Endophytic streptomycetes from higher plants with biological activity
a technology of endophytic streptomycetes and plants, which is applied in the field of endophytic streptomycetes from higher plants, can solve the problems of increased incidence of parasitic protozoan infections, toxic and ineffective antifungal agents currently available, such as amphotericin b, and weakened immune systems
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example 1
Isolation and Identification of the Endophytes of Kennedia nigriscans
[0162] Stems (0.5-1.0 cm is diameter) of Kennedia nigriscans were obtained near the Aborginal village of Manyallaluk, southeast of Katherine, Northern Territory, Australia at 14° 16′ 33″ South and 132° 49′ 45″ East. The stems were thoroughly treated with 70% ethanol and then the outer bark removed with a sterilized sharp blade. The inner pieces of the stem, containing the cambium, phloem, and xylem tissues, were plated on water agar in Petri plates. After incubation for at least 7-10 days at 23° C., individual fungal and bacterial colonies were removed with a sterile fine tipped needle and transferred onto potato dextrose agar (PDA) composed per liter of 39 g of potato dextrose agar.
[0163] The stems of Kennedia nigriscans (snakevine) yielded at least 24 endophytic microorganisms (FIG. 1). At least three of these were Pestalotiopsis spp., which is common for plants growing in tropical or semi-tropical environments...
example 2
Isolation of Other Biologically Active Endophytic Streptomycetes
[0168] The procedure described in Example 1 for selecting endophytic Streptomyces spp. was applied to other plants. Plates were continuously monitored for spore formation by stereo and light microscopy. In some cases, individual Streptomycete colonies were obtained only after 2-3 weeks, because of their small size. They also tended to have close proximity to the plant material and care was needed to obtain them in a pure culture. In other cases they were located beneath the plant material that was placed on the water agar plate. Because of the extended incubation time and their small size, these colonies were easily overlooked.
[0169] Using the methodology described, it was possible to acquire a number of other Streptomyces spp. Each of these was shown by microscopic techniques to be a Streptomycete primarily based on small spore size and the occurrence of spores in chains. In addition each of these has been cultured a...
example 3
Isolation Procedures for the Munumbicins
[0172] Small blocks of PDA containing Streptomyces munumbi were inoculated into 500 ml of PD broth (39 g of potato dextrose per liter) in a 2 liter Erlenmeyer flask and incubated for 3 weeks at 23° C. without shaking. The culture filtrate was then extracted three times with ½ volumes of methylene chloride. The extracts were pooled and then taken to dryness under flash evaporation at 40° C. The yield of dry material per liter was about 250 mg. Approximately 5 mg of the material was subjected to high pressure liquid chromatography (HPLC) on a Microsorb 60-8 Dynamax C-18 column, 250×10 mm (5 micron). The elution solvent was 20% tetrahydrofuran (THF):80% water programmed for 60 minutes to a final concentration of 40% THF:60% water and then held isocratically for 95 minutes. The elution stream was continuously monitored at 260 nm with a flow rate of 2 ml / minute.
[0173] Individual fractions were subjected to a bioassay test by placing them on a Pet...
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