Effects of salt stress on plant growth, abscisic acid and salicylic acid in own-rooted cultivars of Vitis vinifera L.

Aim of study: In the most areas of vineyards worldwide, cultivars are frequently grafted on specific rootstocks to avoid Daktulosphaira vitifoliae pest attack. Nevertheless, the absence of this pest in Canary Islands allowed the chance to conserve and cultivate traditional or new own-rooted genotypes without the requirement of the rootstocks. To investigate the responses of own-rooted genotypes of Vitis vinifera L. to salt stress conditions, ‘Castellana Negra’ (‘CN’) and ‘Negramoll’ (‘Ne’) were used with the aim to characterize their morphological and physiological responses. Area of study: Canary Islands, Spain Material and methods: The effects of NaCl stress on growth, abscisic acid (ABA), salicylic acid (SA) and proline were assessed in ‘CN’ and ‘Ne’ under greenhouse conditions. Main results: In ‘CN’, the decrease of leaf number in stressed plants was lower and started eleven days later than in ‘Ne’. Salt stress also reduced stomatal conductance (gs), although such decrease took place earlier in ‘CN’ than in ‘Ne’. ABA and SA concentrations in ‘CN’ leaves were 2-fold higher than those of ‘Ne’. Salt stress increased leaf ABA and SA content in both genotypes, compared to control. In conclusion, ABA and SA appear to be involved in grapevines responses to salinity and suggest that exogenous SA could be useful to mitigate the stress impacts. Research highlights: ‘CN’ exhibited a better response than ‘Ne’ through the delay of salt injury establishment, and the dissimilar responses between ‘CN’ and ‘Ne’ seem to be associated to the higher accumulation of ABA and SA under salt stress.

Potential Global Distribution of Daktulosphaira vitifoliae under Climate Change Based on MaxEnt

Grape phylloxera, Daktulosphaira vitifoliae, is a small, invasive, sap-sucking pest that is widely present in most viticulture regions all over the world. It is originally from North America and feeds on grapevine roots and leaves. In the current study, the potential distribution area of the leaf-feeding population was investigated with MaxEnt based on population occurrence data under different environmental variables. Results suggested that under current climatic conditions, Europe, East and North China, Japan, the Eastern USA, Uruguay, and the Southeast of South America are highly suitable areas for the occurrence of phylloxera leaf populations. The results showed that isothermality and precipitation of coldest quarter were major factors which contribute more than 60% of the model under current climate conditions. Our results provide important information for governmental decision makers and famers to develop control and management strategies against D. vitifoliae, and can also be used as a reference for studies on other invasive pest.

A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)

Grape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

Characterization of genetic determinants of the resistance to phylloxera, Daktulosphaira vitifoliae, and the dagger nematode Xiphinema index from muscadine background

Background: Muscadine (Muscadinia rotundifolia) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index, vector of the Grapevine fanleaf virus (GFLV) was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 (Muscadinia-Vitis) derived from the muscadine R source ‘NC184-4’ and V. vinifera cv. ‘Cabernet-Sauvignon’ (CS). Results: In this pseudo-testcross, parental maps were constructed using simple-sequence repeats markers and single nucleotide polymorphism markers from a GBS approach. For the VRH8771 map, 2271 SNP and 135 SSR markers were assembled, resulting in 19 linkage groups (LG) and an average distance between markers of 0.98 cM. Phylloxera resistance was assessed by monitoring root nodosity number in an in planta experiment and larval development in a root in vitro assay. Nematode resistance was studied using 10-12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7. Additional QTLs were detected on LG 3 and LG 10, depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulk Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant phenotype. QTL analysis confirmed the results, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18. Conclusions: Based on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to Daktulosphaira vitifoliae and to Xiphinema index were identified from a muscadine source. All together these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.

Characterization of genetic determinants of the resistance to phylloxera, Daktulosphaira vitifoliae, and the dagger nematode Xiphinema index from muscadine background

Background: Muscadine (Muscadinia rotundifolia) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index, vector of the Grapevine fanleaf virus (GFLV) was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 (Muscadinia-Vitis) derived from the muscadine R source ‘NC184-4’ and V. vinifera cv. ‘Cabernet-Sauvignon’ (CS). Results: In this pseudo-testcross, parental maps were constructed using simple-sequence repeats markers and single nucleotide polymorphism markers from a GBS approach. For the VRH8771 map, 2271 SNP and 135 SSR markers were assembled, resulting in 19 linkage groups (LG) and an average distance between markers of 0.98 cM. Phylloxera resistance was assessed by monitoring root nodosity number in an in planta experiment and larval development in a root in vitro assay. Nematode resistance was studied using 10-12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7. Additional QTLs were detected on LG 3 and LG 10, depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulk Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant phenotype. QTL analysis confirmed the results, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18. Conclusions: Based on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to Daktulosphaira vitifoliae and to Xiphinema index were identified from a muscadine source. All together these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.

Use of DNA Markers for Grape Phylloxera Population and Evolutionary Genetics: From RAPDs to SSRs and Beyond

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major pest of cultivated grapevines (Vitis spp.), occurring in virtually all viticultural regions around the world. Different grape phylloxera strains can be found at varying levels on leaves and roots on both own-rooted plants and in plants grafted onto partially resistant rootstocks. Considering its relevance for the adequate management of the pest in infested vineyards, the analysis of its genetic diversity has received considerable attention from the scientific community in the last decades. Here, we review 25 years of DNA-based molecular markers applied to the analysis of the genetic structure and the reproductive mode of grape phylloxera in its native range and in different introduced regions. The use given to RAPD, AFLP, mtDNA sequencing and microsatellite (SSR) genetic markers for the analysis of grape phylloxera diversity is discussed, and an overview of the main findings obtained after their application to different populations collected in diverse regions all around the world is shown. Lastly, we explore how recent advancements in molecular biology and in modern high throughput genotyping technologies may be applied to better understand grape phylloxera natural diversity at a molecular level.