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2020_02_06_ExtractsAndFractions_TidyData.xlsx (38.94 kB)

Antimicrobial testing of the endophytic fungus Neofusicoccum australe

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posted on 2020-02-18, 23:27 authored by Siouxsie WilesSiouxsie Wiles

The ascomycete fungus Neofusicoccum australe (ICMP 21498) was isolated from diseased grapevines in Aotearoa New Zealand. Forty-two Potato Dextrose Agar plates were inoculated with ICMP 21498 and incubated at room temperature for 3 weeks. Fully grown fungal plates were freeze-dried (23.74 g, dry weight) and extracted with MeOH (2 x 800 mL) for 4 hours followed CH2Cl2 (800 mL) overnight. Combined organic extracts were concentrated under reduced pressure to afford an orange oil (1.69 g). The crude product was subjected to C8 reversed-phase column chromatography eluting with a gradient of H2O/MeOH to afford five fractions (F1–F5). Following extraction and fractionation, broth microdilution testing of the extracts was performed to ascertain the antimicrobial activity of each of the fractions against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. Tests were performed in a clear, flat bottom 96 well plate (Thermo Fisher, NUN167008) and bacterial growth determined within each well by measuring absorbance at 600 nm using an Enspire plate reader (Perkin Elmer). Extracts were obtained as dried samples which were dissolved in DMSO to make a 25 mg/mL solution and then further diluted into Mueller Hinton broth II (MHB) (Becton Dickinson, 212322) to achieve a maximum concentration of 2 mg/mL. Each extract (200 µL) was added to two adjacent wells along the top of the 96-well plate. MHB (100 µL) was then added to the remaining wells and extract solution (100 µL) serially diluted two-fold down the plate and discarded, leaving the last row as a growth control. Aliquots of bacteria at an optical density at 600 nm of 0.01 (approximately 1 x 106 colony forming units [CFU]/mL) were then added to all the wells. This gave a maximum concentration of 1 mg/mL and a minimum concentration of 16 µg/mL. An additional plate contained a sterile control row and a negative control well containing DMSO. The maximum volume/volume concentration of DMSO in all extracts was 4%, therefore the negative control was tested at an identical concentration. Plates were incubated at 37 °C with shaking at 100 rpm and absorbance was measured at various time points.



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