Phd in ENVIRONMENTAL AND EVOLUTIONARY BIOLOGY

Thesis title: Use of green substances to control infections by systemic pathogens and their vectors

The control of potyvirus infections in cucurbit crops is a significant challenge as no curative treatments are currently available. Furthermore, the emergence of insecticide-resistant populations of aphid vectors, combined with the limitations imposed by European Union regulations on the availability of active ingredients targeting aphids, make the disease management even more complex. Considering the binomial Potyvirus cucurbitaflavitesselati (zucchini yellow mosaic virus, ZYMV) infecting zucchini plants (Cucurbita pepo L.) as case study, this research aims to identify new approaches that could potentially contribute to the sustainable management of ZYMV infections. In this context, a first objective of this research study was the improvement of the detection tools available for ZYMV. Accurate and reliable detection methods are crucial to facilitate early diagnosis and prevent disease outbreaks, particularly in cases of mixed infections with other potyviruses such as WMV. A one-step reverse transcription real-time PCR (real-time RT-PCR) and droplet-digital PCR (RT-ddPCR) assays were developed for detecting WMV and ZYMV in samples of the main cucurbitaceous crops. Both the assays showed a high sensitivity, and the RT-ddPCRs also allowed the direct estimation of the virus concentrations, opening to a broad range of applications in disease management Besides the accurate detection of ZYMV, this research study was focused on the evaluation of the potential contribution of commercial biostimulants to virus disease management. The biostimulants are recommended for the improvement of the plant nutrient uptake efficiency, but are also known to enhance plant tolerance to pathogens and pests. In this context, a mixture of alfalfa, brown algae, and molasses extracts applied in combination with micronized zeolite, was tested for its effects on ZYMV infection, plant defence responses and aphid vector behaviour. Several crop traits were compared between healthy and ZYMV-infected zucchini plants, which were treated or untreated with biostimulant. Treated plants showed increased growth and higher values of reproductive parameters (number of flowers and fruits) even in infected conditions. After treatment, a decrease of ZYMV titre and a progressive slowdown of symptom severity were observed in plant, together with the up-regulation of some defence genes. The effect of biostimulant on the settling preference and life traits of the aphid vector Myzus persicae was also investigated. Aphid choice test experiments using detached zucchini leaves showed that fewer specimens settled on biostimulant-treated plants, with and without ZYMV. Volatile organic compounds (VOCs) were collected from treated and untreated zucchini plants, both healthy and ZYMV-infected, analysed by GC-MS and used in olfactometer experiments. When allowed to choose freely between VOCs blends from treated and untreated plants, aphids preferred untreated plants independently of their healthy status, confirming a potential role of biostimulants in repelling the vector. Aphids caged on both infected and healthy potted plants, showed a reduction in both survival and offspring production in presence of biostimulant, regardless of the infection conditions. These findings suggest that biostimulant treatment can reduce disease risk in C. pepo crops by potentially contributing to the control of ZYMV and its vector. By integrating advanced molecular diagnostics with biostimulant applications, this study provides a multidisciplinary contribution to the understanding of plant-virus-vector interactions. The obtained results offer new insights into virus detection, plant resilience, and vector control that can be useful for implementing a sustainable disease management.