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      Engineered Orange Ectopically Expressing the Arabidopsis β-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing

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          Abstract

          Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species ( Candidatus Liberibacter spp.) or their insect vectors ( Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit ( E)-β-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Rapid isolation of high molecular weight plant DNA.

            A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
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              Current epidemiological understanding of citrus Huanglongbing .

              Huanglongbing (HLB) is the most destructive citrus pathosystem worldwide. Previously known primarily from Asia and Africa, it was introduced into the Western Hemisphere in 2004. All infected commercial citrus industries continue to decline owing to inadequate current control methods. HLB increase and regional spatial spread, related to vector populations, are rapid compared with other arboreal pathosystems. Disease dynamics result from multiple simultaneous spatial processes, suggesting that psyllid vector transmission is a continuum from local area to very long distance. Evolutionarily, HLB appears to have originated as an insect endosymbiont that has moved into plants. Lack of exposure of citrus to the pathogen prior to approximately 100 years ago did not provide sufficient time for development of resistance. A prolonged incubation period and regional dispersal make eradication nonviable. Multiple asymptomatic infections per symptomatic tree, incomplete systemic distribution within trees, and prolonged incubation period make detection difficult and greatly complicate disease control.
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                Author and article information

                Contributors
                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Media S.A.
                1664-462X
                02 March 2021
                2021
                : 12
                : 641457
                Affiliations
                [1] 1Laboratório de Biotecnologia Vegetal, Pesquisa & Desenvolvimento, Fundo de Defesa da Citricultura (Fundecitrus) , Araraquara, Brazil
                [2] 2Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC), Universidad Politécnica de Valencia (UPV) , Valencia, Spain
                [3] 3Chemistry Department, Universidade Federal de São Carlos (UFSCar) , São Carlos, Brazil
                [4] 4Institute of Chemistry, São Paulo State University (UNESP) , Araraquara, Brazil
                [5] 5Institute of Plant Biology, National Taiwan University , Taipei, Taiwan
                [6] 6Swammerdam Institute for Life Sciences, Green Life Sciences Cluster, University of Amsterdam , Amsterdam, Netherlands
                Author notes

                Edited by: Paulo Arruda, State University of Campinas, Brazil

                Reviewed by: Fengqi Li, Beijing Academy of Agriculture and Forestry Sciences, China; Jorge Maurício Mondego, Instituto Agronômico de Campinas (IAC), Brazil

                *Correspondence: Leandro Peña, lpenya@ 123456ibmcp.upv.es

                This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science

                Article
                10.3389/fpls.2021.641457
                7982956
                33763099
                fd487257-8e98-4993-8d08-81af08b6e2a7
                Copyright © 2021 Alquézar, Volpe, Magnani, de Miranda, Santos, Marques, de Almeida, Wulff, Ting, de Vries, Schuurink, Bouwmeester and Peña.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 14 December 2020
                : 27 January 2021
                Page count
                Figures: 8, Tables: 0, Equations: 0, References: 94, Pages: 15, Words: 0
                Funding
                Funded by: Fundo de Defesa da Citricultura 10.13039/501100010414
                Funded by: Fundação de Amparo à Pesquisa do Estado de São Paulo 10.13039/501100001807
                Funded by: H2020 European Research Council 10.13039/100010663
                Categories
                Plant Science
                Original Research

                Plant science & Botany
                asian citrus psyllid,biotechnology,hlb,sesquiterpenes,chemical ecology,citrus sinensis,transgenic,volatiles

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