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Cucumber green mottle mosaic virus (CGMMV), genus Tobamovirus, is a major pathogen of cucurbits that primarily affects cucumber, melon, and watermelon crops. The aim of this study was to reveal the contribution of CGMMV-infected female flowers to disease spread. Using a fluorescent in situ hybridization (FISH) technique, we show that ovaries and ovules of CGMMV-infected cucumber and melon plants showed a CGMMV-specific fluorescence signal prior to and following anthesis. The fluorescence signal was prominent but sporadic. Ripe fruits of infected melon plants showed strong signals in the funiculus, the seed stalk, which connects the developing seed to the interior ovary wall. Importantly, in seeds, a strong fluorescence signal was observed in the perisperm-endosperm (PE) envelope, which underlies the seed coat and surrounds the embryo. Interestingly, the fluorescence signal was not uniformly distributed in the PE envelope but was localized to a specific envelope layer. These results have important epidemiological implications for CGMMV management and commercial seed production, particularly regarding the improvement of seed disinfection methods that will contribute to limit the global distribution of the virus.

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single‐molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis. [ABSTRACT FROM AUTHOR]