Root growth and belowground interactions in spring wheat - faba bean intercrops

Background and Aims: Intercrops offer multiple advantages over sole crops. However, little is known about the mechanisms that govern the belowground interactions between mixed species. The aim of our study was to characterize root growth and interactions in spring wheat-faba bean intercrops for different sowing densities and spring wheat cultivars, evaluating the potential belowground interactions that governs resource capture. Material and Methods: A �eld experiment was conducted with one faba bean cultivar and two spring wheat cultivars sown at three sowing densities. Destructive root coring was conducted (0-100 cm) in the intercrops and sole crops at two development stages. The root samples were processed to assess the root mass, root length density, and root carbon and nitrogen concentrations. FTIR spectroscopy was used to discriminate the species’ root masses. Results: Root mass advantage of intercrops was observed for both cultivars and both development stages especially in the topsoil layers (0-20cm). A negative impact of intercropping on root mass was observed in the treatment with high total sowing density. Complementarity and cooperation were stronger than competition in the topsoil, but competition dominated root growth in the deeper soil layers. Lower sowing densities encouraged deep rooting of wheat (both cultivars) in intercropping. The early root dominance of one spring wheat cultivar impacted negatively faba bean root growth. Conclusion: Total root mass was affected more by sowing density and less by the spring wheat cultivar in this experiment. An improved understanding of root growth in intercrops can help to support yield advantages and the design of sustainable intercropping systems.


Introduction
Crop mixtures or intercrop (or intercropping is the practice of cultivating two or more crops with different rooting abilities, canopy structure, height, and nutrient requirements simultaneously (Hauggaard-Nielsen et al. 2008; Lithourgidis et al. 2011).Various terminologies for characterizing the yield advantages in intercrops exist in the literature, namely, mixing effect (Hof-Kautz and Rauber 2003), overyielding (Li et (Willey and Osiru 1972), which is identical to 'Land Equivalent Ratio' (LER) de ned by De Wit and Van den Bergh (1965).In the context of our study, we use the term root mass advantage to characterize the positive effect of intercrops on root biomass.To study interactions in intercrops, different experimental designs can be applied.A common one is the replacement (substitutive) design, in which the densities of the partners relative to the respective densities of the sole crops add up to 1t00%.In the additive design, the intercrop is formed by adding the plants of both species in the same densities as in their sole crops; as a result, the total density of the intercrop is higher than the density of sole crops (Snaydon 1991).
The mixture mechanisms that affect intercrop performance are (resource use) complementarity (e.g. through different rooting habits/structures), competition (for light, soil water, and nutrients), and facilitation (e.g. of phosphorus and micronutrient acquisition via root-root interactions) (Vandermeer 1992;Brooker et al. 2015;Stomph et al. 2020;Zhang et al. 2021).The behavior and performance of intercrops is governed by complex interactions and the 4C approach proposed by Justes et al. ( 2021) is both a pedagogical and scienti c way to represent the combination of four joint effects of the competition, complementarity, cooperation, and compensation occurring simultaneously and dynamically between species interactions over the whole cropping cycle.Competition occurs when one species has a greater ability to use limiting resources (e.g., nutrients, water, space, light) than others.According to Justes et al. (2021), complementarity occurs when intercropped plants have different requirements for abiotic resources in space, time, or form.Cooperation (or facilitation) is observed when the modi cation of the environment by one species is bene cial to the other(s).Compensation occurs when the failure of one species is compensated by the other(s) because they differ in their sensitivity to abiotic or biotic stress (Döring and Elsalahy 2022).The 4C approach (Justes et al. 2021) classi es the effects of interspeci c plant-plant interactions, and linked underlying ecological processes as nal effects on yield, and can then be used to assess the plant-plant interactions effects on root density, depth ,and biomass.It has been developed to study intercrops in replacement design.
Due to the mentioned interactions, intercrops offer the possibility of increasing the productivity of a de ned piece of land (Lithourgidis et al. 2011), limiting the use of synthetic fertilizers (Jensen et al. 2020), suppressing weeds (Den Hollander et al. 2007), as well as increasing biodiversity and maintaining and regenerating ecosystem services (Kremen and Miles 2012).Intercrops also minimize risks related to volatile market prices, drought, and/or oods (Brooker et al. 2015;Bedoussac et al. 2015).Further ecosystem services offered by intercrops include belowground biomass advantage which is directly linked to better nitrogen (N) mineralization and carbon (C) sequestration (Cong et al. 2015) and soil stability which decreases soil erosion (Obalum and Obi, 2010, Sharma et al., 2017).
To optimize the intercrop cultivation (e.g.choice of partners, sowing density) and to enhance ecosystem services (e.g.root-based C input for enhanced C sequestration), a better understanding of the underlying mechanisms responsible for belowground growth and interactions in species mixtures and of other ecosystem services is needed (Li et al. 2006; Tosti and Thorup-Kristensen 2010; Bargaz et al. 2015;Brooker et al. 2015;Shao et al. 2019).As root studies are generally laborious, particularly in (in-row) species mixtures, little is known about the effect of intercrop management practices on belowground growth especially under eld conditions and in temperate climatic zones.Several methods for root species identi cation in mixtures have been applied.Methods based on DNA, 13  One important aspect in studying intercrop performance and the linkage of root traits in species mixtures is to understand the effect of management practices such as sowing density and cultivar (cv.) selection as a way to improve intercrop design and cultivation (Demie et al. 2022;Yu et al. 2022).The sowing density is important because it dictates the number of intraspeci c and interspeci c neighbors (Homulle et al. 2022).Sowing density affects aboveground productivity mainly through intra-and interspeci c competition for resources capture (Yu et al. 2016).Belowground, studies on the impact of sowing density on root growth are still scarce, especially when sowing densities of both species are varied.To the best of our knowledge, only Wang et al. (2018) evaluated the effect of increasing total sowing density in a maize-spring wheat strip intercropping system on root growth.They found that with increasing sowing density of maize in species mixtures, root growth of the intercropped maize was increased signi cantly in comparison to the maize sole crop.
The genotype plays an essential role in determining the root traits and eventually the complementarity and/or competition between intercropped species.Shao et al. (2019) found that genotypes with less variation in root size, as well as medium root size, medium to broad root system, and more inter-row root distribution, help to reduce root-to-root competition and tend to have higher yield at high planting densities in a strip intercropping system.Currently, knowledge of the root systems contribution to intercrop yield advantage and the related effects of cultivar choice and sowing density is scarce.Speci c belowground processes between the species should be considered to improve interspeci c facilitation in future species mixture designs (Yu et al. 2022).The aim of this study was therefore to investigate the effect of faba bean and spring wheat intercropping on root and shoot growth and to study the effects of different sowing densities and cultivars on belowground growth.

