Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) first reported in India and its neighbouring countries. ToLCNDV severely affects zucchini crop (Cucurbita ...pepo) in the main production areas of Southern Spain since 2012. This emerging begomovirus is a serious threat to this and other cucurbit crops. Breeding resistant cultivars is the most promising method for disease control, but requires the identification of sources of resistance in the Cucurbita genus. In this work, we screened for ToLCNDV resistance a large collection of Cucurbita spp. accessions, including landraces and commercial cultivars of the main cultivated species, C. pepo, Cucurbita moschata and Cucurbita maxima and wild species. The screening was performed using mechanical and whitefly inoculation. The level of resistance was assessed by scoring symptom severity and by measuring the virus content with quantitative polymerase chain reaction in selected genotypes. Diversity in the response was observed within and among species. Severe symptoms and high viral amounts were found at 30 days after mechanical and whitefly inoculation in C. pepo, in all accessions belonging to the Zucchini morphotype and to other morphotypes of both subspecies, pepo and ovifera, and even in the wild relative Cucurbita fraterna. C. maxima was also highly susceptible. This species showed characteristic symptoms of leaf decay and intense yellowing, different from those of mosaic, curling and internode shortening found in C. pepo. The only species showing resistance was C. moschata. Four accessions were symptomless or had some plants with only mild symptoms after three independent rounds of mechanical inoculation with different inoculum sources. Two of them also remained symptomless after virus inoculation with viruliferous whiteflies. ToLCNDV was detected in these asymptomatic accessions at 15 and 30 days post inoculation, but viral amounts were much lower than those found in susceptible genotypes, suggesting a high level of resistance. The symptoms in the susceptible accessions of this species were also different, with a characteristic leaf mottling, evolving to a severe mosaic. The newly identified C. moschata resistant accessions are good candidates for breeding programmes to avoid the damage caused by ToLCNDV.
Tomato leaf curl New Delhi virus is an emerging whitefly‐borne species of begomovirus in Mediterranean regions that poses a severe threat to cucurbit crops of the genus Cucurbita. Until now, only two ...sources of resistance have been identified in Cucurbita spp., these being PI604506 (cv. Large Cheese) and PI381814 (Indian landrace), both of C. moschata. The resistance of cv. Large Cheese is conferred by a single recessive gene located on chromosome 8. The objective of the present investigation was to screen for tomato leaf curl New Delhi virus (ToLCNDV) resistance among 105 accessions drawn from five species of Cucurbita and, if high resistance was found in any of them, determine the mode of inheritance. Screening was conducted using whitefly‐mediated inoculation on all 105 accessions. The accessions showing some resistance were further screened by mechanical inoculation as well as by quantitative PCR‐based diagnostics. The results showed that, overall, the accessions of C. pepo and C. maxima were the most susceptible, those of C. argyrosperma and C. ecuadorensis intermediate, and those of C. moschata most resistant to ToLCNDV. Only one accession of C. moschata, BSUAL‐252, originating from Japan, was highly resistant to ToLCNDV, showing no symptoms after either method of inoculation, and absence of virus accumulation. Upon crossing BSUAL‐252 with a susceptible accession of C. moschata, BSUAL‐265, the resistance was observed to be conferred by a single dominant gene. This gene is not linked to the genomic region on chromosome 8 where the locus of the previously identified recessive gene for ToLCNDV resistance resides.
The Cucurbita moschata BSUAL‐252 is a new source of resistance to ToLCNDV discovered in a massive screening of the genus, which will be useful to produce resistant lines in breeding programmes of Cucurbita species.
Barley mild mosaic virus
(BaMMV), transmitted by the soil-borne protist
Polymyxa graminis
, has a serious impact on winter barley production. Previously, the BaMMV resistance gene
rym15
was mapped on ...chromosome 6HS, but the order of flanking markers was non-collinear between different maps. To resolve the position of the flanking markers and to enable map-based cloning of
rym15
, two medium-resolution mapping populations Igri (susceptible) × Chikurin Ibaraki 1 (resistant) (I × C) and Chikurin Ibaraki 1 × Uschi (susceptible) (C × U), consisting of 342 and 180 F
2
plants, respectively, were developed. Efficiency of the mechanical inoculation of susceptible standards varied from 87.5 to 100% and in F
2
populations from 90.56 to 93.23%. Phenotyping of F
2
plants and corresponding F
3
families revealed segregation ratios of 250 s:92r (I × C,
χ
2
= 0.659) and 140 s:40r (C × U,
χ
2
= 0.741), suggesting the presence of a single recessive resistance gene. After screening the parents with the 50 K Infinium chip and anchoring corresponding SNPs to the barley reference genome, 8 KASP assays were developed and used to remap the gene. Newly constructed maps revealed a collinear order of markers, thereby allowing the identification of high throughput flanking markers. This study demonstrates how construction of medium-resolution mapping populations in combination with robust phenotyping can efficiently resolve conflicting marker ordering and reduce the size of the target interval. In the reference genome era and genome-wide genotyping era, medium-resolution mapping will help accelerate candidate gene identification for traits where phenotyping is difficult.
