Marisia - Maros Megyei Múzeum Évkönyve 35/2. (2015)
Botany
Antibacterial activity ofcompouds derivedfrom bidroethanolic extracts of Juglans nigra Pseudomonas syringae, Gram negative bacteria, phytopathogenic, facultative anaerobic, has been isolated from soil as a saprophyte on decomposing organisms. Syringae denomination derives from the name of tree lilac (Syringa vulgaris) from which was first isolated. For an optimal development, it was needed a high humidity and temperature between 12—25 °C.Its action is manifested by staining wood and producing ulcers compared with Pseudomonas tumefaciens that produce tumors wood. In some cases, the pathogenesis produced by these bacteria can easily be confused with the one produced by fungi such as Cytospora spp. or Dothichiza spp. alsoproducing wood staining olive green and then yellowish-orange neconturate. On these spots appear fructification but without producing suppuration. Due to the bacterian attack, the wood is split thus enabling the fungal and xylophagous attack. The bacteria do not attack cellulose. These bacteria act only on the pectin. Due tocambios destruction, deformation is produced of wood [5]. Materials and methods In order to study the antibacterial activity of the extract of Juglans nigra Hydroethanolic, we selected two species of Gram-positive bacteria (Bacillus pumilus and B. subtilis) and Gram-negative species (Pseudomonas syringae), which were isolated on the surface of contaminated wood. Strains identification was made by means of automatic method VITEK® 2. Detection of soluble virulence factors We used specific mediums to highlight the external enzyme production. Also, we used to detect gelatinase nutrient agar medium with gelatin. The positive reaction occurs as a result of precipitation marked by a halo around the colonies due to hydrolysis of gelatin.In order to detect caseinase, we used simple agar medium with the addition of milk. The test is considered positive when around the colonies an ivory precipitate forms to calcium paracaseinat. DN-ase acts on the microbial DNA releasing mono- or dinucleotides. It is used the DNA agar with or without toluidine blue. Agar is inoculated into streaks or spots, and the hydrolysis of the DNA is revealed by the appearance of a pink halo around the spot of culture, while the remainder of the medium remains colored in blue. We also use agar medium with the addition ofTWEEN 80, 60 or 40, to detect lecithinase. The presence of lecithinase lead to the appearance of insoluble oleate crystals Ca24 (crystals formed from the released fatty acids and Ca2+). Hydroethanolic extraction of Juglans nigra Juglans nigra leaves were harvested in May and June in Botanical Garden Bucharest. After drying, 300 g of milled plant were macerated in 700 mL of ethanol-water (v / w 5: 2). The solutions were prepared in an amber glass which was stored in the dark at 4°C for a period of 10 days, being agitated every day. After 10 days, the macerate of the plant was placed in a rotary evaporator for 10—15 minutes after which the supernatant was removed using a filter Watthmann no. 41. Lastly we obtained stock solution of which we made dilutions. To calculate the MIC (minimum inhibitory concentration), we used sets of sterile plastic plates, disposable, containing 96 wells, spread over 12 rows, flat bottom and a capacity of approximately 300 ml. As a medium for growth of the bacterial suspension, we used BHI medium. The chemical characterization Hydroethanolic extracts was performed using thin layer chromatography. Thin-layer chromatography (TLC) and high performance (HPTLC) is a 43
