Raman imaging-chemometrics analysis of cell wall biopolymer dynamics in Pseudomonas syringae pv. actinidiae-infeicted kiwifruit stems

• A label-free high - throughput cell imaging method is proposed for kiwifruit stem canker • The method is based on confocal Raman imaging microscopy and chemometrics. • The spatial-temporal changes of five substances in cellular level are investigated. • Early-stage detection of Pseudomonas-infected kiwifruit stems is accomplished. Pseudomonas syringae pv. actinidiae (Psa), the pathogen that causes bacterial canker disease in kiwifruit, has brought about substantial losses to the kiwifruit industry. Investigating the infection process at both the cellular and microscopic levels is of great significance for the formulation of effective control strategies against this disease. Thus, the establishment of appropriate chemical imaging analysis methods becomes essential. Confocal Raman microspectral imaging (CRMI), combined with chemometrics, provides an intuitive means to visualize and characterize the spatiotemporal changes of biopolymers in the cell walls of both healthy and infected kiwifruit stems. Raman spectra of different infected stems exhibit clustering effects in Principal component analysis (PCA), and a classification model constructed using Support vector machines (SVM) achieves an accuracy of 97%. Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) is utilized to resolve spectral matrices and concentration profiles from raw Raman imaging signals. The reconstructed concentration data yields accurate molecular imaging maps of High-methylated pectin (HMP), Low-methylated pectin (LMP), cellulose, hemicellulose, and lignin. The results indicate that, three days after Psa infection, the content of cellulose and HMP in the cell wall increases, while the changes in hemicellulose, lignin, and LMP are minimal. However, five days after infection, the contents of HMP, LMP, cellulose, hemicellulose, and lignin decrease significantly, resulting in the disruption of the cell-wall structure. The chemical imaging method proposed in this study shows great promise as an effective means for studying the bacterial infection process in kiwifruit stems at the cellular level.