Site description, eld design, and crop management
The research facility Campus Klein-Altendorf (CKA) of the University of Bonn, Germany, is located in Rheinbach near Bonn (50° 37' 31'' N, 6° 59' 21'' E).The soil at the experimental station was classi ed as Haplic Luvisol, derived from loess and characterised by a silty-loamy texture with clay accumulation in the subsoil between about 45 and 95 cm soil depth (Barej et al. 2014).The climate at the experimental station can be described as moderately humid with maritime in uences.The mean annual air temperature and precipitation are 10.3°C and 669 mm (1991 to 2020), respectively.In 2021, an in-row mixture trial of spring wheat (Triticum aestivum L.) and faba bean (Vicia faba L.) with different spring wheat and faba bean cv. and sowing densities was established.Each cultivar was also sown as a sole crop.In a subset of these plots, the presented root observations were conducted (Table 1).The sowing densities are given in Table 2. Due to a sowing error, the intended eld design could not be fully implemented and there were therefore less than four eld replicates available (Table S1).Therefore, root sampling was repeated four times in the selected plot (one for each treatment).The plot size was 15m 2 (1.5 x 10 m) with a row distance of 21 cm and 6 rows per plot.

Root sampling
Root samples were taken with a soil auger with an inner diameter of 9 cm down to 100 cm soil depth in the selected plots on 09/06/2021 and on 05-06/07/2021.The root sampling in the intercrop treatments covered always one faba bean and one wheat plant and the core was placed not exactly above a row but next to the row (from the row to 1.5 cm from the middle of the row).On 09/06/2021, the BBCH stages of wheat and faba bean were 39 (end of shooting) and 63 (full owering), respectively.On 05-06/07/2021, the BBCH stages of wheat and faba bean were 69 (end of owering) and 71 (approx.10% of the pods have a species or variety-speci c size achieved), respectively.Samples were taken in eight plots (three sole crops and ve intercrops) replicated four times per plot (Table 1).Soil cores were split into ten centimetre sections and stored separately in plastic bags and dried under a plastic crop tunnel before sample preparation and evaluation performed at the University of Göttingen, Germany.

