High rates of nitrogen fixation and transfer by Cajanus cajan to associated crop in a semi-arid agroforestry system

High rates of nitrogen fixation and transfer by Cajanus cajan to associated crop in a semi-arid agroforestry system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High rates of nitrogen fixation and transfer by Cajanus cajan to associated crop in a semi-arid agroforestry system RAKOTOZAFY Sarah, BORDRON Bruno, RAZAFIMBELO Tantely, RAZAFINDRAKOTO Malalatiana, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7170753/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract In semi-arid regions where nitrogen deficiency limits crop productivity, the integration of leguminous tree species into agroforestry systems (AFS) is likely to enhance nitrogen availability for associated crops. We tested this hypothesis in southern Madagascar, a region characterized by erratic and low rainfall, and nutrient-poor soils where the pigeon pea grown as a pure crop or mixed with cassava. Over a 3-month period, we estimated the atmospheric nitrogen fixation rate (%Ndfa) by two-year-old pigeon pea ( Cajanus cajan ) grown as a pure crop and in association with cassava ( Manihot esculenta ) using N dilution method, and the transfer of N from pigeon pea to cassava (%Ndft) in the mixed pigeon pea-cassava plots. Six-month-old pure cassava plots were used as reference. Each system was replicated three times. 15 N soil labeling (98 atom% 15 N) was used to estimate %Ndfa and %Ndft. %Ndfa was 100% in both pigeon pea systems. Considering %Ndfa of 100% from planting date, the quantity of N fixed was two times higher in pure pigeon pea plot than in mixed pigeon pea-cassava plots, with values of 17.1 kg N ha-¹ and 8.5 kg N ha-¹, respectively. This finding was consistent with the 2.8 times higher planting density in pure pigeon pea plots compared to mixed pigeon pea-cassava plots. Nitrogen transfer from pigeon pea to cassava was greater (72.9%) when the cassava plant was positioned farther from pigeon pea rows (in the middle) compared to when it was planted next to pigeon pea row (42.4%). Our results suggest that integrating pigeon pea into semiarid agroforestry systems could enhance nitrogen status of the associated crop, particularly in unfertilized AFS. leguminous shrubs pigeon pea cassava symbiotic N2 fixation N-transfer Figures Figure 1 Figure 2 1. Introduction Agroforestry systems (AFS) propose a sustainable and productive ecosystem by combining trees and/or shrubs with crops. These perennial cropping systems require less tillage and provide more organic matter to the soil than annual cropping system thereby improving crops nutrition (Bekele-Tesemma 2007 ; Mafongoya et al. 2007 ; Sileshi et al. 2020 ). In tropical areas, the majority of trees in these systems are nitrogen fixing species (NFS)to improve soil fertility and crop nutrition (Crews 1999 ; Menge et al. 2014 ), whereas in semi-arid climates, NFS not only improve the availability of nitrogen where it is frequently deficient, but also help to create favorable micro-environments for crop development (Nair et al., 2021 ). The loss of nitrogen in cropping systems through export at harvest (Kim and Isaac 2022 ), ammonia (NH 3 ) volatilization, and nitrate (NO₃) and ammonium (NH 4 + ) leaching (Cameron et al. 2013 ; Kim and Isaac 2022 ) is a frequent problem in AFS cropping systems. On the one hand, NFS are likely to reduce nitrogen loss within AFS and on the other hand, they can supply nitrogen to the system through atmospheric fixation (Kim & Isaac, 2022 ; Nygren et al., 2012 ). While fixation of atmospheric N 2 in NH₄ + form, through symbiosis with Rhizobium bacteria, is a major source for nitrogen input in agroecosystems (Nygren et al. 2012 ), environmental factors can reduce the N 2 fixation (Bordeleau and Prévost 1994 ; Paula et al. 2015 ). Several studies on soybean indicated that drought stress (Freitas et al. 2022 ; Serraj et al. 1999 ),soil nutrient deficiency such as low P availability (Yao et al. 2022 ) can decrease N 2 fixation as a consequence of photosynthesis reduction and decrease in photosynthates supply to the nodules (Nandwal et al. 1991 ; Sheoran et al. 1988 ). Crops associated with NFS can benefit by direct transfer through root exudates and common mycorrhizal networks (CMN), or indirectly through litter mineralization, and fine root and nodule turnover (Munroe and Isaac 2014 ; Nygren et al. 2012 ; Paula et al. 2015 ; Thilakarathna et al. 2016 ). Previous studies in tropical environments have suggested that N-transfer may be stronger when the associated crop is grown close to the NFS (Paula et al., 2015 ). Zayed et al. ( 2023 ) reported that under nutrient deficiency, especially N deficiency, plants may generally activate physiological and molecular mechanisms to improve their nitrogen uptake efficiency. Eucalyptus trees grown in association with acacia have been shown to develop more extensive root systems and increase root exudation (Oliveira et al. 2021 ). These responses contribute to improve N-transfer within mixed species where the associated crop increase their demand for available N and the NFS respond (Oliveira et al. 2021 ). Among the NFS used in agroforestry systems, pigeon pea ( Cajanus cajan (L.) Millsp) is one of the species recommended for semi-arid areas (Daniel and Ong 1990 ; Mula and Saxena 2010 ; Saxena et al. 2019 ). This leguminous shrub is adapted to harsh climatic and environmental conditions such as low rainfall and nutrient poor soil (Mula and Saxena 2010 ; Odeny 2007 ; Saxena et al. 2019 ). In addition, pigeon pea is efficient to fix atmospheric N 2 (Bopape et al. 2022 ; Kumar Rao and Dart 1987 ; Sameer Kumar et al. 2017 ). In semi-arid Kenya, the percentage of nitrogen derived from fixation (%Ndfa) of five pigeon pea varieties were estimated over 80% in roots, leaves, stover, grains (Kwena et al. 2019 ). The southern Androy region of Madagascar, is characterized by a semi-arid climate, frequent drought, and severe aeolian erosion. These environmental constraints, combined with widespread poverty, have led to deforestation, poor soil fertility and low agricultural productivity (Violas 2020 ). To address this issue, a land-use strategy design described as agroecological blocks has been implemented since 2014. These blocks combine physical and biological components over a minimum area of 10 ha (Violas 2020 ). AFS play a central role in these blocks, with the aim of improving environmental protection and soil fertility by integrating ground cover species and nitrogen-fixing trees, thereby reducing erosion, strengthening the resilience of agro-ecosystems and supporting food production (Fahad et al. 2022 ; Pancholi et al. 2023 ; Octavia et al. 2023 ; Violas 2020 ). Pigeon pea is one of the key species used in these AFS, either as sole crop or in associated crop. These systems are mainly adopted by resource-limited farmers, consequently, no fertilizers are added, which likely lead to progressive soil nutrient depletion (Vanlauwe et al. 2023 ; Violas 2020 ). The integration of pigeon pea into these AFS is therefore expected to promote nitrogen inputs and improve the N status of associated crops. To optimize the management of AFS in this semi-arid area, this study aims to estimate the amount of atmospheric nitrogen fixed and transferred to the associated crops. The results are intended to guide spatial configurations of this agroforestry system to enhance biological nitrogen inputs and improve soil fertility without external inputs. 2. Materials and methods 2.1 Study site The study was carried out in the Karoke Mahandrorano agroecological block (25°15'15"S, 46°1'21"E), located in the Androy region of South Madagascar near the city of Ambovombe, at 200 m asl. Established in 2013, this is one of the oldest agroecological blocks in the region, and serves as a reference site for the other blocks. The site is located on a coastal area, on limestone rock. The relief was characterized by gently undulating topography with slope < 5%. The soil texture was uniform below a depth of 30 cm with sand content around 80%. The soil pH H2O ranged from 7.2 to 9.1 depending on the plots. Soil chemical analyses down to a depth of 0.30 m are given in Table 1 . The climate of the region is classified as BSh (semi-arid hot) according to Köppen-Geiger classification (Beck et al. 2018 ), with wet season from December to April and dry season from May to November (Fig. 1 ). The average annual temperature is 24.4°C, with temperature varying from 19°C (minimum in June) to 40°C (maximum in January). Annual rainfall ranges from less than 300 and 600 mm, occurring mainly during wet season. Sporadic and limited rainfall may also occur in May and June. However, precipitation is highly erratic, with frequent droughts and irregular rainfall patterns. For instance, in 2020, no rainfall was recorded for 18 consecutive months, and in other years, only 3 to 4 rainfall events per season (UNICEF 2024 ). Potential evapotranspiration is 518 mm. year − 1 , which generally exceeds rainfall throughout the year (JICA 2006 ). From August to October, violent soil-eroding winds occur (Morlat and Castellanet 2012 ; Violas 2020 ). The study was conducted during 90 days from January 2024 to April 2024. During this period, the average air temperature was 22.5° C and the cumulative rainfall was 224 mm ( https://www.historique-meteo.net ). Table 1 Main chemical soil attributes of the top soil without pigeon pea at the experimental area (n = 4) Soil depth (cm) 0–10 10–20 20–30 pH 8.92 8.62 8.70 Organic C (g.kg − 1 ) 2.83 ± 0.69 2.46 ± 0.58 1.95 ± 0.61 Total soil N (g.kg − 1 ) 0.29 ± 0.08 0.21 ± 0.06 0.20 ± 0.05 Available P (mg.kg − 1 ) 5.07 ± 2.24 2.58 ± 1.32 2.88 ± 1.45 K (cmol+.kg − 1 ) 0.03 ± 0.01 0.02 ± 0.01 0.02 ± 0.02 Ca (cmol + kg − 1 ) 1.65 ± 1.04 1.68 ± 1.03 1.70 ± 1.24 Na (cmol + kg − 1 ) 0.01 ± 0 0.01 ± 0 0.02 ± 0 2.2 Experimental layout The 15 N isotope dilution method was used to estimate the %Ndfa. The method was applied to pure pigeon pea, mixed pigeon pea-cassava (Manihot esculenta Crantz), and pure cassava plots. In total, nine farmer plots were selected within the AFS agroecological block for the experiment, corresponding to three cropping system with three replicates each: pure pigeon pea, mixed pigeon pea-cassava, and pure cassava plots. All pigeon pea plants were two years old, while the cassava plants were six months old at the time of the experiment. Cassava, a non- N fixing species, was chosen as the reference plant for %Ndfa estimation (Boddey et al. 1990 ). 2.3 15 N soil labeling An area of 100 m 2 was delimited in each plot. Ammonium chloride (Cl- 15 NH 4 ) with 98 atom% 15 N was applied in this area at a rate of 0.8 kg N ha − 1 on 15th, 16th, and 17th January 2024. The fertilizer was diluted in water and applied uniformly to the soil using a watering can. We assumed that the low amount of applied N did not affect the N 2 fixation of pigeon pea (Bouillet et al. 2008 ). 