{"paper_id":"2ae4ec4b-9cff-4247-bf3c-d612be9d3af8","body_text":"Quantifying the effects of arbuscular mycorrhizal fungi and potato cyst 1 \nnematodes on root system architecture using X-ray computed tomography 2 \n 3 \nEric C. Pereira, Saoirse Tracy* 4 \nSchool of Agriculture and Food Science, University College Dublin, Belfield, Dublin, 4, 5 \nIreland 6 \n 7 \n*Correspondence: saoirse.tracy@ucd.ie 8 \n 9 \nAbstract 10 \nCrop root systems develop in biologically complex soils where beneficial symbionts 11 \nand pathogenic organisms can jointly influence root architecture and, consequently, 12 \nbelowground function. In this work , we used X -ray computed tomography (CT) to 13 \nassess how colonisation by the arbuscular mycorrhizal fungus Rhizophagus irregularis 14 \n(AMF) and infection by the potato cyst nematode Globodera pallida (PCN) influence 15 \nroot system architecture in soil -grown tomato and potato plants. Root architectural 16 \ntraits, including root volume and root surface area, were quantified non -destructively 17 \nfrom intact root systems to evaluate the individual and combined effects of AMF 18 \ncolonisation and PCN infection over time. AMF inoculation increased root volume and 19 \nsurface area, whereas PCN infection caused pronounced reductions in these traits, 20 \nparticularly during early development. AMF -associated increases in root system size 21 \nwere maintained in both PCN-free and PCN-infected plants, indicating largely additive 22 \neffects of beneficial and pathogenic soil biota on root architectural outcomes. These 23 \nfindings show that soil organisms can independently reshape crop root development 24 \nin ways likely to influence soil exploration and resource acquisition under biologically 25 \ncomplex conditions. More broadly, the study highlights the value of X-ray CT as a non-26 \ndestructive approach for linking belowground biotic interactions with functionally 27 \nrelevant root traits in sustainable agroecosystems. 28 \n 29 \nKeywords: Arbuscular mycorrhizal fungi, Potato cyst nematodes, X -ray computed 30 \ntomography, Plant-microbe interactions, Root architecture,  31 \n 32 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nINTRODUCTION 33 \nRoot system architecture plays a central role in plant performance by regulating water 34 \nand nutrient acquisition, anchorage, and interactions with the surrounding soil 35 \nenvironment ( Lynch, 1995 ; Lynch, 2022; Freschet  et al., 2021). Beyond providing 36 \nstructural support, roots define the spatial interface through which plants explore soil 37 \nresources, and variation in root system organisation strongly influences plant growth, 38 \nvigour, and resilience under both favourable and stressful conditions. Interactions with 39 \nthe broader root microbiota also play a key role in modulating architecture and stress 40 \nresponses (Balestrini et al., 2024). Despite its importance, quantitative analysis of root 41 \narchitecture remains challenging under soil -grown conditions, where roots are 42 \nembedded within a heterogeneous matrix and interact dynamically with biotic and 43 \nabiotic factors. Traditional root phenotyping approaches, including destructive 44 \nharvesting and washing, disrupt native root–soil relationships and often fail to capture 45 \nthe three-dimensional organisation of intact root systems. Consequently, there is a 46 \npersistent methodological gap in our ability to quantify root architecture in situ. 47 \nX-ray computed tomography (CT) has emerged as a powerful non -invasive imaging 48 \napproach for three-dimensional visualisation of roots growing in soil (Tracy et al., 2010; 49 \nMairhofer et al., 2013; Hou et al., 2022). By reconstructing intact soil cores, X-ray CT 50 \nenables direct observation of the spatial distribution and temporal development of 51 \nroots without disturbing the soil –root interface (Hou et al., 2022 ; Ghosh et al., 2023). 52 \nImportantly, X-ray CT-based methods also enable quantitative extraction of 53 \narchitectural traits (Figure 1), such as root volume and surface area, thereby enabling 54 \nrobust comparisons across treatments and developmental stages (Tracy et al., 2012). 