Estimation of root biomass, root carbon, and nitrogen concentrations
The root samples were washed in a root washing machine (custom made, mesh size 1mm) and cleaned of soil residues and non-root particular organic matter manually.The root samples were frozen in a tea bag between different cleaning, scanning, and drying steps.Roots were scanned with a at-bed scanner (Expression 12000XL, Epson, Suwa, Japan) and analysed with WinRhizo 2016a software (Régent Instruments Inc., Quebec, QC; Canada) to estimate the root length density (RLD, cm cm − 3 soil).After scanning, all roots were oven-dried at 40°C for 48 h and weighted.The samples were ground with an ultra-centrifugal mill (Retsch, ZM 200, Haan, Germany) and stored in glass vials for the next analysis (see Section 2.4).
Due to low absolute weights in deeper soil layers, the root mass samples of the subsoil layers were pooled for weighing and for the carbon (C) and N content determination (after the FTIR analyses) resulting in samples soil depths of 0-10 cm, 10-20 cm, 20-30 cm, 30-60 cm, and 60-100 cm.Root C and N were measured according to ISO 13878 and ISO 10694 standards with an elemental analyzer VarioMAX cube (Elementar Analysensysteme GmbH, Langenselbold, Germany).

Discrimination between species
Fourier Transform Infrared Spectroscopy (FTIR) The roots of the sole crops of the two spring wheat cultivars (SU Ahab and Anabel) and one faba bean cultivar (Fanfare) were used to evaluate the species' root proportion in the intercrop samples.Absorption spectra of the ground root samples of the sole crops, as well as of the intercrops, were measured by the FTIR-ATR spectrometer (Alpha-P with a diamond crystal attenuated total re ection (ATR) device, Bruker Optics, Ettlingen, Germany) with a resolution of 4 cm − 1 and 32 scans in the spectral range of 4000 − 400 cm − 1 .Each sample was measured 3 to 5 times.The evaluation of the FTIR-ATR spectra was conducted with the Opus software Quant 2 (version 7.2, Bruker Optics, Ettlingen, Germany).The FTIR spectra of the sole crop sample species were used for a cluster analysis (Opus software, version 7.2, Bruker Optics) to allow for species discrimination.For the cluster analyses, the spectra were pre-processed by second derivative and vector normalization, the frequency range was reduced and the Euclidian's distance and Ward's algorithm was applied (Fig. S1, S2 and S3).The interspeci c heterogeneity for both species was higher than the intraspeci c heterogeneity permitting a separation of the two species.Both spring wheat cultivars separately but also combined were clearly separable from faba bean via cluster analysis (Fig. S4).Since the average FTIR spectra of both spring wheat cultivars were very similar, both spring wheat cultivars were combined for the second sampling date analyses (Fig. S4 and S5).

Model establishment
For the quanti cation of the root proportion of each species in the intercrops root samples, the FTIR spectra of the single species samples were used to generate a model.For establishing a two-species model, a calibration set of 35 "arti cial mixtures" was generated in 3% steps from 0-100% for spring wheat and faba bean, respectively.These mixtures covered the complete calibration range.20 additional "arti cial mixtures" with known species composition were generated to be used for external calibration of the model.With the FTIR spectra of these calibration mixtures, a model was calculated on the basis of multivariate calibrations with the method of partial least square (PLS) regression using the software Quant 2 (Opus, version 7.2, Bruker Optics, Ettlingen, Germany).The absorption of infrared radiation is correlated to the concentration of compounds in a multi-compound system.The established model was evaluated by an internal validation (cross validation) and was subsequently optimized by the Quant 2 software.This optimization process detects the best data preparation and the best frequency range to explain the actual mixtures of the calibration samples.Six to eight of the proposed optimized models were veri ed by an external calibration (20 additional "arti cial mixtures").Both internal validation and external calibration were compared with the calculated statistical parameters of each calibration.For the rst sampling date for each wheat cultivar, a separate model was generated.The statistical parameters of the model (calibration/internal validation and external calibration) are shown in Tables S2 (date one) and S3 (date two).With the chosen model, the FTIR spectra of the mixed species samples were evaluated with the associated model.The output of this evaluation was the percent share of each species within the mixed species root mass samples which were used for further calculations.Values outside the calibration range (below 0% or above 100%) were corrected to 0% and 100%.

Data analysis and statistics ( 1)
The soil volume of each layer is equal to 636.173 cm 3 (core diameter: 9 cm, sample height: 10 cm).
Root mass (t ha − 1 ) was calculated according to the Eq. ( 2): 2 The surface area of cylinder (core auger) is equal to 63.62 cm 2 .Speci c root length (SRL; m g − 1 ) was calculated as follows: ( 3) The root to shoot ratio (R:S) was estimated by dividing the dry matter root mass by the dry matter shoot biomass.
The LER for the faba bean and spring wheat mixtures was calculated for belowground (LER root ) biomass according to Equations 4-6.The LER was only calculated for the treatments with fully replacement designs.The LER for bean and wheat in intercrops is the sum of the partial LER for bean (pLER Bean ) and wheat (pLER Wheat ): The expected values of root mass, RLD and SRL were estimated based on the Eq. ( 7): Where p is the sowing density of the species in the intercrop divided by the sowing density in the sole crop and M is either the root mass, SRL or the RLD of the sole crop.The data were also analyzed according to the 4C approach (Justes et al., 2021).
Shoot biomass, root mass and RLD were analysed by a one-factorial analysis of variance (Anova) (factor treatment), as well as two-factorial analysis of variance (factors cultivar and sowing density) for all treatments.Mean values of treatments were compared with a Tukey post-hoc test at a signi cance level of α = 0.05.Outliers were detected for each of the response variables (root mass, RLD, FTIR predictions) using the package rstatix in the programme R. Values above-Q3 + 1.5 x IQR or below Q1-1.5 x IQR were considered as outliers and were deleted.Q1 and Q3 are the rst and third quartile, respectively.IQR is the interquartile range (IQR = Q3 -Q1). 3 Results

