Reduced belowground allocation of freshly assimilated C contributes to negative plant-soil feedback in successive winter wheat rotations

Abstract


Introduction
Winter wheat (WW) is the most cultivated staple crop in the world and a staple food for billions of people worldwide, contributing signi cantly to many national economies (Shewry and Hey, 2015;Enghiad et al., 2017).Following a linear increase for many decades, annual WW yield growth is currently stagnating, not meeting the forecasted global demand (Calderini and Slafer, 1998;Ray et al., 2013;Moore and Lobell, 2015;Schauberger et al., 2018).A 2.4% increase in crop yields is required annually to achieve food security by 2050.For WW, the current 0.9% annual yield growth is markedly lagging behind this target (Ray et al., 2013;Crespo-Herrera et al., 2018).Brisson et al. (2010) attributed this observation to agronomic (cereal rotations with more oilseed rape and fewer legumes, lower N fertilization), climatic (drought stress), and political causes (agricultural policies), while genetic progress did not appear to be an underlying cause.
Plant community diversity and succession has been associated with distinct changes in the abiotic and biotic parameters of the soil exerting positive or negative plant-soil feedbacks (PSF, van der Putten et al., 2013; de Vries et al., 2023).Among those, changes in nutrient input as well as contrasting quantity and quality (C:N ratio) of plant litter cause distinct changes in the microbial community diversity and composition (Bennet and Klironomos, 2019; Thakur et al., 2021;De Long et al., 2023).Linking the PSF theory to arable farming, the bene cial effect of a non-cereal pre-crop on WW productivity has been well established and yet it is estimated that up to 40% of the global WW cultivation is grown successively (Angus et al., 2015;Yin et al., 2022), and this trend is expected to continue in the future due to the focus of agrochemical and breeding companies on the staple crops such as WW (Hegewald et al., 2018).This practice is associated with a high risk of soil-borne pathogens and speci cally the necrotrophic fungus Gaeumannomyces graminis var.tritici (Ggt), which causes early root senescence, rotting, and yield decline (Cook, 2003; Kwak and Weller, 2013).Ggt can persist in the soil as a saprotroph after WW has been harvested and its severity increases with increasing frequency of WW self-succession (Palma-Guerrero et al., 2021).However, the soil legacy of wheat monocropping is not limited to Ggt as this has been observed in years without obvious Ggt symptoms suggesting that other soil microbes might contribute to the observed effect (Donn et al., 2015).The inclusion of oilseed rape in crop rotations has been widely appreciated for its importance to soil structure, suppression of WW pathogens, and high post-harvest residual N, among other bene ts (Sieling et al., 2005;Weiser et al., 2017;Hegewald et al., 2018).Therefore, the soil legacy of the WW preceding crop to the following WW can be expected to exert major control over the productivity of WW.
