As a matter of fact, the levels of protein oxidation were increased in these worms. The life extension effect of over expression of SOD-1 was due to activation of longevity-promoting transcriptional factors, such as DAF-16/FoxO, heat shock factor-1 protein. Similarly, over expression of mitochondrial Mn-SOD also increased lifespan of the worms, which was dependent on daf-16. It was suggested that over expression of SOD-1 may trigger a daf-16 and HSF-1-dependent stress response that extends lifespan and not by removing superoxide. It is interesting to note that over expression of SOD causes increased oxidation of protein and no change in lipid peroxidation. This would imply that proteins are more susceptible to oxidative damage than membrane lipids. It is possible that over expression of SOD causes translocation of this antioxidant enzyme from the cytoplasm to the membrane where it protects lipid against oxidative damage in C. elegans. It has been difficult to deliver single antioxidants orally in C. elegans. Using a newly devised technique for an oral administration, it was demonstrated that supplementation with lipid soluble antioxidants tocotrienol, astaxanthin or gamma-tocopherol prolonged the lifespan of C. elegans. In contrast, the same study revealed that adding these antioxidants to the growth medium or the plate did not enhance the lifespan. These results suggest that adding antioxidant directly to the media may no yield consistent results in extending the lifespan. Treatment of worms during pre-reproductive and young adult stages with astaxanthin, a carotenoid present in marine animals and sea weeds,blueberry packaging box significantly extended the lifespan; this treatment was not effective in worms lacking daf-16.

The results also showed that astaxanthin treatment of wild-type worms increased expression of SOD and catalasein two weeks after hatching and daf-16 protein was translocated to the nucleus. It was proposed that the effect of astaxanthin on extension of lifespan was in part mediated via an Ins/Gf-1 signaling pathway. Treatment of C. elegans with cypermethrin increased ROS and H2O2. Elevation of peptide carbonylation suggested free radical damage to proteins, and decreased lifespan. Treatment with alphatocopherol prevented CYP-induced increase in oxidative stress and decline in lifespan . Supplementation of C. elegans media with a mixture of polyphenols derived from blueberries increased lifespan and age-related declines in C. elegans. This treatment also increased survival during acute heat stress. The blueberry extract used contained 3 major fractions all of which exhibited antioxidant activity, but only the fraction containing proanthocyanidins enhanced lifespan and thermotolerance. Treatment with crude Ginkgo biloba extract increased the lifespan of C. elegans. However, purified ingredients of G. biloba especially flavonoids and tamarixetin were most effective in extending lifespan .G. biloba extract also increased resistance to oxidative stress and thermal stress in wild-type and premature aging C. elegans mutant. Supplementation of C. elegans incubation media with a strain of lactic acid bacteria lactobacillus rhamnosus CNCMI-3690, which exhibits antioxidant activity, increased lifespan and protected against oxidative stress. This strain of lactic acid producing bacteria also reduced inflammation. These studies suggest that increased levels of antioxidants extend the lifespan by reducing oxidative stress and possibly by other mechanisms, such as preventing deregulation of protective transcriptional factors. In another study, it was reported that superoxide generators paraquat and plumbagin reduced the length of lifespan.

