Phase-contrast microtomography reveals vascular colonization of tomato roots

Soil-borne vascular pathogens are among the most destructive threats to global agriculture, yet the mechanisms by which they invade and colonize plant roots remain poorly understood. Fusarium oxysporum (Fo), the causal agent of vascular wilt diseases in tomato and many other crops, penetrates host tissues and progressively disrupts the root vascular system, ultimately leading to embolism formation and plant collapse. Understanding these infection dynamics in three dimensions is essential for deciphering fungal pathogenicity and developing innovative disease-control strategies.

In this study, synchrotron radiation phase-contrast X-ray microtomography (SR PC-microCT) was employed to visualize infected tomato roots at submicrometric resolution. The experiments were performed at the SYRMEP beamline of the Elettra synchrotron, enabling non-destructive three-dimensional imaging of fungal colonization within intact root tissues. By combining SR PC-microCT with fluorescence microscopy and image analysis, the study reveals how Fo progressively invades cortical and vascular tissues while disrupting the root architecture. 

The SR PC-microCT reconstructions highlight extensive fungal proliferation inside the vascular cylinder together with the formation of elongated air-filled embolisms aligned with the root axis. Dense structures observed within the stele correspond to fungal biomass colonizing apoplastic compartments and xylem vessels. The volumetric nature of SR PC-microCT allows entire root segments, typically extending over approximately 1.8 mm, to be reconstructed in three dimensions while preserving the native organization of the tissues.

Complementary fluorescence microscopy and confocal imaging analyses demonstrate that Fo develops specialized penetration structures and invasive hyphae, capable of traversing highly confined plant spaces. Remarkably, the fungus can reduce its hyphal diameter by more than twenty-fold to colonize nanoscale apoplastic environments. 

Beyond visualization of fungal invasion, the study demonstrates the power of synchrotron-based phase-contrast imaging for plant pathology. The technique enables simultaneous observation of root anatomy, embolism development, and pathogen distribution without destructive sectioning, overcoming major limitations of conventional optical microscopy in thick plant tissues (Figures 1). Quantitative analyses further revealed a direct correlation between fungal proliferation and progressive loss of root integrity during infection. 

Figure 1: SR PC-microCT longitudinal rendering of a tomato root infected with Fusarium oxysporum. Dense structures (labelled in green) within the stele correspond to fungal biomass colonizing vascular and apoplastic tissues. Scale bar: 0.2 mm. 

Overall, these findings establish SR PC-microCT as a powerful tool for investigating host–pathogen interactions in planta and provide unprecedented insight into the three-dimensional colonization strategies employed by vascular fungal pathogens.

Valentino Maria Guastaferro^1,2, Stefania Vitale^1,3, Lorenzo D’Amico^4,5, Elena Longo⁴, Giuliana Tromba⁴, Morgan Delarue⁶, Matteo Lorito^1,7, Luigi Di Costanzo¹, David Turrà^1,3,7,8
1 Department of Agricultural Sciences, University of Naples “Federico II”, Portici, Naples, Italy 
² Institute for Sustainable Plant Protection – National Research Council, Portici, Naples, Italy 
³ Bioelectronics Task Force, University of Naples “Federico II”, Naples, Italy 
⁴ Elettra – Sincrotrone Trieste S.C.p.A., Basovizza, Trieste, Italy
⁵ School of Physics and Astronomy, Monash University, Clayton, Victoria, Australia 
⁶ LAAS-CNRS, Université de Toulouse, CNRS, Toulouse, France
⁷ Center for Studies on Bioinspired Agro-Environmental Technology, University of Naples “Federico II”, Portici, Naples, Italy
⁸ Computational and Quantitative Biology Task Force, University of Naples “Federico II”, Naples, Italy

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