General characteristics of the growth period
The growing season in 2021 can be characterized as chilly in April and May with a normal rainfall pattern, however, a storm with a heavy rainfall occurred on 14-15/07/2021 with about 120 mm of rainfall.In the growth period from 30/03/2021 to 25/08/2021, total rainfall was 395 mm and the mean air temperature was 14°C (Fig. S6).

Root growth in intercrops
Characterisation of root mass The cumulated root mass over the soil pro le (all soil depths measured) increased from the rst to second date by 19% (mean of the two cultivars) for the sole crop wheat and 34% for the sole crop faba bean (Table S5).For the intercrops, the greatest increase between the two sampling dates were estimated in treatments FB_50_SW_Ana_50 (46%) and FB_100_SW_SUAh_100 (41%) and the lowest were estimated for the treatments FB_50_SW_SUAh_50 (21%) and FB_33_SW_Ana_33 (20%).On sampling date one, the signi cantly highest mean values of total root mass (0-1 m) were observed in the intercrop with wheat cv.SU Ahab with TSD = 66% (FB_33_SW_SUAh_33) and 100% (FB_50_SW_SUAh_50) TSD with 2.11 t ha − 1 and 2.03 t ha − 1 , respectively (Table S5).
At the rst sampling date (Fig. 1a), the lowest root mass values in the topsoil (0-30 cm) were estimated for the wheat sole crops.The highest sowing density (TSD = 200%) showed lower total root mass as compared to the two other sowing densities in intercropping.For the upper subsoil (30-60 cm), the sole wheat root mass was signi cantly higher than all intercrop treatments.The intercropping of faba bean with the wheat cv.Anabel at the lowest sowing density achieved the lowest root mass value, while the faba bean sole crop achieved the second lowest total root mass at this soil depth.For the deeper subsoil layers (60-100 cm), the faba bean sole crop presented the lowest value.At the rst sampling date, spring wheat cv.Anabel developed more roots in deeper soil layers as a sole crop and in intercropping in comparison to cv.SU Ahab (Fig. 1a).
At the second sampling date, no signi cant differences between the treatments with regard to topsoil root mass were observed (Fig. 1b).The intercrops with low sowing density (FB_33_SW_SUAh_33 and FB_33_SW_Ana_33) achieved the signi cantly lowest values of root mass cultivars in the upper subsoil (30-60 cm).In the deeper soil layer (60-100 cm), faba bean reached the lowest root mass.
Results of a two-way Anova (α = 0.05) indicated that the cultivar choice had no signi cant effect on root mass but sowing density had.Also, no signi cant interactions between the sowing density and cultivar for root mass were found (Table S6).

Proportion of faba bean and spring wheat root in intercrops
The results of discrimination between species using the FTIR showed that wheat root mass dominated in the subsoil (20/30-100 cm, Fig. 2).In general, there were no signi cant differences in faba bean root mass proportions between the different treatments.Only in the rst sampling date signi cant differences in 0-10 cm (the very high sowing density led to low faba bean root proportions) and in 60-100 cm depth (the intercrop treatments with wheat cv.Anabel had low faba bean root proportions) were observed.The quick and deep rooting ability of the cv.Anabel in comparison to cv.SU Ahab is illustrated by the greater proportion of faba in intercrops with cv.SU Ahab in the deeper soil depths (60-100cm) at both sampling dates (although the differences were only signi cant at the rst sampling date).

Root mass advantage in intercropping
At the rst sampling date (09/06/2021) in the topsoil and upper subsoil layers (0-40 cm for intercrops with wheat cv.Anabel and 0-30 cm for intercrops with wheat cv.SU Ahab), a positive root mass LER was observed (Table 3).Around owering of spring wheat (second sampling date), the root mass LER was above one for the layers 0-20 cm for the intercrop with cv.SU Ahab and above one from the layers 0-50 cm for the intercrops with cv.Anabel (Table 3).The partitioning of dry matter biomass between the roots (0-100 cm) and shoots is illustrated in Fig. S7.
As expected, the root to shoot ratio R:S decreased from the rst to the second sampling date.The highest ratio was estimated for the intercrop treatments with low sowing density at both sampling dates.
There was a tendency for decreasing R:S with increasing density sowing.The highest R:S was observed in the intercroppng treatments with wheat cv.SU Ahab.The very high sowing density (TSD = 200%) lead to a low R:S in the second sampling date, indicating an investment in shoot rather than in roots.Sole crop faba bean had higher R:S ratio compared to both sole crop spring wheat cultivars.