This is consistent with oxidative theory of aging. It was further reported that treatment that treatment with synthetic SOD mimetics EUK-8 or EUK-134, increased the activities of SOD, but not the levels of SOD proteins. Treatment with EUK-8 or EUK-134 in combination with a superoxide generator increased the lifespan of wild type worms; however, in the absence of a superoxide generator, it failed to increase the lifespan. It is possible that superoxide generator induces adaptive responses led by catalase and glutathione peroxidase and other longevity factors such as transcriptional regulators of antistress proteins, and repair mechanisms. These adaptive responses help SOD mimetic in extending the lifespan. However, in the absence of these adaptive responses produced by treatment with a superoxide generator, SOD mimetic is ineffective in extending the lifespan.The role of chronic inflammation on the lifespan has not been adequately investigated in C. elegans. In order to assess the role of chronic inflammation on the lifespan, antiinflammatory agents have been used. Treatment with aspirin and salicylate treatment increased lifespan and delayed agerelated declines, such as learning behavior , motor activity, thermal tolerance, osmotic resistance, brood size and intracellular protein aggregation in C. elegans by reducing ROS and increasing expressions of antioxidants genes encoding for SOD , catalase and glutathione peroxidase. Aspirin and salicylate treatment also reduced intracellular protein aggregation that is associated with increased aging. Aspirin treatment did not extend lifespan but improved resistance to stressors in worms lacking daf-16. Supplementation with a potent specific inhibitor of cyclooxygenase-2 celecoxib increased lifespan and age-related decline in C. elegans. This effect of celecoxib was not related to its inhibitory effect on COX-2 activity because an analog of celecoxib, which lacks the COX-2 inhibitory effect, also enhanced lifespan of C.elegans .

The life-extending effect of celecoxib appears to be mediated via insulin/IGF-1 signaling pathways]. Thus the effect of celecoxib on C. elegans life span resembles the consequence of SOD-3 over expression in the daf-2 mutant. Lifespan can be extended by multiple pathways that include reduced oxidative stress and activation of longevitypromoting transcriptional factors, such as Daf-16 and heat shock factor-1 protein. Oxidative stress can be reduced by multiple pathways which include reduced activity of mitochondria by inhibiting activities of complexes, increased transcriptional factor Nrf2 which regulate antioxidant enzymes and elevated antioxidant levels which directly scavenge free radicals as well as enhance antioxidant enzymes levels. It is now established that low levels of superoxide is required to activate and translocate Nrf2 from the cytoplasm to the nucleus which reduces oxidative stress by upregulating antioxidant enzymes. In C-elegans, the presence of increased levels of superoxide by deleting SOD-2 decreases the oxidative stress and extends the lifespan. It is possible that increased levels of superoxide triggers activation of Nrf2 that decreases oxidative stress by up-regulating other antioxidant enzymes. The role of Nfr2 in regulating antioxidant enzymes has not been evaluated. Although deletion of all 5 SODs had no impact on the lifespan of C. elegans, the worms lacking all SODs showed reduced fertility, slow development,blueberry packaging containers slower defecation cycle and decreased movement. These data suggest that adaptive responses by other antioxidant enzymes and transcriptional factors may have prevented reduction in lifespan. These data also suggest that all SODs are needed for normal growth and healthy life of worms.The genus Vitiviruswas created in 1997 for the classification of type member grapevine virus A , a plant virus discovered in grapevine with a filamentous flexuous particle differing from trichoviruses in its genomic arrangement. Vitiviruses have a single-stranded RNA genome encoding five genes: replicase , movement protein, coat protein , nucleic-acid-binding protein and a 20 kDa protein of unknown function. In the 2018 International Committee of Virus Taxonomy Master Species List , nine species of vitivirus infecting grapevine are recognized: Grapevine virus A, Grapevine virus B, Grapevine virus D, Grapevine virus E,Grapevine virus F, Grapevine virus G, Grapevine virus H, Grapevine virus I and Grapevine virus J. Since 2019, two more proposed vitiviruses were discovered in grapevine. Grapevine virus L was initially identified in RNAseq data and later detected in multiple plants in Croatia, New Zealand and the United States. Grapevine virus M was also discovered by high throughput sequencing in an American hybrid grapevine. Three different vitiviruses have been associated with the etiology of rugose wood disease in grapevine, a disease with world-wide distribution. GVA is associated with stem grooving on the variety Kober 5BB, grapevine virus B was identified as the causal agent of corky bark in the variety LN33, and grapevine virus D was implicated in growth reduction in the root stock Freedom. Additionally, these vitiviruses are frequently detected in coinfection with grapevine leaf roll viruses, resulting in synergistic interactions that can lead to lethal effects in several scion and root stock combinations. The potential pathogenic role of the remaining grapevine vitiviruses, including proposed members, is still unknown. Reliable diagnostic methods are critical in determining the viral infection status of a grapevine.

