Pezicula ericae

Doyama, Kohei, Haruma, Toshikatsu, Hishiyama, Shojiro, Kato, Atsushi, Masuya, Hayato & Yamaji, Keiko, 2023, Isoavenaciol and 7 - hydroxy-isoavenaciol: Zn-chelating metallophores produced by root-endophytic Pezicula ericae in a Zn-accumulating plant, Aucuba japonica, Phytochemistry (113547) 206, pp. 1-7 : 6

4.5. Cultivation of P. ericae w12-25 and isolation of Zn-chelating metallophores

The fungal mycelia of P. ericae w12-25 were cultured in a 300-mL Erlenmeyer flask containing 200 mL of 1% malt extract (Difco) liquid media. 10 flasks were prepared and each flask was inoculated with 10 mycelial disks (5.5 mm i. d.) of P. ericae w12-25 freshly grown in 1% MA. The flasks were cultured for 31 d at 23 ◦ C with shaking at 150 rpm. After incubation, the culture filtrate (1850 mL) was extracted with ethyl acetate (617 mL × 3). The combined ethyl acetate extract was washed twice with a saturated solution of NaCl, dried over Na 2 SO 4, and evaporated to obtain 343 mg of a brown oily residue. For Zn-chelating metallophore detection, CAS-Zn agar media was prepared according to the procedure described by Alexander and Zuberer (1991), with the exception that ZnCl 2 (10 μmol L 1) was used instead of FeCl 3 ⋅6H 2 O (10 μmol L 1). For the culture filtrate and water layer, 200 μL of the concentrated samples (equivalent to 2000 μL of culture filtrate) were filtered through a 0.22-μm filter and aseptically applied to a sterile ring (stainless steel, 8 mm in diameter) placed on CAS-Zn media. For the ethyl acetate extract, 20 μL of the concentrated ethyl acetate layer (equivalent to 2000 μL of culture filtrate) was aseptically applied to a paper disk (8 mm in diameter), which was dried to remove the solvent and placed on CAS-Zn media. After 24 h of incubation under dark conditions at 23 ◦ C, the clear zone diameter (mm) was measured. Each test was conducted three times and the values were averaged.

Zn-chelating metallophores were isolated using column chromatography and preparative HPLC in combination with the CAS-Zn assay. Zn-chelating activity was mainly detected in the ethyl acetate layer; therefore, a portion of the crude extract (295 mg) was applied to a silica gel column (Wako sil C-200, 22 g, Wako, Osaka, Japan) and eluted with ethyl acetate-hexane (1:1) to separate the compounds (1 mL each). Each fraction was applied to a silica gel thin-layer chromatography (TLC) plate (TLC Silica gel 60 F 254, Merck, Frankfurt, Germany) and the TLC plate placed onto CAS-Zn media to detect the Zn-chelating metallophores. We also used UV light (254 nm) and 5% sulfuric acid in ethanol to confirm the presence of the metallophores in each fraction. Fractions 31–43, which exhibit Zn-chelating activities, were combined (150.6 mg, fraction B). Fractions 1–30 and 44–100 were combined as fractions A (1.4 mg) and C (38.2 mg), respectively. The Zn-chelating activities of fractions A, B, and C were also studied using paper disks, as described beforehand. A portion of fraction B (23.5 mg) was separated into three fractions (fraction a, 0.3 mg; fraction b, 11.7 mg; fraction c, 9.15 mg) using preparative HPLC (Prominence UFLC series: Shimadzu, Kyoto, Japan; Fraction collector: FRC 10A, Shimadzu) equipped with a diode array detector (SPD-M20A, Shimadzu) under the following conditions: Column, Inertsustain C18 (4.6 mm i. d. × 250 mm; GLSciences, Tokyo, Japan); eluent, water (A) and acetonitrile (B); flow rate, 1.0 mL/ min; temperature, 40 ◦ C; detection wavelength, 210 nm. The following gradient was used for the eluent system: 0 min, 50% A and 50% B; 0–20 min, 0% A and 100% B; 20–30 min, 0% A and 100% B. We collected three fractions (fraction a, RT 3.0–4.0 min; fraction b, RT 10.5–11.5 min; fraction c, RT 13.0–14.0 min). The Zn-chelating activities of fractions a, b, and c were evaluated using CAS-Zn media using paper disks as described beforehand. As fractions b and c exhibited Zn-chelating activities, we analyzed fraction c (compound 1) and fraction b (compound 2). The other small peaks did not exhibit Zn-chelating activities. In each step, we confirmed that the Zn-chelating activity returned to the same level as the original culture filtrate by mixing each fraction.