Cell f program

The investigations were carried out in 2016–2017 on a private highbush blueberry plantation in the West Pomeranian Voivodship and on commercial plantations in 2019–2020 in the Mazovian and Lublin Voivodships, Poland. The total area of the inspected crops was over 50 ha. Monitoring the development of disease symptoms and the appearance of etiological signs was carried out every two weeks from mid-December to the end of October of the next year.
Blueberry (cv ‘Duke’) stem fragments with visible etiological signs were collected and investigated under a dissecting microscope (SZ11, Olympus, Tokyo, Japan). Next, stem fragments with visible pycnidia were rinsed with sterile distilled water (5 min) and kept in a humid chamber (2–5 days). Exuded conidia from the pycnidia were transferred onto a PDA medium in Petri dishes (10 cm dia) and incubated under white light at 20 °C. Based on morphology and an average size of 150 conidia (randomly selected from 24 obtained isolates), identification of the pathogen was performed. The conidia were examined under a BX50 light microscope (Olympus). Measurements of the size of the conidia were made using the CellF program (Olympus).

For the determination of sphere forming activity, adherently growing NSCLC cell lines H1299 and A549 were harvested and re-suspended in sphere-forming medium, containing 0.4% bovine serum albumin (Sigma-Aldrich), 10 ng/ml Epidermal Growth Factor (Sigma-Aldrich), 20 ng/ml Basic Fibroblast Growth Factor (VWR, Søborg, Denmark) and, in the case of H1299, 0.25 μg/ml human insulin (Sigma-Aldrich). To ensure a single cell suspension, the cells were filtered through 20 μm Steriflip filters (Millipore, Hellerup, Denmark), before being counted on a Cedex XS cell counter (Roche). The cells were seeded at densities of 250, 500, 1000, 2000, 4000 or 8000 cells/well in 150 μl medium in Corning^™ Ultra-Low Attachment 96-Well Plates (Sigma-Aldrich). At day 6, spheres were counted under the microscope. Three pictures were taken per well, and the diameter of spheres was determined with the Cell^F program (Olympus Europe). For the sphere-based viability assay, 20μl CellTiterBlue^™ (Promega, Nacka, Sweden) was added per well, and the plate incubated for 23 hours prior to viability readout with a Wallac Victor3 1420 Multilabel Counter (Perkin Elmer, Skovlunde, Denmark) at 560_ex/590_em nm. Experiments were performed with 10 wells per setting, and repeated three times.

Forty-one individual G. p. palpalis flies collected at the border region in Cameroon had wings in a state suitable for morphometric analysis. Wings were secured on a microscope slide using a drop of 1:100 diluted Faure’sche solution (33.3 mL distilled water, 13.3% v/v glycerol, 20 g gum arabicum, 33.3% v/v chloralhydrate) on the wing root. They were covered with a cover glass and dried overnight. Pictures were taken using a Olympus SZX16 binocular (Olympus, Hamburg, Germany) with upward light (Olympus KL1500 LCD, Olympus, Hamburg, Germany) employing 10× magnification and scale bar added using Cell^F program (Olympus, Hamburg, Germany). Nine landmarks were selected for analysis using the CLIC package [36 ] as described previously [37 (link)]. COO was used to digitalize the landmarks. Procrustes superimposition (centring of configuration of landmarks, scaling, rotation) and comparison of centroid sizes was done using MOG. Samples were segregated according to their genetic group or sex.