New Molecular Tool for a Quick and Easy Detection of Apple Scab in the Field

Abstract

Venturia inaequalis, an agent of apple scab, is the most important pathogen of Malus x domestica. Control measures against this pathogen rely on intensive phytosanitary programs based on predictive models to identify the meteorological conditions conducive to the primary infection. The detection of the pathogen in field, both in naturally infected symptomatic and asymptomatic leaves, is desirable. Loop-mediated isothermal amplification (LAMP) assays are profitable molecular diagnostic tools for the direct detection of pathogens in field. A LAMP assay for V. inaequalis has been designed on the elongation factor 1-alpha sequence. The validation of the LAMP assay was carried out following the international EPPO standard PM 7/98 in terms of specificity, sensitivity, repeatability and reproducibility. Specificity testing was performed using target and non-target species, such as phylogenetically related Venturia species and other pathogens commonly found in apple, resulting in positive amplification only for the target with a time to positive ranging from 20 to 30 min. Sensitivity testing was performed with serial dilutions of DNA of the target and by artificial inoculation of young apple leaves. The reliability of the LAMP assay as an early-detection tool and its user-friendly application make it suitable for the diagnosis of apple scab in the field.

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

“…Despite, the end-point PCR assays showed a high specificity of 82.0% with an LOD of 110 fg/µL of DNA. In terms of sensitivity, the LAMP assay designed here, showed a limit of detection of 100-999 pg/µL of DNA, like the limit of detection previously described in other assays [66,67]. However, Niessen et al [50] also developed a LAMP assay that focused on the detection of aflatoxigenic strains of Aspergillus section Flavi, with primers designed on the nor1 gene, with a lower detection limit of 9.03 pg of genomic DNA per reaction and based on the detection of the positive results by a colorimetric change or by a fluorescence dye.…”

“…To quantify naturally infected samples, the standard curve generated with the A. flavus FS7 was used, as A. flavus is the most common aflatoxigenic species found in hazelnuts [14]. In order to calculate the number of cells in the inoculated hazelnuts, the elution volume of the commercial DNA (30 µL) of the inoculated hazelnuts was also taken into consideration, as described in other in vivo tests [67].…”

Guidelines for improving statistical analyses of validation datasets for plant pest diagnostic tests

“…Diagnostic sensitivity (DSE) and diagnostic specificity (DSP) are widely used criteria to evaluate the performance of plant pest detection tests (Franco Ortega et al, 2020, 2021; Renvoisé et al, 2019). Diagnostic sensitivity represents the proportion of infested samples that correctly tested positive using a specific test, while diagnostic specificity corresponds to the proportion of samples free from the target pest that correctly tested negative.…”

“…The performance criteria gathered in this section have been used in the framework of the VALITEST test performance studies for plant pests (https://www.valitest.eu/work_packages/index#wp1) and correspond to internationally used recommendations (EPPO standard PM 7/122, 2014; EPPO Standard PM 7/98, 2019; ISF, 2020; ISTA, 2019). Some of these performance criteria have also been referred into scientific publications in intra‐laboratory validation studies and test performance studies (Chabirand et al, 2017; Franco Ortega et al, 2020, 2021; Massart et al, 2009a, 2009b; Renvoisé et al, 2019).…”

Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens

“…Although designing and validating LAMP primers is laborious, LAMP does not require expensive equipment and it is fast and highly specific even when low amounts of pathogen DNA are present in a sample. Notably, when combined with on-site DNA extraction methods, LAMP can be used for molecular detection of plant pathogens directly in the field [ 35 , 36 , 37 , 38 , 39 ]. Recently developed nanotechnologies have also provided a new method of detecting nucleic acid sequences [ 40 , 41 ].…”