Plants allocate photosynthetic C belowground for root growth and maintenance, as indicated by biomass buildup and root respiration (Jones et al., 2009).A part of that C is exuded from the roots into the rhizosphere, which is a hotspot for microbe-root interactions.This process is termed rhizodeposition and, in combination with root litter and dissolved organic C, it constitutes a readily available energy source for soil microorganisms (Kuzyakov and Domanski, 2000;Loeppmann et al., 2019).Rhizodeposition may provide positive or negative feedback for plant nutrient acquisition as indicated by accelerated or decelerated nutrient mineralization by rhizosphere microorganisms (Cheng and Kuzyakov, 2005;Tian et al., 2013;Meier et al., 2017).It has been established that the rhizodeposition-to-root biomass ratio shows very small variation, meaning that factors affecting root growth are also expected to affect C allocation belowground (Pausch et al., 2013;Heinemann et al., 2023).The combination of heterotrophic microbial respiration of rhizodeposits with autotrophic root respiration constitutes the root-derived CO 2 and can be used to estimate the fate of freshly assimilated C in plants (Loeppmann et al., 2019;Henneron et al., 2022).Whether successively grown WW invests more C belowground to stimulate root growth and/or microbial activity, or whether it allocates less C belowground as a result of a negative soil legacy effect remains unknown. 13C labeling of plants is a common and valuable approach to distinguish and quantify the rhizodeposited C from native soil organic C (SOC).It allows for the investigation of C allocation patterns throughout the soil and within the different plant parts (Bahn et al., 2013).In addition, recording the time lag between 13 C xation and rhizodeposition belowground provides important information regarding the C use within plants as well as the availability of photosynthates to soil microorganisms (Brüggemann et al., 2011).Studying C partitioning to the different plant parts and to rhizodeposition has great potential to improve our understanding of C allocation patterns in high-input agricultural systems that are governed by intense microbial interactions and often abiotic stress factors.C allocation dynamics vary depending on plant species, plant developmental stage (higher exudation during earlier growth stages), biotic and abiotic factors (Pausch and Kuzyakov, 2018; Williams and de Vries, 2020; Chai and Schachtman, 2022).In WW, higher exudation for increased nutrient uptake is observed from early growth until owering with a decreasing trend thereafter until full maturity (Sun et al., 2018).At this late developmental stage, C is transported to the head during starch synthesis of the grains (Sun et al., 2019) In light of no Ggt-resistant WW cultivars (Palma-Guerrero et al., 2021), the projected adverse climatic conditions for WW both at European and global scale (Senapati et al., 2021;Zhu et al., 2022) and the premature status of the Ggt-speci c biocontrol research (Osborne et al., 2018;Zhao et al., 2023), there is an urgent need to decipher the mechanisms by which the rotational positions of WW in uences its productivity.Here, we investigated how different rotational positions of WW in uence the allocation of freshly assimilated C in above-and belowground plant parts and its subsequent translocation to the rhizosphere of WW.We hypothesized that WW self-succession results: I. in a limited assimilate supply to the root system and the associated soil microorganisms in successively grown WW due to negative soil legacy feedback.II.reduced storage of freshly assimilated C in aboveground plant parts and especially sink organs (grains) due to reduced root performance.
To test these hypotheses, a greenhouse rhizotron experiment was set up with three contrasting rotational positions of WW.The plants were pulse-labeled with enriched 13 CO 2 , and the allocation of freshly assimilated C was traced in the top-and subsoil over a 25-day period, spanning from owering until grain lling stage.

Experimental design
Soil was collected in September 2020 from the experimental farm Hohenschulen (54°19′05″N, 9°58′38″E), Faculty of Agricultural and Nutritional Sciences, Christian-Albrechts-University of Kiel, Germany (Table 1), from the topsoil (0-30 cm) and subsoil (30-50 cm), from plots after oilseed rape cultivation (W1), rst (W2) and third wheat (W4) after oilseed rape cultivation, and sieved to 2 mm.The soil is a Cambic Luvisol of sandy loam texture (44% sand, 35% silt and 21% clay; Sieling et al., 2005) with no carbonate.Hereafter, they are referred to as rotational positions.On the plots from which soil was collected, the WW cultivar "Nordkap" was sown and fertilized with nitrogen (240 kg N ha -1 ).The residues of the preceding crop were not removed from the soil, and the eld was not ploughed before sampling.We conducted a greenhouse rhizotron experiment (May 10, 2021 to November 12, 2021), using newly designed rhizotrons with a height of 100 cm, width of 35 cm and inner thickness of 2.5 cm (Reichel et al., 2022).The greenhouse was located on the campus of Forschungszentrum Jülich, Germany.The experiment was organized in a full factorial and completely randomized design (CRD), consisting of the three rotational positions W1, W2 and W4 with four replicates each, resulting in 12 experimental units (rhizotrons).The rhizotrons were rotated randomly on a weekly basis.For the online isotopic measurements that are described below, we took measurements of W1 and W4 but not W2, as W1 and W4 comprised the most extreme rotational positions.Three replicates from W1 and W4 were used for the isotopic measurements.Those rhizotrons were equipped with gas-permeable tubing (KM-PPMF_O-2020-KF-0201, Katmaj Filtration, Poland; 35 cm length, 0.155 cm wall thickness, 0.55 cm i.d., 0.86 cm o.d., 0.2 µm pore size) and polyethylene/aluminum tubing (Syn ex® 1300, ¼" o.d., Eaton, Bonn, Germany).The gas-permeable tubing was positioned horizontally in the soil at six depths (5, 15, 25, 35, 65 and 85 cm) and connected to the sampling system with the Syn ex® tubing.The tubing was sealed until it was used for the isotopic measurements.In this way, air exchange between the inner volume of the gas-permeable tubing and the ambient air in the greenhouse could be avoided and water vapor loss minimized.