2.4 Plant sampling and analyzes Aboveground leaf biomass of pigeon pea plants In each pure pigeon pea and mixed plots, a pigeon pea inventory was carried out by counting individual plants and measuring their height. To estimate aboveground leaf biomass, all leaves were harvested randomly from 10 shrubs per plot, replicated four times, resulting in a total of 80 shrubs sampled (Table 2 ). Both fresh and dry masses were recorded. Leaf sampling for N fixation measurements For nitrogen analysis, leaf samples were separately collected in each system and for each species at two time points: before soil labeling in January 2024 and 90 days after soil labeling in April 2024. In the pure pigeon pea plots, leaf sampling was carried out on five shrubs arranged diagonally in a cross pattern (Fig. 2 .A). For each shrub, twenty leaves from the upper part of the crown on two pairs of opposite branches (north-south and east-west) were collected and pooled to make a composite sample representative of the plot (Table 2 ). In the pure cassava plots, this sampling was carried out on 10 plants arranged diagonally and in a cross pattern (Fig. 2 .A): four leaves were sampled per plant, one in each direction (Table 2 ). In the mixed pigeon pea - cassava plots, pigeon pea leaves were collected from five shrubs in a row, with 20 leaves per shrub (Table 2 ), while the cassava, leaves were collected at three different distances from the pigeon pea row: the first plant close to the shrub (near), the second at a medium distance (middle), and the last between two pigeon pea rows (far) (Fig. 2 .B). The sampled leaves were then dried at 60°C and ground, and sent to SILVATECH Laboratory ( https://www6.nancy.inrae.fr/silva/Plateformes/SilvaTech ) for analysis. For this purpose, 10 mg of leaf powder dry matter were placed in tin capsules for isotopic analyzes. N concentrations and δ 15 N values of the leaves were determined using an elemental analyzer (vario ISOTOPE cube, Elementar, Langenselbold, Germany) coupled, via a gas box interface, to a continuous flow isotope ratio mass spectrometer (Isoprime100, IRMS, Elementar UK, Cheadle, United Kingdom). The precision of δ 15 N measurement was 0.5‰. Table 2 Number of pigeon pea sampled for leaf biomass and leaf samples for nitrogen analysis in pure and mixed plots Pigeon pea sample for leaf biomass Leaf sample for N analyzes Pure crop Pigeon pea 10 100 Cassava - 40 Mixed crop Pigeon pea 10 100 Cassava - 30 Replicates 4 3 Total 80 810 2.5 N derived from atmospheric fixation (%Ndfa) The percentage of N derived from N 2 fixation (%Ndfa) for each plot during the experiment is estimated by examining ¹⁵N Atom Excess (AE) using the following equation (Bouillet et al. 2008 ): $$\:\varvec{\%}\varvec{N}\varvec{d}\varvec{f}\varvec{a}=100\left(1-\frac{\frac{\left({\varvec{N}}_{\varvec{F}\varvec{f}\varvec{i}\varvec{n}\varvec{a}\varvec{l}}\:{\varvec{A}\varvec{E}}_{\varvec{F}\varvec{f}\varvec{i}\varvec{n}\varvec{a}\varvec{l}}-{\varvec{N}}_{\varvec{F}\varvec{i}\varvec{n}\varvec{i}\varvec{t}\varvec{i}\varvec{a}\varvec{l}}{\:\varvec{A}\varvec{E}}_{\varvec{F}\varvec{i}\varvec{n}\varvec{i}\varvec{t}\varvec{i}\varvec{a}\varvec{l}}\:\right)}{\left({\varvec{N}}_{\varvec{F}\varvec{f}\varvec{i}\varvec{n}\varvec{a}\varvec{l}\:}-{\varvec{N}}_{\varvec{F}\varvec{i}\varvec{n}\varvec{i}\varvec{t}\varvec{i}\varvec{a}\varvec{l}}\right)}}{\frac{\left({\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{f}\varvec{i}\varvec{n}\varvec{a}\varvec{l}}\:{\varvec{A}\varvec{E}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{f}\varvec{i}\varvec{n}\varvec{a}\varvec{l}}\:-{\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{i}\varvec{n}\varvec{i}\varvec{t}\varvec{i}\varvec{a}\varvec{l}}\:{\varvec{A}\varvec{E}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{i}\varvec{n}\varvec{i}\varvec{t}\varvec{i}\varvec{a}\varvec{l}}\:\right)}{\left({\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{f}\varvec{i}\varvec{n}\varvec{a}\varvec{l}}-{\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{i}\varvec{n}\varvec{i}\varvec{t}\varvec{i}\varvec{a}\varvec{l}}\right)}}\right)$$ 1 where: AE = 15 N x 100 / ( 15 N + 14 N) − 0.003663, AE Ffinal , AE Finitial , AE REFfinal , and AE REFinitial are the percentage atom excess of pigeon pea 90 days after soil labeling, the percentage atom excess of pigeon pea at soil labeling, the percentage atom excess of cassava 90 days after soil labeling, and the percentage atom excess of cassava at soil labeling, respectively. N Ffinal , N Finitial , N REFfinal , and N REFinitial are the N content of pigeon pea 90 days after soil labeling, the N content of pigeon pea at soil labeling, the N content of cassava 90 days after soil labeling, and the N content of cassava at soil labeling, respectively. 2.6 N derived from transfer The proportion of cassava N derived from transfer (%Ndft) from pigeon pea during the experiment period was estimated using Eq. 2 (Snoeck et al. 2000 ) : $$\:\varvec{\%}\varvec{N}\varvec{d}\varvec{f}\varvec{t}=100\times\:\:\frac{{\varvec{\delta\:}}^{15}{\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}}-\:{\varvec{\delta\:}}^{15}{\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}\varvec{a}\varvec{s}\varvec{s}}}{{\varvec{\delta\:}}^{15}{\varvec{N}}_{\varvec{R}\varvec{E}\varvec{F}}-\:{\varvec{\delta\:}}^{15}{\varvec{N}}_{\varvec{f}\varvec{i}\varvec{x}\varvec{a}\varvec{t}\varvec{i}\varvec{o}\varvec{n}}}$$ 2 where: δ 15 N REF is the δ 15 N value of cassava leaves in pure crop, δ 15 N REFass is the δ 15 N of cassava leaves in mixed crop, and δ 15 N fixation is the δ 15 N of pigeon pea leaves in pure crop. 2.7 Statistical analyses One-way analysis of variance (ANOVA) was used to compare the means between the N content, δ 15 N and AE of the different system as fixed factor and, %Ndft values of cassava in mixed crop with distance as fixed factor. Tukey’s post-hoc tests were used to examine pairwise differences. Assumptions of normality and homoscedasticity of residuals were tested using Shapiro-Wilk and Bartlett tests, respectively. Statistical analyses were performed using R 4.0.3 et RStudio 4.1.1, with a significance level α of 0.05. 3. Results The experimental season was characterized by low rainfall (224 mm) and low topsoil fertility. According to Boyer. (1982), the critical deficiency levels for tropical plants grown on ferralitic soils are 0.5 g-kg-¹ for total N and 3 mg-kg-¹ for available P, while the deficiency thresholds are 1.5 g-kg-¹ and 10 mg-kg-¹ respectively. Our soil analysis (depth 0–30 cm) showed total N levels below both the critical level and the deficiency threshold, while available P levels were comparable to the critical level but still above the deficiency threshold. This following section presents data from shrub inventory, pigeon pea biomass assessments, and leaf N analysis, with a focus on N derived from atmospheric fixation (%Ndfa), their transfer in mixed plots (%Ndft), and the overall contribution of fixed N to the system. 3.1 15 N values and percentage of N derived from atmospheric fixation (%Ndfa) No significant difference in AE mean values between the different crop systems was observed at the beginning of the experiment ( p-value = 0.23), with AE initial ranging from 8.42–9.39% (Table 3 ). After 90 days, the AE of cassava leaves in the pure crop was significantly higher than that of pigeon pea leaves in the pure and mixed crops ( p-value = 0.008). No significant difference in N content of the cassava and pigeon pea leaves in pure and mixed crops was found at the beginning of the experiment ( p-value = 0.18) or after 90 days ( p-value = 0.78). N initial ranged from 3.56–4.09%, while N final ranged from 4.08–4.25%. The %Ndfa was estimated to be 100% during the 90 days experiment both in the pure pigeon pea and in mixed pigeon pea-cassava plots. Table 3 N content, 15 N atom excess of leaf samples (AE) in pigeon pea and cassava, and %Ndfa of pigeon pea (Mean values, n = 3) Systems Leaf samples Observed parameters N initial AE initial N final AE final %Ndfa Pure cassava Cassava 4.09a 8.42a 4.08a 9.95a Pure pigeon pea Pigeon pea 3.56a 9.39a 4.14a 8.58b 100 Mixed pigeon pea - cassava Pigeon pea 3.78a 8.92a 4.25a 8.18b 100 *N initial and N final are the N content of leaf samples on soil labeling and after 90 days respectively, expressed in %. AE initial and AE final are the values of atom excess at soil labeling and after 90 days respectively, expressed in %. %Ndfa is the percentage of nitrogen derived from fixation atmospheric. Letters (a–b) indicate significant differences ( p < 0.05) between systems. 3.2 Nitrogen input from N 2 fixation The planting density of pigeon pea was higher in pure system (3464 individuals ha − 1 ) compared to the mixed pigeon pea-cassava system (1216 individuals ha − 1 ), due to inter-row spacing constraints in the mixed plots. Despite this, the average height of pigeon pea shrubs was around 180–190 cm in both systems, suggesting comparable vertical growth regardless of planting arrangement. (Table 4 ). Considering individual plants for each system, there was more N fixed in the mixed pigeon pea-cassava plots (6.97 g N plant − 1 ) than in the pure pigeon pea plots (4.94 g N plant − 1 ), which is expected given the higher leaf biomass per plant in the mixed plot. However, taking planting density into account, the total N input over two years was higher in the pure pigeon pea plots, reaching 17.11 kg N ha − 1 , compared with 8.48 kg N ha − 1 in the mixed pigeon pea -cassava plots (Table 4 ). Table 4 Aboveground pigeon pea growth parameters and nitrogen supply from %Ndfa in 2-year-old pigeon pea plants, in both pure and mixed pigeon pea plots plots (Mean values ± SE, n = 4) Systems Pure pigeon pea Mixed pigeon pea - cassava Pigeon pea planting density (individuals ha − 1 ) 3464 1216 Height (cm) 196.5 ± 44.9 188.3 ± 49.7 Leaf biomass (kg) 0.13 ± 0.11 0.17 ± 0.08 %Ndfa 100 100 g N plant − 1 from %Ndfa 4.94 6.97 kg N ha − 1 from %Ndfa 17.11 8.48 3.3 N transferred from pigeon pea to cassava After 90 days, the mean values of 15 N final varies slightly but significantly from 0.37–0.44% between the three systems (Table 5 ). The highest value of 15 N final was recorded in leaves of the pure cassava plots (0.44%), while the lowest value was observed in the pigeon pea leaves of the mixed pigeon pea-cassava plots (0.38%). Cassava leaves located at different distances from pigeon pea (near, at ¼ inter-row and ½ inter-row) showed similar and non-significant 15 N final values (0.41%, 0.42% and 0.40% respectively), (Table 5 ). However, the percentage of N transferred from pigeon pea to cassava (%Ndft) varied significantly with increasing distance from the pigeon pea plants ( p-value = 0.0009). Cassava plants located in the ½ inter-row received the highest proportion of nitrogen transferred (72.95%), while those positioned near to the pigeon pea (51.73%) or slightly further in the ¼ inter-row (42.45%) received less N. These results suggest that spatial arrangement plays a key role in promoting N transfer. No significant difference in %Ndft was found between the last two positions. Table 5 Mean leaf samples 15 N values in the three cropping system and %Ndft in the mixed plots in relation to the distance between the non-leguminous crop (cassava) and the leguminous crop (pigeon pea) System Leaf samples Distance from pigeon pea 15 N final %Ndft Pure pigeon pea Pigeon pea - 0.39d - Pure cassava Cassava - 0.45a - Mixed pigeon pea - cassava Pigeon pea - 0.38d - Mixed pigeon pea- cassava Cassava Near (S1) 0.41bc 51.73b Mixed pigeon pea - cassava Cassava ¼ inter-row (S2) 0.42b 42.45b Mixed pigeon pea - cassava Cassava ½ inter-row (S3) 0.40cd 72.95a 15 N final is the value of δ 15 N expressed in % of leaf samples after 90 days. %Ndft is the proportion of N derived from transfer. Letters (a–d) indicate significant differences ( p < 0.05) between systems. 4. Discussion 4.1 High efficiency of pigeon pea to fix atmospheric N 2 The nitrogen fixation rate of pigeon pea was estimated at 100% in both, pure pigeon pea and mixed pigeon pea-cassava plots over the 90-day experimental period. These results indicate that, over this period, all the N in pigeon pea leaves was derived from N 2 fixation. This also suggests that, at least during the second vegetative season, pigeon pea relied entirely on biological nitrogen fixation, regardless of the cropping system. According to Dovrat and Sheffer ( 2019 ), biological N fixation is generally optimal during wet season. Soil N deficiency (Dovrat et al. 2018 ) and high P availability (Alon et al. 2021 ) has been shown to increase nitrogen fixation rates by NFS. In our study, although P availability was close to the critical level but likely sufficient to support the energy costs for the N 2 fixation. In addition, the low levels of N in the soil and the fact that the experiment was conducted during the rainy season probably contributed to maximizing symbiotic nitrogen fixation. It is consistent with previous studies showing that biological N fixation is more efficient under unfertilized conditions (Figueiredo et al., 2025). Similar high levels of %Ndfa have been reported in other semi-arid regions: Rao et al. ( 1987 ) recorded 88% in sole pigeon pea and 96% in mixed pigeon pea–sorghum systems in India, while Kwena et al ( 2019 ) found values exceeding 80% in pigeon pea–maize intercropping in Kenya without rhizobia inoculation. Mhango et al ( 2017 ) reported %Ndfa about 76% and no significant difference between pure pigeon pea and mixed pigeon pea-maize crop system. However, short rainfall season means low biomass production and lower N fixation. Paula et al ( 2018 ) showed variability in N 2 fixation rates between seasons in mixed eucalyptus-acacia cropping system in Brazil. Water availability plays a critical role in improving biological N fixation by reactivating or enhancing biomass nodules of NFS. Pigeon pea is also known for its water use efficiency, allowing it to sustain biological N 2 fixation even under tropical dryland conditions (Berriel and Perdomo 2023 ). Consequently, it will be important to assess the dynamics of N 2 fixation from the early stages of pigeon pea growth and across different seasons, as seasonal variations in %Ndfa could occur. 