Persistent plant viruses usually depend on insects for their transmission; they cannot be transmitted between plants or through mechanical inoculation. However, the mechanism by which persistent ...viruses become pathogenic in insect vectors remains unknown. In this study, we used Rice stripe virus (RSV), its insect vector Laodelphax striatellus and host plant (Oryza sativa) to explore how persistent viruses acquire pathogenicity from insect vectors. RSV acquired phytopathogenicity in both the alimentary tract and the salivary gland of L. striatellus. We mechanically inoculated RSV into rice O. sativa leaves through midrib microinjection. Insect‐derived RSV induced a typical stripe symptom, whereas plant‐derived RSV only produced chlorosis in rice leaves. Insect‐derived RSV had higher expression of genes rdrp, ns2, nsvc2, sp and nsvc4 than plant‐derived RSV, and the latter had higher expression of genes cp and ns3 than the former in rice leaves. Different from plant‐derived RSV, insect‐derived RSV damaged grana stacks within the chloroplast and inhibited photosynthesis by suppressing the photosystem II subunit psbp. This study not only presented a convenient method to mechanically inoculate RSV into plants, but also provided insights into the different pathogenic mechanisms of RSV from the insect vector and from viruliferous plants.
Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict ...standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed.
Virus infection on shallots may cause mosaic of leaves and plant dwarf. It is very important to know the identity of the virus as a requirement in determining viral disease management strategy. The ...research is subjected to identify seed- transmitted viruses from shallot bulbs. Detection of virus from bulb was conducted by growing on test, followed by mechanical inoculation on shallot cultivar Biru Bantul, Chenopodium amaranticolor, and Nicotiana tabacum and morphological observation of virus particle by electron microscope. About 2 weeks after inoculation symptoms was observed, i.e. mosaic and dwarf on shallot cultivar Biru Bantul, local necrotic on C. amaranticolor, but symptomless on N. tabacum. Filamentous particles of 650 nm and 800 nm in length was observed under electron microscope from plants with mosaic and dwarfing symptom, respectively. This indicated the presence of 2 different seed-transmitted viruses on infected shallots. Two species of shallot viruses, i.e. Shallot latent carlavirus (SLV) and Onion yellow dwarf potyvirus (OYDV) was most likely the main seedborne viruses on shallot bulb. Identification based on nucleic acid and protein sequence analysis is necessary for further confirmation
Vegetable cowpea is eaten mainly fresh, in the form of young, immature pods, tender and sweet like the common bean. However, like cowpea with seeds, vegetable cowpea experience yield losses due to ...the cowpea aphid-borne mosaic virus (CABMV). This study aims to improve yields through the development of vegetable cowpea varieties resistant to CABMV. The study focused on ten varieties of vegetable cowpea, carried out in a greenhouse at the Kamboinsé research station using a randomized complete block design with three replications, all inoculated with CABMV. The data collection concerned resistance parameters. Mechanical inoculation made it possible to observe various symptoms of CABMV, thus highlighting the existence of variability within the varieties tested. Strong correlations were observed between several variables. Thus, the varieties of vegetable cowpea IT85F-2089-5, UG-CP-8, IT85F-2805-5 and Telma were identified as resistant, because belonging to the low severity classes and having a low value of area under the disease progress curve. On the other hand, the varieties RW-CP-5, UG-CP-6, IT83S-911, niébé baguette grimpant possessing a high severity class were judged to be susceptible. These resistant varieties will thus be able to contribute to the improvement of production and the protection of cowpea resources in Burkina.
Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict ...standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed.
The protocol described is intended to be used alongside molecular methods in order to reveal the relationship between the genome sequence and the biological properties of a single isolate of Citrus ...tristeza virus complex (CTV). It enables the phenotypic profile of the isolates to be defined and to infer the associated tristeza diseases (decline, seedling yellows, or stem pitting), to assess their aggressiveness or potential cross protectiveness (if any), and to monitor their movement into the host plants and the transmissibility by aphids.
Coccinia grandis
is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in
C. grandis
...have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for
C. grandis
, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in
C. grandis.
The viral infection was achieved through mechanical inoculation of ALSV-infected
Chenopodium quinoa
leaf extract onto the cotyledons of
C. grandis
. ALSV-VIGS mediated knockdown of
CgPDS
gene was successfully achieved in
C. grandis
by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of
C. grandis
and shortened the time-frame by skipping viral propagation in
C. quinoa
. Typical yellow-leaf phenotype was observed in
C. grandis
plants agroinfiltrated with ALSV-
CgSU
constructs, indicating robust silencing of
CgSU
gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious
C. grandis
and it can help us understand the mechanism of sex expression.
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