Multiple tests are available for the detection of vitiviruses, including biological indexing, real-time or end-point reverse transcription PCR and HTS. Biological indicators do not show symptoms for all vitiviruses infecting grape, and RT-PCR assays can fail to detect vitivirus variants containing nucleotide differences at critical primer binding locations. HTS is the most effective means of detecting all vitiviruses but its high cost at large scale limits its use as a screening tool. HTS data is helpful to inform and update RT-PCR primer design as new virus strains are continually being characterized. In this study, a universal end-point RT-PCR assay involving degenerate primers with the capacity of detecting all the known grapevine vitiviruses was developed. To validate the new assay, eleven grapevines each infected with one of the vitiviruses were tested. Moreover, a field survey was conducted of known vitivirus-infected grapevines. Following the first reports of vitiviruses in grapevine, several vitiviruses have been discovered in other hosts ; consequently, we investigated if the universal assay can detect these vitiviruses.With the aim of designing a generic RT-PCR assay for all the known grapevine vitiviruses, all the currently available complete and near-complete genome sequences in GenBank were analyzed. Likewise, available amino acid sequences for the REP and CP of different grapevine vitiviruses were obtained from GenBank. Thus, similar sequences were aligned using MUSCLE, and Geneious was used to identify a conserved region suitable for assay design. During the REP analysis including sixty-three sequences , multiple highly conserved regions were observed among the alignment and further investigated. Two of these conserved regions, henceforth referred to as motif A and motif B , were back translated using the EMBOSS Backtranambig program for degenerate primer design. This program takes an aa sequence and creates the nucleic acid sequence it could have come from, utilizing nucleotide ambiguity codes to represent all possible codons for each aa. As a result, primers Vitivirus-RdRp-F and Vitivirus-RdRp-R were generated, which amplify a 219-bp product.To empirically validate the new assay, grapevine material infected by ten different vitiviruses was obtained from the Foundation Plant Services pipeline of domestic and foreign introductions. A previous study in this grapevine collection revealed the presence of multiple vitiviruses. In addition, GVMinfected material was received from Texas A&M AgriLife Research and Extension Center, Weslaco, Texas. Consequently, eleven grapevines infected by GVA to GVM were included in the assay validation. Finally, a healthy grapevine and plants carrying grapevine Pinot gris virus and grapevine rupestris stem pitting-associated virus were also analyzed to test for assay exclusivity. Like vitiviruses, trichoviruses and foveaviruses belong to the family Betaflexiviridae.A universal assay able to detect all known grapevine vitiviruses and potentially other members of the genus Vitivirus was developed here based on sequence data available in GenBank. The presence of highly conserved motifs in the REP protein allowed the design of end-point RT-PCR primers, providing an alternative assay to reduce the work associated with the diagnosis of vitiviruses. The extensive sequence divergence existing among grapevine vitiviruses, observed at the nucleotide and aa levels, makes it difficult to design a test with broad-range detection. RT-PCR with degenerate primers is a simple strategy that is frequently used for the specific and simultaneous detection of multiple viruses. Assays involving degenerate primers targeting grapevine vitiviruses have been described before, however, these studies were conducted in the pre-HTS era, when fewer vitiviruses were known and sequence data was limited. Although Vitis spp. is recognized as the main host associated with the genus Vitivirus, vitiviruses have been identified in other perennial hosts, the majority of which are woody plants. For example, vitiviruses have been reported in blackberry, mint, agave and recently in blueberry. The universal assay successfully detected MV-2 in mint, however failed to detect BGMaV in blueberry. Additional investigation revealed a variation in motif A of BGMaV , and a similar scenario was observed in AVV. Based on our PCR results we predict that the universal assay will miss AVV during diagnosis, though, the rest of the known vitiviruses do not display any aa discrepancy in motifs A or B and they should be detected by the assay.