4.2 Nitrogen transfer dynamics in mixed cropping systems Between 42% and 72% of N was transferred from pigeon pea to cassava. Therefore, the association of pigeon pea benefited the crop system by improving the N nutrition of the associated plant. Monroe and Isaac (2014) indicated that N from NFS can be a source of easily available N for the associated crop via fine root decomposition and nodule turnover or N rhizodeposition. Nitrogen could also be transferred from NFS to companion species via common mycorrhizal networks (Ingleby et al. 2007 ; Oliveira et al. 2021 ). We found that nitrogen transfer dynamics between pigeon pea and cassava were influenced by cassava localization, with higher %Ndft observed at greater distances from pigeon pea. This invalidates our hypothesis of higher %Ndft at closer distance of cassava from pigeon pea, as observed for N transfer from Acacia mangium to Eucalyptus grandis in mixed-species plantations (Paula et al. 2015 ). This could be due to root spatio-temporal dynamics of the fine roots of pigeon pea. Actually, it has been reported that shrubs often have significant lateral root spread, which can favor their interaction with neighboring crops and exploit the surface layers of the soil (Schenk and Jackson 2002 ). In legumes more generally, an increased root density leads to greater nitrogen release through rhizodeposition (Thilakarathna et al. 2016 ). A field study revealed, that at two years, pigeon pea roots could reach up to 4 m laterally in the Androy region of Madagascar (Rakotozafy et al. 2024 ). These observations were not in line with the findings of Ito et al. ( 1996 ) who found that the roots of pigeon pea spread more vertically than horizontally in semi-arid conditions. This discrepancy might be due to the soil characteristics in our study area with a rocky layer found at a depth of 150 cm (Rakotozafy et al., personal communication, 2024). However, our results highlight that the distance of 6–8 m between rows of pigeon pea, as adopted by farmers in the Ambovombe region allows effective N transfer from this NFS to cassava. 4.3 Nitrogen input to the system At two years after planting, N input was at most 17.1 kg N ha − 1 in pure pigeon pea plots and 8.5 kg N ha − 1 in mixed pigeon pea-cassava plots, which was consistent with the respective planting density between the systems. Keston et al ( 2017 ) also found higher N inputs through biological N fixation in pure crops (85.7 kg N ha⁻¹) than in mixed pigeon pea–cowpea crops (57.4 kg N ha⁻¹) after one growing season in Central Malawi. Similarly, Mhango et al ( 2017 ) reported greater nitrogen fixation in pure pigeon pea crop (32 kg N ha⁻¹) than in mixed pigeon pea–groundnut crop (15 kg N ha⁻¹) during a dry year in Northern Malawi. Berriel and Perdomo ( 2023 ) also observed high N input through biological nitrogen fixation of pigeon pea used as a cover crop in Uruguay (253 kg N ha − 1 ). In contrast, our study showed relatively lower values of N input, may be due to harsh ecological conditions and soil constraints. Both studies reported higher levels of available P in the topsoil (0–15 cm depth) than those observed at our site, where low P availability may have limited symbiotic fixation capacity. However, the nitrogen contribution is non negligeable considering that there is no fertilizers applied by the farmers in this region (Violas et al. 2018). Mapfumo et al ( 1999 ) reported similar findings in pigeon pea, further supporting this observation. 5. Conclusion This study provides key insights into nitrogen fixation by pigeon pea as well as into nitrogen transfer dynamics to the associated crop. Our results showed that 2-year old pigeon pea largely relied on biological nitrogen fixation, regardless of the cropping system, which is of great importance in soils with high N deficiency. The high %Ndfa observed aligns with previous findings in semi-arid regions, reinforcing the role of pigeon pea as a highly efficient N-fixing species. These findings highlight the potential of agroecological blocks using pigeon pea to maintain nitrogen supply through biological fixation under semi-arid conditions and without external fertilization, thereby contributing to the resilience of low-input farming systems. Overall, our work highlights the potential of pigeon pea as a sustainable nitrogen source in unfertilized agroecosystems. However, further studies are needed to assess seasonal variations in N 2 fixation and long-term effects on soil fertility and crop productivity. In addition, research should focus on identifying pigeon pea varieties best suited to semi-arid region with sandy soil, and on the use of phosphorus fertilizers to improve the N 2 fixation efficiency of this NFS. Declarations Acknowledgements The authors would like to thank the staff of CTAS and GRET, as well the producers at the Karoke Mahandrorano agroecological block for their facilitation and technical support during experimentation and data collection; and gratefully acknowledge the LRI laboratory for soil analyses, SILVATECH for plant N and 15 N analyses. Funding This study was funded by the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH, through the Action ProSilience Madagascar. ProSilience has been jointly funded by the European Union (EU) and the Federal Ministry for Economic Cooperation and Development (BMZ). The contents are the sole responsibility of the authors and do not necessarily reflect the views of the GIZ, EU and BMZ. Conflicts of interest The authors declare no competing interests Author contribution RAKOTOZAFY Sarah: Investigation, formal analysis, writing original draft BORDRON Bruno: Methodology, funding acquisition, formal analysis, writing - review & editing RAZAFIMBELO Tantely: Validation, writing - review & editing RAZAFINDRAKOTO Malalatiana: Validation, writing - review & editing VOM BROCKE Kirsten: Validation, writing - review & editing HAJASOA Mosa Redida: Investigation BOUILLET Jean-Pierre : Conceptualization, methodology, formal analysis, writing original draft References Alon M, Dovrat G, Masci T, Sheffer E (2021) Soil nitrogen regulates symbiotic nitrogen fixation in a legume shrub but does not accumulate under it. Ecosphere 12:e03843. https://doi.org/10.1002/ecs2.3843 Beck HE, Zimmermann NE, McVicar TR, et al (2018) Present and future Köppen-Geiger climate classification maps at 1-km resolution. 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Symbiosis 27:305–318 Menge DNL, Lichstein JW, Ángeles-Pérez G (2014) Nitrogen fixation strategies can explain the latitudinal shift in nitrogen‐fixing tree abundance. Ecology 95:2236–2245. https://doi.org/10.1890/13-2124.1 Mhango WG, Snapp S, Kanyama-Phiri GY (2017) Biological Nitrogen Fixation of Pigeonpea and Groundnut: Quantifying Response Across 18 Farm Sites in Northern Malawi. In: Sutton MA, Mason KE, Bleeker A, et al. (eds) Just Enough Nitrogen. Springer International Publishing, Cham, pp 139–153 Morlat L, Castellanet C (2012) Intervenir dans une région “à l’écart du développement”. L’action du Gret dans l’Androy au Sud de Madagascar. Coopérer aujourd’hui N° 75. Mula MG, Saxena KB (2010) Lifting the level of awareness on pigeonpea - A global perspective. International Crops Research Institute for the Semi-Arid Tropics, Andhra Pradesh , India Munroe JW, Isaac ME (2014) N2-fixing trees and the transfer of fixed-N for sustainable agroforestry: a review. Agron Sustain Dev 34:417–427. https://doi.org/10.1007/s13593-013-0190-5 Nair PKR, Kumar BM, Nair VD (2021) Biological Nitrogen Fixation and Nitrogen Fixing Trees. In: An Introduction to Agroforestry Nandwal AS, Bharti S, Sheoran IS, Kuhad MS (1991) Drought Effects on Carbon Exchange and Nitrogen Fixation in Pigeonpea (Cajanus cajan L.). J Plant Physiol 138:125–127. https://doi.org/10.1016/S0176-1617(11)80744-3 Nygren P, Fernández MP, Harmand J-M, Leblanc HA (2012) Symbiotic dinitrogen fixation by trees: an underestimated resource in agroforestry systems? Nutr Cycl Agroecosystems 94:123–160. https://doi.org/10.1007/s10705-012-9542-9 Octavia D, Murniati, Suharti S, et al (2023) Smart agroforestry for sustaining soil fertility and community livelihood. For Sci Technol 19:315–328. https://doi.org/10.1080/21580103.2023.2269970 Odeny DA (2007) The potential of pigeonpea ( Cajanus cajan ( L .) Millsp .) in Africa. Nat Resour Forum 31:297–305 Oliveira IR, Bordron B, Laclau JP, et al (2021) Nutrient deficiency enhances the rate of short-term belowground transfer of nitrogen from Acacia mangium to Eucalyptus trees in mixed-species plantations. For Ecol Manag 491:119192. https://doi.org/10.1016/j.foreco.2021.119192 Pancholi R, Yadav R, Gupta H, et al (2023) The Role of Agroforestry Systems in Enhancing Climate Resilience and Sustainability- A Review. Int J Environ Clim Change 13:4342–4353. https://doi.org/10.9734/ijecc/2023/v13i113615 Paula RR, Bouillet J-P, De M. Gonçalves JL, et al (2018) Nitrogen fixation rate of Acacia mangium Wild at mid rotation in Brazil is higher in mixed plantations with Eucalyptus grandis Hill ex Maiden than in monocultures. Ann For Sci 75:14. https://doi.org/10.1007/s13595-018-0695-9 Paula RR, Bouillet J-P, Ocheuze Trivelin PC, et al (2015) Evidence of short-term belowground transfer of nitrogen from Acacia mangium to Eucalyptus grandis trees in a tropical planted forest. Soil Biol Biochem 91:99–108. https://doi.org/10.1016/j.soilbio.2015.08.017 Rakotozafy S, Bordron B, Bouillet JP, Razafindrakoto M (2024) Impact de Cajanus cajan sur les propriétés physico-chimiques et biologiques du sol, exploration racinaire et fixation symbiotique comme processus marquants pour le succès des pratiques agroécologiques dans la région Androy à Madagascar Rao JVDKK, Thompson JA, Sastry PVSS, et al (1987) Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer. Plant Soil 101:107–113. https://doi.org/10.1007/BF02371037 Sameer Kumar CV, Satheesh Naik SJ, Mohan N, et al (2017) Botanical Description of Pigeonpea [ Cajanus Cajan (L.) Millsp.]