Table 1.
List of fungi and plants.
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.
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.
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.
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.
Table 2.
Validation of the limit of detection and repeatability of the qPCR method for Septoria pistaciarum detection.
Table 3.
Results of qPCR assay and fungal isolations from pistachio samples symptomatic and not.