation. Additionally, 8 L of non-chlorinated water were added to dissolve the components and provide moisture. Each soil type was diluted and filtered (0.05 mm) and then mixed with molasses. Solid ingredients were blended separately and later combined with the liquid suspension. The final mixture was compacted and fermented anaerobically for 22 days at ~22 °C in sealed 200 L plastic drums.

2.2. Greenhouse Experiment

The treatments for the experiments, conducted in duplicate, were as follows: T1: water (control); T2: oxamyl nematicide (24% a.i.) in liquid suspension (LS) applied at 400 cc with 100 mg/LL a.i./plant on the day of transplant; T3: ensiled biostimulant with conductive soil (EBCS) applied at 320 g of dry matter/plant; and T4: ensiled biostimulant with suppressive soil (EBSS), applied at 320 g of dry matter/plant. The soil used for both EBCSS preparation and as a substrate for the two experimental replicates was collected from the canton of Talamanca, Limón Province (09°37′354″ N 082°50′051″ W). It had a silty loam texture, consisting of 27% sand, 55% silt, and 18% clay, with a pH of 6.53 and organic matter (OM) content of 4.48%. The conductive soil condition was determined based on the historical population data of R. similis in the sampling area, where nematode counts consistently exceeded 10,000 individuals per 100 g of root tissue at 90 days following nematicide application. The soil used in the preparation of EBSS was collected in Guácimo Canton, Limón Province (10°15′52″ N and 83°38′15″ W). It had a loamy sand texture, consisting of 78% sand, 4% silt, and 18% clay, with a pH of 5.48 and organic matter (OM) content of 3.54%. The suppressive soil condition was determined based on historical records showing that R. similis populations remained below 2000 nematodes per 100 g of root tissue at 90 days following nematicide application. However, following experimental sterilization, populations exceeded 10,000 nematodes per 100 g of root tissue. The texture of the substrates was measured by an adaptation of the Bouyoucos methodology [23]. The experimental design followed a randomized complete block structure with six replicates per treatment. The first evaluation was conducted on 18 June 2021, and the second on 23 February 2022.
To apply the EB treatments, the unsterilized soil substrate was mixed with either biostimulant of conductive soil (EBCS) or biostimulant of suppressive soil (EBSS) at 10% w/w ratio. This corresponded to the same soil/EB ratio used in field applications for the top 10 cm of soil. The prepared substrates were placed into pots, each planted with a stage IV in vitro banana plant (10 cm tall, bearing three fully developed leaves). Pots measured 22–24 cm in upper diameter and 18–20 cm in height, and were filled with approximately 6.5 kg of soil. To protect the nematofauna from UV radiation and maintain substrate humidity, black saran netting (80% mesh) was placed over the substrate surface surrounding the pseudostem.
Twenty days after planting, Mancozeb was applied as a preventive treatment against black sigatoka Pseudocercospora fijiensis Morelet. The fungicide was administered at 60% a.i. in encapsulated suspension (ES) at a concentration of 30 g a.i./L, with applications occurring every 15 days. Fertilization was conducted once a week using Hoagland’s solution, applied at a rate of 100 mL per pot.

2.3. Nematofauna Sampling, Extraction, and Identification

Sample collection for nematofauna extraction was conducted in five distinct stages. The first stage, performed before filling the pots, aimed to determine the basal nematofauna. To achieve this, the substrate was homogenized, and four subsamples were taken from different points within it, until a 100 g of sample was obtained. Subsequent samplings were carried out in the pots at 30, 60, 90, and 120 days after transplant (dat).
To collect each sample after transplanting, four subsamples were taken per pot using a mini stainless-steel auger (2.5 cm in diameter and 35 cm long). These subsamples were collected at four equidistant points on the substrate surface within a 5 cm circular radius, using the pseudostem of each in vitro plant as the central reference point. The soil from the subsamples was homogenized to obtain a final sample of 100 g for each treatment. Nematodes from each sample were extracted using the blended, sieved, and centrifuged method with dense solutions, following the methodology described by [24]. Briefly, after nematode and debris separation using sieves of 500 µm (nematodes passed it) followed by 50 µm (recovery of nematodes) the suspension was centrifuged in two steps. First centrifugation was performed for 4 min at 1800 g and was concentrated on a 10 µm sieve. A total of 484 g of sugar/L was used to obtain a specific density of 1.18 g/mL. The nematode pellet was mixed with this sugar solution (1.18 g/mL) and was centrifuged for 3 min at 1800 g. The supernatant was filtered in a 10 µm sieve, and nematodes were recovered using water from the sieve.
The extracted nematodes were exposed to 5% formalin at 80 °C to induce high-temperature death and aldehyde fixation. The treated nematodes were then stored in 10 mL Gosselin™ polystyrene test tubes, Corning Incorporated (Waltham, MA, USA). One week later, the nematodes in each subsample were counted and identified. For this process, the entire nematode suspension was poured onto the base of a 6 cm diameter Petri dish with a gridded bottom. Then, using the 20× lens of an Olympus CK2 inverted microscope, all nematodes present were counted, and the first 100 individuals observed were identified. To ensure systematic identification, reading began from the upper to the lower visual field of the plate, following the grids horizontally from left to right in a zigzag pattern. Nematodes were classified into five trophic groups (phytoparasites, bacterivores, fungivores, omnivores, and predators) at the family or genus level, based on the taxonomic descriptions of [25,26].

