Evaluation of genetic diversity of Lactobacillus plantarum isolated from alfalfa silage using the BOX-polymerase chain reaction

The objective of this study was to evaluate the genetic diversity of isolates of Lactobacillus plantarum obtained from wilted and nonwilted alfalfa silage. Alfalfa was harvested at 50 d of regrowth and wilted for 6 h. Alfalfa was chopped into particles of 1.5 cm, packed in plastic bags of 25 by 35 cm, and sealed under vacuum. Lactic acid bacteria (LAB) were isolated from samples of fresh alfalfa plants without wilting, fresh forage (Day 0) wilted for 6 h, and its both nonwilted and wilted silages in different fermentation periods (1, 3, 7, 14, 28, and 56 d). The DNA of the strains of LAB was extracted by using a commercial kit (Wizard Genomic DNA Purification kit; Promega). The sequences of the 16S rRNA gene were amplified by PCR using the primers P027F and 1492R. The sequences of the isolates were compared with those available in the GenBank database and aligned using the BLASTn algorithm (basic local alignment search tool) for nucleotides. Of the 138 isolates identified, 58 were L. plantarum; therefore, the BOX-PCR was used to evaluate the diversity of these isolates using the primer BOX-A1. The PCR products were separated on 1.6% agarose gel at 60 V for approximately 2 h. The fingerprint of BOX-PCR was documented using the image display system (Kasvi, K33-312). Ten well-defined banding patterns of polymorphism were obtained between the evaluated isolates, with the same distributed in 10 distinct clades. In each clade, L. plantarum was considered to be present when clones showed a percent similarity equal to or greater than 90%. Only three L. plantarum strains presented no clones with a percentage of less than 90% similarity. No pattern of days of fermentation was observed and no wilting effect on the grouping of the isolates in the clades was observed. Supported by Fapemig, CNPq, and INCT-CA.