<figure><img src="image_1.png" /></figure>

# Earthworms enhance the performance of organic amendments in improving rice growth and nutrition in poor ferralsols

O. Ratsiatosika<sup>a</sup>, J. Trap<sup>b</sup>, V. Herinasandratra<sup>a</sup>, T. Razafimbelo<sup>a</sup>, L. Bernard<sup>b</sup>, E. Blanchart<sup>b,*</sup>

## ARTICLE INFO

**Keywords:**

P nutrition

Pontoscolex corethrurus

Biochemical quality

Ferralsols

Poultry droppings

Pig manure

## ABSTRACT

In the Highlands of Madagascar, providing organic amendments (OAs) is the main farmer fertilization practice in upland rainfed agrosystems dominated by nutrient-depleted Ferralsols. Therefore, enhancing the performance of OAs on plant functions is particularly important for improving both plant productivity and agrosystem sustainability. Earthworms are well-known ecosystem engineers involved in the decomposition of organic matter and play a critical role in nutrient turnover. Inoculating earthworms could be a promising approach to improve the fertilizer performance of OAs. However, there is limited knowledge regarding the interaction between earthworms and OAs of varying biochemical qualities on plant growth and nutrition. In this microcosm experiment, we assessed the effect of different types of OAs (i.e., unamended control, conventional farmyard manure, kraal manure, improved farmyard manure, compost, vermicompost, urban waste, taroka, poultry droppings, pig manure) and earthworm inoculation (with and without earthworms) on the growth and nutrition of rice. The endogeic species *Pontoscolex corethrurus* (Rhinodrilidae) was used in the experiment. After eight weeks of growth, the poultry droppings had the best fertilizer performance, followed by improved farmyard manure and pig manure in treatments without earthworms. The main driver of fertilizer performance appeared to be the quality of OAs. However, other mechanisms, such as an increase in soil pH, may also play a significant role. The inoculation of earthworms significantly enhanced the fertilizer performance of OAs but the magnitude of this enhancement depends on the OAs. Overall, OAs with low fertilizer performance showed a more pronounced response to earthworms. Additionally, earthworms improved the performance of high-quality OAs likely by enhancing the availability of P and Mg. Our findings emphasize the synergistic effects of earthworms and OAs of various qualities on plant growth and nutrition, offering valuable insights for improving fertilization practices in nutrient-poor Ferralsols of Madagascar.

## 1. Introduction

The detrimental impacts of the industrial model of agriculture on soil, water, air, biodiversity, and human health have given rise to the agroecological transition which is based on the intensification of ecological processes in agrosystems (Bommarco et al., 2013; Duru et al., 2015). The shift toward this transition implies the need of relying more on natural functions, biotic interactions, and ecological processes, to increase biodiversity and complexity within agrosystems while amplifying the services provided by living organisms (Altieri, 1999; Barrios, 2007), with continuous involvement of farmers (Trap and Blanchart 2023). For such an agroecological transition, numerous practices that

optimize aboveground functional biodiversity are proposed while relatively little attention has been given to practices that drive the intensification of belowground biodiversity and processes. However, soil organisms interact in intricate networks to deliver key functions at the basis of ecosystem services: (i) soil organic matter dynamics, (ii) nutrient cycling, (iii) maintenance of soil structure, and (iv) pest regulation (Brussaard et al., 2007; Kibblewhite et al., 2008). Plant functions are directly or indirectly affected by these soil ecological processes (De Deyn et al., 2004). Consequently, there is an urgent challenge to design agricultural practices aimed at intensifying soil ecological functions (Barrios, 2007; De Vries et al., 2013).

The inoculation of soil-plant mutualists (referred to as

<footnote><sup>a</sup> Laboratoire des Radio-Isotopes, University of Antananarivo, BP 3383, Route d'Andraisoro, 101 Antananarivo, Madagascar</footnote>

<footnote><sup>b</sup> Eco&Sols, Univ Montpellier, CIRAD, INRAe, IRD, Institut Agro, 2 Place Viala, 34060 Montpellier, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: onja.ratsiatosika@gmail.com (O. Ratsiatosika), jean.trap@ird.fr (J. Trap), vh.nayash@gmail.com (V. Herinasandratra), tantely.razafimbelo@gmail.com (T. Razafimbelo), laetitia.bernard@ird.fr (L. Bernard), eric.blanchart@ird.fr (E. Blanchart).</footnote>
biofertilization) emerges as a potential solution to address this issue. However, this approach is still underdeveloped, except for microorganisms considered beneficial, such as plant growth promoting rhizobacteria or mycorrhizal fungi. Among soil organisms, earthworms are well-known as ecosystem engineers involved in many soil functions and impacting plant growth through five mechanisms: (i) increased mineralization of soil organic matter (Coq et al., 2007; Bernard et al., 2012); (ii) production of plant growth substances via stimulation of microbial activity (Blouin, 2018); (iii) control of pests and parasites (Loranger-Merciris et al., 2012; Blanchart et al., 2020); (iv) stimulation of symbionts (Huhta, 2007) and (v) modifications of soil porosity, aggregation and water retention (Blanchart et al., 1999). In degraded agrosystems, the inoculation of earthworms can thus be crucial to improve the soil functions and to increase ecosystem services (Ratsiatosika and Blanchart, 2021).