. Springer Int Publ 17–29. https://doi.org/10.1007/978-3-319-63797-6 Saxena RK, Saxena KB, Varshney RK (2019) Pigeonpea (Cajanus cajan L. Millsp.): an ideal crop for sustainable agriculture. Adv Plant Breed Strateg Legum 7:1–522. https://doi.org/10.1007/978-3-030-23400-3 Schenk HJ, Jackson RB (2002) Rooting depths, lateral root spreads and below‐ground/above‐ground allometries of plants in water‐limited ecosystems. J Ecol 90:480–494. https://doi.org/10.1046/j.1365-2745.2002.00682.x Serraj R, Sinclair TR, Purcell LC (1999) Symbiotic N2 fixation response to drought. J Exp Bot 50:143–155 Sheoran IS, Kaur A, Singh R (1988) Nitrogen Fixation and Carbon Metabolism in Nodules of Pigeonpea (Cajanus cajan L.) Under Drought Stress. J Plant Physiol 132:480–483. https://doi.org/10.1016/S0176-1617(88)80067-1 Sileshi GW, Mafongoya PL, Nath AJ (2020) Agroforestry Systems for Improving Nutrient Recycling and Soil Fertility on Degraded Lands. In: Agroforestry for Degraded Landscapes. Springer Singapore, Singapore, pp 225–253 Snoeck D, Zapata F, Domenach A-M (2000) Isotopic evidence of the transfer of nitrogen fixed by legumes to coffee trees. Biotechnol Agron Soc Environ 4:95–100 Thilakarathna MS, McElroy MS, Chapagain T, et al (2016) Belowground nitrogen transfer from legumes to non-legumes under managed herbaceous cropping systems. A review. Agron Sustain Dev 36:1–16. https://doi.org/10.1007/s13593-016-0396-4 UNICEF (2024) Bulletin de monitoring de la sècheresse dans le Grand Sud et Sud-Est de Madagascar Vanlauwe B, Amede T, Bationo, André, et al (2023) Fertilizer and Soil Health in Africa: The role of fertilizer in building soil health to sustain farming and address climate change, IFDC. USA Violas D (2020) Stratégie de développement de l’agroécologie dans le grand Sud malgache: retour d’expériences autour des blocs agroécologiques. Éditions du GRET, Nogent-sur-Marne Yao Y, Yuan H, Wu G, et al (2022) Nitrogen fixation capacity and metabolite responses to phosphorus in soybean nodules. Symbiosis 88:21–35. https://doi.org/10.1007/s13199-022-00882-9 Zayed O, Hewedy OA, Abdelmoteleb A, et al (2023) Nitrogen Journey in Plants: From Uptake to Metabolism, Stress Response, and Microbe Interaction. Biomolecules 13:1443. https://doi.org/10.3390/biom13101443 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7170753","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493174103,"identity":"6ec7c260-9a3a-441e-8e3e-e9230a4513d1","order_by":0,"name":"RAKOTOZAFY Sarah","email":"","orcid":"","institution":"Université d’Antananarivo","correspondingAuthor":false,"prefix":"","firstName":"RAKOTOZAFY","middleName":"","lastName":"Sarah","suffix":""},{"id":493174104,"identity":"639a4dcb-31ee-410a-a7e1-75a02fe7cc95","order_by":1,"name":"BORDRON Bruno","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACAwh1AIgTgLiCgQcsWEG8ljNQLWeI1sLYBhXEp8Vc+ozZgw8Md+TM23MfPi6cZydj3n54A8PBPbi1WPblmBvOYHhmLHPmubHxzG3JPDJn0goYDjzD47AzPGbSPAyHE2dIpLFJ8247wCPBkGPA/OEA0VrmALXwvzFgOEC8lgagFokc/Fose9jKJGcYPDOW4HnGbMxzLBmo5VnBAXxazHmYt0l8qLgjJ8GexviYp8bOXoI/eeMDfFqgzkPjE9QwCkbBKBgFowA/AADszko/lL781gAAAABJRU5ErkJggg==","orcid":"","institution":"CIRAD, UMR Eco\u0026Sols","correspondingAuthor":true,"prefix":"","firstName":"BORDRON","middleName":"","lastName":"Bruno","suffix":""},{"id":493174105,"identity":"12c07ecf-b19f-4210-8d80-28beeae81c21","order_by":2,"name":"RAZAFIMBELO Tantely","email":"","orcid":"","institution":"Université d’Antananarivo","correspondingAuthor":false,"prefix":"","firstName":"RAZAFIMBELO","middleName":"","lastName":"Tantely","suffix":""},{"id":493174106,"identity":"43e6b014-8cc8-446c-a0a3-dd1567a1ef52","order_by":3,"name":"RAZAFINDRAKOTO Malalatiana","email":"","orcid":"","institution":"Université d’Antananarivo","correspondingAuthor":false,"prefix":"","firstName":"RAZAFINDRAKOTO","middleName":"","lastName":"Malalatiana","suffix":""},{"id":493174107,"identity":"6a035618-7d84-4350-81d0-9a8af6049fed","order_by":4,"name":"VOM BROCKE Kirsten","email":"","orcid":"","institution":"CIRAD, UMR AGAP","correspondingAuthor":false,"prefix":"","firstName":"VOM","middleName":"BROCKE","lastName":"Kirsten","suffix":""},{"id":493174108,"identity":"f847c893-a5a0-45f6-85ce-94c3bc2b651d","order_by":5,"name":"HAJASOA Mosa Redida","email":"","orcid":"","institution":"Centre Technique Agro-écologique du Sud, CTAS","correspondingAuthor":false,"prefix":"","firstName":"HAJASOA","middleName":"Mosa","lastName":"Redida","suffix":""},{"id":493174109,"identity":"17cf51de-f010-47ff-bc0a-c9f7369c98fb","order_by":6,"name":"Jean-Pierre BOUILLET","email":"","orcid":"","institution":"CIRAD, UMR Eco\u0026Sols","correspondingAuthor":false,"prefix":"","firstName":"Jean-Pierre","middleName":"","lastName":"BOUILLET","suffix":""}],"badges":[],"createdAt":"2025-07-20 15:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7170753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7170753/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88013512,"identity":"2e48bf06-7dcb-4c84-84ef-0b504583c251","added_by":"auto","created_at":"2025-07-31 12:31:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76945,"visible":true,"origin":"","legend":"\u003cp\u003eRainfall (vertical bars) and mean temperature (circle) in Ambovombe from 2014 to 2024 (data source https://www.historique-meteo.net).\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7170753/v1/c8396b602c9cfe3aae3c09c9.png"},{"id":88012551,"identity":"9a676035-63ce-4b92-b522-dbe897094a59","added_by":"auto","created_at":"2025-07-31 12:23:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":235742,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf sampling method in plots for nitrogen analysis. \u003cstrong\u003eA)\u003c/strong\u003e Sampling point for leaves in the pure cassava or in pure pigeon pea plots. \u003cstrong\u003eB)\u003c/strong\u003e Sampling point for leaves in the mixed pigeon pea-cassava plots. S1: near pigeon pea, S2: ¼ of the inter-row, and S3: ½ of the inter-row\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7170753/v1/43ce3a363637a9f3a05722b1.png"},{"id":88014268,"identity":"7122ef6d-62b7-4425-8f9e-f8a14a1f2ad7","added_by":"auto","created_at":"2025-07-31 12:39:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1241419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7170753/v1/1f7bc60d-3c26-4a7d-bb7d-d0401c31fe30.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High rates of nitrogen fixation and transfer by Cajanus cajan to associated crop in a semi-arid agroforestry system","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAgroforestry systems (AFS) propose a sustainable and productive ecosystem by combining trees and/or shrubs with crops. These perennial cropping systems require less tillage and provide more organic matter to the soil than annual cropping system thereby improving crops nutrition (Bekele-Tesemma \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mafongoya et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sileshi et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In tropical areas, the majority of trees in these systems are nitrogen fixing species (NFS)to improve soil fertility and crop nutrition (Crews \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Menge et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), whereas in semi-arid climates, NFS not only improve the availability of nitrogen where it is frequently deficient, but also help to create favorable micro-environments for crop development (Nair et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The loss of nitrogen in cropping systems through export at harvest (Kim and Isaac \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), ammonia (NH\u003csub\u003e3\u003c/sub\u003e) volatilization, and nitrate (NO₃) and ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) leaching (Cameron et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kim and Isaac \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) is a frequent problem in AFS cropping systems. On the one hand, NFS are likely to reduce nitrogen loss within AFS and on the other hand, they can supply nitrogen to the system through atmospheric fixation (Kim \u0026amp; Isaac, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nygren et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile fixation of atmospheric N\u003csub\u003e2\u003c/sub\u003e in NH₄\u003csup\u003e+\u003c/sup\u003e form, through symbiosis with Rhizobium bacteria, is a major source for nitrogen input in agroecosystems (Nygren et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), environmental factors can reduce the N\u003csub\u003e2\u003c/sub\u003e fixation (Bordeleau and Pr\u0026eacute;vost \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Paula et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several studies on soybean indicated that drought stress (Freitas et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Serraj et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1999\u003c/span\u003e),soil nutrient deficiency such as low P availability (Yao et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) can decrease N\u003csub\u003e2\u003c/sub\u003e fixation as a consequence of photosynthesis reduction and decrease in photosynthates supply to the nodules (Nandwal et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Sheoran et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCrops associated with NFS can benefit by direct transfer through root exudates and common mycorrhizal networks (CMN), or indirectly through litter mineralization, and fine root and nodule turnover (Munroe and Isaac \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nygren et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Paula et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Thilakarathna et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previous studies in tropical environments have suggested that N-transfer may be stronger when the associated crop is grown close to the NFS (Paula et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Zayed et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that under nutrient deficiency, especially N deficiency, plants may generally activate physiological and molecular mechanisms to improve their nitrogen uptake efficiency. Eucalyptus trees grown in association with acacia have been shown to develop more extensive root systems and increase root exudation (Oliveira et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These responses contribute to improve N-transfer within mixed species where the associated crop increase their demand for available N and the NFS respond (Oliveira et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the NFS used in agroforestry systems, pigeon pea (\u003cem\u003eCajanus cajan\u003c/em\u003e (L.) Millsp) is one of the species recommended for semi-arid areas (Daniel and Ong \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Mula and Saxena \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Saxena et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This leguminous shrub is adapted to harsh climatic and environmental conditions such as low rainfall and nutrient poor soil (Mula and Saxena \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Odeny \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Saxena et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, pigeon pea is efficient to fix atmospheric N\u003csub\u003e2\u003c/sub\u003e (Bopape et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kumar Rao and Dart \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Sameer Kumar et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In semi-arid Kenya, the percentage of nitrogen derived from fixation (%Ndfa) of five pigeon pea varieties were estimated over 80% in roots, leaves, stover, grains (Kwena et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe southern Androy region of Madagascar, is characterized by a semi-arid climate, frequent drought, and severe aeolian erosion. These environmental constraints, combined with widespread poverty, have led to deforestation, poor soil fertility and low agricultural productivity (Violas \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To address this issue, a land-use strategy design described as agroecological blocks has been implemented since 2014. These blocks combine physical and biological components over a minimum area of 10 ha (Violas \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). AFS play a central role in these blocks, with the aim of improving environmental protection and soil fertility by integrating ground cover species and nitrogen-fixing trees, thereby reducing erosion, strengthening the resilience of agro-ecosystems and supporting food production (Fahad et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pancholi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Octavia et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Violas \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pigeon pea is one of the key species used in these AFS, either as sole crop or in associated crop. These systems are mainly adopted by resource-limited farmers, consequently, no fertilizers are added, which likely lead to progressive soil nutrient depletion (Vanlauwe et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Violas \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The integration of pigeon pea into these AFS is therefore expected to promote nitrogen inputs and improve the N status of associated crops. To optimize the management of AFS in this semi-arid area, this study aims to estimate the amount of atmospheric nitrogen fixed and transferred to the associated crops. The results are intended to guide spatial configurations of this agroforestry system to enhance biological nitrogen inputs and improve soil fertility without external inputs.