Quantifying the effects of arbuscular mycorrhizal fungi and potato cyst nematodes on root system architecture using X-ray computed tomography

10 Crop root systems develop in biologically complex soils where beneficial symbionts 11 and pathogenic organisms can jointly influence root architecture and, consequently, 12 belowground function. In this work , we used X -ray computed tomography (CT) to 13 assess how colonisation by the arbuscular mycorrhizal fungus Rhizophagus irregularis 14 (AMF) and infection by the potato cyst nematode Globodera pallida (PCN) influence 15 root system architecture in soil -grown tomato and potato plants. Root architectural 16 traits, including root volume and root surface area, were quantified non -destructively 17 from intact root systems to evaluate the individual and combined effects of AMF 18 colonisation and PCN infection over time. AMF inoculation increased root volume and 19 surface area, whereas PCN infection caused pronounced reductions in these traits, 20 particularly during early development. AMF -associated increases in root system size 21 were maintained in both PCN-free and PCN-infected plants, indicating largely additive 22 effects of beneficial and pathogenic soil biota on root architectural outcomes. These 23 findings show that soil organisms can independently reshape crop root development 24 in ways likely to influence soil exploration and resource acquisition under biologically 25 complex conditions. More broadly, the study highlights the value of X-ray CT as a non-26 destructive approach for linking belowground biotic interactions with functionally 27 relevant root traits in sustainable agroecosystems. 28 29

Arbuscular mycorrhizal fungi, Potato cyst nematodes, X -ray computed 30 tomography, Plant-microbe interactions, Root architecture, 31 32 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint

33 Root system architecture plays a central role in plant performance by regulating water 34 and nutrient acquisition, anchorage, and interactions with the surrounding soil 35 environment ( Lynch, 1995 ; Lynch, 2022; Freschet et al., 2021). Beyond providing 36 structural support, roots define the spatial interface through which plants explore soil 37 resources, and variation in root system organisation strongly influences plant growth, 38 vigour, and resilience under both favourable and stressful conditions. Interactions with 39 the broader root microbiota also play a key role in modulating architecture and stress 40 responses (Balestrini et al., 2024). Despite its importance, quantitative analysis of root 41 architecture remains challenging under soil -grown conditions, where roots are 42 embedded within a heterogeneous matrix and interact dynamically with biotic and 43 abiotic factors. Traditional root phenotyping approaches, including destructive 44 harvesting and washing, disrupt native root–soil relationships and often fail to capture 45 the three-dimensional organisation of intact root systems. Consequently, there is a 46 persistent methodological gap in our ability to quantify root architecture in situ. 47 X-ray computed tomography (CT) has emerged as a powerful non -invasive imaging 48 approach for three-dimensional visualisation of roots growing in soil (Tracy et al., 2010; 49 Mairhofer et al., 2013; Hou et al., 2022). By reconstructing intact soil cores, X-ray CT 50 enables direct observation of the spatial distribution and temporal development of 51 roots without disturbing the soil –root interface (Hou et al., 2022 ; Ghosh et al., 2023). 52 Importantly, X-ray CT-based methods also enable quantitative extraction of 53 architectural traits (Figure 1), such as root volume and surface area, thereby enabling 54 robust comparisons across treatments and developmental stages (Tracy et al., 2012). 55 While X-ray CT has been widely applied to characterise root growth responses to soil 56 structure and physical constraints, its use for quantifying the effects of interacting soil 57 biota on root system architecture remains comparatively limited (Rogers et al., 2016; 58 Van Harsselaar et al., 2021; Zhang et al., 2022). 59 60 61 Figure 1 – Three-dimensional X -ray computed tomography reconstruction of a potato root system 62 grown in soil, illustrating the spatial distribution of roots (white) within the soil matrix (orange). Scale bar 63 = 10 cm. 64 65 Solanum crops, including tomato ( Solanum lycopersicum L.) and potato ( Solanum 66 tuberosum), are globally important due to their high nutritional and economic value 67 (Raiola et al., 2014; Jansky et al., 2019). Their production , however, is strongly 68 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint constrained by soil-borne pests and pathogens. Among these, potato cyst nematodes 69 (PCN) are among the most damaging threats, causing substantial yield losses in 70 potato and other solanaceous crops worldwide (Moens et al., 2018; Price et al., 2021). 