55 \nWhile X-ray CT has been widely applied to characterise root growth responses to soil 56 \nstructure and physical constraints, its use for quantifying the effects of interacting soil 57 \nbiota on root system architecture remains comparatively limited (Rogers et al., 2016; 58 \nVan Harsselaar et al., 2021; Zhang et al., 2022). 59 \n 60 \n 61 \nFigure 1 – Three-dimensional X -ray computed tomography reconstruction of a potato root system 62 \ngrown in soil, illustrating the spatial distribution of roots (white) within the soil matrix (orange). Scale bar 63 \n= 10 cm. 64 \n 65 \nSolanum crops, including tomato ( Solanum lycopersicum  L.) and potato ( Solanum 66 \ntuberosum), are globally important due to their high nutritional and economic value 67 \n(Raiola et al., 2014; Jansky et al., 2019). Their production , however, is strongly 68 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nconstrained by soil-borne pests and pathogens. Among these, potato cyst nematodes 69 \n(PCN) are among the most damaging threats, causing substantial yield losses in 70 \npotato and other solanaceous crops worldwide (Moens et al., 2018; Price et al., 2021). 71 \nSpecies such as Globodera pallida are obligate biotrophs that persist in soil as long -72 \nlived cysts and impair plant performance by altering root development and nutrient 73 \nuptake (Turner & Rowe, 2006; De Ruijter & Haverkort, 1999). In contrast, arbuscular 74 \nmycorrhizal fungi (AMF) form widespread mutualistic associations with Solanum 75 \nspecies and contribute to plant nutrition by extending the effective absorptive capacity 76 \nof the root system, particularly for phosphorus acquisition (Smith et al., 2011; Chen et 77 \nal., 2018). AMF colonisation has also been shown to influence root system 78 \narchitecture, including changes in root length, branching patterns, surface area, and 79 \nvolume (Chen et al., 2021; Zhang et al., 2021).  80 \nBoth PCN infection and AMF colonisation are therefore known to modify root 81 \ndevelopment, yet their combined effects on root system architecture within intact soil 82 \nenvironments remain poorly quantified. This is an important gap because, in 83 \nagricultural soils, crop root systems develop within biologically complex environments 84 \nwhere mutualists and pests act simultaneously to influence resource capture, stress 85 \ntolerance, and plant performance. Understanding how beneficial and pathogenic soil 86 \norganisms jointly shape root system architecture is therefore important not only for 87 \nroot biology but also for predicting crop function under realistic soil conditions and for 88 \ndeveloping biologically informed management strategies in agroecosystems. Here, we 89 \nused X -ray computed tomography as a non -destructive phenotyping approach to 90 \nquantify how colonisation by the arbuscular mycorrhizal fungus R. irregularis  and 91 \ninfection by the potato cyst nematode G. pallida independently and jointly alter root 92 \nsystem architecture in soil -grown tomato and potato. By measuring root volume and 93 \nsurface area from intact three-dimensional root systems across developmental stages, 94 \nwe aimed to determine how beneficial and pathogenic soil biota reshape crop root 95 \narchitecture under controlled soil -based conditions and to establish a framework for 96 \nlinking these interactions to crop function in agroecosystems. 97 \n 98 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nMaterial and methods 99 \nPlant material and experimental design 100 \nGreenhouse experiments were conducted independently for tomato ( Solanum 101 \nlycopersicum L.) and potato (Solanum tuberosum L.) using a fully factorial design with 102 \ntwo factors: arbuscular mycorrhizal fungi (AMF; uninoculated or inoculated) and potato 103 \ncyst nematode (PCN; non -infected or infected). Each treatment combination was 104 \nreplicated [n = 10] times per species. 