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. 2016 Oct 27:6:35801.
doi: 10.1038/srep35801.

Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

Hengyu Lu  1 , Bree A L Wilson  2   3 , Gavin J Ash  2   3 , Sharon B Woruba  4 , Murray J Fletcher  5 , Minsheng You  1 , Guang Yang  1 , Geoff M Gurr  1   5
Affiliations

Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media

Hengyu Lu et al. Sci Rep. .

Abstract

Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems.

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Figures

Figure 1
Figure 1. Example of loop-mediated isothermal amplification (LAMP) detection of phytoplasma showing positives in the heads of field-collected insects.
(1) = positive control; (2) = Zophiuma pupillata; (3) = Lophops saccharicida (nymph); (4) = Colgar sp.; and (5) = negative control. Time axis uses minutes.
Figure 2
Figure 2. Example of loop-mediated isothermal amplification (LAMP) detection of phytoplasma showing positives in the sucrose media fed upon by field-collected insects.
(1) = positive control; (2) = unidentified Zoraidini species; (3) = Zophiuma pupillata; and (4) = negative control. Time axis uses minutes.
Figure 3
Figure 3. Example of nested PCR with primers R16F2n/R2 for the detection of phytoplasma in the heads of field-collected insects.
Numbers 1–7 represent unidentified Zoraidini species; Numbers 8–16 represent Colgar sp.; (17) = Zophiuma pupillata; (18) = unidentified Ricaniidae species 2, Numbers 19–22 represent unidentified Ricaniidae species 1; M = DL 2000 marker; (+) = positive control; and (−) = negative control.
Figure 4
Figure 4. Example of nested PCR with primers R16F2n/R2 for the detection of phytoplasma in the sucrose media fed upon by field-collected insects.
Numbers 1–3 represent unidentified Zoraidini species; Numbers 4–8 represent Lophops saccharicida (nymph); Numbers 9–14 represent Colgar sp.; Numbers 15–17 represent Zophiuma pupillata; M = DL 2000 marker; (+) = positive control; and (−) = negative control.
Figure 5
Figure 5. Phylogenetic distance tree of phytoplasmas constructed using the neighbor-joining method by comparing 16S rDNA sequences selected from GenBank and 16S rDNA sequence of sampled.
Multiple alignments were performed with ClustalW and edited to a specific length of 1339 bp before constructing the distance tree. Numbers above the branches are confidence values obtained from 1000 bootstrap replicates. Sampled phytoplasma in PNG is shown by arrow as a member of 16SrIV group.
Figure 6
Figure 6. Map of the geographic locations of collection sites in Papua New Guinea.
Drawn using ArcGIS10.0 (http://www.esri.com/software/arcgis/).
See this image and copyright information in PMC

References

    1. Kelly P. et al.. First report of a phytoplasma identified in coconut palms (Cocos nucifera) with lethal yellowing-like symptoms in Papua New Guinea. New Dis. Rep. 23, 9 (2011).
    1. Harrison N., Cordova I., Richardson P. & Dibonito R. In Current Advances in Coconut Biotechnology Vol. 35 (eds Oropeza C. et al..) 183–196 (Springer, 1999).
    1. Danyo G. Review of scientific research into the Cape Saint Paul Wilt Disease (CSPWD) of coconut in Ghana. Afr. J. Agr. Res. 6, 4567–4578 (2011).
    1. Nipah J., Jones P. & Dickinson M. Detection of lethal yellowing phytoplasma in embryos from coconut palms infected with Cape St Paul wilt disease in Ghana. Plant Pathol. 56, 777–784 (2007).
    1. Kembu A., Yasinge W., Tenakanai D. & Bangulass N. Delimiting Survey of an Infectious Coconut Syndrome in Bogia District of Madang Province: a Preliminary Report. 43pp (PNG Cocoa Coconut Institute, 2009).

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