In the Highlands of Madagascar, crops are mostly cultivated on Ferralsols in upland agrosystems. Ferralsols have multiple nutrient deficiencies (especially P, but also N, Ca and Mg) which represent a major constraint for agricultural productivity and sustainability (Raminoarison et al., 2020). Adequate quantities of mineral fertilizers could alleviate nutrient deficiencies but smallholder farmers lack financial means and have low access to mineral inputs. In 2018, it was estimated that the rates of annual fertilizer application on cropland in Madagascar by farmers were as low as 8.0 kg N ha⁻¹, 1.3 kg P₂O₅ ha⁻¹ and 1.2 kg K₂O ha⁻¹ (FAO, 2020). As a result, farmers rely on organic amendments (OAs) as their current fertilization practices to increase crop yield. Traditionally, soil is annually and manually tilled, and OAs are mixed with soil in the seed holes.

Earthworms are widely recognized for their ability to enhance the decomposition of organic matter and play a critical role in nutrient turnover (Lavelle and Spain, 2001). Inoculating earthworms could therefore be a key solution to improve the decomposition of OAs and their effectiveness in enhancing soil nutrient availability. As shown by Brown et al. (2004), the effect of earthworms on the growth of a plant is more pronounced when the soil is of poor quality (in terms of texture, nutrient content and amount of organic matter) than when the soil is of good quality. Similarly, in a meta-analysis, van Groenigen et al. (2014) noted that the contribution of earthworms on plant growth decreases when the nitrogen content in the soil increases after fertilizer application. They also note that the effect of earthworms is stronger after an addition of organic fertilizer than after an addition of inorganic fertilizer. Moreover, the application of OAs in agroecosystems is crucial for the success of earthworm inoculation (Bertrand et al., 2015). Indeed, OAs provide favorable conditions for earthworm communities as organic matter serves as food source for these animals. In cultivated soils of the Madagascar Highlands, earthworm densities vary from 30 ind m⁻² in traditional plowed situations up to 150 ind m⁻² under agroecological practices (conservation agriculture or OM inputs) (Rakotomanga et al., 2016; Ratsiatosika et al., 2021).

The ecological intensification using both biofertilizer (earthworm) and OAs in the upland rice systems in the Highlands of Madagascar is thus of great interest to address the issue of multiple nutrient deficiencies of Ferralsols and to increase crop yield and sustainability. A wide range of OAs from farms (such as manures, animal droppings, household fermentable composts), or commercial products (vermicompost, compost) are available in the Malagasy highlands (Raminoarison et al., 2022). Nevertheless, these authors reported a strong variability in chemical and biochemical composition of these OAs. However, there is a notable gap in scientific knowledge concerning the effect of the interaction between OAs of varying qualities and earthworms on plant performance. To address these challenges, we conducted an eight-week laboratory experiment to test the effect of earthworms on the performance of nine different OAs on rice growth and nutrition. Our study aimed (i) to examine which OA was more effective in improving rice growth in Ferralsols with multiple nutrient deficiencies, (ii) to investigate whether the inoculation of earthworms improve the fertilizing

performance of OAs and (iii) to analyze if the plant response to earthworms depends on the quality of OAs, i.e. their chemical and biochemical characteristics. We hypothesized that (i) the inputs of OAs should increase rice growth; the magnitude of the increase will be greater for OAs of high quality than of low quality (ii) the inoculation of earthworms may increase rice growth and nutrition, (iii) the positive effects of earthworm inoculation are dependent upon the quality of OAs, with the assumption that the relative effect of earthworm would be larger with OAs of lower quality.

## 2. Materials and methods

### 2.1. Soil type and sampling

The upper 10 cm layer of a Ferralsol (FAO classification) under a natural grassland was collected at a site near of Imerintsiatosika, 40 km West of Antananarivo (19°03'14.3"S 47°15'24.5"E, 1400 m altitude, Madagascar). The soil is characterized by a sandy-clay texture (40% clay, 20% silt and 40% sand), an acidic pH (water) of 4.7, a total organic C content of 29.2 g kg⁻¹. The total iron and aluminum oxide contents are high (31.4% Fe₂O₃ and 28.2% Al₂O₃) while those of silica are low (10% SiO₂). The total soil N and available (resin) P contents are 1.30 g kg⁻¹ and 1.31 mg kg⁻¹, respectively. The soil sample was air-dried until constant weight, gently hand-crushed, mixed thoroughly and sieved at 2 mm before use. Most of the roots and vegetation debris were removed.

### 2.2. Biological and organic materials

We used the endogeic earthworm Pontoscolex corethrurus (Rhino-drilidae) which is the most abundant earthworm species in the agricultural areas in the Highlands of Madagascar (Razafindrakoto et al., 2010). The inoculation of this species in field conditions has been recently shown to affect positively different soil functions and agro-system services (Ratsiatosika et al., 2021). Individuals of *P. corethrurus* were sampled near the experimental site of Imerintsiatosika and were stored in the same fresh soil before use. The rice cultivar Chhomrong dhan which is the most cropped cultivars in the Imerintsiatosika region was used in the present study.

Nine OAs of different quality were collected from both farmers (conventional farmyard manure, kraal manure, improved farmyard manure, poultry droppings, pig manure) and commercial suppliers (compost, vermicompost, urban waste and industrial organic waste with bacterial leaven constituents, locally called 'taroka'). These OAs were chosen as they were among the available organic resources in the Highlands of Madagascar (Raminoarison et al., 2022). Chemical and biochemical properties of each OA are given in Table 1. Description and origin of OAs are detailed in Table S1.