2.4. Nematode Communities Analysis

For community analysis, nematodes were first classified taxonomically and assigned to one of the five main trophic groups: phytonematodes, bacterivores, fungivores, omnivores, and predators [13]. Subsequently, they were further categorized based on their survival strategy, the c-p scale for free-living nematodes and the p-p scale for herbivores, following the classification proposed by [27,28]. This classification system arranges nematodes along a continuum with two extremes: (A) Colonizing nematodes, which reproduce rapidly, are opportunistic, and thrive in enriched environments. These are assigned a value of 1 on the c-p or p-p scale. (B) Persistent nematodes, which produce fewer offspring, are highly sensitive to environmental disturbances, and serve as indicators of ecosystem stability and complex food webs [29]. These are assigned a score of 5 on the c-p or p-p scale. Nematodes exhibiting intermediate survival characteristics received a score of 2, 3, or 4, depending on how closely they align with either colonizing or persistent strategies.
Subsequently, the percentage composition of the nematofauna trophic groups present in the experimental conductive soil was determined, and the average of the most frequently observed taxa in the samples by treatment was compared. Additionally, the Shannon diversity index was calculated [30], along with several food web indices, including the maturity index, channel index, basal index, and enrichment index, which integrate the c-p scale with nematode trophic groups to define functional guilds [27,31,32]. Furthermore, various metabolic footprints were assessed. These measurements, based on functional groups, the biomass of each taxonomic category, and nematode body size, allowed for the estimation of carbon consumption throughout their life cycle [33].
At the end of the experiments, 120 days after transplant, several growth parameters were measured. The fresh weight (g) of the root, as well as the fresh and dry weight of the pseudostem and foliage (g), were recorded using an electronic scale (E-ACCURA® Dolphin, New Taipei City, Taiwan). The plant height (cm) was measured from the base to the intersection point of the sheaths of the last two leaves, and the pseudostem diameter (cm) was recorded at its base. Additionally, the number of R. similis was quantified. These nematodes present within the banana plant were extracted and counted following the methodology outlined by [34].

2.5. Statistical Analysis

The comparison of nematode numbers in 100 g of soil by treatment and sampling time was specifically analyzed for the eight most abundant taxa, which comprised 90% of the total nematofauna sampled and for the trophic nematofauna groups as well. For each experiment, a repeated measures analysis was conducted to evaluate the effect of treatments over time using a random intercept generalized linear mixed model. The analysis was performed in R (version R 4.4.1, 2024) utilizing the statistical package “glmmTMB” [35] and the nbinom2 function [36]. This function models the residual variance (V = µ + µ2/φ, link = log), where V = variance of the residuals, µ = population mean, and φ = dispersion parameter. Analysis of some nematofauna groups was needed, however, for the Poisson or nbinom1 functions instead of nbinom2 to attain a better model fit. The Shannon diversity index was analyzed with the “geepack” package using the geeglm function and an “exchangeable” correlation structure [37]. Metabolic indices and fingerprints were assessed using the “nlme” package [38], utilizing the lme function and the pdDiag model of the variance–covariance matrix, accounting for heterogeneous variances across Treatments × Days while treating replicates as a random factor. Comparisons between treatments, time points, and their interactions for all variables analyzed were performed using the emmeans function between the “emmeans” package [39].
At the conclusion of the experiment (120 days after transplant), compliance with the assumptions for the biometric variables was verified using the lm function of R [40]. Additionally, nematode counts were analyzed using generalized linear models (glm) within the “glmmTMB” package and modeling residuals via the nbinom2 function [35]. Following ANOVA, mean comparisons among treatments were conducted using the Tukey test after normality and homoscedasticity tests, and were implemented using the emmeans package [39].

3. Results

3.1. Proportional Composition of the Trophic Groups of the Nematofauna Present in the Experimental Conductive Soil

Overall, the most abundant trophic groups in both experiments were bacterivores, fungivores, and herbivores. Omnivores and predators were scarce, averaging fewer than ten individuals across all four sampling times post-planting (Figure 1). Bacterivores were proportionally dominant in soils treated with EBSS and EBCS, reaching 46% and 41% in the first experiment and 40% and 47% in the replicate, compared to oxamyl (8% and 6%) and the control (5% and 7%). This bacterivore dominance following EB application (p ≤ 0.0001) remained consistent across all sampling times (0, 30, 60, 90, and 120 dat). Similarly, fungivores predominated in EB-treated soils, with 63% and 35% in the first experiment and 47% and 51% in the replicate, versus 1% in both oxamyl and control treatments. Their dominance (p ≤ 0.0001) was also stable throughout all sampling points (Figure 1).
In contrast, herbivores were proportionally more abundant in control soils (12% and 66%) and oxamyl-treated soils (17% and 68%) across all sampling times, compared to EB-treated soils (2% and 27%).