Effect of sowing density on root mass of intercrops
The analysis based on the comparison between the attained and the expected values revealed that under high sowing density (TSD = 200%, additive design) the expected values of root mass in 0-1m soil depth were higher than the attained values (Fig. 4).In contrast, for the lower sowing densities (TSD = 66%, partial replacement design and TSD = 100%, full replacement design), the attained values were higher than the expected one.

Root length density
On both sampling dates and in all soil layers, the RLD of the tap rooted sole faba bean was lowest (Fig. 6a and 6b).In the upper subsoil (30-60 cm), mostly signi cant differences were found between RLD of faba bean and spring wheat in sole cropping.For the mixed cropping treatments, the RLD in the upper subsoil was higher for the fully replacement treatments (TSD = 100%) as compared to the partial replacement ones (TSD = 66%) and vice versa in the deeper subsoil (60-100 cm).Thus, lower sowing densities encouraged deep rooting in mixtures.
No signi cant differences in RLD were observed for the wheat cv.SU Ahab for all sowing densities on either sampling date in any soil layer.For the wheat cv.Anabel, RLD in the upper subsoil was signi cantly higher in the 50%-50% treatment as compared to the 33-33% treatment (both dates).
For deep subsoil (60-100 cm) and for all treatments, the RLD decreased with soil depth.However, the mean RLD for the subsoil (60-100cm) was found to be highest in the 33%-33% mixture with the wheat cv.SU Ahab.Additionally, in both treatments with TSD 66%, the mean RLD from 60-100cm was higher in comparison to the mean RLD of 30-60 cm.Both the intercrops and the spring wheat sole crops attained slightly higher cumulative RLD values than the faba bean, with a mean value over all intercrops and sole crop spring wheat treatments of around 18 cm cm − 3 compared to 5 cm cm − 3 for the faba bean (0-1m soil depth) (Table S7).

Speci c root length
The mean SRL (all depths) was lower in faba bean compared to spring wheat (Table S8).An enhanced SRL (more ne roots in 0-100 cm) in intercrops as compared with the expected SRL from sole crops was observed.A trend for decreasing mean SLR values with increasing TSD in the mixtures was observed.

Root carbon content
The root C content, calculated as C concentrations (mean: 45%) multiplied by root dry matter, did not change signi cantly across the treatments for both sampling dates.However there was a trend of higher root C contents in the intercrop treatments compared with the sole crops, with the exception of the treatment with TSD = 200% (Fig. 7).For the intercrop treatments with wheat cv.SU Ahab, there was a decrease of root C content with increasing TSD.The opposite trend was observed for the wheat cv.Anabel.

Root nitrogen content
The mean root N concentrations were 2.3% (sole faba bean), 0.7% (sole wheat), and 1.2% (intercrop).As expected, the lowest values of root N were estimated in sole spring wheat (Fig. 8).Root N in several intercrop treatments was comparable to the sole crop faba bean treatment.On the second sampling date, no signi cant differences were observed between the intercropping treatments and sole faba bean.However, in faba bean, the root N was also found to be higher in the deeper soil layers (20-60 cm).

Soil mineral N
Before the establishment of the crops, the initial Nmin was 16 kg ha − 1 in the topsoil (0-30 cm), 27 kg ha − 1 in the upper subsoil (30-60 cm) and 55 kg ha − 1 in the deeper soil (60-90 cm).After harvest, lower Nmin values over the whole soil layers were found in the spring wheat sole crop treatments.The topsoil Nmin values were lower in sole cropping (wheat and bean) as compared to the intercropping treatments (Fig. 9).The highest topsoil value (25 kg ha − 1 ) was determined in the treatment FB_100_SW_SUAh_100.
In the upper subsoil 30-60 cm, the lowest values of 7.7 kg ha − 1 was estimated in the intercropping treatment with highest total grain yield and with lowest sowing density (FB_33_SW_Ana_33) followed by both spring wheat sole treatments.Again, the highest value in 30-60 cm soil depth of 18 kg ha − 1 was estimated in the treatment with the highest sowing density FB_100_SW_SUAh_100.In the deeper subsoil (60-90 cm), soil Nmin was lowest in the intercrop treatments FB_100_SW_SUAh_100 and FB_50_SW_SUAh_50.
Higher topsoil N but low subsoil N were observed in the intercrop treatments with wheat cultivar SU Ahab (slower root growth) as compared to the intercrop treatments with cv.Anabel (fast early root growth).Especially in the upper soil layers there was a trend for a higher N depletion (lower Nmin values) in the low sowing density as compared to the high density intercrop treatments.