105 \nTomato seeds and potato tubers were planted in 60 mL and 2000 mL pots, 106 \nrespectively, containing a sand–soil mixture (1:1, v/v). The substrate was autoclaved 107 \nat 121 °C for 30 min prior to planting to minimise background microbial activity.  AMF 108 \ninoculation was performed at planting using an inoculum of Rhizophagus irregularis. 109 \nUninoculated controls received an equivalent volume of sterilised inoculum material to 110 \ncontrol for substrate effects. 111 \nPCN infection was established at planting by incorporating Globodera pallida inoculum 112 \ninto the substrate at a density of 50 cysts per pot , determined prior to planting using 113 \nstandard extraction and counting procedures. Control plants received PCN -free 114 \nsubstrate. Plants were harvested at 2 and 4 weeks after planting. At each time point, 115 \nplants were subjected to X -ray computed tomography (CT) scanning prior to 116 \ndestructive sampling for biomass measurements and confirmation of AMF colonisation 117 \nand PCN infection. 118 \n 119 \nX-ray CT analysis 120 \nTo determine the effects of AMF and PCN on the root architecture of tomato plants, 121 \nall pots were analysed using the GE Nanotom M X-ray CT machine (GE Measurement 122 \nand Control Solutions). The v|tome|x M was set at a voltage of 65 kV and a current of 123 \n300 μA to optimise contrast between background soil and roots. The 'Fast Scan option' 124 \nachieved a voxel resolution of 1.60 μm. 1,078 projection images were taken per scan 125 \nat 200 m/s per image. Once scanning was complete, the images were reconstructed 126 \nusing Phoenix datos|×2 rec reconstruction software, combining the scans into a single 127 \n3D volume representing the entire core.  128 \n 129 \nImage processing 130 \nImage analysis of X-ray CT images was performed using VGStudioMax® (Version 3.2; 131 \nVolume Graphics GmbH, Heidelberg, Germany) to segment cyst nematodes. Cysts 132 \nwere segmented by setting seed points and using selected threshold values in the 133 \nRegion grower , thereby selecting grey -scale pixels associated with  root materials. 134 \nOnce the cysts were segmented from the image, the Erosion and Dilation tool was 135 \nselected with a 1 -pixel radius, and the Region Growing tool was used . Root system 136 \narchitecture parameters , including root length, volume, and surface area, were 137 \nmeasured from segmented root systems. 138 \n 139 \n 140 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nDetection of AMF and PNC in Inoculated and Infected Plants 141 \nDetection of AMF and PCN in plants was conducted by microscopic examination. Root 142 \nsamples were harvested from inoculated and infected plants at harvest to facilitate this 143 \nanalysis. For assessment of fungal structure by microscopy, root specimens were 144 \nprepared using either the potassium hydroxide (KOH) clearing method or non-clearing 145 \nmethods, followed by staining with Chinese ink or aniline blue , as described by  146 \nVierheilig et al. (2005). Conversely, for PCN analysis, cleared root preparations were 147 \nstained with fuchsin acid, as detailed in the method established in  the relevant 148 \nliterature (Byrd et al., 1983). 149 \n 150 \nStatistical Analysis 151 \nThe datasets were evaluated for ANOVA assumptions using the Shapiro –Wilk 152 \nnormality test and the Brown –Forsythe test for equal variances . Then, the effects of 153 \nAMF and PCN on tomato and potato parameters were analysed by two-way ANOVA. 154 \nDifferences between means were evaluated using Tukey’s test. All statistical analyses 155 \nwere performed using SPSS v24. 156 \n 157 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nRESULTS 158 \nX-ray CT imaging of intact root systems in soil 159 \nX-ray computed tomography enabled clear visualisation of intact root systems within 160 \nthe soil matrix for both tomato and potato plants (Figure 2). Three -dimensional 161 \nreconstructions allowed discrimination between soil, root tissue, and air -filled pores 162 \nbased on grayscale intensity, with roots exhibiting lower X -ray attenuation than the 163 \nsurrounding mineral substrate. These reconstructions provided a basis for consistent 164 \nsegmentation and quantitative analysis of root system architecture without disturbing 165 \nthe soil–root interface. 