3.2. Percentage Composition of the Trophic Groups of the Nematofauna Present in the Experimental Conductive Soil

In the first experiment, the total nematofauna was distributed across 13 genera and 10 families, while in the second experiment, 11 genera and 10 families were identified (Table 1). In both assessments, community composition varied depending on the treatment and the sampling period (30, 60, 90, and 120 dat). Although up to twelve taxa were identified in the samples, only the eight taxa were considered in the present analysis—four free-living taxa and four of the most abundant and frequently occurring plant-parasitic taxa in the treatments of both experiments—as they collectively represented approximately 90% of the nematofauna found.
In both experiments, mean nematode abundances ranged from 1 to 16,396 nematodes per 100 g of soil. Treatments that included EBCS and EBSS exhibited the highest abundance, within the same range (from 1 to 16,396 nematodes per 100 g of soil) (Table 1). The most abundant free-living nematode genera in both experiments were as follows: Aphelenchus (Aphelenchidae family) with abundances ranging from 4 to 31% in the first experiment and from 12 to 52% in the second experiment. Aphelenchoides (Aphelenchoididae family) ranging from 4 to 37% in the first experiment and 4 to 28% in the second experiment. Cephalobidae (from 17 to 43% in the first experiment and from 10 to 36% in the second. Rhabditidae (from 3 to 11% in the first experiment and from 3 to 10% in the second). These taxa predominated in substrates where the two EBs were incorporated. Notably, although the genus Aphelenchoides includes herbivorous nematodes, for this analysis, all individuals in this genus were assumed to be fungivores. Regarding the phytoparasitic or herbivorous nematodes in both experiments, the most prominent taxa were the Hoplolaimidae family (from 0 to 17% in the first experiment and from 0 to 8% in the second), Criconematidae (from 0 to 15% in the first experiment and from 0 to 2% in the second), and Meloidogyne genus (Meloidogynidae family), with abundances ranging from 1 to 12% in the first experiment and from 1 to 9% in the second. Additionally, Radopholus (Pratylenchidae family) was identified in the first experiment (1% abundance), while nematodes of the Tylenchidae family were found in the second experiment (4% abundance).
In general, plant-parasitic nematode taxa were predominant in the control treatment and, to a lesser extent, in the oxamyl treatment. Nematodes of the genera Meloidogyne and Radopholus, as well as those from the Hoplolaimidae family, such as Helicotylenchus, are economically significant plant parasites in banana crops. Their recurrent presence in the substrate nematofauna was therefore expected. The genera Aphelenchus and Aphelenchoides were significantly more abundant in the EBCS and EBSS treatments compared to the control and oxamyl treatments (p ≤ 0.05), from 30 to 120 dat in both experiments (Figure 2).
On the other hand, the Cephalobidae family maintained its predominance in the EB treatments, but only from 30 to 60 dat in both experiments and at 120 dat in Experiment 1. At 90 dat in both experiments and at 120 dat in Experiment 2, the abundance of this genus was similar (p > 0.05) across all four treatments. Nematodes of the Rhabditidae family were significantly more abundant in soils treated with the two EBs compared to the control and oxamyl treatment (p ≤ 0.05). However, in Experiment 1 at 30 dat, their abundance was statistically similar across all four treatments.
In contrast, with few exceptions, all four genera of herbivorous nematodes were significantly more abundant (p ≤ 0.05) in the control and oxamyl-treated soils than in either EB treatment. The application of EBs resulted in a reduction in plant-parasitic nematode populations.

