The soil was first fertilized with 3 l per m2 of aged bovine manu

The soil was first fertilized with 3 l per m2 of aged bovine manure and four L. sidoides genotypes (LSID003, LSID006, LSID0104 and LSID0105) showing Geneticin cost differences in their origin and the composition of the essential oils produced were planted in each row. The chemical composition of the essential oil produced by each genotype has been previously described by Blank et al. [16] and is summarized in Table 1. Drip irrigation was conducted daily. Table 1 Genotypes

of pepper-rosmarin ( Lippia sidoides Cham.), their origins, and the major constituents and yield of their essential oils Major chemical constituents (%)* Genotype Origin Thymol Carvacrol Oil yield (ml plant-1) LSID003 S63845 molecular weight Mossoró – RN (05° 08′ 28.3’’ S; 37° 23′ 58.0’’ W) 70.9 – 90.8 0.2 – 0.0 5.79 LSID006 Tabuleiro do Norte – CE (05° 14′ 05.4’’ S; 38° 11′ 35.0’’ W) 66.4 – 81.1 0.4 – 0.3 4.95 LSID104 Poço Redondo – SE (09° 58′ 09.2’’ S; 37° 51′ 50.3’’ W) 7.5 – 8.2 45.3 – 56.1 2.83 LSID105 Poço Redondo – SE (09° 58′ 12.9’’ S; 37° 51′ 49.2’’ W) 69.6 – 79.3 0.2 – 0.2 1.71 * These values correspond to individual measures performed in two consecutive

years [16]. Three plants of each L. sidoides genotype were harvested in the morning period with the plants in full flower, and 20 pieces of stems (approximately 30 cm in length) with leaves were sampled from each plant. Stem and leaf samples were Dorsomorphin surface sterilized by rinsing with 70% ethanol for 2 min, 2.5% sodium hypochlorite for 5 min, 70% ethanol for 30 sec and then washing three times with sterile distilled water. Only the stem samples were subjected to UV light exposure for 5 min prior to the final water wash. To check the efficiency of the disinfection

procedure, 100 μl of the water used in the last wash was plated onto Trypticase Phosphatidylinositol diacylglycerol-lyase Soy Broth (TSB) agar-containing plates and incubated for 5 days at 32°C. Samples that were not contaminated according to the culture-dependent sterility test were cut into pieces of approximately 5 cm, 3 g of each stem and leaf samples were homogenized with 10 ml of sterile distilled water in a sterilized mortar and pestle and used for counting and isolation of endophytic bacterial strains and for DNA isolation. Counting, isolation and DNA extraction of endophytic bacterial strains To determine the colony forming units per ml (CFU ml-1) in the stems and leaves of the different L. sidoides genotypes, each macerated sample (1 ml) obtained after disinfection was mixed with 9 ml of distilled water, and serial dilutions of these samples were plated onto TSB agar plates containing 1% nystatin (50 μg ml-1) and incubated for 5 days at 32°C. Colonies presenting different morphological characteristics in each plate used were selected for further purification. Bacterial cultures were stored at −80°C in TSB with 10% glycerol. All isolates were first divided based on their Gram staining characteristics. Genomic DNA was extracted from all bacterial strains using the protocol described by Pitcher et al. [17].

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