All rhizotrons were kept inclined at 45° to facilitate root growth along the lower side of the rhizotrons.Bulk density was adjusted to 1.45 g cm − 3 using topsoil (collected from 0-30 cm) for the rst 30 cm and subsoil (collected from 30-50 cm) for the following 70 cm.Deionized water was added to reach 70% water-holding capacity (WHC, 215 g H 2 O soil kg − 1 ) at the onset of the experiment.Thereafter, soil moisture was readjusted gravimetrically every 2-3 days to 70% WHC to ensure well-watered conditions.Winter wheat seeds (cultivar "Nordkap") were germinated on petri dishes with sterile lter paper for 24 h in the dark at 20 C. Subsequently, one germinated seed was sown into each rhizotron.Each plant was fertilized with 0.78 g of calcium ammonium nitrate fertilizer (13.5% NO 3 -N, 13.5 NH 4 + -N, 4% CaO, 1% Mn, YaraBela® CAN™, YARA GmbHand Co. KG, Dülmen, Germany) applied at a rate of 240 kg N ha

CO 2 labeling during owering
In order to quantify the C allocation pattern above-and belowground in the contrasting rotational positions W1 and W4, we conducted 13 CO 2 pulse labeling during late owering (BBCH 69).The plants were labeled with highly enriched 99 atom-% 13 C-CO 2 (Campro Scienti c GmbH, Berlin, Germany).
Custom-made polymethyl methacrylate plant chambers (Fig. S1), constructed by the workshop of Forschungszentrum Jülich were tted onto the rhizotrons shortly before the labeling.The chamber was comprised of a 55° triangle-shaped base (opposite of 5 cm × hypotenuse of 6 cm × adjacent 5 cm, wall thickness of 1 cm) with a rubber seal and the plant compartment (height of 60.7 cm, length of 40.Prior to 13 CO 2 pulse labeling, we monitored the decay rate of unlabeled CO 2 inside the chamber by injecting 20 ml of pure unlabeled CO 2 to reach a mixing ratio of 1000 ppm CO 2 in the chamber.This was done to adjust the timing of the 13 CO 2 injections as well as to accurately estimate the 13 CO 2 assimilation time by the plants without the need to keep the gas exchange analyzer connected to the plant chamber during the labeling.Prior to the pulse labeling, the soil surface was covered with thick gas-impermeable foil to minimize diffusion of 13 CO 2 into the soil.Air temperature, relative humidity, and mixing ratio of unlabeled CO 2 was monitored with an infrared gas exchange analyzer (Li-8100, Li-COR, Lincoln, NE, USA).
When the plants had assimilated most of the CO 2 and its concentration had dropped to sub-ambient levels, another injection was made to reach a CO 2 mixing ratio of 1500 ppm inside the chamber.We repeated this procedure for a different set of environmental factors (temperature range: 25.5-29.5°C,relative humidity range: 34-50%, light intensity range: 243-618 µmol m − 2 s − 1 ) to obtain accurate information on how the decay rate of the unlabeled CO 2 would change with changes in abiotic conditions.For 13 CO 2 labeling, we made four injections of 20 ml of 13 CO 2 each in 20-min intervals to ensure that adequate amounts of 13 C were xed by the plants.
