Malt extract agar medium

The antifungal activity of the L. plantarum strains was assessed using the overlay method described by Magnusson et al. [55 (link)] with some modifications. The LAB strains were cultured in De Man, Rogosa and Sharpe (MRS) broth (Oxoid Ltd., Hampshire, UK) at 37 °C for 12 h. Then, they were inoculated with a central single streak of 2 cm on MRS agar plates, which were then incubated at 37 °C for 24 h under anaerobic conditions (GasPack anaerobic system, Sigma–Aldrich; St. Louis, MO, USA).
Fungal cultures from A. apis DSM 3116 and A. apis 3117 were cultured in Malt Extract Agar (MEA) medium (Oxoid Ltd., Hampshire, UK) under aerobic conditions at 28 °C for 5 days. Then, a 6 mm-diameter mycelial disc was removed, dissolved in physiological solution (0.9% NaCl) and vortexed for 5 min; 1 mL of the fungal suspension was then inoculated in a tube containing 10 mL of MEA soft agar (0.7% agar), which had been overlaid on the MRS agar plates previously inoculated with the LAB strains as described above. As a control, a plate containing MEA with the fungal suspension but without bacteria was used. After 72 h of incubation at 37 °C, the inhibitory activity of the L. plantarum strains was measured as the diameter (mm) of the clear zone around the bacterial streaks [56 (link)]. The tests were performed in triplicate.

The plant leaves and stems were washed with running tap water; leaf segments were equally cut by sterilized scalpel from the mid portions of healthy leaves to include the midrib. The cut segments were surface sterilized by immersing into the following series of solutions: sterile dis. H₂O for 60 s, 70% ethanol for 60 s, 2.5% sodium hypochlorite for 4 min, 70% ethanol for 30 s, and a final rinsing in sterile dis. H₂O three times. About 100 µL of the final rinse water was inoculated on Malt Extract Agar (MEA) medium (Oxoid, UK) to success the surface sterilization.
The sterilized plant leaves were cut into five segments (5 mm), and 20 leaf segments per individual plant were placed on the surface of MEA plate (five segment for each plate), supplemented with 0.05 g of streptomycin sulfate per 100 mL of medium to inhibit bacterial growth and incubated at 28 °C ± 2 °C. The plates were checked daily for any fungal growth; single isolates grown out from the tissues were re-inoculated on fresh MEA plates and maintained at 4 °C in MEA slants [20 (link)].

F. oxysporum f.sp. cubense race 4 (Foc TR4, isolate B2) strain, isolated in Hainan of China, was maintained on the PDA medium or Malt extract agar medium (Oxoid, Basingstoke, England) at 28 °C. For liquid culture, modified Czapeck liquid medium in which glucose was replaced with apple pectin were employed.