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

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

Mounira Inas Drais  1 , Giorgio Gusella  2 , Angelo Mazzaglia  1 , Giancarlo Polizzi  2
Affiliations

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

Mounira Inas Drais et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Alignment of primers designed on the β-tubulin gene of Septoria pistaciarum and the closest available sequences of species of different genera.
The mismatching nucleotides of primers to the sequences of S. pistaciarum (first line) are reported. The numbers above the alignment refer to positions in S. pistaciarum strains; The accession numbers of the sequences from NCBI Database are reported below: Septoria pistaciarum MZ285918.1, MZ285917.1, MZ285914.1, MZ285913.1, MZ285915.1, KF442739.1, KF442737.1, KF442741.1, KF442740.1, KF442738.1; Septoria hippocastani KF252907.1, KF253031.1; Septoria dispori MT984358.1, MT984357.1; Septoria linicola MZ073925.1; Septoria astralagi: KF252821.1; Septoria protearum: MT984349.1; Septoria rumicum: KF252998.1. Septoria rudbeckiae: MN105980.1; Septoria longipes: MT984351.1; Septoria passifloricola: MK643050.1, MK643054.1, MK643053.1, Septoria sanguisorbigena: MT984352.1; Septoria pileicola: MT984354.1; Septoria aegopodina: KU921453.1; Septoria tormentillae: KT861479.1; Septoria cannabis: MW556608.1; MW556606.1; Septoria anthrisci: KY853401.1; Cercospora sp.: KF252781.1.
Fig 2
Fig 2. Results of optimization experiments.
A) Results of the gradient PCR to select the best annealing temperature from 55 to 61.7°C for the qPCR reaction. B) Optimization of primer concentrations for the qPCR assay for Septoria pistaciarum; the best performing concentration for the target gene is highlighted in bold.
Fig 3
Fig 3. Amplification curves.
A) Amplification curves of the qPCR sensitivity test using 10-fold serial dilutions of Septoria pistaciarum pure DNA ranging from 10 ng/rxn to 10 fg/rxn. B) Amplification curves of the qPCR sensitivity test using 10-fold dilutions of spiked fungal DNA with Septoria pistaciarum with 50 ng of plant DNA.
Fig 4
Fig 4. Standard curves.
A) Standard curve obtained with 10-fold dilutions of pure DNA extracted from Septoria pistaciarum strain S7 (6 replicates) and related statistics. B) Standard curve obtained with 10-fold dilutions of fungal DNA spiked with 50 ng of plant DNA (6 replicates) and related statistics.
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