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study site\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe study was carried out in the Karoke Mahandrorano agroecological block (25\u0026deg;15'15\"S, 46\u0026deg;1'21\"E), located in the Androy region of South Madagascar near the city of Ambovombe, at 200 m asl. Established in 2013, this is one of the oldest agroecological blocks in the region, and serves as a reference site for the other blocks. The site is located on a coastal area, on limestone rock. The relief was characterized by gently undulating topography with slope\u0026thinsp;\u0026lt;\u0026thinsp;5%. The soil texture was uniform below a depth of 30 cm with sand content around 80%. The soil pH\u003csub\u003eH2O\u003c/sub\u003e ranged from 7.2 to 9.1 depending on the plots. Soil chemical analyses down to a depth of 0.30 m are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe climate of the region is classified as BSh (semi-arid hot) according to K\u0026ouml;ppen-Geiger classification (Beck et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), with wet season from December to April and dry season from May to November (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average annual temperature is 24.4\u0026deg;C, with temperature varying from 19\u0026deg;C (minimum in June) to 40\u0026deg;C (maximum in January). Annual rainfall ranges from less than 300 and 600 mm, occurring mainly during wet season. Sporadic and limited rainfall may also occur in May and June. However, precipitation is highly erratic, with frequent droughts and irregular rainfall patterns. For instance, in 2020, no rainfall was recorded for 18 consecutive months, and in other years, only 3 to 4 rainfall events per season (UNICEF \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Potential evapotranspiration is 518 mm. year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which generally exceeds rainfall throughout the year (JICA \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). From August to October, violent soil-eroding winds occur (Morlat and Castellanet \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Violas \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe study was conducted during 90 days from January 2024 to April 2024. During this period, the average air temperature was 22.5\u0026deg; C and the cumulative rainfall was 224 mm (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.historique-meteo.net\u003c/span\u003e\u003cspan address=\"https://www.historique-meteo.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMain chemical soil attributes of the top soil without pigeon pea at the experimental area (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoil depth (cm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u0026ndash;10\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u0026ndash;20\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u0026ndash;30\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrganic C (g.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal soil N (g.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAvailable P (mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK (cmol+.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCa (cmol\u0026thinsp;+\u0026thinsp;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNa (cmol\u0026thinsp;+\u0026thinsp;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Experimental layout\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe \u003csup\u003e15\u003c/sup\u003eN isotope dilution method was used to estimate the %Ndfa. The method was applied to pure pigeon pea, mixed pigeon pea-cassava (Manihot esculenta Crantz), and pure cassava plots.\u003c/p\u003e\u003cp\u003eIn total, nine farmer plots were selected within the AFS agroecological block for the experiment, corresponding to three cropping system with three replicates each: pure pigeon pea, mixed pigeon pea-cassava, and pure cassava plots. All pigeon pea plants were two years old, while the cassava plants were six months old at the time of the experiment. Cassava, a non- N fixing species, was chosen as the reference plant for %Ndfa estimation (Boddey et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 \u003csup\u003e15\u003c/sup\u003eN soil labeling\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAn area of 100 m\u003csup\u003e2\u003c/sup\u003e was delimited in each plot. Ammonium chloride (Cl-\u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e) with 98 atom% \u003csup\u003e15\u003c/sup\u003eN was applied in this area at a rate of 0.8 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e on 15th, 16th, and 17th January 2024. The fertilizer was diluted in water and applied uniformly to the soil using a watering can. We assumed that the low amount of applied N did not affect the N\u003csub\u003e2\u003c/sub\u003e fixation of pigeon pea (Bouillet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Plant sampling and analyzes\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eAboveground leaf biomass of pigeon pea plants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn each pure pigeon pea and mixed plots, a pigeon pea inventory was carried out by counting individual plants and measuring their height. To estimate aboveground leaf biomass, all leaves were harvested randomly from 10 shrubs per plot, replicated four times, resulting in a total of 80 shrubs sampled (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both fresh and dry masses were recorded.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLeaf sampling for N fixation measurements\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor nitrogen analysis, leaf samples were separately collected in each system and for each species at two time points: before soil labeling in January 2024 and 90 days after soil labeling in April 2024.\u003c/p\u003e\u003cp\u003eIn the pure pigeon pea plots, leaf sampling was carried out on five shrubs arranged diagonally in a cross pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A). For each shrub, twenty leaves from the upper part of the crown on two pairs of opposite branches (north-south and east-west) were collected and pooled to make a composite sample representative of the plot (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the pure cassava plots, this sampling was carried out on 10 plants arranged diagonally and in a cross pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A): four leaves were sampled per plant, one in each direction (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the mixed pigeon pea - cassava plots, pigeon pea leaves were collected from five shrubs in a row, with 20 leaves per shrub (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while the cassava, leaves were collected at three different distances from the pigeon pea row: the first plant close to the shrub (near), the second at a medium distance (middle), and the last between two pigeon pea rows (far) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e.B).\u003c/p\u003e\u003cp\u003eThe sampled leaves were then dried at 60\u0026deg;C and ground, and sent to SILVATECH Laboratory (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www6.nancy.inrae.fr/silva/Plateformes/SilvaTech\u003c/span\u003e\u003cspan address=\"https://www6.nancy.inrae.fr/silva/Plateformes/SilvaTech\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for analysis. For this purpose, 10 mg of leaf powder dry matter were placed in tin capsules for isotopic analyzes. N concentrations and δ\u003csup\u003e15\u003c/sup\u003eN values of the leaves were determined using an elemental analyzer (vario ISOTOPE cube, Elementar, Langenselbold, Germany) coupled, via a gas box interface, to a continuous flow isotope ratio mass spectrometer (Isoprime100, IRMS, Elementar UK, Cheadle, United Kingdom). The precision of δ\u003csup\u003e15\u003c/sup\u003eN measurement was 0.5\u0026permil;.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNumber of pigeon pea sampled for leaf biomass and leaf samples for nitrogen analysis in pure and mixed plots\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePigeon pea sample for leaf biomass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLeaf sample for N analyzes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePure crop\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMixed crop\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eReplicates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e810\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 N derived from atmospheric fixation (%Ndfa)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe percentage of N derived from N\u003csub\u003e2\u003c/sub\u003e fixation (%Ndfa) for each plot during the experiment is estimated by examining \u0026sup1;⁵N Atom Excess (AE) using the following equation (Bouillet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e):\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{\\%}\\varvec{N}\\varvec{d}\\varvec{f}\\varvec{a}=100\\left(1-\\frac{\\frac{\\left({\\varvec{N}}_{\\varvec{F}\\varvec{f}\\varvec{i}\\varvec{n}\\varvec{a}\\varvec{l}}\\:{\\varvec{A}\\varvec{E}}_{\\varvec{F}\\varvec{f}\\varvec{i}\\varvec{n}\\varvec{a}\\varvec{l}}-{\\varvec{N}}_{\\varvec{F}\\varvec{i}\\varvec{n}\\varvec{i}\\varvec{t}\\varvec{i}\\varvec{a}\\varvec{l}}{\\:\\varvec{A}\\varvec{E}}_{\\varvec{F}\\varvec{i}\\varvec{n}\\varvec{i}\\varvec{t}\\varvec{i}\\varvec{a}\\varvec{l}}\\:\\right)}{\\left({\\varvec{N}}_{\\varvec{F}\\varvec{f}\\varvec{i}\\varvec{n}\\varvec{a}\\varvec{l}\\:}-{\\varvec{N}}_{\\varvec{F}\\varvec{i}\\varvec{n}\\varvec{i}\\varvec{t}\\varvec{i}\\varvec{a}\\varvec{l}}\\right)}}{\\frac{\\left({\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{f}\\varvec{i}\\varvec{n}\\varvec{a}\\varvec{l}}\\:{\\varvec{A}\\varvec{E}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{f}\\varvec{i}\\varvec{n}\\varvec{a}\\varvec{l}}\\:-{\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{i}\\varvec{n}\\varvec{i}\\varvec{t}\\varvec{i}\\varvec{a}\\varvec{l}}\\:{\\varvec{A}\\varvec{E}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{i}\\varvec{n}\\varvec{i}\\varvec{t}\\varvec{i}\\varvec{a}\\varvec{l}}\\:\\right)}{\\left({\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{f}\\varvec{i}\\varvec{n}\\varvec{a}\\varvec{l}}-{\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{i}\\varvec{n}\\varvec{i}\\varvec{t}\\varvec{i}\\varvec{a}\\varvec{l}}\\right)}}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ewhere: AE\u0026thinsp;=\u0026thinsp;\u003csup\u003e15\u003c/sup\u003eN x 100 / (\u003csup\u003e15\u003c/sup\u003eN + \u003csup\u003e14\u003c/sup\u003eN) \u0026minus;\u0026thinsp;0.003663, AE\u003csub\u003eFfinal\u003c/sub\u003e, AE\u003csub\u003eFinitial\u003c/sub\u003e, AE\u003csub\u003eREFfinal\u003c/sub\u003e, and AE\u003csub\u003eREFinitial\u003c/sub\u003e are the percentage atom excess of pigeon pea 90 days after soil labeling, the percentage atom excess of pigeon pea at soil labeling, the percentage atom excess of cassava 90 days after soil labeling, and the percentage atom excess of cassava at soil labeling, respectively. N\u003csub\u003eFfinal\u003c/sub\u003e, N\u003csub\u003eFinitial\u003c/sub\u003e, N\u003csub\u003eREFfinal\u003c/sub\u003e, and N\u003csub\u003eREFinitial\u003c/sub\u003e are the N content of pigeon pea 90 days after soil labeling, the N content of pigeon pea at soil labeling, the N content of cassava 90 days after soil labeling, and the N content of cassava at soil labeling, respectively.