Early Detection of Fungal Infection of Arabidopsis and Brassica by Raman Spectroscopy

SONG-YI KUO¹Ling Ying Chiu¹Ekta Jain²Gajendra Pratap Singh²Muhammad Nabil Syafiq Bin Jamaludin¹Rajeev J. Ram^2,3*Nam-Hai Chua^1,2*

• ¹Temasek Life Sciences Laboratory, Singapore, Singapore
• ²Singapore-MIT Alliance Disruptive and Sustainable Technologies for Agricultural Precision, Singapore, Singapore
• ³Massachusetts Institute of Technology Research Laboratory of Electronics, Cambridge, United States

Here, we used Raman spectroscopy to characterize the effects of chitin treatment and fungal inoculations on Arabidopsis thaliana and Brassica vegetables. Chitin, a recognized fungal pathogen-associated molecular pattern (PAMP), elicited a dose dependent positive Elicitor Response Index (ERI) in wild-type Arabidopsis. Mutant plants lacking chitin receptors (cerk1 and lyk4/5) displayed minimal ERI, whereas fls2 mutant deficient in the bacterial-specific flg22 receptor was hyper-responsive. These results confirm critical role of chitin receptors in activating downstream pathways and highlighting distinct responses in two separate pattern-triggered immunity (PTI) systems. Inoculations of Colletotrichum higginsianum and Alternaria brassicicola induced significant changes in Infection Response Index (IRI) values, with the former giving positive IRI at 12-48 hours post-inoculation whereas the latter exhibited a transient negative IRI before transitioning to positive values. Notably, Raman shifts could predict fungal infection before the appearance of visible symptoms, establishing Raman shifts as a potential early diagnostic marker. Comparative analyses of infected Brassica vegetables revealed varied sensitivity to fungal pathogens and a correlation between symptom severity and IRI values. Furthermore, randomized controlled trials validated the reliability of Raman technology for early, pre-symptomatic detection of fungal infections, achieving an accuracy rate of 76.2% in Arabidopsis and 72.5% in Pak-Choy (Brassica rapa chinensis). Principal component analysis differentiated Raman spectral features associated with fungal and bacterial infections, emphasizing their unique profiles and reinforcing the utility of Raman spectroscopy for early detection of pathogen-related plant stress. Our work supports the application of noninvasive diagnostic techniques in agricultural practices, enabling timely intervention against crop diseases.