166 \n 167 \n 168 \nFigure 2 – Representative X-ray computed tomography slice of a pot containing a potato plant grown 169 \nin a soil:sand (1:1, v:v ) substrate. Different constituents are visible based on grayscale intensity: (A) 170 \nsoil, (B) root tissue, and (C) air-filled pores. Scale bar = 10 cm. 171 \n 172 \nEffects of AMF and PCN on root system architecture in tomato 173 \nThree-dimensional CT reconstructions revealed clear differences in root system 174 \narchitecture among treatments in tomato plants at both 2 and 4 weeks after planting 175 \n(Figure 3). Quantitative analysis showed that both AMF inoculation and PCN infection 176 \nsignificantly influenced root volume and root surface area, whereas no significant AMF 177 \n× PCN interaction was detected at either time point (Figure 4; Table 1). 178 \n10 cm \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\n 179 \nFigure 3 - Three-dimensional X-ray computed tomography reconstructions of tomato root systems at 2 180 \nand 4 weeks after planting. Roots are shown for uninoculated and AMF -inoculated plants under PCN-181 \nfree and PCN -infected conditions, illustrating treatment -dependent differences in root system 182 \narchitecture. 183 \n 184 \nAt two weeks, AMF inoculation resulted in a significant increase in root volume relative 185 \nto uninoculated plants, both in the absence (+50%) and presence (+28%) of PCN 186 \ninfection (Figure 4A). A similar pattern was observed for root surface area, which 187 \nincreased by 52% in PCN -free plants and by 23% in PCN -infected plants following 188 \nAMF inoculation (Figure 4C). At four weeks, AMF-associated increases in root volume 189 \nremained significant, with increases of 40% in PCN -free plants and 60% in PCN -190 \ninfected plants (Figure 4B). Root surface area also increased significantly at this stage, 191 \nby 36% in PCN-free plants and 68% in PCN-infected plants (Figure 4D). 192 \nPCN infection had a strong negative effect on tomato root architecture at both 193 \ndevelopmental stages. At two weeks, PCN infection significantly reduced root volume 194 \nin both uninoculated (−48%) and AMF -inoculated plants (−65%) (Figure 4A). At four 195 \nweeks, PCN-induced reductions in root volume were even more pronounced, reaching 196 \n75% in uninoculated plants and 62% in AMF -inoculated plants (Figure 4B). Similar 197 \ntrends were observed for root surface area, with significant reductions at both time 198 \npoints (Figures 4C and 4D). Despite these reductions, AMF -inoculated plants 199 \nconsistently maintained larger root systems than their uninoculated counterparts 200 \nunder PCN infection. 201 \n 202 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\n 203 \nFigure 4 – Root system architectural traits of tomato plants quantified by X-ray CT. Root volume (A, B) 204 \nand root surface area (C, D) are shown for uninoculated (green) and AMF -inoculated (orange) plants 205 \nunder PCN-free and PCN -infected conditions at 2 weeks (A, C) and 4 weeks (B, D) after planting. 206 \nValues represent means ± SD (n = 5). Asterisks indicate significant differences (*p < 0.05; **p < 0.01). 207 \n 208 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nEffects of AMF and PCN on root system architecture in potato 209 \nX-ray CT imaging also revealed treatment -dependent differences in root system 210 \narchitecture in potato plants at both 2 and 4 weeks (Figure 5). As observed in tomato, 211 \nboth AMF inoculation and PCN infection significantly affected root architectural traits, 212 \nwhile their interaction was not significant (Figure 6; Table 1). 213 \n 214 \nFigure 5 - Three-dimensional X-ray computed tomography reconstructions of potato root systems at 2 215 \nand 4 weeks after planting. Roots are shown for uninoculated and AMF -inoculated plants under PCN-216 \nfree and PCN-infected conditions, highlighting treatment-dependent differences in root architecture. 