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 N derived from transfer\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe proportion of cassava N derived from transfer (%Ndft) from pigeon pea during the experiment period was estimated using Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (Snoeck et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) :\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{\\%}\\varvec{N}\\varvec{d}\\varvec{f}\\varvec{t}=100\\times\\:\\:\\frac{{\\varvec{\\delta\\:}}^{15}{\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}}-\\:{\\varvec{\\delta\\:}}^{15}{\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}\\varvec{a}\\varvec{s}\\varvec{s}}}{{\\varvec{\\delta\\:}}^{15}{\\varvec{N}}_{\\varvec{R}\\varvec{E}\\varvec{F}}-\\:{\\varvec{\\delta\\:}}^{15}{\\varvec{N}}_{\\varvec{f}\\varvec{i}\\varvec{x}\\varvec{a}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ewhere: δ\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003eREF\u003c/sub\u003e is the δ\u003csup\u003e15\u003c/sup\u003eN value of cassava leaves in pure crop, δ\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003eREFass\u003c/sub\u003e is the δ\u003csup\u003e15\u003c/sup\u003eN of cassava leaves in mixed crop, and δ\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003efixation\u003c/sub\u003e is the δ\u003csup\u003e15\u003c/sup\u003eN of pigeon pea leaves in pure crop.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analyses\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOne-way analysis of variance (ANOVA) was used to compare the means between the N content, δ\u003csup\u003e15\u003c/sup\u003eN and AE of the different system as fixed factor and, %Ndft values of cassava in mixed crop with distance as fixed factor. Tukey\u0026rsquo;s post-hoc tests were used to examine pairwise differences. Assumptions of normality and homoscedasticity of residuals were tested using Shapiro-Wilk and Bartlett tests, respectively. Statistical analyses were performed using R 4.0.3 et RStudio 4.1.1, with a significance level α of 0.05.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe experimental season was characterized by low rainfall (224 mm) and low topsoil fertility. According to Boyer. (1982), the critical deficiency levels for tropical plants grown on ferralitic soils are 0.5 g-kg-\u0026sup1; for total N and 3 mg-kg-\u0026sup1; for available P, while the deficiency thresholds are 1.5 g-kg-\u0026sup1; and 10 mg-kg-\u0026sup1; respectively. Our soil analysis (depth 0\u0026ndash;30 cm) showed total N levels below both the critical level and the deficiency threshold, while available P levels were comparable to the critical level but still above the deficiency threshold. This following section presents data from shrub inventory, pigeon pea biomass assessments, and leaf N analysis, with a focus on N derived from atmospheric fixation (%Ndfa), their transfer in mixed plots (%Ndft), and the overall contribution of fixed N to the system.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 \u003csup\u003e15\u003c/sup\u003eN values and percentage of N derived from atmospheric fixation (%Ndfa)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eNo significant difference in AE mean values between the different crop systems was observed at the beginning of the experiment (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.23), with AE\u003csub\u003einitial\u003c/sub\u003e ranging from 8.42\u0026ndash;9.39% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After 90 days, the AE of cassava leaves in the pure crop was significantly higher than that of pigeon pea leaves in the pure and mixed crops (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008). No significant difference in N content of the cassava and pigeon pea leaves in pure and mixed crops was found at the beginning of the experiment (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18) or after 90 days (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.78). N\u003csub\u003einitial\u003c/sub\u003e ranged from 3.56\u0026ndash;4.09%, while N\u003csub\u003efinal\u003c/sub\u003e ranged from 4.08\u0026ndash;4.25%. The %Ndfa was estimated to be 100% during the 90 days experiment both in the pure pigeon pea and in mixed pigeon pea-cassava plots.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eN content, \u003csup\u003e15\u003c/sup\u003eN atom excess of leaf samples (AE) in pigeon pea and cassava, and %Ndfa of pigeon pea (Mean values, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSystems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLeaf samples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e\u003cp\u003eObserved parameters\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003einitial\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eAE\u003c/b\u003e\u003csub\u003e\u003cb\u003einitial\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eN\u003c/b\u003e\u003csub\u003e\u003cb\u003efinal\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eAE\u003c/b\u003e\u003csub\u003e\u003cb\u003efinal\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e%Ndfa\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.09a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.42a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.08a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.95a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure pigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.56a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.39a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.14a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.58b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixed pigeon pea - cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.78a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.92a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.25a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8.18b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e*N\u003csub\u003einitial\u003c/sub\u003e and N\u003csub\u003efinal\u003c/sub\u003e are the N content of leaf samples on soil labeling and after 90 days respectively, expressed in %.\u003c/p\u003e\u003cp\u003eAE\u003csub\u003einitial\u003c/sub\u003e and AE\u003csub\u003efinal\u003c/sub\u003e are the values of atom excess at soil labeling and after 90 days respectively, expressed in %.\u003c/p\u003e\u003cp\u003e%Ndfa is the percentage of nitrogen derived from fixation atmospheric.\u003c/p\u003e\u003cp\u003eLetters (a\u0026ndash;b) indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between systems.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Nitrogen input from N\u003csub\u003e2\u003c/sub\u003e fixation\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe planting density of pigeon pea was higher in pure system (3464 individuals ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to the mixed pigeon pea-cassava system (1216 individuals ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), due to inter-row spacing constraints in the mixed plots. Despite this, the average height of pigeon pea shrubs was around 180\u0026ndash;190 cm in both systems, suggesting comparable vertical growth regardless of planting arrangement. (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsidering individual plants for each system, there was more N fixed in the mixed pigeon pea-cassava plots (6.97 g N plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than in the pure pigeon pea plots (4.94 g N plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), which is expected given the higher leaf biomass per plant in the mixed plot. However, taking planting density into account, the total N input over two years was higher in the pure pigeon pea plots, reaching 17.11 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, compared with 8.48 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the mixed pigeon pea -cassava plots (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAboveground pigeon pea growth parameters and nitrogen supply from %Ndfa in 2-year-old pigeon pea plants, in both pure and mixed pigeon pea plots plots (Mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSystems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePure pigeon pea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMixed pigeon pea - cassava\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePigeon pea planting density (individuals ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3464\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1216\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHeight (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e196.5\u0026thinsp;\u0026plusmn;\u0026thinsp;44.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e188.3\u0026thinsp;\u0026plusmn;\u0026thinsp;49.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf biomass (kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%Ndfa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg N plant\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from %Ndfa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ekg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from %Ndfa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 N transferred from pigeon pea to cassava\u003c/h2\u003e\u003cp\u003eAfter 90 days, the mean values of \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003efinal\u003c/sub\u003e varies slightly but significantly from 0.37\u0026ndash;0.44% between the three systems (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The highest value of \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003efinal\u003c/sub\u003e was recorded in leaves of the pure cassava plots (0.44%), while the lowest value was observed in the pigeon pea leaves of the mixed pigeon pea-cassava plots (0.38%).\u003c/p\u003e\u003cp\u003eCassava leaves located at different distances from pigeon pea (near, at \u0026frac14; inter-row and \u0026frac12; inter-row) showed similar and non-significant \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003efinal\u003c/sub\u003e values (0.41%, 0.42% and 0.40% respectively), (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the percentage of N transferred from pigeon pea to cassava (%Ndft) varied significantly with increasing distance from the pigeon pea plants (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0009). Cassava plants located in the \u0026frac12; inter-row received the highest proportion of nitrogen transferred (72.95%), while those positioned near to the pigeon pea (51.73%) or slightly further in the \u0026frac14; inter-row (42.45%) received less N. These results suggest that spatial arrangement plays a key role in promoting N transfer. No significant difference in %Ndft was found between the last two positions.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean leaf samples \u003csup\u003e15\u003c/sup\u003eN values in the three cropping system and %Ndft in the mixed plots in relation to the distance between the non-leguminous crop (cassava) and the leguminous crop (pigeon pea)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSystem\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeaf samples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDistance from pigeon pea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003efinal\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%Ndft\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure pigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.39d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.45a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixed pigeon pea - cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigeon pea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.38d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixed pigeon pea- cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNear (S1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.41bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e51.73b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixed pigeon pea - cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026frac14; inter-row (S2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.42b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42.45b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMixed pigeon pea - cassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCassava\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026frac12; inter-row (S3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.40cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e72.95a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003efinal\u003c/sub\u003e is the value of δ\u003csup\u003e15\u003c/sup\u003eN expressed in % of leaf samples after 90 days. %Ndft is the proportion of N derived from transfer. Letters (a\u0026ndash;d) indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between systems.