3.3. Comparison of Ecological Indices and Metabolic Footprints Shannon Diversity Index

The application of EBs increased the abundance of fungivorous (Aphelenchus and Aphelenchoides) and bacterivorous (Cephalobidae and Rhabditidae) trophic groups while reducing plant-parasitic nematodes. The predominance of certain taxa and the decline of others influenced the diversity following amendment application. This effect is reflected in the decrease in the soil nematofauna diversity with both EBs (p ≤ 0.0001) compared to the control and the chemical treatment (Figure 3A,B). Overall, this trend remained consistent across all time points (30, 60, 90, and 120 dat) and in both experiments (Figure 3C,D).
The average maturity index did not exceed two points in any of the experiments, treatments, or sampling times. This could indicate a recovery phase following the disturbance experienced by the substrate from the banana plantation until its harvest and establishment in both experiments. Alternatively, it may reflect a temporary increase in the availability of assimilable nutrients for all plants (Figure 4). Initially, this greater nutrient availability was observed in the control and in the oxamyl treatment (p = 0.0148 in Experiment 1 and p = 0.0482 in Experiment 2) compared to the EBs. However, between 90 and 120 dat, this difference diminished (p = 0.0615 and p = 0.3059) across these treatments for both experiments (Figure 5).
In the first experiment, no significant differences were found between treatments (p = 0.0698). In the second experiment, however, a difference was observed between the control and EBSS, with EBSS presenting a higher channel index (p = 0.0273). The other treatments showed similar values for this variable. Overall, no variation in this index was detected over time across treatments in either experiment. This trend was particularly evident between 60 and 90 dat, where all treatments exhibited statistically similar results (p = 0.1737 in Experiment 1 and p = 0.3407 in Experiment 2).
All treatments in both experiments exhibited a basal index above 50% (Figure 4). In addition to the initial disturbance of the experiment soil, this index reflects a depleted and deteriorated soil food web, which is characteristic of intensively managed soils, such as those in commercial banana plantations. However, in both experiments, the control and oxamyl treatments showed a higher basal index than the two EB treatments (p = 0.0014 in Experiment 1 and p = 0.0044 in Experiment 2). Over time, the variation in this index displayed erratic behavior, making it difficult to identify a clear trend in this parameter (Figure 5). Additionally, all treatments in both experiments exhibited intermediate enrichment indices (Figure 4). On average, the two EB treatments had higher enrichment indices than the control and the oxamyl treatment in both experiments (p = 0.0016 in experiment 1 and p = 0.0182 in experiment 2). This trend remained consistent over time in both experiments (Figure 5).
On average, EBs presented a higher enrichment index (p = 0.0433) and fungivore (p ≤ 0.0001) and bacterivore (p ≤ 0.0190) footprints compared to the control and oxamyl in both experiments (Figure 5). In contrast, the control and the oxamyl treatments showed a higher herbivory footprint compared to EBs in the first experiment (p = 0.0015), although this trend was not significant in Experiment 2 (p = 0.2867). The differences in the metabolic enrichment (p = 0.0702) and bacterivores footprint (p = 0.0636) between EBs and the other treatments decreased between 90 and 120 dat (Figure 5). Meanwhile, the herbivory and fungivore footprints demonstrated erratic behavior over time in both experiments. Regarding herbivory footprint, the differences between treatments diminished from 120 dat in the first experiment (p = 0.2522), whereas in the second experiment, they remained significant (p = 0.0010). The opposite pattern was observed with the fungivore footprint: differences between treatments remained unchanged at 120 dat in the first experiment (p = 0.0001), but completely disappeared in the second experiment (p = 0.5457) (Figure 5).

3.4. Comparison of Biometric Variables of the Root and Population of Radopholus similis

EBCS and BBSS exhibited a growth-promoting effect and effectively suppressed plant-parasitic nematodes. This was reflected in the increased biometric variables compared to the control in both experiments: Plant height increased by 47% in both treatments (p = 0.0008) in Experiment 1 and by 28 and 38%, respectively (p ≤ 0.0001), in Experiment 2. Pseudostem diameter increased by 35% in both treatments (p ≤ 0.0001) in Experiment 1 and by 29% and 39%, respectively (p ≤ 0.0001), in Experiment 2. Pseudostem fresh weight increased by 92% and 93% (p ≤ 0.0001) in Experiment 1 and by 91% and 116% (p ≤ 0.0001) in Experiment 2. Foliage fresh weight increased by 71% and 67% (p = 0.0154) in Experiment 1 and by 82% and 113% (p ≤ 0.0001) in Experiment 2. Pseudostem dry weight increased by 55% and 60% (p ≤ 0.0001) in Experiment 1 and by 63% and 80% (p = 0.0025) in Experiment 2. Foliage dry weight increased by 53% and 50% (p = 0.0002) in Experiment 1 and by 55% and 90% (p ≤ 0.0001) in Experiment 2. Furthermore, both EBs significantly reduced R. similis levels compared to the absolute control by 99 and 90% (p ≤ 0.0001) in Experiment 1 and by 99 and 99% (p≤ 0.0001) in Experiment 2 (Figure 6). Additionally, a significant difference in root fresh weight was observed between the control plants and EBs in Experiment 1 (p ≤ 0.0001). However, in Experiment 2, no differences were found between treatments (p = 0.3111).
Both EBs significantly increased two biometric variables compared to the oxamyl treatment in both experiments: Pseudostem fresh weight increased by 40% and 38% (p ≤ 0.0001) in the first experiment and by 25% and 41% (p ≤ 0.0001) in the second experiment; and Foliage fresh weight increased by 39% and 36% (p = 0.0154) in the first experiment and by 24% and 45% (p ≤ 0.0001) in the second experiment (Figure 6).

