Rapid identification of tomato Sw-5 resistance-breaking isolates of <i>Tomato spotted wilt virus</i> using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques

<div><p>In tomato, resistance to <i>Tomato spotted wilt virus</i> (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50–70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.</p></div

Occurrence and Distribution of Major Viruses Infecting Eggplant in Lebanon and Molecular Characterization of a Local Potato Virus X Isolate

This research was carried out in order to evaluate the presence and distribution of viral infections causing severe disease in eggplant plants collected from different districts in Bekaa valley, Lebanon. Most infected plants showed virus-like symptoms consisting predominantly of leaf blotch, mottling chlorotic and ring spots; leaf twisting and plant dwarf were also observed in the visited fields. Symptomatic and asymptomatic plants were collected and screened by ELISA test for the presence of several different pathogenic viruses potentially present in the area. Results showed that potato virus Y (PVY) was the most prevalent virus found by ELISA (detected in the 15.3% of the tested plants), followed by eggplant mottled dwarf virus (EMDV, 2.9%) and cucumber mosaic virus (CMV, 1.2%), while tomato spotted wilt virus (TSWV), alfalfa mosaic virus (AMV) and pepper mottle virus (PepMoV) were not detected. Biological indexing of symptomatic ELISA-negative plants, followed by electron microscopy, indicated the presence of virus-like particles of the genus Potexvirus, which was subsequently confirmed as potato virus X (PVX) by RT-PCR and Sanger sequencing. PVX was found in 35.3% of the tested plants, all sampled in the northern Bekaa area. In a phylogenetic analysis, the partial coat protein gene sequence of a selected Lebanese isolate, PVX-AK1, clustered together with other PVX isolates from Asia. Furthermore, the 124-aa sequence of PVX-AK1 shared 100% identity with PVX-UK3, an isolate which is known as avirulent in potato genotypes carrying either Nx or Rx resistance genes. This work revealed a picture of the previously uninvestigated phytosanitary status of eggplant crops in an important horticultural area of Lebanon

Occurrence and Distribution of Major Viruses Infecting Eggplant in Lebanon and Molecular Characterization of a Local Potato Virus X Isolate

This research was carried out in order to evaluate the presence and distribution of viral infections causing severe disease in eggplant plants collected from different districts in Bekaa valley, Lebanon. Most infected plants showed virus-like symptoms consisting predominantly of leaf blotch, mottling chlorotic and ring spots; leaf twisting and plant dwarf were also observed in the visited fields. Symptomatic and asymptomatic plants were collected and screened by ELISA test for the presence of several different pathogenic viruses potentially present in the area. Results showed that potato virus Y (PVY) was the most prevalent virus found by ELISA (detected in the 15.3% of the tested plants), followed by eggplant mottled dwarf virus (EMDV, 2.9%) and cucumber mosaic virus (CMV, 1.2%), while tomato spotted wilt virus (TSWV), alfalfa mosaic virus (AMV) and pepper mottle virus (PepMoV) were not detected. Biological indexing of symptomatic ELISA-negative plants, followed by electron microscopy, indicated the presence of virus-like particles of the genus Potexvirus, which was subsequently confirmed as potato virus X (PVX) by RT-PCR and Sanger sequencing. PVX was found in 35.3% of the tested plants, all sampled in the northern Bekaa area. In a phylogenetic analysis, the partial coat protein gene sequence of a selected Lebanese isolate, PVX-AK1, clustered together with other PVX isolates from Asia. Furthermore, the 124-aa sequence of PVX-AK1 shared 100% identity with PVX-UK3, an isolate which is known as avirulent in potato genotypes carrying either Nx or Rx resistance genes. This work revealed a picture of the previously uninvestigated phytosanitary status of eggplant crops in an important horticultural area of Lebanon

<i>Cucumber mosaic virus</i>/satellite RNA infections alter <i>prosystemin</i> expression in tomato plants.

<p>Expression of <i>prosystemin</i> transcript measured with RT-qPCR in tomato plants cv. Moneymaker at 9 days post-inoculation with <i>Cucumber mosaic virus</i> (CMV-Fny) in combination with its satellite RNAs, either necrogenic (FN) or non-necrogenic mutant (FNNmut). Gene expression is relative to mock and normalized by <i>ubiquitin</i> gene (<i>ubi3</i>). Bars on the columns represent the standard deviation (n = 6). Columns with different letters are significantly different according to LSD test (P<0.05).</p

High resolution melting and custom TaqMan SNP Genotyping assays for the detection and discrimination of Sw-5 non infecting (SNI) and Sw-5 resistant breaking (SRB) alleles.

<p>(a) The temperature-shifted difference curves discriminated the two reference samples and their mixture in three separated clusters. The two lines correspond to two technical replicates for each sample. (b) Scatter plot of fluorescence data from Custom TaqMan SNP Genotyping assays. Each dot denotes individual replicates (four technical replicates per sample) and corresponds to FAM and VIC fluorescence levels associated to SNI- and SRB-specific probes, respectively. Allele-specific boundaries defined four non-overlapping areas corresponding to SNI, SRB, SNI/SRB mix and no template control (NTC).</p

Severity of disease symptoms induced by <i>Cucumber mosaic virus</i>/satellite RNA infections is reduced in transgenic tomato plants overexpressing <i>prosystemin</i>.

<p>Severity of symptoms caused by <i>Cucumber mosaic virus</i> (CMV) combined with two satellite RNAs, either necrogenic (77-satRNA) or non-necrogenic (the mutant variant NNmut-satRNA), on tomato plants cv. Better Boy, wild type (BB) or overexpressing the <i>prosystemin</i> transgene (BBP+). In each graph, the disease severity scores observed on 14 plants is shown. In the BBP+/FN graph (bottom right), two groups of plants that displayed significantly different disease development are indicated with a red (8 plants showing rapid necrosis or RN) and a blue (6 plants showing delayed necrosis or DN) line.</p

Primers and probes used in the described experimental procedures.

<p>Primers and probes used in the described experimental procedures.</p

List of isolate name, plant host, and SNP discrimination by sequencing, HRM and Taqman-SNP Genotyping assay.

<p>List of isolate name, plant host, and SNP discrimination by sequencing, HRM and Taqman-SNP Genotyping assay.</p

Disease symptoms induced by <i>Cucumber mosaic virus</i>/satellite RNA infections in transgenic tomato plants overexpressing <i>prosystemin</i>.

<p>Disease phenotypes in transgenic plants cv. Better Boy overexpressing <i>prosystemin</i> (BBP+) 21 days post-inoculation with CMV/satRNA combinations. Top Left panel, mock-inoculated healthy control (Mock); Top Right, plants inoculated with CMV/NNmut-satRNA showing suppression of symptoms (FNNmut); Bottom Left, plants inoculated with CMV/77-satRNA showing rapid and lethal necrosis [FN (RN)]; Bottom Right, plants inoculated with CMV/77-satRNA showing partial resistance and delayed necrosis [FN (DN)];</p