Screening of herbicidal compounds using B. chinensis
The dicot B. chinensis was selected for phytotoxicity testing to screen the fungal strains that produce herbicidal compounds. Briefly, 5 high-quality seeds were transferred to each well of the 96-well plate, with 50 μL water containing crude extract or purified compounds in the concentration range 0.2–2 mg mL−1 or 5–25 nM. Cultures were incubated at 25 °C under light/dark cycle of 16 h/8 h in a growth chamber. Each condition was triplicated and repeated at least three times. The germination rate was examined for visual signs of phytotoxicity. 2,4-Dichlorophenoxyacetic acid (2,4-D) was used as positive control.
Isolation and identification of harzianum A and B
The fungal strain used in this study was T. brevicompactum CGMCC19618. Preparation of fermentation extracts, analysis by liquid chromatography-mass spectrometry (LC–MS). HPLC-HRESIMS and MS–MS spectra were acquired on an Agilent 1290 Infinity II HPLC coupled with an Agilent QTOF 6530 instrument. 1H and 13C NMR spectra were obtained on an Agilent DD2 spectrometer at 600 MHz for 1H NMR and 150 MHz for 13C NMR. Two-liter fermentation broth was extracted using ethyl acetate. The extracts were first subjected to silica gel (25 g) column chromatography and eluted with a gradient of chloroform/methanol to yield five fractions (Fraction A, v/v 100:0, 250 mL; Fraction B, v/v 99:1, 250 mL; Fraction C, v/v 98:2, 250 mL; Fraction D, v/v 95:5, 250 mL; Fraction E, v/v 90:10, 250 mL). Each of these fractions was analyzed by HPLC–MS. Fractions containing the target compounds were subsequently purified by semi-preparative HPLC on an Agilent Eclipse XDB-C18 reversed-phase column (5 mm, 9.4 mm × 250 mm) using an Agilent 1260 Infinity II system.
Harzianum A (1): 1H NMR (400 MHz, methanol-d4) δ 7.90 (1H, dd, J = 15.1, 11.6 Hz, H-4′), 7.39 (1H, dd, J = 15.3, 11.3 Hz, H-6′), 6.78 (1H, t, J = 11.4 Hz, H-3′), 6.71 (1H, dd, J = 15.0, 11.3 Hz, H-5′), 6.07 (1H, d, J = 15.3 Hz, H-7′), 5.82 (1H, d, J = 11.4 Hz, H-2′), 5.70 (1H, dd, J = 7.9, 3.5 Hz, H-4), 5.38 (1H, d, J = 5.3 Hz, H-10), 3.78 (1H, d, J = 5.2 Hz, H-2), 3.70 (1H, d, J = 5.3 Hz, H-11), 3.11 (1H, d, J = 3.9 Hz, H-13α), 2.91 (1H, d, J = 3.9 Hz, H-11β), 2.57 (1H, dd, J = 15.4, 7.8 Hz, H-3α), 2.04 (1H, m, H-3β), 2.00 (2H, m, H-8), 1.94 (1H, m, H-7β), 1.71 (3H, s, H-16), 1.48 (1H, m, H-7α), 0.97 (3H, s, H-15), 0.73 (3H, s, H-14); 13C NMR (100 MHz, methanol-d4) δ 169.8 (C-8′), 167.1 (C-1′), 145.0 (C-6′), 144.3 (C-3′), 141.2 (C-9), 139.4 (C-5′), 136.9 (C-4′), 126.1 (C-7′), 121.5 (C-2′), 119.8 (C-10), 80.4 (C-2), 76.6 (C-4), 72.0 (C-11), 66.6 (C-12), 50.5 (C-5), 48.7 (C-13), 41.5 (C-6), 37.6 (C-3), 28.9 (C-8), 25.5 (C-7), 23.3 (C-16), 16.3 (C-15), 6.5 (C-14); (+)-HRESIMS m/z 401.2003 [M+H]+ (calcd. for C23H29O6, 401.1964).
