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Direct detection of plant pathogens in the field

Project(current): EPANEK 2020-2023 

Projects (previous): RIS3Crete (2022), Flagship Olive-roads (2022)

The direct detection of plant pathogens like fungi, bacteria, viruses, viroids is a matter of great importance. These pathogens affect field plants with huge economic impact, causing plant diseases that usually cannot be treated. Therefore, early diagnosis is necessary, in order to avoid financial loss to the agricultural products. In the last years we have been developing assays based on isothermal amplification for plantborne pathogens detection directly in crude samples and in the field.

Within the last 3 years, we have been developing methods for the isothermal amplification of genetic material of different plant pathogens and detection via the use of colorimetric indicators or acoustic biosensors. The main targets we have been working with include the fungus Verticillium dahliae in olive leaves (in collaboration with ELGO-Dimitra and the Agriculture University of Athens); the Citrus tristeza virus (CTV) in citrus leaves (in collaboration with ELGO-Dimitra); the Potato spindle tuber viroid (PSTVd) in N. benthamiana and tomato leaves; the Cucumber mosaic virus (CMV) in N. benthamiana leaves; and the Avocado sunblotch viroid (ASBVd) in avocado leaves (the last three in collaboration with Prof. Kalantidis lab in IMBB-FORTH).  

The colorimetric detection of the amplified target is realized thanks to a novel portable device (IRIS) developed and patented within the lab. The main goal of this work is the creation of a diagnostic system, in which both the amplification of minimum DNA/RNA copies (<10 copies) and real-time monitoring of the process by detecting the color change of the samples (quantitative colorimetric qcLAMP) will be achieved. In order to perform real-time detection, the device heats the samples to an appropriate temperature while visualizing the color change of a pH-sensitive dye by a mini-camera. In addition, our assays are developed in order to detect the target DNA/RNA directly in a crude plant tissue without extraction. Finally, the ultimate goal is the ability to complete the analysis in less than 30 min. The control of the device as well as the visualization occur through a smart-phone or tablet. The results of the detection can be uploaded to a relevant database (Cloud) to which all end-users can have access to, in order to decide for further disease-management actions. We are currently performing tests in the field for the detection of CTV in citrus trees and Verticillium dahliae in olive leaves.

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