title: Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.
abstract: A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Maria I
authors.initials: MI
authors.lastname: Prigigallo
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abdelfattah
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Santa O
authors.initials: SO
authors.lastname: Cacciola
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Roberto
authors.initials: R
authors.lastname: Faedda
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Simona M
authors.initials: SM
authors.lastname: Sanzani
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: David E L
authors.initials: DE
authors.lastname: Cooke
authors.affiliation: First, second, and seventh authors: Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy; third and fourth authors: Dipartimento di Agricoltura, Alimentazione e Ambiente, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy; fifth author: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi Aldo Moro, Via G. Amendola 165/A, 70126, Bari, Italy; and sixth author: The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: L
authors.initials: L
authors.lastname: Schena
mesh_terms: D020132:Anticipation, Genetic; D058893:DNA Barcoding, Taxonomic; D004271:DNA, Fungal / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D021141:Nucleic Acid Amplification Techniques; D010838:Phytophthora / Q000145:classification / Q000235:genetics
chemical_list: D004271:DNA, Fungal
keywords: metagenomic analyses; ornamental nurseries