Antimicrobial testing of the endophytic fungus Neofusicoccum australe

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.

从新西兰奥塔哥受病害影响的葡萄藤中分离得到的子囊菌真菌Neofusicoccum australe (ICMP 21498) 被用于本实验。将 ICMP 21498接种于42块马铃薯葡萄糖琼脂平板上，并在室温下培养3周。待真菌平板完全生长后，进行冷冻干燥（干重23.74克）并使用甲醇（2 x 800毫升）提取4小时，随后以氯仿（800毫升）过夜提取。将混合有机提取物在减压条件下浓缩，得到1.69克的橙色精油。对粗产品进行C8反相柱层析，以H2O/甲醇梯度洗脱，得到五个组分（F1-F5）。在提取和分馏之后，对各个组分进行了针对金黄色葡萄球菌ATCC 29213和埃希氏菌ATCC 25922的抗菌活性进行 broth microdilution 测试。测试在Thermo Fisher生产的透明、平底96孔板（NUN167008）上进行，通过Enspire酶标仪（Perkin Elmer）在600纳米处测量吸光度以确定每个孔中的细菌生长情况。提取物作为干燥样品获得，并溶解于DMSO制备成25毫克/毫升的溶液，然后进一步稀释至Mueller Hinton broth II (MHB) (Becton Dickinson, 212322)中，以实现最大浓度为2毫克/毫升。每个提取物（200微升）被加入96孔板顶部相邻的两个孔中。随后，将MHB（100微升）加入剩余的孔中，并将提取物溶液（100微升）以两倍稀释的方式沿板向下稀释，最后留出最后一行作为生长对照。在600纳米处的细菌光密度为0.01（约1 x 10^6 菌落形成单位[CFU]/毫升）的细菌稀释液被加入到所有孔中，这提供了最大浓度为1毫克/毫升和最小浓度为16微克/毫升。另有一块平板含有无菌对照行和一个含有DMSO的阴性对照孔。所有提取物中DMSO的最大体积比浓度为4%，因此阴性对照在相同浓度下进行测试。平板在37°C下以100 rpm的振荡条件下培养，并在不同时间点测量吸光度。