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      Molecular and Morphological Identification of Mealybug Species (Hemiptera: Pseudococcidae) in Brazilian Vineyards

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          Abstract

          Mealybugs ( Hemiptera: Pseudococcidae) are pests constraining the international trade of Brazilian table grapes. They damage grapes by transmitting viruses and toxins, causing defoliation, chlorosis, and vigor losses and favoring the development of sooty mold. Difficulties in mealybug identification remain an obstacle to the adequate management of these pests. In this study, our primary aim was to identify the principal mealybug species infesting the major table grape-producing regions in Brazil, by morphological and molecular characterization. Our secondary aim was to develop a rapid identification kit based on species-specific Polymerase Chain Reactions, to facilitate the routine identification of the most common pest species. We surveyed 40 sites infested with mealybugs and identified 17 species: Dysmicoccus brevipes (Cockerell), Dysmicoccus sylvarum Williams and Granara de Willink, Dysmicoccus texensis (Tinsley), Ferrisia cristinae Kaydan and Gullan, Ferrisia meridionalis Williams, Ferrisia terani Williams and Granara de Willink, Phenacoccus baccharidis Williams, Phenacoccus parvus Morrison, Phenacoccus solenopsis Tinsley, Planococcus citri (Risso), Pseudococcus viburni (Signoret), Pseudococcus cryptus Hempel, four taxa closely related each of to Pseudococcus viburni, Pseudococcus sociabilis Hambleton, Pseudococcus maritimus (Ehrhorn) and Pseudococcus meridionalis Prado, and one specimen from the genus Pseudococcus Westwood. The PCR method developed effectively identified five mealybug species of economic interest on grape in Brazil: D. brevipes, Pl. citri, Ps. viburni, Ph. solenopsis and Planococcus ficus (Signoret). Nevertheless, it is not possible to assure that this procedure is reliable for taxa that have not been sampled already and might be very closely related to the target species.

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          The family Closteroviridae revised.

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            DNA barcodes for two scale insect families, mealybugs (Hemiptera: Pseudococcidae) and armored scales (Hemiptera: Diaspididae).

            Although DNA barcode coverage has grown rapidly for many insect orders, there are some groups, such as scale insects, where sequence recovery has been difficult. However, using a recently developed primer set, we recovered barcode records from 373 specimens, providing coverage for 75 species from 31 genera in two families. Overall success was >90% for mealybugs and >80% for armored scale species. The G·C content was very low in most species, averaging just 16.3%. Sequence divergences (K2P) between congeneric species averaged 10.7%, while intra-specific divergences averaged 0.97%. However, the latter value was inflated by high intra-specific divergence in nine taxa, cases that may indicate species overlooked by current taxonomic treatments. Our study establishes the feasibility of developing a comprehensive barcode library for scale insects and indicates that its construction will both create an effective system for identifying scale insects and reveal taxonomic situations worthy of deeper analysis.
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              A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae).

              Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                25 July 2014
                : 9
                : 7
                : e103267
                Affiliations
                [1 ]Embrapa Uva e Vinho, Bento Gonçalves, Brazil
                [2 ]Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, Brazil
                [3 ]Institut National de la Recherche Agronomique, Institut Sophia Agrobiotech, UMR INRA / UNSA / CNRS BP 167. 06903, Sophia-Antipolis, France
                [4 ]ANSES, Laboratoire de la Santé des Végétaux, Unité d'entomologie et Plantes Invasives Campus International de Baillarguet, CS 30016, Montferrier-sur-Lez, France
                State University of Maringá/Universidade Estadual de Maringá, Brazil
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: TM MB. Performed the experiments: A. Bertin A. Blin J-FG DB GR. Analyzed the data: VCPS A. Blin J-FG TM. Contributed reagents/materials/analysis tools: TM MB. Wrote the paper: VCPS J-FG TM.

                Article
                PONE-D-14-04488
                10.1371/journal.pone.0103267
                4111580
                25062012
                c5398ca0-7356-4b83-9d0e-829aab77734d
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 April 2014
                : 28 June 2014
                Page count
                Pages: 13
                Funding
                This work was supported by the European Union Seventh Framework Programme FP7-KBBE #265865 “PURE”, FP7-IRSES #269196 “IPRABIO”and FP7-IAPP #324475 “COLBICS” and by the French grants Agropolis Fondation ( http://www.agropolis-fondation.fr/) (RTRA – Montpellier, BIOFIS project number 1001-001) and ‘Bibliothèque du Vivant’. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Agriculture
                Pest Control
                Computational Biology
                Genome Analysis
                Genomic Databases
                Sequence Assembly Tools
                Evolutionary Biology
                Population Genetics
                Haplotypes
                Genetics
                Genomics
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Zoology
                Entomology

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                Uncategorized

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