Determination of tryptoquialanines A and C produced by Penicillium digitatum in oranges: Are we safe?

A method was developed and validated for determination of tryptoquialanines A and C in orange samples on epicarp (exterior peel), mesocarp (white peel), and endocarp (fruit juice) based on QuEChERS extraction and LC-MS/MS analysis. The method showed an excellent linearity over a range of 5-400 µg kg-1, with r2 ≥ 0.998. The limits of detection (LOD) and quantification (LOQ) were 5 and 10 µg kg-1, respectively. Recoveries showed values between 57 and 101%, with RSD ≤ 12%. Analysis of infected oranges showed diffusion of the alkaloids between the orange layers after 4 days post infection in concentrations > LOQ. Mycotoxin diffusion to healthy oranges after direct contact with infected oranges for 48 h, showed alkaloid concentrations ≥10 µg kg-1 on epicarp layer. The developed method can be easily applied for quality control in routine analysis of orange fruit due to the high risk that these tremorgenic alkaloids represent to human health.

Monitoring indole alkaloid production by Penicillium digitatum during infection process in citrus by Mass Spectrometry Imaging and molecular networking.

Green mold, caused by Penicillium digitatum, is the most destructive post-harvest disease in citrus. Secondary metabolites produced by fungal phytopathogens have been associated with toxicity to their respective host through the interaction with a wide range of cell targets. Natural products have also been described as important molecules for biocontrol and competition in their respective environment. For P. digitatum, the production of indole alkaloids, tryptoquialanines A and B, have been reported. However, their biological role remains unknown. Mass Spectrometry Imaging (MSI) technique was applied here for the first time to monitor the secondary metabolites produced on the orange surface during infection in order to gain insights about the P. digitatum-citrus interaction mechanisms. Through the combination of MSI and molecular networking it was possible to report, for the first time, the production of tryptoquivalines and fumiquinazolines by P. digitatum and also the accumulation of tryptoquialanines on the fruit surface from 4 to 7 d post inoculation. P. digitatum was also evaluated concerning the ability to sinthesize indole alkaloids in vivo in the different citrus hosts. The biological role of tryptoquialanines was investigated and tryptoquialanine A was submitted to insecticidal bioassays that revealed its high toxicity against Aedes Aegypti, suggesting an important insecticidal action during orange decay.