### 2.3. Experimental design

The experiment was carried out in January–March 2019 at the Radio-Isotopes Laboratory (LRI) of the University of Antananarivo (Madagascar). Two independent factors were tested in this experiment: (i) the inoculation of earthworms with two modalities: without earthworms (EW-) and with earthworms (EW+), and (ii) the inputs of OAs with ten modalities: unamended control (C), conventional farmyard manure (CFM), kraal manure (KM), improved farmyard manure (IFM), compost (COM), vermicompost (VCT), urban waste (UW), taroka (TK), poultry droppings (PD) and pig manure (PIM). We set up 20 treatments and each was replicated 3 times to give a total of 60 experimental units.

### 2.4. Microcosm set-up

The experiment was conducted in microcosms consisting of 1 L plastic pots. The bottoms of the microcosms were first perforated with two holes allowing water drainage and then, they were covered with a
<caption>**Table 1**</caption>

Chemical and biochemical characteristics of organic amendments. CFM: Conventional farmyard manure; KM: Kraal manure; IFM: Improved farmyard manure; COM: Compost; VCT: Vermicompost; UW: Urban waste; TK: Taroka; PD: Poultry droppings; PIM: Pig manure.

<table><thead><tr><th>Variables</th><th>Units</th><th>CFM</th><th>KM</th><th>IFM</th><th>COM</th><th>VCT</th><th>UW</th><th>TK</th><th>PIM</th><th>PD</th></tr></thead><tbody><tr><td colspan="11"><strong>Chemical characteristics</strong></td></tr><tr><td>Total C</td><td>g.kg<sup>-1</sup></td><td>195</td><td>141</td><td>220</td><td>166</td><td>231</td><td>113</td><td>72</td><td>241</td><td>298</td></tr><tr><td>Total N</td><td>g.kg<sup>-1</sup></td><td>14.6</td><td>10.3</td><td>16.9</td><td>13.6</td><td>20.0</td><td>6.0</td><td>4.1</td><td>27.4</td><td>21.1</td></tr><tr><td>Total P</td><td>g.kg<sup>-1</sup></td><td>3.5</td><td>1.6</td><td>2.9</td><td>4.7</td><td>3.7</td><td>3.7</td><td>3.8</td><td>29.8</td><td>15.5</td></tr><tr><td>Total K</td><td>g.kg<sup>-1</sup></td><td>36.8</td><td>6.9</td><td>19.9</td><td>5.3</td><td>17.1</td><td>2.6</td><td>4.8</td><td>16.4</td><td>12.0</td></tr><tr><td>Total Ca</td><td>g.kg<sup>-1</sup></td><td>6.7</td><td>3.6</td><td>10.7</td><td>44.0</td><td>7.6</td><td>9.0</td><td>28.3</td><td>14.9</td><td>37.3</td></tr><tr><td>Total Mg</td><td>g.kg<sup>-1</sup></td><td>3.0</td><td>2.6</td><td>3.3</td><td>9.1</td><td>3.8</td><td>1.9</td><td>2.9</td><td>16.9</td><td>4.4</td></tr><tr><td>C:N ratio</td><td>unitless</td><td>13.4</td><td>13.6</td><td>13.0</td><td>12.2</td><td>11.6</td><td>18.8</td><td>17.4</td><td>8.8</td><td>14.1</td></tr><tr><td>C:P ratio</td><td>unitless</td><td>55.7</td><td>87.8</td><td>75.8</td><td>35.4</td><td>62.5</td><td>30.5</td><td>18.8</td><td>8.1</td><td>19.2</td></tr><tr><td>N:P ratio</td><td>unitless</td><td>4.2</td><td>6.4</td><td>5.8</td><td>2.9</td><td>5.4</td><td>1.6</td><td>1.1</td><td>0.9</td><td>1.4</td></tr><tr><td>pH</td><td>unitless</td><td>8.7</td><td>8.7</td><td>9.4</td><td>8.1</td><td>8.1</td><td>7.8</td><td>8.0</td><td>8.2</td><td>6.0</td></tr><tr><td colspan="11"><strong>Biochemical characteristics</strong></td></tr><tr><td>Soluble</td><td>%</td><td>28.9</td><td>13.8</td><td>21.3</td><td>26.7</td><td>24.5</td><td>29.2</td><td>29.6</td><td>61.8</td><td>59.9</td></tr><tr><td>Hemicellulose</td><td>%</td><td>0.7</td><td>11.4</td><td>3.1</td><td>4.7</td><td>6.4</td><td>1.7</td><td>7.4</td><td>12.9</td><td>12.7</td></tr><tr><td>Cellulose</td><td>%</td><td>54.3</td><td>56.2</td><td>55.5</td><td>55.2</td><td>46.3</td><td>59.5</td><td>50.8</td><td>17.2</td><td>19.6</td></tr><tr><td>Lignin</td><td>%</td><td>16.1</td><td>18.6</td><td>20.1</td><td>13.4</td><td>22.9</td><td>9.6</td><td>12.2</td><td>8.1</td><td>7.8</td></tr></tbody></table>

thin mosquito net to prevent earthworms from escaping. Each microcosm was filled with 1 kg of sieved dry soil and moistened with 380 ml of water at the beginning of the experiment to reach field capacity. Bulk density was about 1.1 g cm⁻³, similar to the one of cropped soils that are regularly tilled. Regarding EW + treatments, three adults of *P. corethrurus* were introduced into each pot. This earthworm density (~150 individuals per m²) corresponds to high field density (Rakotomanga et al., 2016). OAs were added in microcosms at an equivalent dose of 6 Mg dry mass ha⁻¹ and mixed with the soil. This amount applied is a common fertilization practice in the Highlands of Madagascar. Three rice seeds were sown in each microcosm. During the experiment, the microcosms were placed outside under natural weather conditions. The soil moisture content was regularly controlled by weighing and irrigating to adjust the soil water content. The spatial placements of the microcosms were randomly rearranged every week. The experiment lasted 8 weeks.

