Rapid Phytopathogen Identification and Metabolomic Profiling of Cucumber Leaves in Response to Garlic Treatment and Fusarium Incarnatum Infection

Cucumber often suffers from various phytopathogen infection which causes great economic loss and waste. Thus, it is important to have rapid identification method to identify the species of phytopathogens to aid in the disease management process by detecting early infection before symptoms occur. Furthermore, in managing phytopathogen infection, the use of biostimulants such as garlic which contains antimicrobial properties is gradually becoming an alternative to synthetic pesticides for a sustainable agricultural practice. However, studies on the application of garlic treatment during the interaction of phytopathogen and plant are scarce. Hence, this study was performed to establish a rapid identification method using Recombinase Polymerase Amplification (RPA) with species-specific primer to identify Fusarium and Curvularia species which show disease symptoms on cucumber crop. Next, this study aimed to quantify the symptoms and the metabolite response of cucumber leaves infected with F. incarnatum after treated with garlic or water by using Liquid Chromatography-Time of Flight-Mass Spectrometry (LC-TOF-MS). Lastly, it was to identify unique signature metabolites of cucumber crop associated with Fusarium incarnatum infection and garlic treatment by coupling LC-TOF-MS data with multivariate analysis. The methodology of this study started with the isolation of phytopathogen on potato dextrose agar (PDA) and followed with pathogenicity test. Then, ITS-5.8S DNA fragment of selected phytopathogens were amplified by using PCR and sequenced for species identification. Phylogenetic analysis was performed to confirm the species identification. Next, the species-specific primer was designed, and RPA method at 37℃ for 20 minutes with 0.1 ng limit of detection was established for rapid detection of Fusarium and Curvularia species. To determine the effect of garlic during the interaction of phytopathogen and plant, 100 ug mL-1 aqueous garlic extract (AGE) in consort with distilled water as control treatment were applied to 14 days old-cucumber plant, followed by infection with 1X105 conidial suspension of F. incarnatum. The results revealed a significant decrease in disease severity index (DSI) of cucumber crops treated and infected with both AGE and F. incarnatum compared to cucumbers infected with F. incarnatum only. Moreover, metabolomic approach by using LC-TOF-MS coupled with multivariate analysis such as PCA, PLS-DA and heatmap reveal that cucumber plants treated and infected with both AGE and F. incarnatum had a lower accumulation of precursor compounds and intermediates of phenylpropanoid pathway like myo-inositol and cinnamic acid compared to garlic-treated cucumber plants. In addition, compared to F. incarnatum-infected plants, cucumber plants treated and infected with both AGE and F. incarnatum showed low concentration of cell wall-associated compounds like sinapic acid and nortracheloside, which lead us to believe that AGE-treated and F. incarnatum-infected cucumber plants encountered less stress compared to cucumber plants infected with F. incarnatum only. In conclusion, this study suggested that application of AGE successfully induced cucumber plant innate immune system for incoming infection by F. incarnatum and successfully alleviated disease severity symptoms caused by Fusarium leaf blight.