In order to facilitate the online isotopic measurements, an automatic valve-switching unit was constructed (Fig. S2) following the setup of Rothfuss et al. (2013Rothfuss et al. ( , 2015)).The time course of soil δ 13 C at the six abovementioned depths was monitored with an isotope ratio infrared spectrometer (IRIS, Delta Ray™, Thermo Fisher Scienti c, Inc., Waltham, MA, USA) ) after the 13 CO 2 pulse labeling.Data was recorded 2 hours after labeling (0 days after labeling, DAL), for two consecutive days after labeling and on the tenth and twenty-fth day after labeling (DAL).Every time we tted the chamber onto the rhizotron, the soil surface was covered with gas-impermeable foil to prevent gas exchange between the soil and the chamber interior.

Plant harvest and analyses
At harvest (BBCH 92) the aerial plant parts were split into pseudo-stems (hereafter called stems), leaves, husks and grains.The rhizotron plates were removed and the soil pro le was then divided into seven layers (0-10, 10-20, 20-40, 40-50, 50-70, 70-80 and 80-100 cm) and samples from all soil depths were taken.Due to the extensive root growth throughout the rhizotron, there was no root-free bulk soil.Therefore, we considered the soil to be root-affected (RA).Within every soil depth, several soil aliquots were pooled to form a composite sample and then split into several samples before the analysis.The roots were retrieved after washing off the soil through a 1-mm sieve and stored in 30% ethanol.All plant material was ball-milled (MM 400, Retsch, Germany) and weighed into tin capsules (HEKAtech, Wegberg, Germany) for determination of 13 C content using an elemental analyzer coupled to an isotope-ratio mass spectrometer (EA-IRMS, Flash EA 2000, coupled to Delta V Plus; Thermo Fisher Scienti c Inc., Waltham, MA, USA).
The chloroform-fumigation extraction (CFE) method was used to estimate microbial biomass C (C mic ) and N (N mic ).Ten grams of fresh soil stored at 4°C were weighed in beakers and placed inside a desiccator.They were incubated with ethanol-free chloroform (80 ml) at room temperature for 24 h.Soil samples were then extracted with 0.01 M CaCl 2 and analyzed with a TOC analyzer (TOC-V + ASI-V + TNM, Shimadzu, Japan).Non-fumigated soil samples were extracted with the same protocol.C mic and N mic were estimated as the difference between the extracted C and N from fumigated and non-fumigated soil samples.
The correction factors, kEC = 0.45 and kEN = 0.4, were used for the calculation of the extractable part of C mic and N mic ) (Wu et al., 1990;Joergensen, 1996).Ten milliliters of extracted fumigated and nonfumigated soil solution were freeze-dried in polypropylene vials and stored in a desiccator until further processing.Then, 120 µl of deionized H 2 O were added into every PP vial to solubilize the precipitate.The solution was then pipetted into 5 mm × 9 mm silver capsules and air-dried for 2 days.The capsules were placed into a desiccator connected to a vacuum pump and incubated with 200 ml HCl for 1 day.After that, they were placed onto a heating plate at 40°C for 8 hours and stored in the freezer at -20°C overnight.They were then freeze-dried again and tted into 10 mm × 10 mm silver capsules before 13 C analysis with the elemental analyzer as described before.The C mic δ 13 C was calculated according to Werth and Kuzyakov (2008).