## 2.5. Plant and soil analyses

At the end of the experiment, plants were cut at the soil surface to measure the shoot biomass. The soil was gently removed from the microcosms and both the roots and surviving earthworms were hand-sorted. The earthworm survival rate was calculated by comparing final to initial earthworm abundance. Roots were separated from the adhering soil by carefully washing the root system under flowing water to remove soil particles. The shoot and root biomasses were measured after oven-drying plant tissues at 65 °C for 48 h. The total N contents in shoot were determined by CHNS/O microanalyser (Flash, 2000 Series, CHNS/O 122 Analysers Thermo Scientific, IRCOF, France). Total P contents were also measured in shoot after calcination at 550 °C for 5 h and digestion with nitric acid at 65% (Murphy and Riley, 1962). Then, a colorimetric dosage was realized using molybdenum blue colorimetric method. The aboveground shoot N and P were calculated by multiplying shoot N and P contents by shoot biomass, respectively. We also calculated the aboveground N uptake efficiency (Aboveground NUpTE = [aboveground shoot N] × [Root biomass]⁻¹) and aboveground P uptake efficiency (Aboveground PUpTE = [aboveground shoot P] × [Root biomass]⁻¹) to determine how efficient a plant can uptake N and P from the soil according to treatments (Liao et al., 2008; Ranoarisoa et al., 2020).

## 2.6. Statistical analyses

### 2.6.1. Individual fertilizer performance of OAs

The performance of OAs alone in enhancing plant growth and nutrition was considered to be the individual fertilizer performance of OAs (Ind_P). For each organic amendment, we computed the mean and

the standard deviation (n = 3) of each plant variable in treatments without earthworms. Considering the nine types of OAs (without control), we then scaled their mean values between 0.1 and 1 by using homothetic transformation: Y = 0.1 + ((x - b)/(a - b)) × 0.9, where Y is the value of a variable after transformation, x is the mean of plant variable to transform, a is the maximum mean and b is the minimum mean among the OAs (Velasquez et al., 2007). The Ind_P was computed as the mean of the transformed values of plant variables. A higher Ind_P indicates that the organic amendment is more effective in improving the plant growth and nutrition among the panel of studied OAs.

### 2.6.2. Earthworm ecological performance of OAs

Earthworm ecological performance (Eco_P) was assessed as the plant response to earthworm inoculation in each type of OA. To evaluate the Eco_P, we calculated the cohen's d effect size as following: ES = (Mi(EW+) - Mi(EW-))/(SDi-pooled) for the organic amendment "i", where Mi(EW+) is the mean of the plant variable for the EW + treatment, Mi(EW-) is the mean of the plant variable for the EW- treatment and SD-pooled is the pooled standard deviation of the plant variable. The cohen's d effect size was calculated for all plant variables. We also used a homothetic transformation to scale the cohen's d effect size between 0.1 and 1, with 1 being the highest effect size. The final Eco_P was computed as the mean of cohen's d of each plant variable.

### 2.6.3. Data analyses

Statistical analyses were performed with the R software (4.2.2 version) (Team, 2022). One way-ANOVA was used on plant parameters to test the effect of OAs alone in treatments without earthworms. This allowed to determine the individual fertilizer performance of OAs (Ind_P). Besides, a two-way ANOVA was performed to test the two main effects (Organic Amendments "OA", earthworm inoculation "E") and their interactions on plant growth and nutrition. We calculated the eta-squared (eta²) from the two-way ANOVA in order to compare the proportion of variance explained by each effect (main effect, interaction, residuals) (Brown, 2008). A post hoc Tukey HSD test was performed for multiple mean comparison. We checked the normality and homogeneity of variance of the residuals by using the Shapiro's test and Levene's test, respectively. Data were transformed if necessary to match the prerequisites for analysis of variance (ANOVA). All tests were achieved with a significance level α = 0.05. Additionally, we performed a principal component analysis (PCA) to visualize the relationship between the individual fertilizer performance, the earthworm ecological performance and the quality of OAs. A second PCA was conducted to evaluate the relationship between the cohen's d values of each variable of interest and the chemical and biochemical characteristics of OAs. This allowed to detect organic amendment traits that could explain the direction and
magnitude of the effect of earthworms on plant growth and nutrition. The PCA was performed with the R software by using "FactomineR" package.

# 3. Results

## 3.1. Earthworm survival rates

At the end of the experiment, the introduced earthworms survived in all treatments, with an average survival rate of 89%. We recorded no earthworms in pots that did not receive *P. corethrurus* at the beginning of the experiment. OAs had a significant effect on the earthworm survival rate in EW + treatments (p-value = 0.012). The highest survival rate (178%) was observed in pots receiving kraal manure (KM) while the lowest (44%) was recorded in pots receiving vermicompost (VCT) (Fig. S1).

