First report of eggplant mottled crinkle virus infecting eggplant in Greece.

During winter 2020-2021, a severe virus-like disease outbreak was observed in eggplant (Solanum melongena L.) hybrids 'Monarca' (F1) and 'Angela' (F1) growing under protected conditions in Heraklion, Crete, Greece. In three greenhouses, the percentage of infected plants reached 100% leading to crop abandonment. Symptoms included leaf mottling and yellowing accompanied with plant stunting and apical necrosis. Extensive fruit damage was due to severe malformation and necrotic lesions on the calyx, peduncle and the endocarp (Sup. Fig. 1). To identify the causal agent, total RNA was extracted from a symptomatic eggplant fruit with PureLink™ RNA Mini Kit (ThermoFisher Scientific, USA), which was subjected to high throughput sequencing (HTS) analysis (Illumina Inc., USA). The de novo assembly of the obtained 25 million, 75 bp, single-end reads with Geneious Prime (Biomatters, New Zealand) and the annotation of the resulting contigs with BLASTn revealed the presence of only eggplant mottled crinkle virus (EMCV, genus Tombusvirus) in the sample. The assembled sequence of EMCV isolate from Greece (EMCV-Gr, GenBank Acc. No. MW716271) was 4764 bp in length, covering the full genome of the virus and showing 96.3 % nucleotide (nt) identity with an isolate identified from calla lilies (Zantedeschia sp.) in Taiwan (AM711119). Five symptomatic and seven asymptomatic 'Monarca' (F1) eggplants, as well as two symptomatic 'Angela' (F1) eggplants were tested by RT-PCR that targeted the capsid protein gene of the virus (Dombrovsky et al., 2009). PCR products of 1184 bp were obtained from the seven symptomatic samples and their Sanger sequencing revealed 100 % nt identity with the respective HTS-derived EMCV sequence. No product was obtained from the analysis of the asymptomatic samples. Mechanical sap transmission of the HTS analysed eggplant sample resulted in necrotic local lesions on Nicotiana rustica and Chenopodium quinoa, necrotic local lesions plus systemic necrosis on N. tabacum cv. Xanthi-nc, cv. Samsun and N. glutinosa, systemic collapse of N. benthamiana, and leaf mottling plus stunting of pepper cv. Yolo Wonder plants (Sup. Fig. 1I). Although no symptoms were observed on tomato plants cv. Ace 55, systemic EMCV infection was detected by RT-PCR. To establish the relationship between the disease and EMCV, infected tissue from N. benthamiana plants was used for the mechanical inoculation of virus-tested negative eggplant seedlings cv. Black beauty. Necrotic spots, shoot necrosis, leaf mottling and mosaic, symptoms were observed (Sup. Fig. J) on the test plants ten days post inoculation and the presence of the virus was confirmed by RT-PCR as described. To the best of our knowledge this is the first report of EMCV infecting eggplant in Greece. The virus was originally described in eggplant in Lebanon (Makkouk et al., 1981) and it is mainly present outside the European Union (EU) territory, including India, Japan, Taiwan, Iran and Israel (Dombrovsky et al., 2009 and references therein). A latent EMCV infection was detected in pear in Italy (Russo et al., 2002) and the virus is considered by the European Food Safety Authority as an exotic virus of the genera Cydonia, Malus, and Pyrus that meets all the criteria to qualify as an EU quarantine pest (Bragard et al., 2019). Τhe severity of the disease observed in Crete leading to the destruction of eggplant greenhouse cultivations, constitutes EMCV as an emerging threat to eggplant and other solanaceous crops for Greece and Europe.

One-Step Multiplex Quantitative RT-PCR for the Simultaneous Detection of Viroids and Phytoplasmas.

A one-step multiplex quantitative reverse transcription polymerase chain reaction protocol is described, for the detection in pome trees of Pear blister canker viroid and Apple scar skin viroid, together with universal detection of phytoplasmas. Total nucleic acids extraction is performed according to a modified CTAB protocol and TaqMan MGB probes are used to surpass high genetic variability of viroids. The multiplex real-time assay is at least ten times more sensitive than conventional protocols and its features make it suitable for rapid and massive screening of pome fruit trees phytoplasmas and viroids in certification schemes and surveys.

Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

Effect of Pyraclostrobin Application on Viral and Bacterial Diseases of Tomato.

Quinone outside inhibitors (QoI) are powerful fungicides, which have been reported, additionally to their fungicide activity, to increase plant capacity to activate cellular defense responses and to promote plant growth. In this work, the effect of the QoI class fungicide pyraclostrobin was examined against Cucumber mosaic virus (CMV), Potato virus Y (PVY) and Pseudomonas syringae pv. tomato in tomato plants following artificial inoculation of the plants with the pathogens. Under controlled environmental conditions, pyraclostrobin delayed viral and bacterial disease development, even if P. syringae pv. tomato internal population levels were not affected significantly. In contrast, under field conditions in commercial greenhouses, a reduced CMV disease incidence throughout the tomato cultivation period was recorded. Gene expression analysis indicated an effect of pyraclostrobin application on tomato MAPKs transcript levels and a possible interference with plant stress responses.

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.