Soil volumetric water content
In general, the soil volumetric water content around the owering of spring wheat in July (second sampling date) was higher than at the early sampling date in June.Soil water content for the spring wheat cultivar Anabel, which indicates the potential to root quickly and deeply, was lower in the sole crop treatment and in mixtures compared to the cultivar SU Ahab at the second sampling date, particularly at deeper soil depths (Fig. 10).However, in the treatment with the cultivar SU Ahab as a sole crop and as intercrop (TSD = 100%) the lowest soil water content values were measured at 30-60cm soil depth.In general, soil water depletion was lower for the low density as compared to the high density intercrop treatments (sampling date 2).

Discussion
Root mass, root length density and belowground interactions Root system extension of wheat often exceeds the one of legumes like faba bean (Gregory et al. 1995;Turpin et al. 2002), though under eld conditions, factors such as phenology, sampling technique and sampling depth may in uence root growth.The faba bean root mass at owering (2.3 t ha) observed in our study is higher than the values reported in the studies from Rengasamy and Reid (1993), who reported average root mass over years and treatments of approximately 1.4 t ha − 1 for a sampling depth of 70 cm.These values are also higher than the values reported by Streit et al. ( 2019) who found values of around 0.7 t ha − 1 for a sampling depth up to 60 cm.This difference can be attributed to the sampling depth (0-100 cm), higher sowing density considered in our study for the sole cropping treatments and also the sampling technique as we always considered a faba bean in the soil core which overrepresented the faba bean as compares the studies of Kemper et al. (2022).Literature revealed high variability for spring wheat root masses ranging from 0.8 t ha − 1 to 1.4 t ha − 1 at owering (Wechsung et al. 1995;Gan et al. 2009).In our study, a spring wheat root mass of 1.4 t ha − 1 was reached at owering over the soil depth of 0 to 1 m.This rather high value can be partly attributed to the enhanced sowing density considered for the sole crops compared to the optimal sowing density recommended for spring wheat. .Many studies reported that intercrops produce signi cantly higher root masses as compared to their sole cropping equivalents (Ma and Chen 2016).Root mass advantage was observed in faba bean-maize (Xia et al. 2013) and faba bean-winter wheat intercrops (Streit et al. 2019).In our study, the mean topsoil root LER was above one indicating a root mass advantage in intercropping versus sole cropping.In the upper subsoil it depended on the spring wheat cultivar, but LER was always below one in the deeper subsoil (60-100 cm).
A combination of tap rooted and brous rooted crops is widely recognized as being one of the mechanisms of overyielding in intercrops due to belowground complementarity which may increase water and nutrient acquisition by niche differentiation and due to resource partitioning (Yu et al. 2022).In line with this nding, the attained values of root mass in the intercrop treatments for both wheat and faba bean (0-1m soil depth) were mostly higher than the expected values (Fig. 4).This applied for both the low density (TSD = 66%) and the nearly optimal sowing density (TSD = 100%), but not for the very high sowing density (TSD = 200%).
In our study, spring wheat roots responded to belowground interactions with faba bean by growing more deeply in the soil pro le (TSD = 66% and 100%) (comparison of attained and expected RLD values, see Fig. S8).Similar changes in the rooting depth have been observed for barley-pea mixtures (Hauggaard-Nielsen et al. 2001) and for maize-wheat mixtures (Yang et al. 2022).The increased RLD of spring wheat intercropped with faba bean compared to sole spring wheat is probably due to changes in above-and belowground allocation of biomass in order to minimize direct resource competition in the same soil volume.Another study found that in comparison to sole cropped faba bean, intercropping faba bean with sa or or mustard enhanced the RLD of faba bean in the subsoil (Schröder and Köpke 2012).
It is assumed that belowground biomass advantage during vegetative stages fosters higher resource availability, as well as shoot and grain overyielding.This was especially reported under stress conditions (Fargione and Tilmann, 2005;Hector et al., 2002).The enhanced root growth and development partially compensated competition for light (Amossé et al. 2013), carbon dioxide (Shili-Touzi et al. 2010) and other resources (Wang et al. 2018).This was not con rmed in our study.The results of the R:S (Fig. S7) as well the aboveground shoot mass and yield data (Table S4) showed a trend to have negative correlation between root growth advantage and aboveground overyielding.The favorable growing conditions characterizing our experimental site and year combination (fertile soil, favorable soil moisture due to plenty of rain) could be a reason behind these observations.Similar studies in contrasted environments should be performed to better assess the relationship between belowground root advantage and aboveground overyielding.

