Figure 3 Denaturing gradient gel electrophoresis (DGGE) fingerpri

Figure 3 Denaturing gradient gel electrophoresis (DGGE) fingerprints of fungal 18S rRNA gene fragments amplified from stem and leaf DNA templates obtained from four genotypes of Lippia sidoides using two sets of primers – EF4/ITS4 [27],[28] and ITS1f/ITS2 [24],[25]. Lanes 1, 2, 3, 4, 1′, 2′, 3′, 4′ – stem samples and 5, 6, 7, 8, 5′, 6′, 7′, 8′ – leaf selleck chemicals llc samples from genotypes LSID003, LSID006, LSID104 and LSID105, respectively. Lanes marked with M correspond to a 1 kb ladder (Promega). The letter F followed by numbers indicates bands that were extracted

from the gels for sequence analysis. The right side shows the corresponding dendrogram obtained after cluster analysis with mathematical averages (UPGMA) and Dice similarity coefficients JNK-IN-8 clinical trial comparing the fungal 18S rRNA gene fragments amplified from stem and leaf DNA templates obtained from four

genotypes of L. sidoides. The genotypes are represented by the three first numbers (LSID – 003, 006, 104 and 105), followed by C or F for stem and leaf samples, respectively, and T1 and T2 corresponding to the replicates. The DGGE gels were analyzed to evaluate the distribution of the species and to correlate the profiles obtained with the L. sidoides essential oil constituents. Principal component analysis (PCA) was used as described previously [31] using the PC-ORD statistical software [32]. Nucleotide sequence accession numbers The nucleotide sequences determined in this study for the culturable bacterial community were deposited in the GenBank database under accession numbers JX471071 – JX471146 and for the DGGE band sequences in the DDBJ database under accession numbers BCKDHA AB778305

to AB778478. Results The bacterial community in the stems and leaves of four L. sidoides genotypes as determined by a cultivation-dependent approach After disinfecting the stems and leaves of the different L. sidoides genotypes, serial dilutions of these samples were plated onto TSB agar plates for counting and selection of bacterial strains. Table 3 shows the determination of the colony selleck chemical forming units (CFU ml-1) in the stems and leaves. Across the four genotypes, the number of bacterial cells varied from zero to 1.6 × 103 CFU ml-1 in the leaves and 1.2 to 3.4 x 105 CFU ml-1 in the stems. Colonies presenting different morphologies in each plate used for counting were selected for further characterization. In total, 145 strains were collected: for stems, 37 were from LSID003, 36 from LSID006, 26 from LSID104 and 29 from LSID105; 17 strains were collected from the leaves of LSID105. The strains were then Gram-stained; 96 of the strains were Gram-negative and 49 were Gram-positive (Table 3). DNA from both Gram-negative and Gram-positive strains was then amplified using ERIC and BOX-PCR, respectively, for a preliminary screening of their diversity.

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