Verticillium wilt resulting from infection by Verticillium dahliae is one of the most devastating soilborne fungi of the olive tree (Olea europaea L.) worldwide. The pathogen infects a wide variety of plants and can survive in the soil for many years, and chemicals cannot control it. Therefore, sustainable disease management strategies are suggested, with the exploitation of host resistance as the most predominant control measure in practice. In addition, disease risk assessment in commonly used plant genotypes is a prominent issue. In this respect, nine commercially grown Greek olive varieties (‘Amfissis’, ‘Atsiholou’, ‘Chalkidikis’, ‘Koroneiki’, ‘Kothreiki’, ‘Koutsourelia’, ‘Mastoidis’, ‘Megaritiki’, and ‘Tragolia’) and one variety of international interest (‘Picual’) were comparatively evaluated for their resistance to V. dahliae. The roots of young plants were immersed in a concentrated conidial suspension in order to perform an artificial inoculation. We evaluated disease reactions in a 140-day assessment period based on external symptoms (disease severity, disease incidence, and mortality) and calculated the relative areas under disease progress curves (relative AUDPC). The process of qPCR was used to evaluate V. dahliae DNA in vascular tissues and plant growth parameters (height and fresh weight). A cumulative stress response was calculated to consider the overall effect of V. dahliae on olive cultivars. The olive varieties resistance to V. dahliae varied significantly, with ‘Koroneiki’, ‘Tragolia’, and ‘Atsiholou’ being the most resistant. Interestingly, most tested varieties showed a significantly low resistance level, suggesting increased risk for the Greek olive industry due to V. dahliae.

In the present study, a novel method was developed and validated for the early, rapid and on-site detection of Botrytis cinerea in crude tomato samples using a newly designed loop-mediated isothermal amplification (LAMP) assay and the portable device PEBBLE. LAMP primers (targeting the IGS region of rDNA gene) were designed, and the sensitivity, specificity and accuracy of the developed method were determined (97.96%, 100% and 98.70%, respectively). The LAMP assay, combined with the portable device, enabled real-time quantitative colorimetric (qcLAMP) detection of B. cinerea in less than 30 min. The limit of detection (LoD) of the qcLAMP assay was 1 pg genomic DNA (≈20 genome copies) per reaction, whereas conventional PCR (utilized as the gold standard method) detected no less than 100 pg of genomic DNA per reaction. In terms of B. cinerea infective propagules, qcLAMP could detect as few as 10² spores in both pure and crude DNA samples extracted from artificially inoculated tomato leaves, whereas PCR detected no fewer than 10⁴ spores in pure DNA samples, failing to achieve detection in crude extracts. In in-vivo bioassays, qcLAMP successfully detected B. cinerea immediately following spore deposition on leaf tissues (0 hours post inoculation) in both pure and crude DNA extracts. In contrast, conventional PCR was successful only in pure DNA samples from leaves collected 2 days post inoculation (dpi) and in crude extracts at 3 dpi, when symptoms were already evident. Evaluations in field samples demonstrated that qcLAMP detected B. cinerea in 100% of crude extracts from symptomatic tissues and in 45% of asymptomatic (latently infected) samples. Conversely, conventional PCR failed to detect the pathogen in crude extracts, regardless of the disease status (symptomatic or asymptomatic). The developed method can serve as a rapid, reliable, and user-friendly tool for the early detection of B. cinerea in the field, enabling prompt assessment of inoculum pressure, thereby facilitating timely management interventions.

In the present study, a novel method was developed and validated for the early, rapid and on-site detection of Botrytis cinerea in crude tomato samples using a loop-mediated isothermal amplification (LAMP) assay adapted for the portable device PEBBLE. Previously reported LAMP primers (targeting the IGS region of rDNA gene) were utilized and optimized in terms of concentration for this workflow, and the sensitivity, specificity and accuracy of the developed method were determined (97.96%, 100% and 98.70%, respectively). The LAMP assay, combined with the portable device, enabled real-time quantitative colorimetric (qcLAMP) detection of B. cinerea in less than 30 min. The limit of detection (LoD) of the qcLAMP assay was 1 pg genomic DNA (≈20 genome copies) per reaction, whereas conventional PCR (utilized as the gold standard method) detected no less than 100 pg of genomic DNA per reaction. In terms of B. cinerea infective propagules, qcLAMP could detect as few as 10² spores in both pure and crude DNA samples extracted from artificially inoculated tomato leaves, whereas PCR detected no fewer than 10⁴ spores in pure DNA samples, failing to achieve detection in crude extracts. In in vivo bioassays, qcLAMP successfully detected B. cinerea immediately following spore deposition on leaf tissues (0 h post-inoculation) in both pure and crude DNA extracts. In contrast, conventional PCR was successful only in pure DNA samples from leaves collected 2 days post-inoculation (dpi) and in crude extracts at 3 dpi, when symptoms were already evident. Evaluations of in-field samples demonstrated that qcLAMP detected B. cinerea in 100% of crude extracts from symptomatic tissues and in 45% of asymptomatic (latently infected) samples. Conversely, conventional PCR failed to detect the pathogen in crude extracts, regardless of the disease status (symptomatic or asymptomatic). The developed method can serve as a rapid, reliable, and user-friendly tool for the early detection of B. cinerea in the field, enabling prompt assessment of inoculum pressure, thereby facilitating timely management interventions.