4. Discussion

In particular, the EBs were made of various raw materials rich in sugars, nutrients, and carbohydrates, which microorganisms can transform for their growth, as constituents or as by-products of digestion or fermentation, such as polysaccharides, oligosaccharides, amino acids, and various other nutrients [10]. Additionally, volatile organic compounds, including ethers, alcohols, ketones, and organic acids, are also synthesized during silage formation [41,42]. If present in the EBs, these compounds could cause toxicity to herbivorous or phytoparasitic nematodes. Furthermore, the oxamyl treatment showed a high percentage of bacterivores, which could be linked to an increase in bacterial populations. These bacteria likely utilized the by-products of oxamyl biodegradation, along with other substrate nutrients, for biomass synthesis [43].
The genera Aphelenchus and Aphelenchoides were the most abundant fungivorous taxa in soil containing EBs. A channel index greater than 50% indicates that organic matter decomposition is primarily driven by fungal activity rather than bacterial activity [44]. Based on this abundance and the obtained channel index, the decomposition and carbon release of the EBs favored fungal growth more than bacterial growth. The resulting fungal population was sufficient to sustain abundant populations of fungivorous nematodes in the substrates throughout the experiment. It is common to find c-p1 nematodes in labile organic matter with a low C/N ratio, such as in EBs; however, c-p2 nematodes, including Aphelenchus and Aphelenchoides, were predominant. This contrasts with the findings of [45,46], who detected c-p2 nematodes in recalcitrant organic matter. At the start of the experiment, bacterivorous nematodes (Cephalobidae and Rhabditidae) also maintained high populations in EB treatments, but these gradually declined over time. This decline may be attributed to the lower concentration of nutrients and prebiotic compounds favorable to bacterial growth compared to those supporting fungi. This trend aligns with the high availability of food (e.g., labile organic carbon), as expressed in the enrichment index proposed by [44], which was higher in the soil mixed with EBs. Moreover, soil treated with EBs exhibited a greater metabolic footprint for both fungivorous and bacterivorous nematodes, indicating an increased accumulation of carbon by these functional groups [44].
Although few omnivorous and predatory nematodes were found in the initial soil before mixing with EBs, they were generally absent across all treatments. This absence may be linked to the initial substrate management before mixing and the depleted conditions of the experiment soil, which was collected from a commercial banana plantation subjected to intensive management. Typically, these groups of nematodes (omnivores and predators) are highly sensitive to soil disturbances [27,32]. The basal index confirmed the exhausted state of the experiment soil, which persisted to varying degrees across all treatments. This absence of c-p4 and c-p5 nematodes in the present study contrasts with findings by [47], who, in evaluations of banana-producing farms in Costa Rica, recorded up to 7% omnivorous nematodes and 2% predators. The same study reported 60% of herbivores, a finding that aligns with the present study. Both omnivores and predators are highly sensitive to environmental disturbances that impact their reproduction and negatively alter their habitat [44]. The pot-based design experiment imposes some artificial constraints, such as possible effects on the most sensible nematode groups, limited soil volume, UV shielding, etc.; however, it is also a way to normalize the diversity and densities of nematodes among treatments and perfect the conditions the experiment is performed under. Field conditions could have some minor differences in comparison to pot-based experiment that could be explored in future in a more detailed experiment.
According to the food web profile graph established by [32], a high diversity of nematofauna is associated with homeostasis (maintaining the balance of soil structure and function from an ecological perspective), particularly when nematodes representing all survival strategies (c-p1 to c-p5) are present. This diversity is also linked to soil fertility and suppressiveness of soil pathogens. However, in the present evaluation, EBs let to a reduction in the diversity of the nematofauna, likely due to two main factors: (i) the proliferation of a few taxa reliant on fungal and bacterial growth, and (ii) the significant decline in herbivorous nematofauna, including R. similis. Despite this decrease in diversity, as confirmed by the maturity index, the rise in fungivorous and bacterivorous nematode taxa suggests that EBs enhanced nutrient availability and promoted prebiotic growth. Additionally, EBs induce organic matter and possible changes in soil texture that could enhance the increase in energy and nutrient flow to the system. Similarly, the decline in both ectoparasitic and endoparasitic herbivorous nematodes highlights the suppressive effect of EBs. This was further supported by the observed increase in foliage and pseudostem biomass in vitro, along with the suppression of R. similis in the root.
The EBs showed a consistent suppressive effect on nematodes and growth promoters, including the banana endoparasite R. similis, which is particularly challenging to control due to its migratory endoparasite nature [48]. Several mechanisms may be associated with these effects, including the following: (i) The synthesis of nematotoxic by-products and metabolites by the rhizospheric microbiota, derived from EBs components such as sugars, amino acids, enzymes, cellulose, lignin, and organic acids. These metabolites may have exerted a suppressive effect on R. similis directly within the intraradical symplast or apoplast, or externally on the limited remaining inoculum surviving outside the root [49,50,51]. (ii) The activation of plant defense mechanisms through elicitors biologically synthesized in the rhizosphere as a result of EB application [52,53]. (iii) The promotion of microbiota capable of producing phytohormones, phosphorus, and potassium solubilizers, as well as free-living nitrogen fixers with biofertilizer or biostimulant properties [54]. The rhizospheric microbiota responsible for these effects may originate from either the soil or the EBs, or result from joint interactions between both sources at different levels.