71 Species such as Globodera pallida are obligate biotrophs that persist in soil as long -72 lived cysts and impair plant performance by altering root development and nutrient 73 uptake (Turner & Rowe, 2006; De Ruijter & Haverkort, 1999). In contrast, arbuscular 74 mycorrhizal fungi (AMF) form widespread mutualistic associations with Solanum 75 species and contribute to plant nutrition by extending the effective absorptive capacity 76 of the root system, particularly for phosphorus acquisition (Smith et al., 2011; Chen et 77 al., 2018). AMF colonisation has also been shown to influence root system 78 architecture, including changes in root length, branching patterns, surface area, and 79 volume (Chen et al., 2021; Zhang et al., 2021). 80 Both PCN infection and AMF colonisation are therefore known to modify root 81 development, yet their combined effects on root system architecture within intact soil 82 environments remain poorly quantified. This is an important gap because, in 83 agricultural soils, crop root systems develop within biologically complex environments 84 where mutualists and pests act simultaneously to influence resource capture, stress 85 tolerance, and plant performance. Understanding how beneficial and pathogenic soil 86 organisms jointly shape root system architecture is therefore important not only for 87 root biology but also for predicting crop function under realistic soil conditions and for 88 developing biologically informed management strategies in agroecosystems. Here, we 89 used X -ray computed tomography as a non -destructive phenotyping approach to 90 quantify how colonisation by the arbuscular mycorrhizal fungus R. irregularis and 91 infection by the potato cyst nematode G. pallida independently and jointly alter root 92 system architecture in soil -grown tomato and potato. By measuring root volume and 93 surface area from intact three-dimensional root systems across developmental stages, 94 we aimed to determine how beneficial and pathogenic soil biota reshape crop root 95 architecture under controlled soil -based conditions and to establish a framework for 96 linking these interactions to crop function in agroecosystems. 97 98 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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99 Plant material and experimental design 100 Greenhouse experiments were conducted independently for tomato ( Solanum 101 lycopersicum L.) and potato (Solanum tuberosum L.) using a fully factorial design with 102 two factors: arbuscular mycorrhizal fungi (AMF; uninoculated or inoculated) and potato 103 cyst nematode (PCN; non -infected or infected). Each treatment combination was 104 replicated [n = 10] times per species. 105 Tomato seeds and potato tubers were planted in 60 mL and 2000 mL pots, 106 respectively, containing a sand–soil mixture (1:1, v/v). The substrate was autoclaved 107 at 121 °C for 30 min prior to planting to minimise background microbial activity. AMF 108 inoculation was performed at planting using an inoculum of Rhizophagus irregularis. 109 Uninoculated controls received an equivalent volume of sterilised inoculum material to 110 control for substrate effects. 111 PCN infection was established at planting by incorporating Globodera pallida inoculum 112 into the substrate at a density of 50 cysts per pot , determined prior to planting using 113 standard extraction and counting procedures. Control plants received PCN -free 114 substrate. Plants were harvested at 2 and 4 weeks after planting. At each time point, 115 plants were subjected to X -ray computed tomography (CT) scanning prior to 116 destructive sampling for biomass measurements and confirmation of AMF colonisation 117 and PCN infection. 118 119 X-ray CT analysis 120 To determine the effects of AMF and PCN on the root architecture of tomato plants, 121 all pots were analysed using the GE Nanotom M X-ray CT machine (GE Measurement 122 and Control Solutions). The v|tome|x M was set at a voltage of 65 kV and a current of 123 300 μA to optimise contrast between background soil and roots. The 'Fast Scan option' 124 achieved a voxel resolution of 1.60 μm. 1,078 projection images were taken per scan 125 at 200 m/s per image. Once scanning was complete, the images were reconstructed 126 using Phoenix datos|×2 rec reconstruction software, combining the scans into a single 127 3D volume representing the entire core. 128 129 Image processing 130 Image analysis of X-ray CT images was performed using VGStudioMax® (Version 3.2; 131 Volume Graphics GmbH, Heidelberg, Germany) to segment cyst nematodes. Cysts 132 were segmented by setting seed points and using selected threshold values in the 133 Region grower , thereby selecting grey -scale pixels associated with root materials. 