## 3.2. Plant response to organic amendments

After the 8-week period of growth, the plant biomass and nutrition increased following the application of OAs in treatments without earthworms. Among OAs, the poultry droppings (PD) showed the highest increase of the shoot biomass (×8), the root biomass (×7), the aboveground shoot N (×8), the aboveground shoot P (×49) and the aboveground PUpTE (×7), in comparison to the unamended control. No significant difference was detected between the poultry droppings (PD), improved farmyard manure (IFM) and pig manure (PIM) regarding final rice shoot and root biomasses. However, the increase in aboveground shoot P in soil receiving IFM (×26) and PIM (×21) were significantly lower than the plant growing in soils receiving PD. A similar trend was observed for the aboveground shoot N. We recorded an increase by 5-fold of the aboveground shoot N in both IFM and PIM treatments (Fig. S2).

## 3.3. Plant response to earthworm inoculation

The inoculation of earthworms had a strong influence on all plant variables (Table 2). We found that the presence of earthworms had positive effect on the shoot biomass (+63%), the root biomass (+30%), the aboveground shoot N (+107%), the aboveground shoot P (+93%), the aboveground NUpTE (+63%) and the aboveground PUpTE (+46%), in comparison to treatments without earthworms (Fig. 1). The highest eta² was found on aboveground NUpTE (48%), followed by the above-ground shoot N (31%) and the aboveground shoot P (30%) (Table 2).

**Table 2**

<table><caption>ANOVA table of eta² and p-value showing the main effects of earthworms and organic amendments and their interaction on plant properties.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Organic amendments</th><th colspan="2">Earthworms</th><th colspan="2">Interaction</th><th>Residuals</th></tr><tr><th>Eta²</th><th>p-value</th><th>Eta²</th><th>p-value</th><th>Eta²</th><th>p-value</th><th>Eta²</th></tr></thead><tbody><tr><td>Shoot biomass</td><td>69%</td><td>***</td><td>18%</td><td>***</td><td>5%</td><td>*</td><td>9%</td></tr><tr><td>Root biomass</td><td>71%</td><td>***</td><td>7%</td><td>***</td><td>4%</td><td>ns</td><td>19%</td></tr><tr><td>Shoot N amount</td><td>55%</td><td>***</td><td>31%</td><td>***</td><td>6%</td><td>**</td><td>7%</td></tr><tr><td>Shoot P amount</td><td>31%</td><td>***</td><td>30%</td><td>***</td><td>6%</td><td>ns</td><td>33%</td></tr><tr><td>NUpTE</td><td>27%</td><td>***</td><td>48%</td><td>***</td><td>11%</td><td>**</td><td>14%</td></tr><tr><td>PUpTE</td><td>67%</td><td>***</td><td>10%</td><td>***</td><td>10%</td><td>**</td><td>13%</td></tr></tbody></table>

## 3.4. Crossing effect of OAs and earthworm inoculation on plant biomass and nutrition

The two-way ANOVA showed a significant interactive effect of OA type and earthworm inoculation on the shoot biomass, the aboveground shoot N, the aboveground NUpTE and the aboveground PUpTE (Table 2). Plants responded positively to earthworms but the magnitude of this response varied according to the OA applied (Fig. 2). For instance, the effect size of the shoot biomass ranged from 1.6 (IFM) to 5.9 (PIM). Similarly, the effect size of the aboveground shoot N varied between 2.2 (TK) and 9.7 (PIM). Concerning the aboveground PUpTE, we found a positive effect of earthworms in all treatments, except in IFM (cohen's d = -0.08). The highest effect size of aboveground PUpTE (3.7) was found in CFM treatment (Fig. 2).

Overall, the earthworm ecological performance of OAs (Eco_P) tended to be negatively correlated with the individual fertilizer performance (Ind_P) (Fig. 3), except for PD and PIM. Indeed, the lowest Ind_P was found for UW, VCT and CFM with values of 0.19, 0.25 and 0.27, respectively. However, they had high Eco_P with values of 0.73, 0.55 and 0.49, respectively. Regarding PD and PIM, they also had high Eco_P (0.44 and 0.76, respectively) despite their high Ind_P (0.99 and 0.54, respectively) (Fig. 3).

## 3.5. Individual fertilizer performance, earthworm ecological performance and quality of OA relationships

The PCA performed on chemical and biochemical characteristics of OAs and their individual fertilizer performance and earthworm ecological performance showed a large dispersion of the nine OAs on three dimensions (total inertia = 81.2%). The first two dimensions of the PCA explained 68.9% of the total inertia (Fig. 4). The first dimension (relative inertia = 47.6%) opposed the PD and PIM with high values of individual fertilizer performance, total P, total organic C, total organic N, total Mg, soluble and hemicellulose contents (positive scores) to the other types of OAs with high cellulose content and C:P ratio (negative scores). The second dimension (relative inertia = 21.4%) mainly explained by the total K and lignin (positive scores) was associated to IFM, VCT and CFM. They were opposed to TK and UW which were characterized by high C:N ratio (negative score). The third dimension (relative inertia = 12.3%) was mainly explained by the earthworm ecological performance. The second PCA performed on the quality of OAs and the cohen's d values showed that the first and the second dimensions accounted for 42.1% and 19.3% of the total variance, respectively (Fig. 5). The first dimension opposed PD and PIM which were characterized by high values of cohen's d of the shoot biomass, cohen's d of the root biomass, total P, total Mg and soluble (positive scores) to IFM and KM with high cellulose, lignin and C:P ratio (negative scores). The second dimension was mainly composed by total N (positive score) and C:N ratio (negative score).