5. Conclusions

In conclusion, EBs modified the flow of carbon, nutrients, and energy, influencing the abundance of fungivorous and bacterivorous taxa while consistently reducing the population of plant-parasitic nematodes throughout the experimental period. As a result, fungivorous and bacterivorous taxa became predominant in the experimental soil. The initial decline in nematofauna diversity could, in the long term, contribute to enhanced soil structure, functionality, plant parasite suppression, and nutrient availability due to the presence of the prebiotics and suppressive compounds in the EBs. Although this study was conducted in pots (introducing additional stress to the native nematofauna and with a limited volume of soil), the study of the temporal evolution of the basal population allowed for the assessment of soil stress, exhaustion, and degradation of the experimental soil, which originated from a commercial banana plantation subjected to intensive management. EBs played a significant role in increasing nutrient enrichment and suppressing plant-parasitic nematodes in a conductive soil environment. They could serve as a valuable component in strategies aimed at enhancing soil resilience, fertility, and natural suppression, provided that more sustainable production practices are implemented.

Author Contributions

Conceptualization, P.E.T.-A. and J.E.P.-R.; methodology, P.E.T.-A., I.V.-B., A.M.C., F.E.-B., F.B. and J.E.P.-R.; formal analysis, P.E.T.-A., I.V.-B., A.M.C., F.E.-B., F.B. and J.E.P.-R.; writing—review and editing, P.E.T.-A., I.V.-B., A.M.C., F.E.-B., F.B. and J.E.P.-R.; supervision, I.V.-B., A.M.C., F.E.-B., F.B. and J.E.P.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded with the support of the National Banana Corporation (CORBANA) of Costa Rica.

Data Availability Statement

The data supporting the findings of this study are not publicly available due to privacy restrictions.