134 Once the cysts were segmented from the image, the Erosion and Dilation tool was 135 selected with a 1 -pixel radius, and the Region Growing tool was used . Root system 136 architecture parameters , including root length, volume, and surface area, were 137 measured from segmented root systems. 138 139 140 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint Detection of AMF and PNC in Inoculated and Infected Plants 141 Detection of AMF and PCN in plants was conducted by microscopic examination. Root 142 samples were harvested from inoculated and infected plants at harvest to facilitate this 143 analysis. For assessment of fungal structure by microscopy, root specimens were 144 prepared using either the potassium hydroxide (KOH) clearing method or non-clearing 145 methods, followed by staining with Chinese ink or aniline blue , as described by 146 Vierheilig et al. (2005). Conversely, for PCN analysis, cleared root preparations were 147 stained with fuchsin acid, as detailed in the method established in the relevant 148 literature (Byrd et al., 1983). 149 150 Statistical Analysis 151 The datasets were evaluated for ANOVA assumptions using the Shapiro –Wilk 152 normality test and the Brown –Forsythe test for equal variances . Then, the effects of 153 AMF and PCN on tomato and potato parameters were analysed by two-way ANOVA. 154 Differences between means were evaluated using Tukey’s test. All statistical analyses 155 were performed using SPSS v24. 156 157 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint

158 X-ray CT imaging of intact root systems in soil 159 X-ray computed tomography enabled clear visualisation of intact root systems within 160 the soil matrix for both tomato and potato plants (Figure 2). Three -dimensional 161 reconstructions allowed discrimination between soil, root tissue, and air -filled pores 162 based on grayscale intensity, with roots exhibiting lower X -ray attenuation than the 163 surrounding mineral substrate. These reconstructions provided a basis for consistent 164 segmentation and quantitative analysis of root system architecture without disturbing 165 the soil–root interface. 166 167 168 Figure 2 – Representative X-ray computed tomography slice of a pot containing a potato plant grown 169 in a soil:sand (1:1, v:v ) substrate. Different constituents are visible based on grayscale intensity: (A) 170 soil, (B) root tissue, and (C) air-filled pores. Scale bar = 10 cm. 171 172 Effects of AMF and PCN on root system architecture in tomato 173 Three-dimensional CT reconstructions revealed clear differences in root system 174 architecture among treatments in tomato plants at both 2 and 4 weeks after planting 175 (Figure 3). Quantitative analysis showed that both AMF inoculation and PCN infection 176 significantly influenced root volume and root surface area, whereas no significant AMF 177 × PCN interaction was detected at either time point (Figure 4; Table 1). 178 10 cm .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint 179 Figure 3 - Three-dimensional X-ray computed tomography reconstructions of tomato root systems at 2 180 and 4 weeks after planting. Roots are shown for uninoculated and AMF -inoculated plants under PCN-181 free and PCN -infected conditions, illustrating treatment -dependent differences in root system 182 architecture. 183 184 At two weeks, AMF inoculation resulted in a significant increase in root volume relative 185 to uninoculated plants, both in the absence (+50%) and presence (+28%) of PCN 186 infection (Figure 4A). A similar pattern was observed for root surface area, which 187 increased by 52% in PCN -free plants and by 23% in PCN -infected plants following 188 AMF inoculation (Figure 4C). At four weeks, AMF-associated increases in root volume 189 remained significant, with increases of 40% in PCN -free plants and 60% in PCN -190 infected plants (Figure 4B). Root surface area also increased significantly at this stage, 191 by 36% in PCN-free plants and 68% in PCN-infected plants (Figure 4D). 