# 4. Discussion

## 4.1. Individual fertilizer performance of organic amendments

Unsurprisingly, providing OAs strongly enhanced rice biomass and nutrition in comparison to the unamended control, i.e. an overall increase of the shoot biomass by 2-fold–8-fold was observed when OAs were applied. Poultry droppings (PD), improved farmyard manure (IFM) and pig manure (PIM) exhibited the highest individual fertilizer performance while the lowest values were observed for urban waste (UW), vermicompost (VCT) and conventional farmyard manure (CFM). In a multiple nutrient-omission experiment conducted with the same soil, it has been found that rice growth was strongly limited by P and co-limited by N-Ca-Mg (Raminoarison et al., 2020). Thus, the difference of individual fertilizer performance of OAs could be attributed to their different effect on the soil nutrient availability, which is primarily

<footnote>***(P-value <0.001); ** (P-value <0.01); * (P-value <0.05); ns (not significant).</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (A) Plant (shoot and root) biomass, (B) nutrient amounts (aboveground shoot N and P), and (C) aboveground nutrient uptake efficiencies (NUptE and PUptE) at the end of experiment according to organic amendments and earthworm inoculation. Vertical bars correspond to standard deviation (SD). Asterisks indicate significant effect between earthworm treatments according to student t-test in each organic amendment, (\* P-value <0.05, \*\* P-value <0.01, \*\*\* P-value <0.001). C: unamended control; CFM: Conventional farmyard manure; KM: Kraal manure; IFM: Improved farmyard manure; COM: Compost; VCT: Vermicompost; UW: Urban waste; TK: Taroka; PD: Poultry droppings; PIM: Pig manure EW-: without earthworms; EW+: with earthworms.</figcaption></figure>

driven by their initial elemental composition (Mohanty et al., 2013). Numerous studies reported that the various qualities of organic materials are an essential factor governing the rate of nutrient mineralization in the soil and the subsequent release of available nutrients for the plants (Palm et al., 2001; Vanlauwé et al., 2002; Mohanty et al., 2013). Regarding P availability which is the most limiting nutrient in Ferralsols, an investigation conducted in lowland rice of weathered tropical P-deficient soils showed that OAs with high total P content and low C:P ratio improved the P uptake and the rice shoot biomass (Rinasoa et al., 2022). Our results are in line with this observation since we found that the individual fertilizer performance was positively correlated to the total P content but negatively correlated to the C: P ratio, especially for PD and PIM. Moreover, PD and PIM displayed high contents of total organic N and total Mg, high soluble content and low cellulose and lignin contents. Consequently, PD and PIM are more susceptible to rapid decomposition by soil microorganisms (Mohanty et al., 2013). Thus, the high individual performance of PD and PIM in improving plant growth and nutrition could be linked to their initial high quality overcoming the high P deficiency and the N-Ca-Mg co-limitation of Ferralsols.

Concerning IFM, we did not find clear evidence of a significant relationship between its high individual fertilizer performance and its initial quality. This material is considered as a low-quality OAs according to its low total organic N content, low total P content, high C:P ratio, high cellulose content and high lignin content. However, its application enhanced the shoot biomass (× 26), the shoot P amount (× 26) and the shoot N amount (× 5) in comparison to the unamended control. This finding suggests that the initial quality of OAs was not necessarily the sole factor explaining the performance of OAs in improving plant growth and nutrition. Besides multiple nutrient

deficiencies, aluminum toxicity caused by high soil acidity and high iron (Fe) and aluminum (Al) oxyhydroxides is an important constraint limiting plant growth in weathered soils (Halder et al., 2021; Haynes and Mokolobate, 2001). Indeed, Al and Fe oxyhydroxides have high potential in adsorbing P onto their surfaces, thus, strongly decreasing P availability (Halder et al., 2021). However, the rice cultivar Chhomrong Dhan used in the present experiment was found as the most sensitive cultivar to Al toxicity, implying its high sensitivity to soil acidity (De Bauw et al., 2021). Haynes and Mokolobate (2001) reported that the application of organic residues increased the soil pH over the first two months of residue decomposition but the magnitude of the rise depended on the type of residue. Hence, we suggest that the high performance of IFM in increasing rice growth is linked to its impact on the increase of the soil pH which may decrease Al toxicity, enhance P availability and increase rice P uptake. In a field study conducted in the Highlands of Madagascar, De Bauw et al. (2021) pointed out that the reduction of Al toxicity by application of organic matter (farmyard manure) outweighed the potential effects of organic P supply on rice yield and P uptake. Known as "master soil variable" due to its important influence on soil chemical, physical and biological properties and processes (De Bauw et al., 2021; Minasny et al., 2016), the soil pH may also increase the rate of mineralization of IFM, which could probably explain the 5-fold increase of the aboveground shoot N in comparison to that of unamended treatment. Previous work of Khalil et al. (2005) showed that the decomposition rate of organic materials was 59% explained by the soil pH and 61% explained by the soil pH coupled with the C:N ratio of the organic materials. Among OAs, IFM had the highest pH (9.4), which may partially increase the initial soil pH (4.7). Further research should be undertaken to test this assumption and to better understand the
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Effect sizes (Cohen's d) of earthworm inoculation on plant variables according to organic amendments. (A) Plant (shoot and root) biomass, (B) aboveground shoot N and P amounts, (C) aboveground nutrient uptake efficiencies (NUptE and PUptE). C: unamended control; CFM: Conventional farmyard manure; KM: Kraal manure; IFM: Improved farmyard manure; COM: Compost; VCT: Vermicompost; UW: Urban waste; TK: Taroka; PD: Poultry droppings; PIM: Pig manure.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Individual fertilizer performance and ecological earthworm performance relationships. CFM: Conventional farmyard manure; KM: Kraal manure; IFM: Improved farmyard manure; COM: Compost; VCT: Vermicompost; UW: Urban waste; TK: Taroka; PD: Poultry droppings; PIM: Pig manure.</figcaption></figure>

underlying mechanisms that explain the high fertilizer performance of IFM treatment.

