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.

Distribution and diversity of Phytophthora across Australia

“…The few metabarcoding studies of Phytophthora communities reveal an ever-increasing diversity. Prigigallo et al(2016) examined soil and roots from eight nurseries, detecting 25 Phytophthoraphylotypes. For each nursery at least twice as many species were detected using metabarcoding compared with using a cloning/Sanger sequencing approach.…”

Peer Review #1 of "Oomycete metabarcoding reveals the presence of Lagenidium spp. in phytotelmata (v0.1)"

“…Manuscript to be reviewed phylotypes (Prigigallo et al 2016). However, strategies based on genus specific primers do not offer the opportunity to globally assess oomycete communities.…”

“…Most of these oomycete barcoding efforts have been restricted to assessing phylum-specific primers on DNA preparations obtained from axenically-grown isolates, and few have transitioned to primer validation assays that (i) incorporated environmental sampling, and (ii) combined primers with specific sequencing strategies/platforms. Pioneer oomycete metabarcoding studies have favored the use of ITS primers, and the production of small size amplicons (Prigigallo et al 2016;Riit et al 2016;Sapkota & Nicolaisen 2015). Oomycete PeerJ reviewing PDF | (2019:06:38661:1:1:NEW 9 Sep 2019)…”

Modern approaches for early detection of forest pathogens are sorely needed in the United States

“…For example, using these approaches, Prigigallo et al (2016) detected several species of Phytophthora, including P. ramorum (causal agent of sudden oak death), in root and soil samples collected from eight nurseries in southern Italy. P. ramorum was previously reported in Italy only twice, once in imported plants and another time in grape vines; in both cases, the pathogen was immediately eradicated (Prigigallo et al, 2016).…”

“…For example, using these approaches, Prigigallo et al (2016) detected several species of Phytophthora, including P. ramorum (causal agent of sudden oak death), in root and soil samples collected from eight nurseries in southern Italy. P. ramorum was previously reported in Italy only twice, once in imported plants and another time in grape vines; in both cases, the pathogen was immediately eradicated (Prigigallo et al, 2016). Similarly, Vettraino et al (2015) detected foliar pathogens on seedlings of three European oak species planted in China on sites of comparable climate and plant community composition to those of their areas of origin in Europe.…”