192 PCN infection had a strong negative effect on tomato root architecture at both 193 developmental stages. At two weeks, PCN infection significantly reduced root volume 194 in both uninoculated (−48%) and AMF -inoculated plants (−65%) (Figure 4A). At four 195 weeks, PCN-induced reductions in root volume were even more pronounced, reaching 196 75% in uninoculated plants and 62% in AMF -inoculated plants (Figure 4B). Similar 197 trends were observed for root surface area, with significant reductions at both time 198 points (Figures 4C and 4D). Despite these reductions, AMF -inoculated plants 199 consistently maintained larger root systems than their uninoculated counterparts 200 under PCN infection. 201 202 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint 203 Figure 4 – Root system architectural traits of tomato plants quantified by X-ray CT. Root volume (A, B) 204 and root surface area (C, D) are shown for uninoculated (green) and AMF -inoculated (orange) plants 205 under PCN-free and PCN -infected conditions at 2 weeks (A, C) and 4 weeks (B, D) after planting. 206 Values represent means ± SD (n = 5). Asterisks indicate significant differences (*p < 0.05; **p < 0.01). 207 208 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint Effects of AMF and PCN on root system architecture in potato 209 X-ray CT imaging also revealed treatment -dependent differences in root system 210 architecture in potato plants at both 2 and 4 weeks (Figure 5). As observed in tomato, 211 both AMF inoculation and PCN infection significantly affected root architectural traits, 212 while their interaction was not significant (Figure 6; Table 1). 213 214 Figure 5 - Three-dimensional X-ray computed tomography reconstructions of potato root systems at 2 215 and 4 weeks after planting. Roots are shown for uninoculated and AMF -inoculated plants under PCN-216 free and PCN-infected conditions, highlighting treatment-dependent differences in root architecture. 217 218 In two-week-old potato plants, AMF inoculation significantly increased root volume by 219 33% in PCN-free plants and by 38% in PCN-infected plants (Figure 6A). At four weeks, 220 AMF continued to exert a positive effect on root volume, with increases of 26% in PCN-221 free plants and 28% in PCN-infected plants (Figure 6B). Root surface area responded 222 similarly to AMF inoculation, increasing by 24% (PCN -free) and 20% (PCN -infected) 223 at two weeks (Figure 6C), and by 21% and 36%, respectively, at four weeks (Figure 224 6D). 225 PCN infection significantly reduced root volume and surface area in potato plants at 226 the two-week stage. Root volume decreased by 22% in uninoculated plants and by 227 16% in AMF -inoculated plants under PCN infection (Figure 6A). Root surface area 228 was also significantly reduced, with decreases of 19% in uninoculated plants and 23% 229 in AMF-inoculated plants (Figure 6C). In contrast, PCN effects on root volume and 230 surface area were not statistically significant at four weeks (Figures 6B and 6D). 231 232 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint 233 Figure 6 – Root system architectural traits of potato plants quantified by X-ray CT. Root volume (A, B) 234 and root surface area (C, D) are shown for uninoculated (green) and AMF -inoculated (orange) plants 235 under PCN-free and PCN -infected conditions at 2 weeks (A, C) and 4 weeks (B, D) after planting. 236 Values represent means ± SD (n = 5). Asterisks indicate significant differences (*p < 0.05; **p < 0.01; 237 ***p < 0.001). 238 239 240 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint Detection of Fungi and PCN in Root Plants 241 Microscopic examination confirmed the presence of R. irregularis structures, including 242 hyphae and spores, in roots of AMF -inoculated tomato and potato plants at both 243 sampling times, irrespective of PCN treatment (Figure 7). Juvenile stages of G. pallida 244 were observed in roots of PCN -infected plants, both in the presence and absence of 245 AMF inoculation (Figure 8). These observations confirm the successful establishment 246 of both AMF colonisation and PCN infection in the respective treatments. 247 248 249 Figure 7 – Representative light microscopy images showing Rhizophagus irregularis structures in roots 250 of tomato and potato plants inoculated with AMF, including hyphae and spores, under PCN -free and 251 PCN-infected conditions. 252 253 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint 254 Figure 8 – Representative light microscopy images showing juvenile stages of Globodera pallida in 255 roots of tomato and potato plants under PCN-infected conditions, in the absence and presence of AMF 256 inoculation. 257 258 Statistical summary 259 Two-way analysis of variance confirmed significant main effects of AMF inoculation 260 and PCN infection on root volume and root surface area in both species, while AMF × 261 PCN interactions were not significant for any trait or time point (Table 1). 