Harzianum B (2): 1H NMR (400 MHz, methanol-d4) δ 7.40 (1H, dd, J = 15.4, 10.0 Hz, H-6′), 7.34 (1H, dd, J = 15.4, 10.0 Hz, H-3′), 6.79 (2H, d, J = 10.0 Hz, H-4′ and H-5′), 6.11 (1H, d, J = 15.4 Hz, H-7′), 6.07 (1H, d, J = 15.4 Hz, H-2′), 5.69 (1H, dd, J = 7.9, 3.5 Hz, H-4), 5.38 (1H, d, J = 5.3 Hz, H-10), 3.75 (1H, d, J = 5.2 Hz, H-2), 3.70 (1H, d, J = 5.3 Hz, H-11), 3.10 (1H, d, J = 3.9 Hz, H-13α), 2.90 (1H, d, J = 3.9 Hz, H-11β), 2.55 (1H, dd, J = 15.4, 7.8 Hz, H-3α), 2.04 (1H, m, H-3β), 2.00 (2H, m, H-8), 1.94 (1H, m, H-7β), 1.71 (3H, s, H-16), 1.48 (1H, m, H-7α), 0.96 (3H, s, H-15), 0.72 (3H, s, H-14); 13C NMR (100 MHz, methanol-d4) δ 169.8 (C-8′), 167.7 (C-1′), 144.9 (C-6′), 144.5 (C-3′), 141.2 (C-9), 138.8 (C-5′), 138.2 (C-4′), 126.2 (C-7′), 125.2 (C-2′), 119.8 (C-10), 80.5 (C-2), 76.8 (C-4), 72.0 (C-11), 66.6 (C-12), 50.5 (C-5), 48.6 (C-13), 41.6 (C-6), 37.4 (C-3), 28.9 (C-8), 25.5 (C-7), 23.3 (C-16), 16.3 (C-15), 6.3 (C-14); (+)-HRESIMS m/z 401.2003 [M+H]+ (calcd. for C23H29O6, 401.1964).
Phytotoxicity assays of harzianum A and B
The seeds of B. chinensis, O. sativa L. cv. Nipponbare, S. viridis L. Beauv. or E. crusgalli L. Beauv. were surface sterilized in 0.5% NaOCl (3 min), and then rinsed six times in distilled water. 1 μL of HA and HB (1–2) was applied to five seeds at doses from 1 ng to 100 μg per seed (Vinale et al. 2009). Equal volume of methanol was added to the control seeds. The treated seeds were sown in Petri dishes (100 × 150 mm) containing agar medium (0.8% w/v). Plates were placed vertically in the growth chamber (25 ± 2 °C temperature, 55% HR, and 16 h/8 h light/dark circle) to promote geotropic root growth. After treatment, seedlings were collected and separated into shoot and root. Growth inhibition was measured as reduced root or shoot length relative to the negative control (1 μL methanol). 2,4-D was used as positive control in all experiments. Each treatment was repeated two times.
Pot assays of harzianum A and B
The herbicidal activity of HA and HB (1–2) mixture was tested against the dicot B. chinensis in pot assays with 2,4-D as positive control. The test compounds were dissolved in 10 μL methanol and diluted with 5 mL water to the required concentrations, and applied to pot-grown plants in a greenhouse. Plastic pots with a diameter of 9.5 cm were filled with garden soil to a depth of 8 cm. Approximately 15 seeds of the tested weeds were sown in the soil at a depth of 1–2 cm and grown at a temperature of 25 °C in a greenhouse. The air relative humidity was 50%. For pre-emergence assays, 5 mL of the HA and HB solution was sprayed to the soil. For post-emergence assays, the B. chinensis was treated at the two-leaf stage with designated dosages with three replicates. The solvent (10 μL methanol in 5 mL water) was used as negative control. Herbicidal activity was evaluated visually 6 days post treatment.