The organic extracts had been concentrated to dryness making use of vacuum evaporator and resuspended in 0. 5 ml of methanol. The 10 fold concentrated extracts had been cen trifuged and 5 ul of each sample was subjected to HPLC on a 5 um Nucleosil C18 column with 0. 1% o phosphoric acid as solvent A and acetonitrile as solvent B at a linear gradient at a movement fee of 0. 85 ml/min. The chromatographic system consisted of the 1090 M liquid chromatograph outfitted by using a diode array de tector in addition to a Kayak XM 600 ChemStation. Numerous wavelengths monitoring was carried out at 210, 230, 260, 280, 310, 360, 435 and 500 nm and UV visible spectra were mea sured from 200 to 600 nm. HPLC ESI MS analysis of Streptomyces secondary metabolites HPLC DAD ESI MS evaluation was carried out with an Agilent 1200 HPLC series equipped which has a binary HPLC pump, autosampler and diode array detector, and an Agi lent LC/MSD Ultra Trap Technique XCT 6330.
The Samples have been sepa rated on the three selleckchem um Nucleosil C18 column and separated by linear gradient elution from 10% eluent B to 100% eluent B in 15 minutes at a flow price of 400 ul/min. Wavelength monitoring was performed at 230 nm, 260 nm, 280 nm, 360 nm and 435 nm. MS Instrument settings have been as follows, Ionization, ESI, Mode, Ultra Scan, Capillary voltage, three. 5 kV, Temperature, 350 C, Tuning mass, m/z 400. The pro duction levels with the following metabolites have been quanti fied determined by the comparison of their peak spot with that obtained by HPLC analysis of recognized amount of pure substance, Acta 2930 B1, actiphenol, cyclohexi mide, ferulic acid. Inoculation of Arabidopsis thaliana with streptomycetes and with Alternaria brassicicola, chlorophyll fluorescence and ailment index measurements Sterile Arabidopsis thaliana Col 0 seeds had been positioned on half power MS medium containing 1% glucose and 0.
8% agar for germination. Soon after 7 days, seedlings were transferred to MS with 2% agar. To develop seed lings in an upright place with leaves free of charge from con tact with the agar find more info surface, the best third of sound medium was eliminated in the Petri dish. Seedlings have been placed with roots on the agar and leaves from the airspace. Petri dishes have been then stored inside a vertical place to permit root development to the agar surface. Plants have been cultivated at 22 C, 200uE/m2s by using a light/dark cycle of 8/16 h. Soon after 7 days, roots were inoculated with AcM 9, AcM11, AcM29, AcM29, AcM30 and constructive control Streptomyces GB 4 two. Bacterial cultures grown in ISP 2 medium for four to 5 days had been separated from development medium by centrifugation, washed 3 times in sterile water and diluted to an OD of 0.