Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Agarwal A

PROVIDER: S-EPMC7718921 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Agarwal Arati A Cunningham J Paul JP Valenzuela Isabel I Blacket Mark J MJ

Scientific reports 20201204 1

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 ...[more]

PMID: 33277555

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Project description:Grape Phylloxera, Daktulosphaira vitifoliae, is a gall-forming insect that feeds on the leaves and roots of many Vitis species. The roots of the cultivated V. vinifera cultivars and hybrids are highly susceptible to grape phylloxera feeding damage. The native range of this insect covers most of North America, and it is particularly abundant in the eastern and central United States. Phylloxera was introduced from North America to almost all grape-growing regions across five of the temperate zone continents. It devastated vineyards in each of these regions causing large-scale disruptions to grape growers, wine makers and national economies. In order to understand the population diversity of grape phylloxera in its native range, more than 500 samples from 19 States and 34 samples from the introduced range (northern California, Europe and South America) were genotyped with 32 simple sequence repeat markers. STRUCTURE, a model based clustering method identified five populations within these samples. The five populations were confirmed by a neighbor-joining tree and principal coordinate analysis (PCoA). These populations were distinguished by their Vitis species hosts and their geographic locations. Samples collected from California, Europe and South America traced back to phylloxera sampled in the northeastern United States on V. riparia, with some influence from phylloxera collected along the Atlantic Coast and Central Plains on V. vulpina. Reproductive statistics conclusively confirmed that sexual reproduction is common in the native range and is combined with cyclical parthenogenesis. Native grape phylloxera populations were identified to be under Hardy-Weinberg equilibrium. The identification of admixed samples between many of these populations indicates that shared environments facilitate sexual reproduction between different host associated populations to create new genotypes of phylloxera. This study also found that assortative mating might occur across the sympatric range of the V. vulpina west and V. cinerea populations.

Project description:BackgroundMuscadine (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 (Vitis-Muscadinia) derived from the muscadine R source 'NC184-4' and V. vinifera cv. 'Cabernet-Sauvignon' (CS).ResultsIn 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 and designated RDV6. Additional QTLs were detected on LG 3 (RDV7) and LG 10 (RDV8), depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulked 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 and QTLs were thus designated respectively XiR2, XiR3 and XiR4, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18.ConclusionsBased on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to D. vitifoliae and to X. 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.

Dataset Information

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

Publications

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

Similar Datasets

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