Comprehensive study of selection-value lines of zucchini on the level of damage by the yellow mosaic virus (ZYMV) and manifestation of other diseases

The work is aimed at the selection of linear samples of zucchini (Cucurbita pepo L. var. Giromontia Duch.) with increased complex resistance to abiotic factors of cultivation – zucchini yellow mosaic virus (ZYMV), powdery mildew, bacteriosis and other viral pathogens. Selection studies on choosing virus-resistant zucchini lines from the United Kingdom, Italy, the United States and Spain have identified a number of important correlations levels of resistance to ZYMV and to harmful microorganisms and viruses, belonging to other genera. The assessment of the resistance of the selected zucchini lines to the complex of studied diseases was performed on a 9-point scale of the classifier of the Council for Mutual Economic Assistance (CMEA). In total, during 2017–2019, 20 lines of zucchini were studied in the field. According to the complex resistance to all pathogens (zucchini yellow mosaic virus, green speckled mosaic, white mosaic and cucumber mosaic, bacteriosis and fusarium wilt), four lines have been identified – LZ 17-11, RVL-19, LZ 17-45 and LZ 17-49, which were resistant at the level of points 7 and 9 on the scale of CMEA. The possibility of selecting potentially resistant linear zucchini genotypes for ZYMV based on correlations with the manifestation of other diseases that occurred in the field has been confirmed. The highest level of correlation 0.59<rp<1.0) is observed when comparing the degree of powdery mildew development and symptoms of ZYMV (17 statistically confirmed values of the pairwise correlation coefficient or 80.95 %). A similar indicator for the pair “bacteriosis/ZYMV” is 14 statistically confirmed values of the pairwise correlation coefficient or 66.67 % (–1.0<rp<0.99), and for the pair “field viruses of other origin/ZYMV” – 11 values, which is 52.38 % (–1.0<rp<0.93). Zucchini samples were identified, in which statistically significant correlations were observed for all pairs of comparative traits. Among them, the variety-standard Chaklun and 4 more lines – LZ 17-1, LZ 17-8, LZ 17-50 and LZ 17-44 (–1.0<rp<0.92). The analysis of correlations shows a complex genetic organization of signs of resistance in zucchini lines to the studied pathogens and a high dependence of the manifestation of these signs on the response of the genotype of the line. One of the proofs of this experimental fact is that in all comparative pairs of indicators of the degree of development of certain diseases there are linear genotypes with both positive and negative values of Pearson correlation coefficients (rp). The high level of statistically significant correlations revealed gives grounds for wide use of correlation analysis in selection work for choosing lines potentially resistant to the complex of the most common diseases in vegetable agrocenoses

The Melon Zym Locus Conferring Resistance to ZYMV: High Resolution Mapping and Candidate Gene Identification

Zucchini yellow mosaic virus (ZYMV; potyviridae) represents a major pathogen of Cucurbitaceae crops. ZYMV resistance in melon PI 414723 is conditioned by a dominant allele at the Zym locus. This resistant accession restricts viral spread and does not develop mosaic symptoms, but necrosis sometimes develops in response to inoculation. In previous studies, Zym has been mapped to linkage group II of the melon genetic map. In the present study, positional cloning of the locus was undertaken, starting from the CM-AG36 SSR marker at approximately 2 cm distance. We utilized five mapping populations that share the same resistant parent, PI 414723, and analyzed a total of 1630 offspring, to construct a high-resolution genetic map of the Zym locus. Two melon BAC libraries were used for chromosome walking and for developing new markers closer to the resistance gene by BAC-end sequencing. A BAC contig was constructed, and we identified a single BAC clone, from the ZYMV susceptible genotype MR-1, that physically encompasses the resistance gene. A second clone was isolated from another susceptible genotype, WMR 29, and the two clones were fully sequenced and annotated. Additional markers derived from the sequenced region delimited the region to 17.6 kb of a sequence that harbors a NAC-like transcription factor and, depending on the genotype, either two or three R-gene homologs with a CC-NBS-LRR structure. Mapping was confirmed by saturating the map with SNP markers using a single mapping population. The same region was amplified and sequenced also in the ZYMV resistant genotype PI 414723. Because numerous polymorphic sites were noted between genotypes, we could not associate resistance with a specific DNA polymorphism; however, this study enables molecular identification of Zym and paves the way to functional studies of this important locus.

