Whole-cell bacterial biosensor applied to identify the presence of Thaumatotibia leucotreta larva in citrus fruits by volatile sensing

Abstract

The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick), is a major pest of citrus fruits and other plants and is defined as a quarantine pest in several countries. Supervision and visual monitoring are crucial for the detection of eggs and larvae. There is a growing need to implement alternative pest control strategies and early detection methodologies in the packing house before shipping. Biosensors may provide a better-suited solution, as they are portable, do not require expensive equipment, and may be used with simple methodologies. In this study, a whole-cell bacterial biosensor was developed to identify the presence of Thaumatotibia leucotreta larvae in citrus fruits. The volatile organic compounds (VOCs) released by the larvae were identified (n = 11), which were not previously tested. The change in the VOCs composition in infected and uninfected mandarin (Citrus reticulata) was examined using gas chromatography-mass spectrometry (GC-MS) and the bioluminescent bacterial panel. During this work, two fixation methods (alginate beads and tablets) were tested for the bioluminescent bacteria cells, and tablets were preferred due to their higher uniformity and consistency of the light signal from the cells. Furthermore, the response of the bacteria to the changes in the concentration of VOCs as a result of a change in the amount of the larvae was examined (larvae n = 3, 6, or 9). In addition, in order to demonstrate the ability of the whole-cell biosensor to detect the presence of larva within the fruit, infected and uninfected mandarins were tested on the 14th day of larval hatching. The results showed that the bioluminescent bacteria could differentiate between mandarins with and without larva. The differences in the light signals (Induction Factor (IF)) between the mandarin fruits can be seen in the results with the larva (cytotoxicity IF = 3.046, genotoxicity IF = 2.643, and quorum sensing IF = 1.954) and without (cytotoxicity IF = 1.589, genotoxicity IF = 1.149 and quorum sensing IF = 1.205). The findings of this study make it possible to further develop a prototype using bioluminescent bacteria in order to detect pests of citrus fruits. The early detection of infected fruits will allow their filtering from the crop and therefore reduce the damage to additional fruits. Even one larva can invalidate the whole stock and the plot for future export.