# Applications of Raman Microscopy/Spectroscopy-Based Techniques to Plant Disease Diagnosis
## 10 Raman Microspectroscopy in Plant Disease Diagnosis  
Raman microspectroscopy has emerged as a powerful tool in the field of plant disease diagnosis due to its ability to provide detailed molecular information without the need for extensive sample preparation (Figure 3). By utilizing this technique, researchers can identify specific biomarkers associated with various diseases in plants (Figure 3b-d). For instance, changes in the spectral profile of plant tissue can reveal alterations in cell composition, allowing for early detection of pathogens or physiological disorders. Additionally, Raman spectroscopy can distinguish between different plant species and specific strains of diseases, providing valuable information for targeted treatment strategies. Raman spectra indicated the presence of lycopene in tomato fruit at the ripe stage with bands, as a peak, at 1156 and 1510 cm\({}^{-1}\) and the presence of \(\beta\)-carotene as a strong Raman peak in the spectral regions at 1100 and 1200 cm\({}^{-1}\), correlated with the C-C bonds [39]. When tomato fruits which were infected with _Alternaria alternata_ and its toxins, such as tenuazonic acid, alterniol and alterniol monomethyl, the lycopene, \(\beta\)-carotene, ascorbic acid and phenolic contents were found to be lowest in the pathogen-infected fruits, concluding that _A. alternata_ affects the nutritional value of tomato fruits the most, alone or due to a combined effect with the toxins [69; 70]. Based on the above-mentioned results, we can conclude that the study of the presence of lycopene and \(\beta\)-carotene in tomato fruit using Raman spectra exhibits strong evidence of tomato health or infection being affected by the pathogen _A. alternata_. In Figure 3, we present plant disease progress flow graphs that illustrate the progression of plant infection and the establishment of fungus in plant tissue (Figure 3a). Among various plant pathology techniques (Figure 3b), Raman spectroscopy can be utilized (Figure 3c) for fast and reliable identification of the infection of the fungus _A. alternata_ (Figure 3d). It is well known that in plants, chlorophyll \(a\) exhibits strong fluorescence, particularly under visible light excitation, which can interfere with Raman signal detection. Researchers [71] have demonstrated that the chlorophyll \(a\) molecule contains an electron donor group (carbonyl at the g-position, abbreviated as \"C\({}_{9}\)=O\") with electronic absorption at 660-665 nm. Additionally, the same authors [71] found that when measuring the Raman spectra of typical chlorophyll forms, the Raman lines in the low-frequency region (below 300 cm\({}^{-1}\)) were sensitive to these forms of chlorophyll. This is an important consideration for our review article when using Raman spectroscopy. Furthermore, many microorganisms contain carotenoids that fluoresce, giving them their bright colors. These pigments can produce strong fluorescence when they are exposed to Raman laser excitation. Raman spectroscopy has been successfully used to detect \(\beta\)-carotene in a wide range of microorganisms, as well as bacterioruberin as a primary carotenoid in halophilic archaea. Another article presented data from Raman spectroscopy for carotenoid detection in microorganisms, with implications for astrobiology researchers, demonstrating that the Raman spectra of various carotenoids from representative species of archaea, bacteria, cyanobacteria and algae, when compared with HPLC extracts, showed that carotenoids can be easily identified using the Raman method. The researchers concluded that using 514 and 532 nm lasers provided excellent results due to the advantage of Raman resonance enhancement. Furthermore, the accumulation of phenolic compounds was significantly increased in all walnut genotypes after inoculation with the bacterium _Xanthomonas arboricola_ pv. _juglands_, the causal pathogen of walnut blight, suggesting that these phenolic compounds contribute to early defense against walnut blight [63].