Sowing density effect on root growth advantage and complementarity
The spatial arrangement in intercropping is an important factor for the above-and belowground growth (Wang et al. 2018;Homulle et al. 2022).In our study, the spatial arrangement was represented by the sowing density that characterized the designs considered in the study, as well as by the completely mixed design or adjacent row design which permitted a high interaction between the species (Homulle et al., 2022;Li et al., 2006).The high sowing density in the additive design resulted in low root biomass and lower root to shoot ratio (Fig. S9).
In a sole cropped spring wheat experiment, Hecht et al. (2016) found that RLD increased with increasing sowing density in the topsoil (0-10 cm), partly due to greater production of ne roots.The authors argued that light competition forced plants to grow more shoot mass at the cost of investment into roots, in our study an increased sowing density fostered RLD only at the rst sampling date and only in 0-10 cm soil depth.However, for the second date there was a decrease of total RLD with increasing TSD.Bulson et al.(1997) reported a signi cant decrease in resource complementarity with increasing wheat and faba bean sowing density.The presented low attained root mass compared to the expected values in the high sowing density treatment (additive design, TSD = 200%) indicates low belowground complementarity under the high sowing density of the additive design.

Cultivar effect on intercrops belowground interactions
Although statistically there was no signi cant effect of the cultivar on the root mass, we observed a difference in rooting ability between both spring wheat cultivars (Fig. 1a and 1b, Fig. 6a and 6b).The ability of cv.Anabel to root quickly and deeply around faba bean owering as compared to cv.SU Ahab resulted in lower root mass proportions of faba bean intercropped with cv.Anabel compared to intercropped with cv.SU Ahab.The application of the 4C method (Justes et al. 2021) as indicated in Fig. 11 con rmed those observations.Different aspects of belowground intercrops interactions were found in our study depending on the soil depth, vegetative stage, and cultivar.According to the 4C approach (Fig. 11), in the rst sampling date (around faba bean owering) the fast rooting cv.Anabel dominated the faba bean in the soil layers between 10-60 cm and compete with faba bean in the deep subsoil (60-100 cm).In contrast, the intercrops with cv.SU Ahab dominated faba bean only in the upper soil layers (0-10 cm).
Across both treatments (fully replacement design) and both sampling dates, the complementarity and cooperation were stronger than competition in the topsoil (0-30cm) meaning that roots of both cultivars and both species are growing better as intercrops than as sole crops.In contrast, there was competitive root growth in the deeper soil layers.
Soil mineral N, soil water, and root carbon and nitrogen mineral N content in the topsoil was greater in the intercrops than in both sole crops, indicating a difference in N uptake rate between intercropping and sole cropping systems.In a long-term experiment, an increase of topsoil organic N content by 11% was observed in intercropping as compared to sole cropping, indicating that increased biological N xation contributed to increased soil N content (Cong et al. 2015).It is widely recognized that N uptake is mainly performed by the ne roots (McCormack et al. 2017).This was also indicated by our study where for the low density treatments with high SRL (higher ne roots compared to the high TSD treatment), the N uptake was greater than in the high density treatments.
Plant diversity also affects soil organic C stocks in deeper soil which is more stable and di cult to access for microbes (Chen et al. 2020).Hence, root-based C inputs in deeper soil layers is the major source of soil organic carbon (Yu et al. 2022).We observed no signi cant effect of root-based C input in the deep soil layer (60-100 cm, date 05/07/2021, Fig. S10).In the deeper soil layers (30-100 cm), total C in roots in the mixtures was on average 22% greater than the average root C in sole faba bean and 18% lower than average root C in sole spring wheat (mean of both cultivars), providing a possible mechanism for the divergence in soil C sequestration between sole crops and intercropping systems.Similar trends were observed by Cong et al.(2015).

Advantages and limitations of study
Taking into consideration different intercropping designs namely the partial replacement design, full replacement design and the additive design to assess the belowground interactions in intercrops and also the factors in uencing the root growth was one of the strengths of this study.Comparing the root growth patterns in intercrops and sole crops on two different growth stages ( owering of wheat and owering of bean), although laborious, permitted to better understand the dynamics of root growth in intercropping.The latter has shown to be affected by the cultivar choice and sowing density.The ability of FTIR to discriminate between intercropped species has been shown.However, due to lack of real eld replicates, a clear relationship between belowground root interactions and aboveground overyielding could not be statistically tested.The analysis based on one shoot biomass replicate considered in the study and presented in the supplementary showed that a high TSD led to high shoot biomass but lower root mass.Additionally, a decrease of R:S with increasing sowing density was (Fig. S9).We assume that in that speci c experimental site and year combination, the resource limitations were too small for a clearer relationship between aboveground overyielding and belowground growth and very strong interactions could not be observed.