Data analysis
Data were checked for normality using the Shapiro-Wilkinson test and for homogeneity of variances using the Levene test.For data not meeting the assumptions of normality the Yeo-Johnson (Yeo and Johnson, 2000), Box-Cox (Box-Cox, 1964) and log transformations were applied.The transformation used for a certain variable is mentioned in the respective table.The factors in the general linear models (GLM) were rotational position (three levels) and soil depth (seven levels).The data on δ 13 C of soil respiration measured on ve dates was analysed with repeated measures ANOVA.Date ( ve levels), rotational position (two levels) and soil depth (three levels) were de ned as xed factors.Bonferroni correction was used for multiple comparisons to identify differences between the contrasted factors at p ≤ 0.05.Data analysis was performed using R and IBM SPSS Statistics for Windows, version 23 (IBM Corp., Armonk, N.Y., USA).Graphs were made with 'ggplot2' (Wickham, 2016) using R Statistical Software (v4.2.1; R Core Team 2022).

Results
Online measurement of pulse-labeled δ 13 C of soil respiration at six soil depths revealed different allocation patterns of freshly assimilated C during owering of WW (Fig. 2).Translocation of photosynthates to greater soil depths (> 30 cm) peaked at two days after labeling (DAL) and decreased gradually thereafter until 25 DAL.Rotational position had a strong effect (Table 2) on root-derived 13 C during the measurement period.Interestingly, the δ 13 C-CO 2 of W4 (373.0‰) was higher than W1 (279.7‰)one DAL.This trend leveled off two DAL and was reversed ten DAL, with 195.4‰ and 109.1‰ δ 13 C-CO 2 traced throughout the soil pro le of W4 and W1 respectively (Fig. 2d,e).Depth speci c contrasts between the rotational positions followed the same trend with lower values of δ 13 C-CO 2 recorded in W1 compared to W4 at 35 cm (232.9‰vs 367.9‰), 65 cm (81.6‰ vs 136.8‰), and 85 cm (33.3‰ vs 56.6‰).However, on the last measurement day there was higher 13 C measured in W1 compared to W4 at 25 cm (173.2‰vs 82.4‰) and 35 cm (162.1‰vs 79.4‰).Rotational position exacerbated a strong in uence (Table 3) on allocation of freshly xed C in WW biomass with a signi cant increase in δ 13 C in W1 (237.1‰)compared to W2 (50.3‰) and W4 (39.1‰).
Table 4 Effect of rotational position (Rot_pos), soil depth (Depth) and their interaction on δ 13 C of microbial biomass C (δ 13 C Cmic ), dissolved organic carbon (δ 13 C DOC ) and root biomass (δ 13 C Root ).The Yeo-Johnson transformation for statistical testing was used for δ 13

Discussion
Using a novel experimental approach, employing real-time quanti cation of root-derived 13 C from 13 CO 2 pulse labeling, we showed that preceding crop legacy is a strong determinant of the above and belowground C allocation in winter wheat (WW).To our knowledge, this is the rst time that such nondestructive and real-time method is used to track the fate of photosynthesized C to understand the soil legacy effect of preceding crops to WW.
Belowground C allocation is a determinant of several processes including C and N mineralization rates, residue turnover and microbial community composition.Freshly assimilated C has a strong stimulatory effect on rhizosphere processes, including rhizosphere priming and nutrient cycling that are governed by microbial activity (Frey, 2019;Wang et al., 2021).C translocation from WW biomass into soil was rapid in our experiment, resulting in high soil δ 13 C-CO 2 observed after 2 hours post 13 CO 2 labeling.This was the case for the soil respiration of both W1 and W4 that were measured with the automatic 13 CO 2 manifold system.rapid C translocation to soil has been previously observed in a mountain grassland labeling study assessing diurnal variations in photoassimilate supply to the roots (Bahn et al., 2009), but also, for example, in beech trees (Gorka et al., 2019).We hypothesized that the soil legacy of successively grown WW will lead to reduced allocation of freshly xed C due to a negative soil legacy feedback.Initially there was higher root-derived 13 C in the rhizosphere of W4 compared to W1.This increase is proportional to the difference in the initial microbial biomass of W4 and W1 (Table 1), indicating that the community of W1 needed a longer time period to consume the rhizodeposits (dissolved organic C, DOC) than that of W4.