262 263 Table 1. Results of two-way analysis of variance (ANOVA) testing the effects of AMF inoculation, PCN 264 infection, and their interaction on root volume and root surface area in tomato and potato plants at 2 265 and 4 weeks after planting. 266 AMF inoculation PCN infection AMF x PCN F P F P F P Tomato plants 2 weeks Root volume 3.418 0.101 7.685 0.024 1.536 0.250 Root surface area 2.597 0.146 8.431 0.020 1.645 0.235 4 weeks Root volume 6.024 0.040 16.83 0.003 0.413 0.538 Root surface area 6.120 0.038 25.16 0.001 0.309 0.593 Potato plants 2 weeks Root volume 36.71 <0.001 8.521 0.019 0.018 0.897 Root surface area 12.88 0.007 11.46 0.010 0.718 0.421 4 weeks Root volume 14.93 0.005 3.461 0.099 0.011 0.918 Root surface area 14.95 0.005 1.678 0.231 0.954 0.357 267 268 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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269 This study shows that beneficial and pathogenic soil organisms can independently and 270 consistently reshape crop root architecture in soil -grown plants. Across tomato and 271 potato, AMF colonisation increased root volume and surface area, whereas PCN 272 infection reduced these traits, particularly during early development. The absence of 273 a significant AMF × PCN interaction indicates that these organisms exerted largely 274 additive effects on root system architecture. These findings are relevant to 275 agroecosystems because root system development underpins soil exploration, 276 resource acquisition, and the capacity of crops to maintain performance under 277 biologically complex soil conditions. 278 Consistent with previous studies, AMF colonisation by R. irregularis was associated 279 with increased root system size, reflected in greater root volume and surface area in 280 both plant species (Begum et al., 2019; Diagne et al., 2020; Ramírez -Flores et al., 281 2019; Shafiq et al., 2023). Broader microbiome studies show that microbial 282 interactions can drive root phenotypic plasticity (Dini -Andreote et al., 2025). These 283 architectural changes likely reflect AMF -induced modulation of root development, 284 including altered branching patterns and expansion of the absorptive root surface, as 285 reported in earlier work employing destructive phenotyping. In contrast, PCN infection 286 by G. pallida exerted a strong negative effect on root system architecture, particularly 287 during early developmental stages. Significant reductions in root volume and surface 288 area were observed in PCN -infected plants, consistent with the well -documented 289 capacity of cyst nematodes to impair root growth and disrupt normal root development 290 (Moens et al., 2018; Palomares-Rius et al., 2017). Together, these results indicate that 291 beneficial and pathogenic belowground organisms can drive contrasting architectural 292 outcomes within the same crop root system. 293 From an agroecosystem perspective, variation in root system size and spatial 294 development can influence how effectively crops explore soil, intercept water and 295 nutrients, and maintain early vigour under biotic and abiotic stress (Freschet et al., 296 2021; Lynch, 2022). Increases in root volume and surface area associated with AMF 297 colonisation may therefore indicate enhanced potential for belowground resource 298 capture (Begum et al., 2019) , whereas reductions caused by PCN infection likely 299 reflect a constrained capacity to exploit soil resources during early development (De 300 Ruijter & Haverkort, 1999) . Although root volume and surface area do not directly 301 measure nutrient uptake or yield, they represent functionally relevant traits through 302 which soil biota may influence crop performance in soil -based production systems 303 (Freschet et al., 2021; Lynch, 2022). 304 Importantly, although AMF inoculation and PCN infection both significantly influenced 305 root architecture, no significant interaction between these factors was detected for the 306 measured traits. This indicates that AMF -associated increases in root volume and 307 surface area occurred consistently in both PCN-free and PCN-infected plants. Rather 308 than reflecting a specific antagonistic or suppressive interaction between the two 309 organisms, the results point to largely additive effects on root system development. 310 Microscopic observations confirmed the simultaneous presence of AMF structures 311 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint and PCN juveniles within roots, supporting previous reports that co-colonisation by R. 312 irregularis and G. pallida does not necessarily result in mutual inhibition (Bell et al., 313 2022). Within this context, the observed architectural responses represent additive 314 outcomes of symbiotic and pathogenic influences on root system development. 315 This distinction is important for biologically informed crop management. The present 316