Genetic Loci Associated with Resistance to Zucchini Yellow Mosaic Virus in Squash

Zucchini Yellow Mosaic Virus (ZYMV) is an aphid-transmitted potyvirus that causes severe yield losses in squash (Cucurbita moschata) production worldwide. Development of resistant cultivars using traditional breeding approaches relies on rigorous and resource-intensive phenotypic assays. QTL-seq, a whole genome re-sequencing based bulked segregant analysis, is a powerful tool for mapping quantitative trait loci (QTL) in crop plants. In the current study, the QTL-seq approach was used to identify genetic loci associated with ZYMV resistance in an F2 population (n = 174) derived from a cross between Nigerian Local (resistant) and Butterbush (susceptible). Whole genome re-sequencing of the parents and bulks of resistant and susceptible F2 progeny revealed a mapping rate between 94.04% and 98.76%, and a final effective mapping depth ranging from 81.77 to 101.73 across samples. QTL-seq analysis identified four QTLs significantly (p < 0.05) associated with ZYMV resistance on chromosome 2 (QtlZYMV-C02), 4 (QtlZYMV-C04), 8 (QtlZYMV-C08) and 20 (QtlZYMV-C20). Seven markers within the QTL intervals were tested for association with ZYMV resistance in the entire F2 population. For QtlZYMV-C08, one single nucleotide polymorphism (SNP) marker (KASP-6) was found to be significantly (p < 0.05) associated with ZYMV resistance, while two SNPs (KASP-1 and KASP-3) and an indel (Indel-2) marker were linked to resistance within QtlZYMV-C20. KASP-3 and KASP-6 are non-synonymous SNPs leading to amino acid substitutions in candidate disease resistant gene homologs on chromosomes 20 (CmoCh20G003040.1) and 8 (CmoCh08G007140.1), respectively. Identification of QTL and SNP markers associated with ZYMV resistance will facilitate marker-assisted selection for ZYMV resistance in squash.

Molecular characterisation of Zucchini yellow mosaic virus infecting Cucurbita pepo in Egypt

The complete nucleotide sequence of Zucchini yellow mosaic virus isolate from Egypt (ZYMV-Egz_MT383108) was determined. The sequence comparisons suggested that the isolate belongs to Group A. The sequence analysis of the Egyptian isolate showed the highest similarity (~96–97%) with the isolates leaf1 (KJ923767.1) and PA_2006 (JQ716413.1) from the USA and the lowest similarity (84%) with an isolate (AF014811.2) from Singapore. The phylogenetic analysis revealed that ZYMV-Egz occupied a distinct clade together with the USA isolates in Group A, known to be the most widespread throughout the world. This is a first record of the complete nucleotide sequence of an Egyptian isolate of ZYMV.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein–protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein–protein interactions.

Carotenoid and tocopherol fortification of zucchini fruits using a viral RNA vector

Carotenoids and tocopherols are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. Here, we demonstrate an alternative biotechnological strategy for carotenoid and tocopherol fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated in zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and β-carotene (pro-vitamin A), lutein and phytoene, in both rind and flesh. Considerably higher accumulation of α- and γ-tocopherol was also detected, particularly in fruit rind. Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.

Development of Bottle Gourd Lines Resistant to Zucchini Yellow Mosaic Virus Using Ethyl Methanesulfonate Mutagenesis

Zucchini yellow mosaic virus (ZYMV) causes serious damage to cucurbit crops worldwide and can be spread by aphids, by mechanical injury, and in seeds. With the popularization of cucurbit grafting, the use of susceptible rootstock has increased the risk of ZYMV infection in cucurbit crops. In China, the bottle gourd (Lagenaria siceraria) is a widely used rootstock in grafted watermelon production. However, few resistant bottle gourds are available commercially. This study developed bottle gourd lines resistant to ZYMV using ethyl methanesulfonate (EMS) mutagenesis. A new mutated bottle gourd population (M1) was generated by treating seeds with EMS. Diverse phenotypes were observed in the seedlings, flowers, and fruit of M2 plants, some of which are of potential commercial interest, such as dwarfing and different fruit shapes. Based on the M2 phenotypes, 106 M3 lines were selected and screened for resistance to ZYMV by mechanical inoculation and agroinfiltration. Nine M3 lines were resistant to ZYMV during three tests. One inbred M4 line (177-8) was developed and showed stable resistance and no virus when tested using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and polymerase chain reaction. These resistant lines are promising materials for developing watermelon rootstock and exploring resistance genes as new ZYMV-resistant resources. EMS induction could be a practical strategy for creating resistant cucurbit crops.