217 \n 218 \nIn two-week-old potato plants, AMF inoculation significantly increased root volume by 219 \n33% in PCN-free plants and by 38% in PCN-infected plants (Figure 6A). At four weeks, 220 \nAMF continued to exert a positive effect on root volume, with increases of 26% in PCN-221 \nfree plants and 28% in PCN-infected plants (Figure 6B). Root surface area responded 222 \nsimilarly to AMF inoculation, increasing by 24% (PCN -free) and 20% (PCN -infected) 223 \nat two weeks (Figure 6C), and by 21% and 36%, respectively, at four weeks (Figure 224 \n6D). 225 \nPCN infection significantly reduced root volume and surface area in potato plants at 226 \nthe two-week stage. Root volume decreased by 22% in uninoculated plants and by 227 \n16% in AMF -inoculated plants under PCN infection (Figure 6A). Root surface area 228 \nwas also significantly reduced, with decreases of 19% in uninoculated plants and 23% 229 \nin AMF-inoculated plants (Figure 6C). In contrast, PCN effects on root volume and 230 \nsurface area were not statistically significant at four weeks (Figures 6B and 6D). 231 \n 232 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\n 233 \nFigure 6 – Root system architectural traits of potato plants quantified by X-ray CT. Root volume (A, B) 234 \nand root surface area (C, D) are shown for uninoculated (green) and AMF -inoculated (orange) plants 235 \nunder PCN-free and PCN -infected conditions at 2 weeks (A, C) and 4 weeks (B, D) after planting. 236 \nValues represent means ± SD (n = 5). Asterisks indicate significant differences (*p < 0.05; **p < 0.01; 237 \n***p < 0.001). 238 \n 239 \n 240 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nDetection of Fungi and PCN in Root Plants 241 \nMicroscopic examination confirmed the presence of R. irregularis structures, including 242 \nhyphae and spores, in roots of AMF -inoculated tomato and potato plants at both 243 \nsampling times, irrespective of PCN treatment (Figure 7). Juvenile stages of G. pallida 244 \nwere observed in roots of PCN -infected plants, both in the presence and absence of 245 \nAMF inoculation (Figure 8). These observations confirm the successful establishment 246 \nof both AMF colonisation and PCN infection in the respective treatments. 247 \n 248 \n 249 \nFigure 7 – Representative light microscopy images showing Rhizophagus irregularis structures in roots 250 \nof tomato and potato plants inoculated with AMF, including hyphae and spores, under PCN -free and 251 \nPCN-infected conditions. 252 \n 253 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\n 254 \nFigure 8 – Representative light microscopy images showing juvenile stages of Globodera pallida in 255 \nroots of tomato and potato plants under PCN-infected conditions, in the absence and presence of AMF 256 \ninoculation. 257 \n 258 \nStatistical summary 259 \nTwo-way analysis of variance confirmed significant main effects of AMF inoculation 260 \nand PCN infection on root volume and root surface area in both species, while AMF × 261 \nPCN interactions were not significant for any trait or time point (Table 1). 262 \n 263 \nTable 1. Results of two-way analysis of variance (ANOVA) testing the effects of AMF inoculation, PCN 264 \ninfection, and their interaction on root volume and root surface area in tomato and potato plants at 2 265 \nand 4 weeks after planting. 266 \n   AMF inoculation PCN infection AMF x PCN \n   F P F P F P \nTomato plants 2 \nweeks \nRoot volume 3.418 0.101 7.685 0.024 1.536 0.250 \nRoot surface area 2.597 0.146 8.431 0.020 1.645 0.235 \n4 \nweeks \nRoot volume 6.024 0.040 16.83 0.003 0.413 0.538 \nRoot surface area 6.120 0.038 25.16 0.001 0.309 0.593 \nPotato plants 2 \nweeks \nRoot volume 36.71 <0.001 8.521 0.019 0.018 0.897 \nRoot surface area 12.88 0.007 11.46 0.010 0.718 0.421 \n4 \nweeks \nRoot volume 14.93 0.005 3.461 0.099 0.011 0.918 \nRoot surface area 14.95 0.005 1.678 0.231 0.954 0.357 \n 267 \n 268 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nDiscussion 269 \nThis study shows that beneficial and pathogenic soil organisms can independently and 270 \nconsistently reshape crop root architecture in soil -grown plants. Across tomato and 271 \npotato, AMF colonisation increased root volume and surface area, whereas PCN 272 \ninfection reduced these traits, particularly during early development. The absence of 273 \na significant AMF × PCN interaction indicates that these organisms exerted largely 274 \nadditive effects on root system architecture. These findings are relevant to 275 \nagroecosystems because root system development underpins soil exploration, 276 \nresource acquisition, and the capacity of crops to maintain performance under 277 \nbiologically complex soil conditions. 278 \nConsistent with previous studies, AMF colonisation by R. irregularis was associated 279 \nwith increased root system size, reflected in greater root volume and surface area in 280 \nboth plant species (Begum et al., 2019; Diagne et al., 2020; Ramírez -Flores et al., 281 \n2019; Shafiq et al., 2023). Broader microbiome studies show that microbial 282 \ninteractions can drive root phenotypic plasticity (Dini -Andreote et al., 2025). These 283 \narchitectural changes likely reflect AMF -induced modulation of root development, 284 \nincluding altered branching patterns and expansion of the absorptive root surface, as 285 \nreported in earlier work employing destructive phenotyping. In contrast, PCN infection 286 \nby G. pallida exerted a strong negative effect on root system architecture, particularly 287 \nduring early developmental stages. Significant reductions in root volume and surface 288 \narea were observed in PCN -infected plants, consistent with the well -documented 289 \ncapacity of cyst nematodes to impair root growth and disrupt normal root development 290 \n(Moens et al., 2018; Palomares-Rius et al., 2017). Together, these results indicate that 291 \nbeneficial and pathogenic belowground organisms can drive contrasting architectural 292 \noutcomes within the same crop root system. 293 \nFrom an agroecosystem perspective, variation in root system size and spatial 294 \ndevelopment can influence how effectively crops explore soil, intercept water and 295 \nnutrients, and maintain early vigour under biotic and abiotic stress  (Freschet et al., 296 \n2021; Lynch, 2022). Increases in root volume and surface area associated with AMF 297 \ncolonisation may therefore indicate enhanced potential for belowground resource 298 \ncapture (Begum et al., 2019) , whereas reductions caused by PCN infection likely 299 \nreflect a constrained capacity to exploit soil resources during early development  (De 300 \nRuijter & Haverkort, 1999) . Although root volume and surface area do not directly 301 \nmeasure nutrient uptake or yield, they represent functionally relevant traits through 302 \nwhich soil biota may influence crop performance in soil -based production systems  303 \n(Freschet et al., 2021; Lynch, 2022). 304 \nImportantly, although AMF inoculation and PCN infection both significantly influenced 305 \nroot architecture, no significant interaction between these factors was detected for the 306 \nmeasured traits. This indicates that AMF -associated increases in root volume and 307 \nsurface area occurred consistently in both PCN-free and PCN-infected plants. Rather 308 \nthan reflecting a specific antagonistic or suppressive interaction between the two 309 \norganisms, the results point to largely additive effects on root system development. 310 \nMicroscopic observations confirmed the simultaneous presence of AMF structures 311 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\nand PCN juveniles within roots, supporting previous reports that co-colonisation by R. 312 \nirregularis and G. pallida does not necessarily result in mutual inhibition (Bell et al., 313 \n2022). Within this context, the observed architectural responses represent additive 314 \noutcomes of symbiotic and pathogenic influences on root system development. 