suggest that AMF-associated changes in root architecture should therefore not 317 be interpreted solely as evidence of direct antagonism against nematodes, but rather 318 as a biologically mediated shift in host root development that persists even when the 319 pest remains present, consistent with broader evidence that mycorrhizal effects on 320 plant defence and performance are mechanistically complex and often host-mediated 321 (Cameron et al., 2013; Vos et al., 2012; Gough et al., 2020). In agroecosystems, such 322 additive outcomes may still be valuable if they help maintain root system function, 323 improve soil exploration, or buffer early developmental damage in infested soils. This 324 is particularly relevant because potato cyst nematodes can impair root growth, nutrient 325 uptake, and crop growth, meaning that partial maintenance of root system 326 development may still have functional benefits even without direct pest suppression 327 (De Ruijter & Haverkort, 1999). More broadly, the findings support the idea that 328 beneficial soil biota may contribute to crop resilience by modulating root traits and host 329 tolerance, even when soil-borne pests remain present (Bell et al., 2022). 330 Within this broader biological context, X -ray CT provides a valuable means of 331 quantifying how interacting belowground organisms reshape crop root systems in 332 intact soil. Its non -destructive, three -dimensional imaging capacity allows root 333 architectural traits to be measured without disrupting root –soil relationships, thereby 334 preserving the spatial context in which plant–microbe and plant–pathogen interactions 335 occur. The suitability of X -ray CT for plant –nematode systems has previously been 336 supported by the direct detection of potato cyst nematodes in soil -grown plants 337 (Pereira et al., 2025), and the present study extends that framework by demonstrating 338 reproducible treatment effects of AMF colonisation and PCN infection on root volume 339 and surface area in tomato and potato. In this sense, X -ray CT serves as a robust 340 intermediate platform for linking controlled mechanistic studies with more complex 341 agroecosystem questions, enabling more precise investigation of how soil biota 342 influence crop root development under realistic soil-based conditions. 343 344

345 This study shows that beneficial and pathogenic soil organisms can independently 346 reshape crop root architecture in soil -grown plants. Across tomato and potato, 347 colonisation by Rhizophagus irregularis increased root volume and surface area, 348 whereas infection by Globodera pallida reduced these traits, particularly during early 349 development. The absence of a significant AMF × PCN interaction indicates that these 350 organisms exert largely additive effects on root system architecture. AMF -associated 351 increases in root system size were maintained even under nematode pressure. Root 352 system size and spatial development influence how effectively crops explore soil, 353 acquire water and nutrients, and maintain function under stress. These findings 354 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint therefore indicate that soil biota can have important consequences for crop 355 performance in biologically complex soils. X -ray CT was central to this analysis by 356 enabling non-destructive quantification of root architectural traits in intact soil, thereby 357 linking belowground biotic interactions with functionally relevant structural outcomes. 358 More broadly, this study provides a mechanistic foundation for biologically informed 359 strategies to improve crop resilience and manage soil-borne constraints in sustainable 360 agroecosystems. 361 362

363 We gratefully acknowledge funding from the Leverhulme Trust (RPG-2019-162). 364 365 Competing interests 366 The authors have declared that no competing interests exist. 367 368 Author contributions 369 Conceptualization: Eric C. Pereira, Saoirse Tracy. 370 Data curation: Eric C. Pereira, Saoirse Tracy. 371 Investigation: Eric C. Pereira. 372 Methodology: Eric C. Pereira, Saoirse Tracy. 373 Supervision: Saoirse Tracy. 374 Writing – original draft: Eric C. Pereira. 375 Writing – review & editing: Eric C. Pereira, Saoirse Tracy. 376 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.09.710487doi: bioRxiv preprint

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