Specificity of resistance and tolerance to cucumber vein yellowing virus in melon accessions and evidence for resistance breaking associated with a single mutation in VPg

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e. very mild symptoms in spite of virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific since many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

Candidate Effectors of Plasmodiophora brassicae Pathotype 5X During Infection of Two Brassica napus Genotypes

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of canola (Brassica napus) in Canada. Disease management relies heavily on planting clubroot resistant (CR) cultivars, but in recent years, new resistance-breaking pathotypes of P. brassicae have emerged. Current efforts against the disease are concentrated in developing host resistance using traditional genetic breeding, omics and molecular biology. However, because of its obligate biotrophic nature, limited resources have been dedicated to investigating molecular mechanisms of pathogenic infection. We previously performed a transcriptomic study with the cultivar resistance-breaking pathotype 5X on two B. napus hosts presenting contrasting resistance/susceptibility, where we evaluated the mechanisms of host response. Since cultivar-pathotype interactions are very specific, and pathotype 5X is one of the most relevant resistance-breaking pathotypes in Canada, in this study, we analyze the expression of genes encoding putative secreted proteins from this pathotype, predicted using a bioinformatics pipeline, protein modeling and orthologous comparisons with effectors from other pathosystems. While host responses were found to differ markedly in our previous study, many common effectors are found in the pathogen while infecting both hosts, and the gene response among biological pathogen replicates seems more consistent in the effectors associated with the susceptible interaction, especially at 21 days after inoculation. The predicted effectors indicate the predominance of proteins with interacting domains (e.g., ankyrin), and genes bearing kinase and NUDIX domains, but also proteins with protective action against reactive oxygen species from the host. Many of these genes confirm previous predictions from other clubroot studies. A benzoic acid/SA methyltransferase (BSMT), which methylates SA to render it inactive, showed high levels of expression in the interactions with both hosts. Interestingly, our data indicate that E3 ubiquitin proteasome elements are also potentially involved in pathogenesis. Finally, a gene with similarity to indole-3-acetaldehyde dehydrogenase is a promising candidate effector because of its involvement in indole acetic acid synthesis, since auxin is one of the major players in clubroot development.

Use of Novel Lab-Assays to Examine the Effect of Pyrethroid-Treated Bed Nets on Blood Feeding Success and Longevity of Highly Insecticide-Resistant Anopheles Gambiae S.I. Mosquitoes.

Background: There is a pressing need to improve understanding of how insecticide resistance affects the functional performance of Insecticide Treated Nets (ITNs). Standard WHO insecticide resistance monitoring assays are designed for resistance surveillance and do not necessarily provide insight into how different frequencies, mechanisms or intensities of resistance affect the ability of ITNs to reduce malaria transmission. Methods: The current study presents some novel laboratory-based assays that attempt to better simulate realistic exposure of mosquitoes to ITNs and to quantify impact of exposure not only on instantaneous mortality, but also blood feeding and longevity, two traits that are central to transmission. The assays evaluated the performance of a standard ITN (Permanet® 2.0), a ‘next generation’ combination ITN that includes a resistance breaking synergist (Permanet® 3.0), and an untreated net (UTN), against field-derived Anopheles gambiae s.l. mosquitoes from Côte d’Ivoire exhibiting 1500-fold pyrethroid resistance. Results: The study revealed that a standard ITN induced negligible instantaneous mortality against the resistant mosquitoes, whereas the resistance breaking net caused high mortality and a reduction in blood feeding. However, the ITNs still impacted long term survival relative to the UTN. The impact on longevity depended on feeding status, with blood-fed mosquitoes living longer than unfed mosquitoes following ITN exposure. The ITNs also reduced the blood feeding success, the time spent on the net, and blood-feeding duration, relative to the untreated net. Conclusion: Thus, while the standard ITN did not have as substantial instantaneous impact as the resistance breaking net, it still had significant impacts on traits important for transmission. These results highlight the benefit of improved bioefficacy assays that allow for realistic exposure and consider sub- or pre-lethal effects to help assess the functional significance of insecticide resistance.

Full Genome Evolutionary Studies of Wheat Streak Mosaic-Associated Viruses Using High-Throughput Sequencing

Wheat streak mosaic (WSM), a viral disease affecting cereals and grasses, causes substantial losses in crop yields. Wheat streak mosaic virus (WSMV) is the main causal agent of the complex, but mixed infections with Triticum mosaic virus (TriMV) and High plains wheat mosaic emaravirus (HPWMoV) were reported as well. Although resistant varieties are effective for the disease control, a WSMV resistance-breaking isolate and several potential resistance-breaking isolates have been reported, suggesting that viral populations are genetically diverse. Previous phylogenetic studies of WSMV were conducted by focusing only on the virus coat protein (CP) sequence, while there is no such study for either TriMV or HPWMoV. Here, we studied the genetic variation and evolutionary mechanisms of natural populations of WSM-associated viruses mainly in Kansas fields and fields in some other parts of the Great Plains using high-throughput RNA sequencing. In total, 28 historic and field samples were used for total RNA sequencing to obtain full genome sequences of WSM-associated viruses. Field survey results showed WSMV as the predominant virus followed by mixed infections of WSMV + TriMV. Phylogenetic analyses of the full genome sequences demonstrated that WSMV Kansas isolates are widely distributed in sub-clades. In contrast, phylogenetic analyses for TriMV isolates showed no significant diversity. Recombination was identified as the major evolutionary force of WSMV and TriMV variation in KS fields, and positive selection was detected in some encoding genomic regions in the genome of both viruses. Furthermore, the full genome sequence of a second Kansas HPWMoV isolate was reported. Here, we also identified previously unknown WSMV isolates in the Great Plains sharing clades and high nucleotide sequence similarities with Central Europe isolates. The findings of this study will provide more insights into the genetic structure of WSM-associated viruses and, in turn, help in improving strategies for disease management.

Resistance-breaking tomato spotted wilt virus variant that recently occurred in pepper in South Korea is a genetic reassortant

Tomato spotted wilt virus (TSWV) is a destructive viral pathogen in various crops, including pepper. While the single dominant gene Tsw has been utilized in pepper breeding to confer resistance to TSWV, the occurrence of TSWV variants that overcome Tsw-mediated resistance has been reported in various countries after several years of growing resistant cultivars. In this study, we determined the complete genome sequence of a resistance-breaking TSWV variant (TSWV-YI) that recently emerged in pepper in South Korea. TSWV-YI infected all the resistant pepper cultivars tested. The phylogenetic and recombination analyses of the complete TSWV-YI genome sequence showed that it is a reassortant that acquired its L and M RNA segments from the existing South Korean TSWV population and its S RNA in an isolate from another country. Given that TSWV-YI is a resistance-breaking variant, it appears that reassortment of the S RNA led to the emergence of this variant that breaks the Tsw gene in pepper grown in South Korea. Our results suggest that resistance-breaking TSWV variants are a potential threat to pepper production in South Korea and that strategies to manage these variants should be developed to ensure sustainable pepper production.