Acknowledgments

This research is part of a study in the Natural Sciences for Development (DOCINADE) program of the first author. We are also grateful to DOCINADE and CORBANA S.A. for their support in carrying out this research project.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Mean number of nematodes per trophic group of nematofauna present in conductive soil from a commercial banana plantation, placed in pots with in vitro banana seedlings. Bacterivores (A,B), fungivores (C,D), herbivores (E,F), omnivores (G,H) and predators (I,J). This variable was determined in duplicate in June 2021 (1) and October 2022 (2). The evaluations were conducted at 0, 30, 60, 90 and 120 days after transplant. The treatments were as follows: Unapplied soil; T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCS) 320 g of DM/plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Bars sharing the same letter within each trophic group and treatment across sampling days are not significantly different (Tukey test, p = 0.05).
Figure 1. Mean number of nematodes per trophic group of nematofauna present in conductive soil from a commercial banana plantation, placed in pots with in vitro banana seedlings. Bacterivores (A,B), fungivores (C,D), herbivores (E,F), omnivores (G,H) and predators (I,J). This variable was determined in duplicate in June 2021 (1) and October 2022 (2). The evaluations were conducted at 0, 30, 60, 90 and 120 days after transplant. The treatments were as follows: Unapplied soil; T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCS) 320 g of DM/plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Bars sharing the same letter within each trophic group and treatment across sampling days are not significantly different (Tukey test, p = 0.05).
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Figure 2. Comparison of the average of the eight most abundant nematode taxa in the conductive soil of a commercial banana plantation, placed in pots with in vitro banana seedlings during assessments carried out at 30 (A,B), 60 (C,D), 90 (E,F), and 120 (G,H) days after transplant. The variables were determined in duplicate in June 2021 (Experiment 1) and October 2022 (Experiment 2). The treatments compared in terms of abundance were as follows: T1: Untreated control; T2 Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCS) 320 g of DM/plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Bars with the same letter within each taxon are statistically equal according to the significance level (p = 0.05) of the Tukey test.
Figure 2. Comparison of the average of the eight most abundant nematode taxa in the conductive soil of a commercial banana plantation, placed in pots with in vitro banana seedlings during assessments carried out at 30 (A,B), 60 (C,D), 90 (E,F), and 120 (G,H) days after transplant. The variables were determined in duplicate in June 2021 (Experiment 1) and October 2022 (Experiment 2). The treatments compared in terms of abundance were as follows: T1: Untreated control; T2 Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCS) 320 g of DM/plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Bars with the same letter within each taxon are statistically equal according to the significance level (p = 0.05) of the Tukey test.
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Figure 3. Averages of the Shannon index determined on the nematofauna of a conductive soil of a commercial banana plantation, placed in pots with in vitro banana seedlings. This variable was determined in duplicate in June 2021 (Experiment 1) and October 2022 (Experiment 2). (A,B) General average of the Shannon index. (C,D) Average of the Shannon index of the evaluations carried out at 30, 60, 90, and 120 days after transplant. The treatments were: T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCSS) 320 g of DM/plant; and T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Bars with the same letter on each sampling day are statistically equal according to the Tukey test (p = 0.05).
Figure 3. Averages of the Shannon index determined on the nematofauna of a conductive soil of a commercial banana plantation, placed in pots with in vitro banana seedlings. This variable was determined in duplicate in June 2021 (Experiment 1) and October 2022 (Experiment 2). (A,B) General average of the Shannon index. (C,D) Average of the Shannon index of the evaluations carried out at 30, 60, 90, and 120 days after transplant. The treatments were: T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCSS) 320 g of DM/plant; and T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Bars with the same letter on each sampling day are statistically equal according to the Tukey test (p = 0.05).
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Figure 4. Average metabolic indices and fingerprints of nematofauna in conductive soil from a commercial banana plantation, placed in pots with in vitro banana seedlings. The evaluation was conducted in duplicate: Experiment 1 (June 2021) and Experiment 2 (October 2022). The treatments compared were as follows: T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i. per plant); T3: Ensiled biostimulant with conductive soil EBCSS) 320 g of dry matter (DM) per plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM per plant. Boxes with the same letter within each variable indicate statistically similarity according to the Tukey test (p = 0.05).
Figure 4. Average metabolic indices and fingerprints of nematofauna in conductive soil from a commercial banana plantation, placed in pots with in vitro banana seedlings. The evaluation was conducted in duplicate: Experiment 1 (June 2021) and Experiment 2 (October 2022). The treatments compared were as follows: T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i. per plant); T3: Ensiled biostimulant with conductive soil EBCSS) 320 g of dry matter (DM) per plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM per plant. Boxes with the same letter within each variable indicate statistically similarity according to the Tukey test (p = 0.05).
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Figure 5. Average of metabolic indices and footprints of nematofauna in conductive soil from a commercial banana plantation, placed in pots with in vitro banana seedlings. The evaluation was conducted in duplicate: Experiment 1 (June 2021) and Experiment 2 (October 2022). The treatments compared were as follows: T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i. per plant); T3: Ensiled biostimulant with conductive soil EBCSS) 320 g of dry matter (DM) per plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM per plant. Boxes with the same letter within each variable indicate statistically similarity according to the Tukey test (p = 0.05).