However, the much higher root-derived 13 C traced in the soil respiration of W1 compared to W4 10 and 25 DAL provides evidence for accelerated senescence of the root system of W4.Indeed, increased belowground C allocation can help plants cope with biotic and abiotic stresses by increasing rhizodeposition and investing more in extensive root systems (Sanders and Arndt, 2012;Chandregowda et al., 2023).In soil of successive WW cultivation, potentially more C has to be invested into the root or soil microbial activities to deal with the soil pathogens and lower soil mineral content, which could lead to a tradeoff with nutrient uptake and overall plant performance.Reduced rhizodeposition could be also complemented by reduced root growth as both can happen simultaneously (Pausch and Kuzyakov 2018;Heinemann et al., 2023).However, separating autotrophic to heterotrophic respiration is experimentally challenging (Kuzyakov and Larionova, 2005).
In this study, the biomass 13 C content of successively grown WW was found to be markedly lower than that of WW grown after oilseed rape, which was partially re ected also in lower C translocation belowground.Obviously, W1 clearly bene ted from the preceding oilseed rape with a markedly higher 13 C content compared to self-successional wheat.The highest enrichment of 13 C was found in the aboveground plant biomass, followed by soil respiration, extractable DOC, and microbial biomass C (C mic ).This is consistent with a 14 CO 2 pulse labeling experiment with WW, in which most of the recovered 14 C was found in plant biomass and soil respiration, whereas root 14 C constituted the smallest pool of the total traced C (Sun et al., 2018).Although we observed relatively low 13 C enrichment in the root biomass C pool, we found a higher 13 C enrichment of the C mic , which is opposite to what has been described previously for wheat (Van De Broek et al., 2020) but also other plant species, such as chicory and alfalfa (Hafner and Kuzyakov et al., 2016).For the experiment of Van De Broek et al. (2020), this could relate to the much higher amount of 13 C label that entered the system with weekly 13 C-pulses as opposed to our single 13 C-pulse.However, in another 13 C-pulse labeling experiment on maize (Meng et al., 2013), the authors found very low recovery rates for root 13 C at grain lling stage compared to elongation phase, while they observed the opposite trend for shoot biomass.This can be attributed to the dynamic C investment strategy of plants that prioritize root elongation during tillering for acquiring nutrients and water over root maintenance, while C translocation to the grain is dominant following anthesis (Sun et al., 2018).Indeed, plants allocated a big portion of the labeled C on aboveground plant parts and especially in grains and husks, with lower levels for stems and nally leaves.This clearly shows a remobilization and increased translocation of the assimilated 13 C towards the reproductive plant organ and thus, the grains of the plants.Thus, the differences in amount and pattern of 13 C allocation between W1 on the one hand, and W2 and W4 on the other hand, suggest a change in the plants' growth strategies depending on the rotational position of WW.
The amount of 13 C traced in the microbial biomass can vary greatly depending on the plant species and variety (Elias et al., 2017;Van De Broek et al., 2020).In addition to autotrophic respiration by roots, heterotrophic (microbial) respiration substantially contributes to total soil respiration (Brüggemann et al., 2011).In the rhizosphere, heterotrophic respiration is a very important sink of fresh photoassimilates.proposed that under conditions of reduced assimilate supply, the lack of carbohydrate reserves in microbes contributes to a faster decline in their respiration rates (Brüggemann et al. 2011).This was not evident in our experiment, as the root-derived 13 C of W4 was signi cantly lower than of W1 during the later growth stage of the plants, while the two rotational positions did not differ in the 13 C content of their C mic .More importantly, we found that C mic of W1 and W4 soil was signi cantly more enriched in 13 C compared to W2.Both W1 and W4 had higher initial C mic values than W2 at the start of the experiment (Table 1), while there was no signi cant difference among the rotational positions at the end of the experiment (Fig. S3).This means that the microbial community of W1 and W4 incorporated more freshly assimilated C into their biomass compared to W2.