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.1 High efficiency of pigeon pea to fix atmospheric N\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eThe nitrogen fixation rate of pigeon pea was estimated at 100% in both, pure pigeon pea and mixed pigeon pea-cassava plots over the 90-day experimental period. These results indicate that, over this period, all the N in pigeon pea leaves was derived from N\u003csub\u003e2\u003c/sub\u003e fixation. This also suggests that, at least during the second vegetative season, pigeon pea relied entirely on biological nitrogen fixation, regardless of the cropping system. According to Dovrat and Sheffer (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), biological N fixation is generally optimal during wet season. Soil N deficiency (Dovrat et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and high P availability (Alon et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) has been shown to increase nitrogen fixation rates by NFS. In our study, although P availability was close to the critical level but likely sufficient to support the energy costs for the N\u003csub\u003e2\u003c/sub\u003e fixation. In addition, the low levels of N in the soil and the fact that the experiment was conducted during the rainy season probably contributed to maximizing symbiotic nitrogen fixation. It is consistent with previous studies showing that biological N fixation is more efficient under unfertilized conditions (Figueiredo et al., 2025). Similar high levels of %Ndfa have been reported in other semi-arid regions: Rao et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) recorded 88% in sole pigeon pea and 96% in mixed pigeon pea\u0026ndash;sorghum systems in India, while Kwena et al (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found values exceeding 80% in pigeon pea\u0026ndash;maize intercropping in Kenya without rhizobia inoculation. Mhango et al (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported %Ndfa about 76% and no significant difference between pure pigeon pea and mixed pigeon pea-maize crop system.\u003c/p\u003e\u003cp\u003eHowever, short rainfall season means low biomass production and lower N fixation. Paula et al (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showed variability in N\u003csub\u003e2\u003c/sub\u003e fixation rates between seasons in mixed eucalyptus-acacia cropping system in Brazil. Water availability plays a critical role in improving biological N fixation by reactivating or enhancing biomass nodules of NFS. Pigeon pea is also known for its water use efficiency, allowing it to sustain biological N\u003csub\u003e2\u003c/sub\u003e fixation even under tropical dryland conditions (Berriel and Perdomo \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, it will be important to assess the dynamics of N\u003csub\u003e2\u003c/sub\u003e fixation from the early stages of pigeon pea growth and across different seasons, as seasonal variations in %Ndfa could occur.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Nitrogen transfer dynamics in mixed cropping systems\u003c/h2\u003e\u003cp\u003eBetween 42% and 72% of N was transferred from pigeon pea to cassava. Therefore, the association of pigeon pea benefited the crop system by improving the N nutrition of the associated plant. Monroe and Isaac (2014) indicated that N from NFS can be a source of easily available N for the associated crop via fine root decomposition and nodule turnover or N rhizodeposition. Nitrogen could also be transferred from NFS to companion species \u003cem\u003evia\u003c/em\u003e common mycorrhizal networks (Ingleby et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe found that nitrogen transfer dynamics between pigeon pea and cassava were influenced by cassava localization, with higher %Ndft observed at greater distances from pigeon pea. This invalidates our hypothesis of higher %Ndft at closer distance of cassava from pigeon pea, as observed for N transfer from \u003cem\u003eAcacia mangium\u003c/em\u003e to \u003cem\u003eEucalyptus grandis\u003c/em\u003e in mixed-species plantations (Paula et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This could be due to root spatio-temporal dynamics of the fine roots of pigeon pea. Actually, it has been reported that shrubs often have significant lateral root spread, which can favor their interaction with neighboring crops and exploit the surface layers of the soil (Schenk and Jackson \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In legumes more generally, an increased root density leads to greater nitrogen release through rhizodeposition (Thilakarathna et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A field study revealed, that at two years, pigeon pea roots could reach up to 4 m laterally in the Androy region of Madagascar (Rakotozafy et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These observations were not in line with the findings of Ito et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) who found that the roots of pigeon pea spread more vertically than horizontally in semi-arid conditions. This discrepancy might be due to the soil characteristics in our study area with a rocky layer found at a depth of 150 cm (Rakotozafy et al., personal communication, 2024). However, our results highlight that the distance of 6\u0026ndash;8 m between rows of pigeon pea, as adopted by farmers in the Ambovombe region allows effective N transfer from this NFS to cassava.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Nitrogen input to the system\u003c/h2\u003e\u003cp\u003eAt two years after planting, N input was at most 17.1 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in pure pigeon pea plots and 8.5 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in mixed pigeon pea-cassava plots, which was consistent with the respective planting density between the systems. Keston et al (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) also found higher N inputs through biological N fixation in pure crops (85.7 kg N ha⁻\u0026sup1;) than in mixed pigeon pea\u0026ndash;cowpea crops (57.4 kg N ha⁻\u0026sup1;) after one growing season in Central Malawi. Similarly, Mhango et al (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported greater nitrogen fixation in pure pigeon pea crop (32 kg N ha⁻\u0026sup1;) than in mixed pigeon pea\u0026ndash;groundnut crop (15 kg N ha⁻\u0026sup1;) during a dry year in Northern Malawi. Berriel and Perdomo (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also observed high N input through biological nitrogen fixation of pigeon pea used as a cover crop in Uruguay (253 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In contrast, our study showed relatively lower values of N input, may be due to harsh ecological conditions and soil constraints. Both studies reported higher levels of available P in the topsoil (0\u0026ndash;15 cm depth) than those observed at our site, where low P availability may have limited symbiotic fixation capacity. However, the nitrogen contribution is non negligeable considering that there is no fertilizers applied by the farmers in this region (Violas et al. 2018). Mapfumo et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) reported similar findings in pigeon pea, further supporting this observation.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study provides key insights into nitrogen fixation by pigeon pea as well as into nitrogen transfer dynamics to the associated crop. Our results showed that 2-year old pigeon pea largely relied on biological nitrogen fixation, regardless of the cropping system, which is of great importance in soils with high N deficiency. The high %Ndfa observed aligns with previous findings in semi-arid regions, reinforcing the role of pigeon pea as a highly efficient N-fixing species. These findings highlight the potential of agroecological blocks using pigeon pea to maintain nitrogen supply through biological fixation under semi-arid conditions and without external fertilization, thereby contributing to the resilience of low-input farming systems.\u003c/p\u003e\u003cp\u003eOverall, our work highlights the potential of pigeon pea as a sustainable nitrogen source in unfertilized agroecosystems. However, further studies are needed to assess seasonal variations in N\u003csub\u003e2\u003c/sub\u003e fixation and long-term effects on soil fertility and crop productivity. In addition, research should focus on identifying pigeon pea varieties best suited to semi-arid region with sandy soil, and on the use of phosphorus fertilizers to improve the N\u003csub\u003e2\u003c/sub\u003e fixation efficiency of this NFS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the staff of CTAS and GRET, as well the producers at the Karoke Mahandrorano agroecological block for their facilitation and technical support during experimentation and data collection; and gratefully acknowledge the LRI laboratory for soil analyses, SILVATECH for plant N and \u003csup\u003e15\u003c/sup\u003eN analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Deutsche Gesellschaft f\u0026uuml;r Internationale Zusammenarbeit (GIZ) GmbH, through the Action ProSilience Madagascar. ProSilience has been jointly funded by the European Union (EU) and the Federal Ministry for Economic Cooperation and Development (BMZ).\u003c/p\u003e\n\u003cp\u003eThe contents are the sole responsibility of the authors and do not necessarily reflect the views of the GIZ, EU and BMZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAKOTOZAFY Sarah: Investigation, formal analysis, writing original draft\u003c/p\u003e\n\u003cp\u003eBORDRON Bruno: Methodology, funding acquisition, formal analysis, writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eRAZAFIMBELO Tantely: Validation, writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eRAZAFINDRAKOTO Malalatiana: Validation, writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eVOM BROCKE Kirsten: Validation, writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eHAJASOA Mosa Redida: Investigation\u003c/p\u003e\n\u003cp\u003eBOUILLET Jean-Pierre : Conceptualization, methodology, formal analysis, writing original draft\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlon M, Dovrat G, Masci T, Sheffer E (2021) Soil nitrogen regulates symbiotic nitrogen fixation in a legume shrub but does not accumulate under it. Ecosphere 12:e03843. https://doi.org/10.1002/ecs2.3843\u003c/li\u003e\n\u003cli\u003eBeck HE, Zimmermann NE, McVicar TR, et al (2018) Present and future K\u0026ouml;ppen-Geiger climate classification maps at 1-km resolution. Sci Data 5:180214. https://doi.org/10.1038/sdata.2018.214\u003c/li\u003e\n\u003cli\u003eBekele-Tesemma A (2007) Profitable agroforestry innovations for eastern Africa : experience from 10 agroclimatic zones of Ethiopia, India, Kenya, Tanzania and Uganda, World Agroforestry Centre (ICRAF). Eastern Africa Region\u003c/li\u003e\n\u003cli\u003eBerriel V, Perdomo CH (2023) Cajanus cajan: a promissory high-nitrogen fixing cover crop for Uruguay. Front Agron 5:1214811. https://doi.org/10.3389/fagro.2023.1214811\u003c/li\u003e\n\u003cli\u003eBoddey RM, Urquiaga S, Neves MCP, et al (1990) Quantification of the contribution of N2 fixation to field-grown grain legumes\u0026mdash;A strategy for the practical application of the 15N isotope dilution technique. Soil Biol Biochem 22:649\u0026ndash;655. https://doi.org/10.1016/0038-0717(90)90011-N\u003c/li\u003e\n\u003cli\u003eBopape FL, Beukes CW, Katlego K, et al (2022) Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils. Agriculture 13:30. https://doi.org/10.3390/agriculture13010030\u003c/li\u003e\n\u003cli\u003eBordeleau LM, Pr\u0026eacute;vost D (1994) Nodulation and nitrogen fixation in extreme environments. Plant Soil 161:115\u0026ndash;125. https://doi.org/10.1007/BF02183092\u003c/li\u003e\n\u003cli\u003eBouillet JP, Laclau JP, Gon\u0026ccedil;alves JLM, et al (2008) Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil. For Ecol Manag 255:3918\u0026ndash;3930. https://doi.org/10.1016/j.foreco.2007.10.050\u003c/li\u003e\n\u003cli\u003eBoyer J (1982) Facteurs de fertilit\u0026eacute; et utilisation des sols, Les sols ferrallitiques / Office de la Recherche Scientifique et Technique Outre-Mer. ORSTOM, Paris\u003c/li\u003e\n\u003cli\u003eCameron KC, Di HJ, Moir JL (2013) Nitrogen losses from the soil/plant system: a review. Ann Appl Biol 162:145\u0026ndash;173. https://doi.org/10.1111/aab.12014\u003c/li\u003e\n\u003cli\u003eCrews TE (1999) The presence of nitrogen fixing legumes in terrestrial communities: Evolutionary vs ecological considerations. https://doi.org/10.1007/BF01007581\u003c/li\u003e\n\u003cli\u003eDaniel JN, Ong CK (1990) Perennial pigeonpea: a multi-purpose species for agroforestry systems. Agrofor Syst 10:113\u0026ndash;129. https://doi.org/10.1007/BF00115360\u003c/li\u003e\n\u003cli\u003eDovrat G, Masci T, Bakhshian H, et al (2018) Drought-adapted plants dramatically downregulate dinitrogen fixation: Evidences from Mediterranean legume shrubs. 