## 4.2. Positive effect of earthworms

Our results showed that the plant biomass and nutrition were increased in the presence of earthworms. Positive effects of earthworms were earlier widely reported in tropical (Brown et al. 1999, 2004; Ratsiatosika et al., 2021) or temperate countries (Scheu, 2003). For cereals,

the general pattern of the plant response to earthworm inoculation is a significant increase of the shoot biomass without significant effect on the root biomass (Kreuzer et al., 2004; Van Groenigen et al., 2014; Coulis et al., 2014). Here, we found that earthworms significantly increase more the shoot biomass (+63%) than the root biomass (+30%). In a review, Bertrand et al. (2015) reported that the effect of earthworms was more pronounced on the aboveground biomass while a variable effect was noticed on the root biomass. Among the mechanisms through which earthworms increase plant growth, the enhancement of N mineralization was the most dominant pathway often cited in the literature (Scheu, 2003; Van Groenigen et al., 2014). Likewise, we found that the presence of earthworms strongly increased the aboveground shoot N (+107%) and the aboveground NUPtE (+63%). Nonetheless, given that the soil used in the present study was strongly limited by P, it is likely that the enhancement of P availability by earthworms is a major pathway to the positive effect of earthworms (Trap et al., 2021). This is supported by the increase of the aboveground shoot P by 93% and the aboveground PUPtE by 43% in the presence of earthworms.

## 4.3. Earthworm ecological performance and quality of OA relationships

The most striking finding of our study is that the magnitude of the positive effect of earthworms on plant performance was dependent on the OAs applied. Previous studies suggested that earthworms had greater effect on plant growth in nutrient-poor than in nutrient-rich soils, this was especially shown for nitrogen (van Groenigen et al., 2014). Following this, in our study, soil receiving nutrient-poor OAs such as UW, VCT and CFM was presumed to have a stronger effect of earthworms while soil amended with nutrient-rich OAs such as PD, IFM and PIM was presumed to have a
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Principal Component Analysis performed on individual fertilizer performance, earthworm ecological performance and chemical and biochemical characteristics of organic amendments. (A) Circle of correlations on the dimensions 1 and 2 of the factorial plan. (B) Individual factorial plan (dimensions 1 and 2). (C) Eigenvalue diagram. (D) correlation plot between variables and dimensions.</figcaption></figure>

C: Total organic carbon content; N: Total organic nitrogen content; P: Total phosphorus content; K: Total potassium content; Ca: Total calcium content; Mg: Total magnesium content; pH: pH in water; Sol: Soluble content; Hem: Hemicelulose content; Cel: Celullose content; Lig: Lignin content; C_N: C:N ratio; C_P: C:P ratio; Ind_P: Individual fertilizer performance; Eco_P: Earthworm ecological performance.

CFM: Conventional farmyard manure; KM: Kraal manure; IFM: Improved farmyard manure; COM: Compost; VCT: Vermicompost; UW: Urban waste; TK: Taroka; PD: Poultry droppings; PIM: Pig manure.

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Principal Component Analysis performed on cohen's d of plant parameters and chemical and biochemical characteristics of organic amendments. (A) Circle of correlations on the dimensions 1 and 2 of the factorial plan. (B) Individual factorial plan (dimensions 1 and 2). (C) Eigenvalue diagram. (D) correlation plot between variables and dimensions.</figcaption></figure>

C: Total organic carbon content; N: Total organic nitrogen content; P: Total phosphorus content; K: Total potassium content; Ca: Total calcium content; Mg: Total magnesium content; pH: pH in water; Sol: Soluble content; Hem: Hemicelulose content; Cel: Celullose content; Lig: Lignin content; C_N: C:N ratio; C_P: C:P ratio; d_Shoot: cohens'd of shoot biomass; d_Root: cohens'd of root biomass; d_Shoot_N: cohen's d of aboveground shoot N; d_Shoot_P: cohen's d of aboveground shoot P; d_NUptE: cohen's d of aboveground N uptake efficiency; d_PUptE: cohen's d of aboveground P uptake efficiency.

CFM: Conventional farmyard manure; KM: Kraal manure; IFM: Improved farmyard manure; COM: Compost; VCT: Vermicompost; UW: Urban waste; TK: Taroka; PD: Poultry droppings; PIM: Pig manure.