Figure 5. Average of metabolic indices and footprints of nematofauna in conductive soil from a commercial banana plantation, placed in pots with in vitro banana seedlings. The evaluation was conducted in duplicate: Experiment 1 (June 2021) and Experiment 2 (October 2022). The treatments compared were as follows: T1: Untreated control; T2: Oxamyl nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i. per plant); T3: Ensiled biostimulant with conductive soil EBCSS) 320 g of dry matter (DM) per plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM per plant. Boxes with the same letter within each variable indicate statistically similarity according to the Tukey test (p = 0.05).
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Figure 6. Averages of biometric parameters and R. similis counts determined in in vitro banana plants grown in pots containing conductive soil from a commercial banana plantation. The treatments applied were: T1: Untreated control; T2: Oxamyl nematicide (24% active ingredient (a.i.)) in liquid suspension (LS) applied at 400 cc with 100 ppm a.i. per plant); T3: Ensiled biostimulant with conductive soil (EBCS) applied at 320 g of dry matter (DM) per plant; and T4: Ensiled biostimulant with suppressive soil (EBSS) applied at 320 g of DM per plant. Evaluations were conducted 120 days after transplanting, during the harvest of the experiment. Bars with the same letter within each variable indicate statistical similarity according to the Tukey test (p = 0.05).
Figure 6. Averages of biometric parameters and R. similis counts determined in in vitro banana plants grown in pots containing conductive soil from a commercial banana plantation. The treatments applied were: T1: Untreated control; T2: Oxamyl nematicide (24% active ingredient (a.i.)) in liquid suspension (LS) applied at 400 cc with 100 ppm a.i. per plant); T3: Ensiled biostimulant with conductive soil (EBCS) applied at 320 g of dry matter (DM) per plant; and T4: Ensiled biostimulant with suppressive soil (EBSS) applied at 320 g of DM per plant. Evaluations were conducted 120 days after transplanting, during the harvest of the experiment. Bars with the same letter within each variable indicate statistical similarity according to the Tukey test (p = 0.05).
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Table 1. Averages of nematodes identified during the June 2021 (1) and October 2022 (2) assessments. The experiments were set up in pots with conductive soil from a commercial banana farm. Samples from repeated measurements per pot were collected at 0, 30, 60, 90, and 120 days after transplant for each treatment.
Table 1. Averages of nematodes identified during the June 2021 (1) and October 2022 (2) assessments. The experiments were set up in pots with conductive soil from a commercial banana farm. Samples from repeated measurements per pot were collected at 0, 30, 60, 90, and 120 days after transplant for each treatment.
TreatmentsBasal Soil Condition1. Control2. Oxamyl3. EBCS4. EBSS
Experiment 1
Family or genus		Trophic group 1c-pp-p0306090120306090120306090120306090120
AphelenchoidesM2 04413531392027210143731081622851392898769
AphelenchusM2 0358131111920308341171146092125311273502429
AporcelaimidaeO5 40000000000000000
CephalobidaeB2 23591582742823021323611237116614036083294750425254
CriconematidaeH 36491153408287871311568011230
DiplogastridaeB1 30000450002603800026900
DiploscapterB1 00100526114102177801133621
DorylaimidaeO4 00002212000000800
FilenchusM2 82822767200000800
GracilacusH 202020000000000000
HelicotylenchusH 3957089172437245000016030
HoplolaimidaeH 3614872054548514911000900200
IotonchusD4 41402000000000060
MeloidogyneH 3068614018258419311511610263618
MonhysteridaeB2 00100000000000000
PanagrolaimidaeB1 001002020000000674
ParatylenchusH 210000000000000000
PseudacrobelesB2 10000000000000000
PsilenchusH 20017001610000001000
RadopholusH 3250333201119000130003
RhabditidaeB1 40313536188402471232334196536860
ScutellonemaH 330020000000000000
TylenchusH 2200001010000035030
SteinernematidaeB1 0000001000650002200
TylenchydaeH 20323131641552050001616
XiphinemaH 510000000000000000
Experiment 2
Family or genus		Trophic group 1c-pp-p0306090120306090120306090120306090120
AphelenchoidesM2 04211122127178936108357012951835711682887
AphelenchusM2 21382610385520362910,20130051341208716,39618941516806
CephalobidaeB2 28172103150889710313510712867413472011714568342119
CriconematidaeH 3001011100119102206741115039
DiploscapterB1 025559289185332458021121273688
DorylaimidaeO4 061024000070001100
FilenchusM2 211215101049000600030
HelicotylenchusH 3779207312971422071200400
HoplolaimidaeH 3437221134421612310704271803
IotonchusD4 09500700100000000
LongidoridaeH 500000000100000000
MacrolaimellusB2 20000000000000000
MeloidogyneH 33108657265264208980033102
MononchidaeD4 20000000000000000
PanagrolaimidaeB 1000000000014000000
ParatylenchusH 230000000000000000
PsilenchusH 2041011532000000000
RadopholusH 37222332136000170400
RhabditidaeB1 03417391511756306704211701367156132133
ScutellonemaH 3120000000000000000
TylenchusH 2100002003000000002
TylenchydaeH 206110021871502114119015
1 Trophic group: bacterivores (B), predators (D), fungivores (M), herbivores (H), and omnivores (O). The survival strategy of free-living nematodes is referred to as c-p, while that of herbivores or phytoparasites is referred to as p-p. The treatments were as follows: T1: Untreated control; T2: Oxamil nematicide 24% a.i. in liquid suspension (LS) (400 cc with 100 ppm a.i./plant); T3: Ensiled biostimulant with conductive soil (EBCS) 320 g of DM/plant; T4: Ensiled biostimulant with suppressive soil (EBSS) 320 g of DM/plant. Sampling after transplanting.
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Torres-Asuaje, P.E.; Varela-Benavides, I.; Cotes, A.M.; Echeverría-Beirute, F.; Blanco, F.; Palomares-Rius, J.E. Impacts of Silage Biostimulants on Nematofauna in Banana Crop Soils: A Sustainable Alternative to Nematicides. Agronomy 2025, 15, 1860. https://doi.org/10.3390/agronomy15081860

AMA Style

Torres-Asuaje PE, Varela-Benavides I, Cotes AM, Echeverría-Beirute F, Blanco F, Palomares-Rius JE. Impacts of Silage Biostimulants on Nematofauna in Banana Crop Soils: A Sustainable Alternative to Nematicides. Agronomy. 2025; 15(8):1860. https://doi.org/10.3390/agronomy15081860

Chicago/Turabian Style

Torres-Asuaje, Pedro E., Ingrid Varela-Benavides, Alba M. Cotes, Fabián Echeverría-Beirute, Fabio Blanco, and Juan E. Palomares-Rius. 2025. "Impacts of Silage Biostimulants on Nematofauna in Banana Crop Soils: A Sustainable Alternative to Nematicides" Agronomy 15, no. 8: 1860. https://doi.org/10.3390/agronomy15081860

APA Style

Torres-Asuaje, P. E., Varela-Benavides, I., Cotes, A. M., Echeverría-Beirute, F., Blanco, F., & Palomares-Rius, J. E. (2025). Impacts of Silage Biostimulants on Nematofauna in Banana Crop Soils: A Sustainable Alternative to Nematicides. Agronomy, 15(8), 1860. https://doi.org/10.3390/agronomy15081860

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