1100 series instrument

All reagents were obtained from commercial sources and used without further purification. Organic solvents were used as purchased. Water was of MilliQ^® quality. Buffers were prepared by standard procedures. A Crison pH-meter equipped with a 5014 Crison electrode was used for pH measurements at room temperature. Analytical thin-layer chromatography (TLC) was performed on 0.25 mm thick pre-coated silica gel plates (60 F254). UV-VIS absorption spectra were registered at room temperature on a V-560 JASCO spectrophotometer, using calibrated 2 mL quartz cuvettes, and for quartz slides, on a Cary 4000 UV-Vis spectrophotometer, using the solid sample holder. Steady-state fluorescence emission spectra were recorded with a FP-750 JASCO spectrofluorometer. ¹H-NMR and ¹³C-NMR spectra were recorded in deuterated solvents at 400 MHz on a Bruker DRX 400; δ values are reported in ppm and coupling constants are given in Hz. HPLC analyses for reaction monitoring were performed on an Agilent 1100 series instrument equipped with an LC-10AD VP pump and a G1314A UV-VIS detector, using a Sphereclone C18 column (4.6 × 150 mm, 5 μm). LC-MS analysis was conducted on an ESI-TOF 1260/6230DA (Agilent Technologies) in positive ion mode. IR spectra were recorded on Nicolet 5700 FT-IR + Smart performer spectrometer, mounting a Continuum FT-IR Microscope and on Bruker Optics TENSOR 27 FT-IR.

To measure the production of FK520 and FK523, 2 ml of fermentation broth was mixed with 3 ml of ethanol. After 30 min of ultrasonic extraction, and 10 min of centrifugation at 8,000 × g, the supernatant was filtered through a 0.2 μm syringe-driven filter (Spartan10463100, Whatman, England), suitable for organic solvents. The concentrations of FK520 and FK523 were quantified by liquid chromatography on a 1100 series instrument (Agilent, United States), equipped with a C-18 column (150 mm × 4.6 mm, 3.5 μm; Agilent). The mobile phase and gradient elution program were same as reported previously (Yu et al., 2019 (link)). The flow rate was 2 mL/min and the detection wavelength was 205 nm. The injection volume was 20 μL and the column temperature was 60∘C.
To measure the biomass concentration, mycelia from 5 ml of fermentation broth were washed once with 0.1 M-HCl solution and twice with Milli-Q water. After centrifugation for 10 min at 8,000 × g, the wet cell pellet was dried in an oven at 80∘C until a constant weight to measure the dry cell weight (DCW). The biomass concentration was defined as the ratio of DCW to the volume of the fermentation broth.

We collected five samples from each fern type every 10 days starting on 10 June (the last samples, on 18 August, were collected only nine days after the previous collection because of time constraints). In all but the last two collections, we took shelter and shelter-adjacent samples only from shelters that contained feeding larvae. In the last two collections, nearly all individuals had pupated, so we took samples from shelters that were not being actively fed upon. We immediately froze the samples at -80°C and then transported them on dry ice. We extracted phenolic compounds from the samples with 80% aqueous methanol and analyzed extracts by reversed phase HPLC on a 1100 series instrument (Agilent, Foster City, CA) equipped with a diode array detector and a C18 column (150 x 4.6mm, 3um, Gemini, Phenomenex, Torrance, CA) as described by Keinanen et al. [22 (link)]. We classified isolated peaks into compound classes based on their UV absorption characteristics and quantified hydroxycinnamic acid derivatives and flavonoids by integration of UV absorption signals at 320 and 360nm, respectively.

The preparative-scale HPLC was performed with an Agilent 1100 series instrument (Santa Clara, CA, USA) consisting of a quaternary pump, a degasser, a thermostated column compartment, a photodiode-array detector, a high-performance auto sampler, and a fraction collector, all controlled by Agilent ChemStation ver. B.01.01 software and equipped with a reversed-phase Luna C₁₈(2) column (Phenomenex, 250 × 21.2 mm, 5 µm, 100 Å). The column was operated at room temperature, the flow rate was maintained at 20 mL/min, using a binary mixture of water–acetonitrile (95:5 v/v) as eluent A and acetonitrile–water (95:5 v/v) as eluent B, both acidified with 0.1% formic acid. A volume of 900 µL 50 mg/mL extract was injected and the following gradients were used: standard gradient to obtain Fr.1: 0 min, 0% B; 30 min, 100% B; 35 min, 100% B; 36 min, 0% B and 5 min of equilibration; gradient VI to isolate active compounds of F. racemosa EtOAc extract: 0 min, 40% B; 50 min, 75% B; 51 min, 100% B; 56 min, 100% B; 57 min, 40% B; and 5 min of equilibration.