Soil respiration of root-derived 13 C decreased in the subsoil especially during the rst two days after the labeling, while this effect was only partly evident 25 DAL.This means that the soil microorganisms were not severely C-limited in the subsoil as indicated also by the insigni cant effect of soil depth on δ 13 C Cmic .
Therefore, they must have utilized similar amounts of rhizodeposited C under these non C-limiting conditions.This is similar to what Van De Broek et al. (2020) reported, with the topsoil layers being more enriched in δ 13 C Cmic compared to the subsoil, but this trend was not signi cant.However, there was a main effect of soil depth on the distribution of δ 13 C DOC .This suggests that the 13 C pool of DOC was largely in uenced by distribution of root biomass and/or rhizodeposits along the soil pro le.In addition, we observed a strong effect of rotational position in the δ 13 C DOC .There was more 13 C traced in the DOC of W1 compared to W2 in both the top-and subsoil as hypothesized.DOC sources include decomposing C compounds from plant residues and litter as well as root exudates, such as organic acids, amino acids and sugars (Kindler et al., 2011;Panchal et al., 2022).Due to its fast turnover time, DOC is an important pool that encompasses changes in old and new C cycling in the soil, and as such is a major determinant of soil respiration (Brüggemann et al., 2011).
Overall, our results on the 13 C traced in soil respiration, plant biomass, labile C and microbial biomass C after 13 C pulse labeling suggest increased incorporation of recently assimilated C into biomass, followed by increased C translocation to the rhizosphere of WW after oilseed rape compared to successively grown WW.More of this translocated C was incorporated into microbial biomass directly through root exudation or indirectly through the heterotrophic utilization of root litter.The ndings of our experiment enhance our understanding on the plant-soil feedback of contrasting WW rotational positions with respect to aboveand belowground allocation of freshly assimilated C. The indirect effect of reduced C allocation in successively grown WW, likely caused by a negative soil legacy effect, results in reduced root performance and thus potentially lower yield compared to more complex crop rotations with higher C allocation below ground.In contrast, this implies that the increased and sustained C investment in the root system of W1 is overcompensated by higher and longer overall plant vigor, ultimately leading to higher yield.How to overcome these negative soil legacy effects in successional wheat cultivation remains the subject of future research.
. Rhizodeposition moderates microbial-plant competition by the provision of labile C and the resulting enhanced nutrient cycling when the nutritional demands of the plants are maximal (Hernández-Calderón et al., 2018; Mohan et al., 2020).
− 1 , split to three doses of 80 kg N ha − 1 each at BBCH 25, BBCH 30/31 and BBCH 50.The plants were harvested at the grain ripening stage (BBCH 92).The environmental conditions during the experiments are shown in Fig. 1.
7 cm and width of 8.7 cm, wall thickness of 0.35 cm; total volume of 19 240 cm 3 ).Two fans (252 N. DC axial fan, 12 V, 25 × 25 × 8 mm, EBM-Papst Mul ngen GmbH and Co. KG, Mul ngen, Germany) were xed at the top corners of the chamber to ensure thorough air mixing.A rubber seal port at the uppermost side of the chamber was used to inject the 13 CO 2 .
Similar to other isotopic tracer experiments, we found a strong link between belowground allocation of freshly xed C and soil respiration, dissolved organic C (DOC) and soil microbial biomass (Bahn et al., 2013; Tavi et al., 2013, Sommer et al., 2016; Weng et al., 2017; Van De Broek et al., 2020).It has been

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Figure 1 Time
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Table 3
Effect of rotational position (Rot_pos), plant part (Plant_part) and their interactions on winter wheat biomass δ 13 C. Signi cant values at p ≤ 0.05 level are indicated in bold.
C Cmic and δ 13 C DOC .Signi cant values at p ≤ 0.05 level are indicated in bold.