Calcium carbonate

For the production of unloaded alginate hydrogels, alginate (Sigma, 180947), D-(+)-gluconic acid δ-lactone (GDL, Sigma, G4750) and calcium carbonate (Fisher Scientific, S25220A and 10617581) were used as received. Alginate hydrogels were produced from aqueous alginate solutions (2–6% w/v) mixed with 10 mg/mL GDL. calcium carbonate (2.5–40 mg/mL) was suspended with distilled water and sonicated at room temperature for 10 min. The calcium carbonate suspension was then added to the alginate – GDL solution to produce different hydrogel formulations, named AC2.5 (Alginate 1%, CaCO₃ 2.5 mg/mL), AC5 (Alginate 1%, CaCO₃ 5 mg/mL), and AC10 (Alginate 1%, CaCO₃ 10 mg/mL). The gelling mixture was placed in 24 well plates (0.5 mL/well) and was left to gel at T = 37°C for 10–60 min. Gelling time was determined with the test-tube inversion assay, by tilting the plates containing the polymeric mixture every 2 min, until no flow was observed.

The blackleg-infected stubble pieces from the spring and fall field samples for each cultivar were cut into 2-mm pieces then surface sterilized in a 10% bleach solution for 2 min. Once rinsed in sterile water, the pieces were incubated on V8 juice agar [200 mL V8 juice (Campbells, Toronto, ON), 800 mL distilled water, 15 g Difco Agar Technical (BD Diagnostics Systems, Sparks, MD), 0.75 g calcium carbonate (Fisher Scientific, Fair Lawn, NJ), and 0.1 g streptomycin sulfate salt (Sigma-Aldrich, Saint Louis, MO)] amended with 10 mL of streptomycin sulfate. Two Petri dishes per stubble sample were placed on a light bench under cool white, fluorescent light at 22–24°C for 4–7 days. Samples of 10–20 stems were plated per field sample to try to achieve the goal of 10 isolates per sample. Around 5 days post plating, a single pycnidia was picked from the conidial ooze using a fine wire under a dissecting microscope and plated onto a fresh V8 juice agar plate as a single spore isolate; this was duplicated to ensure isolates were gathered from each stem sample. The pycnidia samples grew for 5–12 days on a light bench under the same conditions as the previous step.

Citric acid, yeast extract, malt extract, calcium lactate pentahydride, tween 80, sodium hydroxide, magnesium sulfate heptahydride, manganese sulfate monohydride, sucrose, glucose, fructose, peptone, calcium carbonate and agar were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cycloheximide was obtained from MP Biomedicals, LLC (Solon, OH, USA) and oxytetracycline dihydrate was obtained from Acros Organics (Springfield Township, NJ, USA). Lactic acid, Folin–Ciocalteu reagent, 4-dimethylaminocinnamaldehyde (DMAC), (±)-catechin, (–)-epicatechin and procyanidin B2 (PCB2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Procyanidin C1 (PCC1) and cinnamtannin A2 (CinA2) were obtained from Planta Analytica (New Milford, CT, USA). Solvents were ACS grade or higher.

Sodium alginate from Millipore Sigma (Cat# A1112) (LV), BASF (Hydagen 558P) (HV1), TIC (Algin 400) (HV2), and DuPont Danisco (GRINDSTED Alginate FD 155) (HV3) were used in this study (Table 1). Calcium hydrogen phosphate, succinic acid, sodium citrate, glacial acetic acid, sodium carbonate, β-D-glucose, sodium bicarbonate, L-serine, sodium hydroxide, ammonium hydroxide, hydrochloric acid, calcium carbonate, and methanol were purchased from Thermo Fisher. Schiff’s fuchsin sulfite reagent, sodium metabisulfate, periodic acid, anthrone, D-(+)-galacturonic acid, concentrated sulfuric acid, disodium 2,2-bicinchoninate (BCA), copper (II) sulfate, chloroform, 1-phenyl-3-methyl-5-pyrazolone (PMP), and D-(+)-mannuronic acid were purchased from Millipore Sigma. The polymannuronic acid (YP31737) and polyguluronic acid (YP03135) was obtained from Carbosynth Ltd. (Berkshire UK) at a minimum of 85% purity and 10% water content with an average molecular weight of 6–8 kDa.

Cargill, Inc. (Wayzata, MN, USA) generously provided raw unfermented cocoa beans, sourced from Hispaniola. The beans were stored in burlap sacks at 3.5 °C prior to use. LC-MS grade acetonitrile (ACN), LC-MS grade methanol (MeOH), citric acid, yeast extract, malt extract, calcium-lactate pentahydride, tween 80, sodium hydroxide, magnesium sulfate heptahydride, manganese sulfate monohydride, sucrose, glucose, fructose, calcium carbonate, agar, and water were obtained from Thermo-Fisher Scientific (Waltham, MA, USA). Glacial acetic acid, methanol, and acetone were obtained from VWR (Radnor, PA, USA). The standards of (−)-epicatechin (EC), (±)-catechin (C), and procyanidin B2 (PCB2) were obtained from ChromaDex (Irving, CA). Standards of procyanidin C1 (PCC1), cinnamtannin A2 (CinA2), and DP 5-9 purified from cocoa (purity: DP 3-5: 93–99%, DP6-9: 80–92%) were obtained from Planta Analytica (New Milford, CT, USA). Ammonium formate, Folin-Ciocalteu reagent, 4-dimethylaminocinnamaldehyde (DMAC), and α-glucosidase (from Saccharomyces cerevisiae) were obtained from Sigma-Aldrich (G5003, St. Louis, MO, USA). The solvents were ACS grade or higher.