Metabarcoding Analysis of Phytophthora Diversity Using Genus Specific Primers and 454 Pyrosequencing

Author , , , , , ,
Date In Press
Publication Phytopathology
Key Words
AbstractA 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 8 nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in 7 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 5 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 diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.
Full Citation Prigigallo, M.I.; Abdelfattah, A.; Cacciola, S.O.; Faedda, R.; Sanzani, S.M.; Cooke, D.E.L.; and Schena, L. In press. Metabarcoding Analysis of Phytophthora Diversity Using Genus Specific Primers and 454 Pyrosequencing. Phytopathology. http://dx.doi.org/10.1094/PHYTO-07-15-0167-R.