Conclusion
In our study, belowground root growth and interactions varied with the different intercropping designs and spring wheat cultivars considered in the study.The belowground intercrop advantage decreased with increasing sowing density.Intercropping of faba bean with a spring wheat cultivar characterized by a rather small root system during faba bean owering fostered a higher belowground intercrop advantage.We found that lower sowing densities i) led to a lower depletion of soil water in the deeper soil layers, ii) fostered deeper rooting, iii) led to a depletion of more N in the upper soil layers, and iv) fostered higher SRL and thus potentially enhanced root N uptake as compared to high density intercrops.A study with contrasting growth conditions with several sampling dates should be conducted to better assess the relationship between above-and belowground overyielding and support the generalization of the obtained results.Moreover, there is a need to explore the effects of mixtures on soil C and N sequestration to mitigate climate change.Points refer to middle point of the investigated soil layer depth (e.g. 15 cm in case of 0-30 cm).Lines were smoothed.and (i-2) indicate a dominance of grain legume on cereal i.e. grain legume suppresses cereal by modifying the environment and vice versa in (l-1) and (l-2), the area j indicate that complementarity and cooperation is higher than competition, area k indicates that competition is higher than complementarity and cooperation.
Cereals are generally considered as strong competitors compared to legumes, mainly due to a larger root system and deeper root distribution(Gregory et al. 1995; Hauggaard-Nielsen et al. 2001; Corre-Hellou and Crozat 2005; Bedoussac et al. 2015)

Figure 6b :
Figure 6b: Mean values ± standard error (n = 4) of root length density (not crop-speci c) in cm cm -3 (RLD), for sole faba bean and sole spring wheat, as well as for the mixtures treatments for cumulated three soil layers in 05/07/2021.Different letters indicate signi cant differences (Anova and Tukey posthoc test, α=0.05).Error bars refer to the standard deviation.

Figure 7 :
Figure 7: Root C (not crop-speci c) in t ha -1 , for sole faba bean and sole spring wheat, as well as for the intercrop treatments for three soil layers on 09/06/2021 (top panel) and 05/07/2021 (bottom panel).Different letters are signi cant differences comparing the cumulative root C over all soil layers (Anova and Tukey post-hoc test, α=0.05).

Figure 11 :
Figure 11: The mean pLER (n=4) of the spring wheat and bean root mass are shown for each soil depth and for the two replacement design treatments FB_50_SW_Ana_50 and FB_50_SW_SUAh_50 for the sampling dates 09/06/2021 (left panel) and 05/07/2021 (right panel).The graphical representation of the pLER of the two species intercropped is based on 4C approach (Justes et al. 2021).The areas (i-1) al. 2013; Streit et al. 2019; Nelson et al. 2021; Yang et al. 2022) or 'Relative Yield Total' Yu et al. (2022)019;Kemper et al. 2022)g and need extensive training(Rewald et al. 2012).The monolith excavation method combined with visual distinction(Li et al. 2011;Yu et al. 2022) is rather simple and cheap but less accurate.Infrared spectroscopy has been proven to be a fast tool to discriminate roots of different over differential depths) ranged from 0.52 to 1.50 depending on the experimental year and the species(Streit et al. 2019;Kemper et al. 2022).Further methods to explore root-root interactions in species mixtures (e.g.tracer techniques, minirhizotrons, or rhizoboxes) are explained inYu et al. (2022).
species such as corn-soybean (White et al. 2011), pea-oat (Naumann et al. 2010), pea-oat and maizebarnyard grass (Legner et al. 2018), faba bean-wheat (Streit et al. 2019), and blue lupin-winter rye (Kemper et al. 2022).Fourier transform infrared (FTIR) spectroscopy can be applied to separate roots of species in mixtures and can also give an estimation of the species speci c proportions within a root sample (Meinen and Rauber 2015; Streit et al. 2019; Kemper et al. 2022).In these studies, mean root mass LER (

Table 1
Treatments with spring wheat (cv.SU Ahab, cv.Anabel) and faba bean (cv.Fanfare) and the respective sowing densities at Campus Klein-Altendorf in 2021.The total sowing density (TSD) is the sum of both sowing densities.

Table 3
Mean values ± standard deviation of root partial land equivalent ratio of bean (pLER Bean, n = 4), wheat (pLER Wheat, n = 4) and root land equivalent ratio (LER, n = 4) based on root mass of the intercrops with wheat for two sampling dates for the replacement treatments FB_50_SW_Ana_50 and FB_50_SW_SUAh_50.For the rst sampling date (60-100 cm), no values were provided for the treatment FB_50_SW_SUAh_50 due to absence or low root mass in all replicates.No standard deviation was provided for the treatment FB_50_SW_Ana_50 due low number of replicates (n = 1)