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Plant Soil 294:125\u0026ndash;136. https://doi.org/10.1007/s11104-007-9239-z\u003c/li\u003e\n\u003cli\u003eIto O, Matsunaga R, Katayama K, et al (1996) Roots and nitrogen in cropping systems of the semi-arid tropics. In: Proceedings of the International Workshop: Dynamics of Roots and Nitrogen in Cropping Systems of the Semi-Arid Tropics. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Pradesh, India\u003c/li\u003e\n\u003cli\u003eJICA (2006) Etude sur l\u0026rsquo;approvisionnement en eau potable, autonome et durable dans la R\u0026eacute;gion du Sud de la R\u0026eacute;publique de Madagascar\u003c/li\u003e\n\u003cli\u003eKeston OWN, Ernest S, Jerome PM, Patson. CN (2017) Biological nitrogen fixation by pigeon pea and cowpea in the doubled-up and other cropping systems on the Luvisols of Central Malawi. 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Nutr Cycl Agroecosystems 76:137\u0026ndash;151. https://doi.org/10.1007/s10705-006-9049-3\u003c/li\u003e\n\u003cli\u003eMapfumo P, Giller KE, Mpepereki S, Mafongoya PL (1999) Dinitrogen fixation by pigeonpea of different maturity types on granitic sandy soils in Zimbabwe. Symbiosis 27:305\u0026ndash;318\u003c/li\u003e\n\u003cli\u003eMenge DNL, Lichstein JW, \u0026Aacute;ngeles-P\u0026eacute;rez G (2014) Nitrogen fixation strategies can explain the latitudinal shift in nitrogen‐fixing tree abundance. Ecology 95:2236\u0026ndash;2245. https://doi.org/10.1890/13-2124.1\u003c/li\u003e\n\u003cli\u003eMhango WG, Snapp S, Kanyama-Phiri GY (2017) Biological Nitrogen Fixation of Pigeonpea and Groundnut: Quantifying Response Across 18 Farm Sites in Northern Malawi. In: Sutton MA, Mason KE, Bleeker A, et al. (eds) Just Enough Nitrogen. Springer International Publishing, Cham, pp 139\u0026ndash;153\u003c/li\u003e\n\u003cli\u003eMorlat L, Castellanet C (2012) Intervenir dans une r\u0026eacute;gion \u0026ldquo;\u0026agrave; l\u0026rsquo;\u0026eacute;cart du d\u0026eacute;veloppement\u0026rdquo;. L\u0026rsquo;action du Gret dans l\u0026rsquo;Androy au Sud de Madagascar. Coop\u0026eacute;rer aujourd\u0026rsquo;hui N\u0026deg; 75.\u003c/li\u003e\n\u003cli\u003eMula MG, Saxena KB (2010) Lifting the level of awareness on pigeonpea - A global perspective. International Crops Research Institute for the Semi-Arid Tropics, Andhra Pradesh , India\u003c/li\u003e\n\u003cli\u003eMunroe JW, Isaac ME (2014) N2-fixing trees and the transfer of fixed-N for sustainable agroforestry: a review. Agron Sustain Dev 34:417\u0026ndash;427. https://doi.org/10.1007/s13593-013-0190-5\u003c/li\u003e\n\u003cli\u003eNair PKR, Kumar BM, Nair VD (2021) Biological Nitrogen Fixation and Nitrogen Fixing Trees. In: An Introduction to Agroforestry\u003c/li\u003e\n\u003cli\u003eNandwal AS, Bharti S, Sheoran IS, Kuhad MS (1991) Drought Effects on Carbon Exchange and Nitrogen Fixation in Pigeonpea (Cajanus cajan L.). J Plant Physiol 138:125\u0026ndash;127. https://doi.org/10.1016/S0176-1617(11)80744-3\u003c/li\u003e\n\u003cli\u003eNygren P, Fern\u0026aacute;ndez MP, Harmand J-M, Leblanc HA (2012) Symbiotic dinitrogen fixation by trees: an underestimated resource in agroforestry systems? Nutr Cycl Agroecosystems 94:123\u0026ndash;160. https://doi.org/10.1007/s10705-012-9542-9\u003c/li\u003e\n\u003cli\u003eOctavia D, Murniati, Suharti S, et al (2023) Smart agroforestry for sustaining soil fertility and community livelihood. For Sci Technol 19:315\u0026ndash;328. https://doi.org/10.1080/21580103.2023.2269970\u003c/li\u003e\n\u003cli\u003eOdeny DA (2007) The potential of pigeonpea ( Cajanus cajan ( L .) Millsp .) in Africa. 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Ann For Sci 75:14. https://doi.org/10.1007/s13595-018-0695-9\u003c/li\u003e\n\u003cli\u003ePaula RR, Bouillet J-P, Ocheuze Trivelin PC, et al (2015) Evidence of short-term belowground transfer of nitrogen from Acacia mangium to Eucalyptus grandis trees in a tropical planted forest. Soil Biol Biochem 91:99\u0026ndash;108. https://doi.org/10.1016/j.soilbio.2015.08.017\u003c/li\u003e\n\u003cli\u003eRakotozafy S, Bordron B, Bouillet JP, Razafindrakoto M (2024) Impact de Cajanus cajan sur les propri\u0026eacute;t\u0026eacute;s physico-chimiques et biologiques du sol, exploration racinaire et fixation symbiotique comme processus marquants pour le succ\u0026egrave;s des pratiques agro\u0026eacute;cologiques dans la r\u0026eacute;gion Androy \u0026agrave; Madagascar\u003c/li\u003e\n\u003cli\u003eRao JVDKK, Thompson JA, Sastry PVSS, et al (1987) Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer. 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J Exp Bot 50:143\u0026ndash;155\u003c/li\u003e\n\u003cli\u003eSheoran IS, Kaur A, Singh R (1988) Nitrogen Fixation and Carbon Metabolism in Nodules of Pigeonpea (Cajanus cajan L.) Under Drought Stress. J Plant Physiol 132:480\u0026ndash;483. https://doi.org/10.1016/S0176-1617(88)80067-1\u003c/li\u003e\n\u003cli\u003eSileshi GW, Mafongoya PL, Nath AJ (2020) Agroforestry Systems for Improving Nutrient Recycling and Soil Fertility on Degraded Lands. In: Agroforestry for Degraded Landscapes. Springer Singapore, Singapore, pp 225\u0026ndash;253\u003c/li\u003e\n\u003cli\u003eSnoeck D, Zapata F, Domenach A-M (2000) Isotopic evidence of the transfer of nitrogen fixed by legumes to coffee trees. Biotechnol Agron Soc Environ 4:95\u0026ndash;100\u003c/li\u003e\n\u003cli\u003eThilakarathna MS, McElroy MS, Chapagain T, et al (2016) Belowground nitrogen transfer from legumes to non-legumes under managed herbaceous cropping systems. A review. Agron Sustain Dev 36:1\u0026ndash;16. https://doi.org/10.1007/s13593-016-0396-4\u003c/li\u003e\n\u003cli\u003eUNICEF (2024) Bulletin de monitoring de la s\u0026egrave;cheresse dans le Grand Sud et Sud-Est de Madagascar\u003c/li\u003e\n\u003cli\u003eVanlauwe B, Amede T, Bationo, Andr\u0026eacute;, et al (2023) Fertilizer and Soil Health in Africa: The role of fertilizer in building soil health to sustain farming and address climate change, IFDC. USA\u003c/li\u003e\n\u003cli\u003eViolas D (2020) Strat\u0026eacute;gie de d\u0026eacute;veloppement de l\u0026rsquo;agro\u0026eacute;cologie dans le grand Sud malgache: retour d\u0026rsquo;exp\u0026eacute;riences autour des blocs agro\u0026eacute;cologiques. \u0026Eacute;ditions du GRET, Nogent-sur-Marne\u003c/li\u003e\n\u003cli\u003eYao Y, Yuan H, Wu G, et al (2022) Nitrogen fixation capacity and metabolite responses to phosphorus in soybean nodules. Symbiosis 88:21\u0026ndash;35. https://doi.org/10.1007/s13199-022-00882-9\u003c/li\u003e\n\u003cli\u003eZayed O, Hewedy OA, Abdelmoteleb A, et al (2023) Nitrogen Journey in Plants: From Uptake to Metabolism, Stress Response, and Microbe Interaction. Biomolecules 13:1443. https://doi.org/10.3390/biom13101443\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"agroforestry-systems","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"agfo","sideBox":"Learn more about [Agroforestry Systems](http://link.springer.com/journal/10457)","snPcode":"10457","submissionUrl":"https://submission.nature.com/new-submission/10457/3","title":"Agroforestry Systems","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"leguminous shrubs, pigeon pea, cassava, symbiotic N2 fixation, N-transfer","lastPublishedDoi":"10.21203/rs.3.rs-7170753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7170753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn semi-arid regions where nitrogen deficiency limits crop productivity, the integration of leguminous tree species into agroforestry systems (AFS) is likely to enhance nitrogen availability for associated crops. We tested this hypothesis in southern Madagascar, a region characterized by erratic and low rainfall, and nutrient-poor soils where the pigeon pea grown as a pure crop or mixed with cassava. Over a 3-month period, we estimated the atmospheric nitrogen fixation rate (%Ndfa) by two-year-old pigeon pea (\u003cem\u003eCajanus cajan\u003c/em\u003e) grown as a pure crop and in association with cassava (\u003cem\u003eManihot esculenta\u003c/em\u003e) using N dilution method, and the transfer of N from pigeon pea to cassava (%Ndft) in the mixed pigeon pea-cassava plots. Six-month-old pure cassava plots were used as reference. Each system was replicated three times. \u003csup\u003e15\u003c/sup\u003eN soil labeling (98 atom% \u003csup\u003e15\u003c/sup\u003eN) was used to estimate %Ndfa and %Ndft. %Ndfa was 100% in both pigeon pea systems. Considering %Ndfa of 100% from planting date, the quantity of N fixed was two times higher in pure pigeon pea plot than in mixed pigeon pea-cassava plots, with values of 17.1 kg N ha-\u0026sup1; and 8.5 kg N ha-\u0026sup1;, respectively. This finding was consistent with the 2.8 times higher planting density in pure pigeon pea plots compared to mixed pigeon pea-cassava plots. Nitrogen transfer from pigeon pea to cassava was greater (72.9%) when the cassava plant was positioned farther from pigeon pea rows (in the middle) compared to when it was planted next to pigeon pea row (42.4%). Our results suggest that integrating pigeon pea into semiarid agroforestry systems could enhance nitrogen status of the associated crop, particularly in unfertilized AFS.\u003c/p\u003e","manuscriptTitle":"High rates of nitrogen fixation and transfer by Cajanus cajan to associated crop in a semi-arid agroforestry system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 12:22:54","doi":"10.21203/rs.3.rs-7170753/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T09:43:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T07:44:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41279922768902622616686878787204120531","date":"2025-08-03T13:10:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114541921176969804615515652602939320691","date":"2025-07-30T12:21:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T13:41:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-28T12:18:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-21T03:13:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Agroforestry Systems","date":"2025-07-20T15:51:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"agroforestry-systems","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"agfo","sideBox":"Learn more about [Agroforestry Systems](http://link.springer.com/journal/10457)","snPcode":"10457","submissionUrl":"https://submission.nature.com/new-submission/10457/3","title":"Agroforestry Systems","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e5d11405-f540-4e73-b836-779828805d47","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T12:41:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 12:22:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7170753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7170753","identity":"rs-7170753","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}