Alternaria alternata and Pseudomonas viridiflava strains belong to the microbial collection of the IPSP-CNR, Florence (Italy). The fungus A. alternata was grown on Potato Dextrose Agar (PDA, Oxoid Ltd., Basingstoke, UK) plates for 1 week at 25 °C. The bacterium P. viridiflava was grown on Nutrient Agar (Oxoid Ltd., UK) amended with 2.5 g/L of glucose (NGA) for 48 h at 27 °C. PDA and NGA media were also used to test the inhibition activity against the two pathogens of the main terpenic and phenolic compounds of rosemary leaf identified in this study. The selected compounds were: (+)-borneol, (−)-borneol, (+)-camphor, (−)-camphor, β-caryophillene, (+)-α-pinene, (−)-α-pinene, (+)-β-pinene, (−)-β-pinene, (−)-verbenone and rosmarinic acid. Copper sulphate was used as a positive control for both pathogens. The agar well-diffusion method was used to evaluate the antimicrobial activity of the different compounds [50].

PDA was prepared according to the manufacturer’s instructions, autoclaved at 121 °C for 15 min and poured into Petri dishes. After the medium solidification, two wells of 0.5 cm diameter were cut at the opposite side of the agar plates using a cork borer and 20 µL of the liquid or 20 µg of the solid compounds were placed into the wells. Twenty µL of sterile distilled water (SDW) was placed into the wells of control plates. Then, a 0.5 cm disk cut from the edge of the A. alternata colony was placed in the middle of each Petri dish. Three plates for each treatment were prepared. Plates were sealed with three layers of parafilm, incubated at 25 °C and monitored for two weeks. To evaluate the inhibition activity of the tested compounds against A. alternata, two diameters were measured for each fungal colony and data were compared to the diameter size of the control (SDW).

The 48 h-old culture of P. viridiflava was used to prepare a bacterial suspension in saline solution (0.8% NaCl). The bacterial concentration of the suspension was measured by a spectrophotometer and adjusted to an optical density of 0.1 at 530 nm, corresponding to 1 × 108 cfu/mL, and 1.5 mL of the suspension was mixed with 15 mL of NGA medium poured into the Petri dishes. After the medium solidification, two wells of 0.5 cm diameter were cut at the opposite side of the agar plates using a cork borer, and then 20 µL of the liquid or 20 µg of the solid compounds were placed into the wells. Plates with 20 µL of SDW in the wells were used as controls of bacterial growth. Three plates for each treatment were prepared. Plates were sealed with three layers of parafilm and then incubated at 27 °C for 48 h. The antibacterial activity of the tested compounds was evaluated measuring the size of the growth inhibition halo surrounding the wells.

A. alternata: The 96-well microplate dilution method reported by Hassan and Cutter [51] was used for (+)-α-pinene, (−)-β-pinene, verbenone, rosmarinic acid and copper sulphate, while for borneol, and the two camphor enantiomers that were not soluble in the liquid growth medium, the protocol described above (Section 4.6.1) was used to determine the MIC. For the microdilution method, the fungus was grown on PDA plates for one week, then conidia were collected by scraping the agar surface and suspended in potato dextrose broth (PDB). Conidia were enumerated by a hemocytometer and concentration was adjusted to 1 × 106 conidia/mL. The antimicrobial compounds were dissolved in methanol to obtain a 10% solution. This solution was serially diluted 1:1 in PDB until the concentration of 0.039% [51]. One hundred µL of each dilution were placed in the microplate wells, then 100 µL of the conidial suspension was added to each well. Each dilution was tested in triplicate. The positive control was represented by 100 µL of the conidial suspension plus 100 µL of PDB, while the negative control consisted of 200 µL of sterile PDB. A control to test methanol inhibition activity was also carried out. Plates were incubated at 25 °C for 48 h and fungal growth was determined both by visual reading and by a spectrophotometer at 450 nm. The MIC value for the two camphor enantiomers and borneol was considered as the highest dilution of each compound that determines a significant reduction of the growth of A. alternaria colony with respect to the control (SDW).

P. viridiflava: The 96-well microplate dilution method was used for all compounds. The bacterium was grown for 48 h in plates containing NGA medium, then the bacterial suspension was prepared in nutrient broth amended with 0.25% glucose (NGB) following the procedure described in Section 4.6.2. On the basis of the results obtained from the test of in vitro antimicrobial activity, only the compounds that were active against P. viridiflava (i.e., (+)-α-pinene, verbenone and rosmarinic acid) were assayed for the MIC determination. Only the enantiomer (+)-α-pinene was tested, since inhibition activity had resulted not different for the two forms of this compound. The dilutions of the different compounds and next steps of the procedure were the same of that previously reported for A. alternata, using NGB as a growth medium. The positive control was represented by 100 µL of the bacterial suspension plus 100 µL of NGB, while the negative control consisted of 200 µL of sterile NGB. A control to test methanol inhibition activity was also carried out. Plates were incubated at 27 °C for 24 h, then the bacterial growth was first recorded by a visual reading of the plates. Then, 30 µL of a resazurin solution (0.015%) was added to each well and plates were incubated for 3 h at 27 °C. The color of the suspensions contained in each well was recorded.

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