lower effect. Our results tended to be consistent with this finding, except for PD and PIM. Laossi et al. (2010) reported that the soil fertility was not necessarily the main proxy determining the performance of earthworms in increasing plant growth. In a study conducted by Blouin et al. (2006), the authors clearly demonstrated that the soil N content is an important factor

determining the effect of earthworms on plant growth. They found that at low soil N levels, the main mechanism responsible for the positive effect of earthworms is the enhanced net N mineralization. This mechanism is excluded at high soil N levels. Thus, the question emerging from our observation is what are the main mechanisms explaining the large effect of
earthworms in PIM and PD treatments despite their high individual potential in increasing the soil nutrient availability. The PCA performed on the earthworm effect sizes (cohen's d values) for each plant variable and OA quality revealed that both the direction and magnitude of the effect of earthworms on the shoot and of the root biomasses were positively correlated with the total P, total Mg and soluble contents of OAs. Conversely, a negative correlation of the earthworm effect sizes on the shoot and root biomasses was observed for the C:P ratio, cellulose and lignin contents. This result suggests that earthworms increased plant biomass through the enhancement of P and Mg availability released during the mineralization of OAs. This finding reinforces the statement of Raminoarison et al. (2020) about the strong P deficiency and the importance of Mg nutrient on crop grown in Malagasy Ferralsols. Specifically for P, four mechanisms have been claimed to be responsible of the increase of available P by earthworms (Le Bayon and Milleret, 2009; Ros et al., 2017; Vos et al., 2022): (i) stimulation of microbial activity in the earthworm gut leading to an increase production of phosphatases and mineralization of organic P; (ii) changes in ortho-P binding to metal (hydr)oxides induced by the competitive binding dissolved organic carbon from the earthworm gut; (iii) increase of pH in earthworm casts affecting P speciation and adsorption; (iv) selective feeding by earthworms on soil particles with a higher P content. Recently, a new mechanism explaining the earthworm-induced P availability in soils dominated with iron-(hydr)oxide was found by Vos et al. (2022). These authors suggested that earthworms decrease the reactive surface area of metal-(hydr)oxides due to the low redox potential in the earthworm gut, decreasing the adsorption of ortho-P. From our observations, we cannot clearly identify the main mechanism involved in the additional release of available P by earthworms. However, it is noteworthy that *P. corethrurus* strongly increased the P availability by 76% through the solubilization of native soil P during its digestive tract (Trap et al., 2021). Thus, it is likely that the stimulation effect of earthworms on microbial activity during the OA ingestion could enhance the rate of mineralization of OAs, resulting in an increase of P availability. Given that PD and PIM had a high total P content and were easily decomposable, the surplus of available P released during the ingestion of these OAs in earthworm gut could explain the huge effect of earthworms. In contrast, in IFM treatment, the stimulatory effect of earthworms in increasing the available P during the digestion tract may be less important due to the low total P content and the high cellulose and lignin contents that are recalcitrant to microbial decomposition. This could probably explain the small effect of earthworm inoculation in the soil receiving IFM. Further research is needed to clearly identify the main mechanisms of earthworm-induced increased plant growth through the increase of soil nutrient availability. A nutrient omission trial combined with earthworm inoculation seems to be relevant to clearly hierarchize the rationale behind the hypothesis of N-induced effect or P-induced effect of earthworms on the improvement of plant growth in highly weathered tropical soils.

### 4.4. Implications for a sustainable agriculture in Madagascar

Our results showed that earthworms are valuable allies for farmers because of their positive effects on plant growth, even without the addition of OAs (Vidal et al., 2023). Moreover, the results revealed that OAs alone can provide less or more nutrients to plants. At first glance, targeting OAs with high fertilizer performance seems the priority strategy to overcome the multiple nutrient deficiencies of Ferralsols. Nevertheless, the study showed that OAs with low fertilizer performance interacted more pronouncedly with earthworms. In addition, earthworms enhanced the fertilizing performance of OAs with high quality. Targeting OAs with high individual fertilizer performance and high earthworm ecological performance appears crucial in the objective of ecological intensification. Depending on the availability of OAs, farmers may have the choice to select materials based on this dual performance optimization. Carrying out a similar study by testing the widest possible range of fertilizer materials potentially available in the highlands of Madagascar would provide a new performance indicator for fertilizer resources.

## 5. Conclusion

In this study, we found that earthworms increased the individual fertilizer performance of OAs. However, the magnitude of the increase was variable according to the type of OA applied, which is consistent with our hypothesis. Overall, OAs with low individual fertilizer performance showed greater effect of earthworms. Nevertheless, earthworms had the potential to increase the performance of OAs of high quality likely through the improvement of P and Mg availability during mineralization. Our findings emphasize the synergistic effect of earthworms and OAs of different qualities on plant growth and nutrition. At the level of family farms where crop fertilization is mainly focused on the supply of OAs, using earthworms as biofertilizers and OAs together could thus be a key solution to improve agricultural productivity and sustainability in Ferralsols with multiple nutrient deficiencies.

### Formatting of funding sources

This study was funded by the project SECuRE (Soil ecological function restoration to enhance agrosystem services in rainfed rice cropping systems in agroecological transition, ID 1605-007), and the project Innov'Earth (Innovative agricultural nature-based solutions to reduce climate change, land degradation and food insecurity: the potential of earthworms and vermicompost in small tropical farms, ID 2101-003), both funded through ANR (the French National Research Agency) under the "Investissements d'avenir" programme with the reference ANR-10-LABX-001-01 Labex Agro and coordinated by Agropolis Fondation.

### CRediT authorship contribution statement

O. Ratsiatosika: Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. J. Trap: Writing – original draft, Validation, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization. V. Herinasandratra: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. T. Razafimbelo: Writing – original draft, Validation, Supervision, Resources, Project administration, Conceptualization. L. Bernard: Conceptualization, Resources, Validation, Writing – original draft. E. Blanchart: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – original draft.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### Acknowledgements

Authors are thankful to Nancy Rakotondrazafy (IRD, UMR Eco&Sols) and Damase Razafimahafaly (LRI) for technical help.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.soilbio.2024.109477.
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