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Nucleobytes 4peaks7/3/2023 The Kimura 2-Parameter model ( Kimura, 1980) was used to estimate evolutionary distance, and the gaps were treated as missing data. Alignments were checked and manually adjusted when necessary. The sequence alignment and phylogenetic analysis were conducted using MEGA version 6.0 ( Tamura et al., 2013). The sequences obtained were compared with rDNA D1/D2 and ITS data sequences from strains available in GenBank ( by using BLASTn, and sequences with ≥98% similarity were downloaded in FASTA format. The nucleotide sequences were visualized and edited using 4Peaks software ( ) and checked manually nucleotides with ambiguous positions were clarified. The amplified products were sent to Macrogen (South Korea) for purification and direct sequencing. Each PCR product (3 μL) was visualized on a 1.5% agarose gel stained with gel-red (Biotium). PCR blank reaction controls were incorporated. Cycling conditions were 5 min at 94 ☌ 35 cycles of 1 min at 94 ☌, 1 min at 55 ☌ and 1 min at 72 ☌ and a final elongation step of 2 min at 72 ☌. The reactions contained 1 μL of DNA extract 5 p moles of each primers 2.5 mM each dNTP 2 mM MgCl2 1X PCR buffer (KCl) 1 unit of Taq DNA polymerase (Thermo Scientific) and sterile distilled water. PCR amplifications of the LSU and ITS rDNA were performed in a final volume of 20 μL. Subsequently, the internal transcribed spacer region (ITS) and the D1/D2 domain of the large subunit ribosomal DNA 28S (LSU rDNA) were amplified using primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3) and ITS1 (5’-TGAACCTGCAGAAGGATCATTA-3’) ( White et al, 1990 Barnes and Szabo, 2007) as well as NL1m (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and NL-4m (5 ’-GGTCCGTGTTTCAAGACG-3 ’) ( O’Donnell, 1993). The acceleration voltage was 15 KV, the tilt was 0° to 90°, and the images were taken with a resolution of 3.024 × 2.304 pixels at a scanning speed of 12 min 54 s.įor molecular identification, dark lesions on foliar pinnae were collected, and DNA extraction was successful using an E.Z.N.A.® Insect DNA Kit (Omega Bio-Tek, Georgia, EEUU) according to the manufacturer's instructions. The working distance (WD) varied depending on the sample type. Sections of approximately 0.25 cm 2 were obtained for environmental scanning electron microscope (SEM) observations in an EVO LS 10 microscope (Carl Zeiss, Germany), placed in aluminum sample holders with carbon-contact-bearing adhesives, and analyzed under vacuum with variable pressure mode (VP) (chamber pressure 150 Pa (under vacuum) and column 2×10 -5 Torr (high vacuum)). Morphometric studies were conducted on additional herbarium samples of diseased leaves, and micrometric leaf sections were obtained for optical microscope observations. Lesions appeared isolated or else grouped on both side of the leaves. Symptoms were characterized initially by very small yellow lesions that turned dark brown in the center with fuzzy edges, affecting primarily the oldest leaves. canariensis was collected in Hanga Roa, Avenue Atanu Tekena (27° 09’08.48”S 109° 25’53.52”W 28 masl). In January 2015, a random sample of diseased leaves from palms of P. Plant Sample and morphologic identification
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