Computational protocol: Stemphylium Leaf Blight of Garlic (Allium sativum ) in Spain: Taxonomy and In Vitro Fungicide Response

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Protocol publication

[…] Fungal mycelia were removed from a 4-day-old PDA culture and genomic DNA was extracted following the method described by . The DNA concentration was determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). The nucleotide sequence of the complete ITS1–5.8S–ITS2 region was obtained using the primer pair ITS1/ITS4 described by .PCR reactions were performed in 25 μl volumes containing 1 μl of each primer (20 μM), 2.5 μl of 10× PCR buffer, 1 μl MgCl2 (50 mM), 0.5 μl dNTPs mix (40 mM; Biotools, Madrid, Spain), 0.15 μl Taq DNA polymerase (5 U/μl) (Biotools), and 2 μl (100 ng) template DNA. The cycling conditions for ITS1 and ITS4 primer pair used were in accordance with those described by . PCR products were detected in 1% agarose ethidium bromide gel in 1× TAE buffer (40 mM Tris-acetate and 1 mM ethylenediaminetetraacetic acid).PCR products were purified with the UltraClean™ PCR Clean-Up Purification kit (Mobio, Carlsbard, CA, USA) following the manufacturer’s instructions. Both strands were sequenced using an ABI 3730xl genetic analyzer (Applied Biosystems, Carlsbard, CA, USA) by Stab Vida Ltd. (Sintra, Portugal). Sequences were processed and edited using CLC 5.0 software (CLC Bio, Boston, MA, USA) and deposited in the GenBank database. [...] Seven isolates of S. vesicarium identified as described above were selected for subsequent studies. The effectiveness of nine fungicides was evaluated () in in vitro assays on PDA medium supplemented with the corresponding concentration of the chemical (1, 10, 100, and 1,000 ppm). Doses were selected according to and . Control assays were also included using PDA medium not supplemented with fungicides.All the plates (including the control) were inoculated with a 1-cm-diameter agar plug excised from the actively growing front of 7-week-old colonies of each isolate. Inoculated plates were incubated for 7 days at 25 ± 1°C in obscurity. Each combination of isolate, fungicide, and concentration was evaluated in six replicate plates. Radial growth was determined using a digital scalimeter at the end of the incubation period by calculating the mean of two colony diameters and then used to determine the percentage inhibition in comparison to the control assays. All data analyses were conducted with IBM SPSS Statistics version 21.0 (IBM Co., Armonk, NY, USA). Data were tested for normality, homogeneity of variances, independence and linearity. The data were also tested for significance of the main effects and interactions. The data were analyzed statistically using analysis of variance (ANOVA) and regression analyses. Post-hoc analyses were performed using Tukey test. In all cases, P < 0.05 was applied as the significance level.Percentage inhibition was plotted against log fungicide concentration for each fungicide and was fitted to a linear regression. Subsequently, the effective concentration at which mycelial growth showed 50% inhibition (EC50) was also calculated. Isolates were classified as showing high sensitivity with EC50 values ranging between 0.1 and 5 ppm, moderate sensitivity, with EC50 values between 5 and 50 ppm, low sensitivity, with EC50 values between 50 and 500 ppm, or insensitive, with EC50 values above 500 ppm. […]

Pipeline specifications

Software tools Biotools, SPSS
Applications Miscellaneous, Population genetic analysis
Chemicals Succinic Acid