315 \nThis distinction is important for biologically informed crop management. The present 316 \nresults suggest that AMF-associated changes in root architecture should therefore not 317 \nbe interpreted solely as evidence of direct antagonism against nematodes, but rather 318 \nas a biologically mediated shift in host root development that persists even when the 319 \npest remains present, consistent with broader evidence that mycorrhizal effects on 320 \nplant defence and performance are mechanistically complex and often host-mediated 321 \n(Cameron et al., 2013; Vos et al., 2012; Gough et al., 2020). In agroecosystems, such 322 \nadditive outcomes may still be valuable if they help maintain root system function, 323 \nimprove soil exploration, or buffer early developmental damage in infested soils. This 324 \nis particularly relevant because potato cyst nematodes can impair root growth, nutrient 325 \nuptake, and crop growth, meaning that partial maintenance of root system 326 \ndevelopment may still have functional benefits even without direct pest suppression 327 \n(De Ruijter & Haverkort, 1999). More broadly, the findings support the idea that 328 \nbeneficial soil biota may contribute to crop resilience by modulating root traits and host 329 \ntolerance, even when soil-borne pests remain present (Bell et al., 2022). 330 \nWithin this broader biological context, X -ray CT provides a valuable means of 331 \nquantifying how interacting belowground organisms reshape crop root systems in 332 \nintact soil. Its non -destructive, three -dimensional imaging capacity allows root 333 \narchitectural traits to be measured without disrupting root –soil relationships, thereby 334 \npreserving the spatial context in which plant–microbe and plant–pathogen interactions 335 \noccur. The suitability of X -ray CT for plant –nematode systems has previously been 336 \nsupported by the direct detection of potato cyst nematodes in soil -grown plants 337 \n(Pereira et al., 2025), and the present study extends that framework by demonstrating 338 \nreproducible treatment effects of AMF colonisation and PCN infection on root volume 339 \nand surface area in tomato and potato. In this sense, X -ray CT serves as a robust 340 \nintermediate platform for linking controlled mechanistic studies with more complex 341 \nagroecosystem questions, enabling more precise investigation of how soil biota 342 \ninfluence crop root development under realistic soil-based conditions. 343 \n 344 \nConclusion 345 \nThis study shows that beneficial and pathogenic soil organisms can independently 346 \nreshape crop root architecture in soil -grown plants. Across tomato and potato, 347 \ncolonisation by Rhizophagus irregularis  increased root volume and surface area, 348 \nwhereas infection by Globodera pallida reduced these traits, particularly during early 349 \ndevelopment. The absence of a significant AMF × PCN interaction indicates that these 350 \norganisms exert largely additive effects on root system architecture. AMF -associated 351 \nincreases in root system size were maintained even under nematode pressure. Root 352 \nsystem size and spatial development influence how effectively crops explore soil, 353 \nacquire water and nutrients, and maintain function under stress. These findings 354 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint \n\ntherefore indicate that soil biota can have important consequences for crop 355 \nperformance in biologically complex soils. X -ray CT was central to this analysis by 356 \nenabling non-destructive quantification of root architectural traits in intact soil, thereby 357 \nlinking belowground biotic interactions with functionally relevant structural outcomes. 358 \nMore broadly, this study provides a mechanistic foundation for biologically informed 359 \nstrategies to improve crop resilience and manage soil-borne constraints in sustainable 360 \nagroecosystems. 361 \n 362 \nAcknowledgements 363 \n We gratefully acknowledge funding from the Leverhulme Trust (RPG-2019-162).  364 \n 365 \nCompeting interests 366 \nThe authors have declared that no competing interests exist. 367 \n 368 \nAuthor contributions 369 \nConceptualization: Eric C. 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