title: Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.
abstract: Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.
authors.affiliation: Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
authors.forename: Maan
authors.initials: M
authors.lastname: Jawhari
authors.affiliation: Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
authors.forename: Peter
authors.initials: P
authors.lastname: Abrahamian
authors.affiliation: Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
authors.forename: Ali Abdel
authors.initials: AA
authors.lastname: Sater
authors.affiliation: Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
authors.forename: Hana
authors.initials: H
authors.lastname: Sobh
authors.affiliation: Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
authors.forename: Patil
authors.initials: P
authors.lastname: Tawidian
authors.affiliation: Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon. Electronic address: abujawyf@aub.edu.lb.
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Abou-Jawdah
mesh_terms: D000818:Animals; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis*; D007313:Insecta / Q000382:microbiology; D045582:Phytoplasma / Q000145:classification* / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D013045:Species Specificity
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Almond witches' broom phytoplasma; Mollicutes; Stone fruit trees; qPCR