A quantitative PCR assay for the detection and quantification of Septoria pistaciarum, the causal agent of pistachio leaf spot in Italy

PLoS One. 2023 May 19;18(5):e0286130. doi: 10.1371/journal.pone.0286130. eCollection 2023.

Abstract

Septoria leaf spot is one of the most widespread diseases affecting pistachio (Pistacia vera) in countries of the Mediterranean region. Septoria pistaciarum was recently confirmed as the causal agent of this disease in Italy. Currently, the detection of S. pistaciarum relies on isolation techniques. These require significant amounts of labor, and time for completion. Also, a reliable identification requires the sequencing of at least two housekeeping genes, in addition to the morphological observations. To accurately detect the presence and quantify S. pistaciarum in pistachio tissues, a molecular tool was necessary. We designed applicable primers that allow reliable amplification of the β-tubulin gene. The amplification of target DNA was highly efficient, with a 100% success rate, and the assay was able to detect as little as 100 fg/rxn of pure fungal DNA. When tested in artificial mixtures of plant and pathogen DNAs, the assay was able to detect the pathogen consistently at a limit of detection of 1 pg/rxn. The assay was also effective in identifying the pathogen in naturally infected samples, providing rapid detection in all symptomatic specimens. The resulting qPCR assay is an improved detection tool for accurate diagnosis of S. pistaciarum that can also contribute to better understand the population dynamics of the pathogen in the orchard.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ascomycota* / genetics
  • Italy
  • Pistacia* / genetics
  • Polymerase Chain Reaction / methods

Supplementary concepts

  • Septoria pistaciarum

Grants and funding

This work has been supported by “Programma Ricerca di Ateneo MEDIT-ECO UNICT 2020-2022 Linea 2-University of Catania (Italy)” and “Innovazioni di prodotto e di processo in campo e in post-raccolta per la valorizzazione del pistacchio in Sicilia”. PSR Sicilia 2014/2020—Misura 16, sottomisura 16.1 Cooperazione CleanPistachio—Innovazioni di prodotto e di processo in campo e in post-raccolta per la valorizzazione del pistacchio in Sicilia; CUP: G66D20000260009”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.