Whatman grade 1 filter paper

Standard 17 glass fiber, Whatman filter paper Grade 1, and nitrocellulose (FF120HP) membranes were acquired from GE Healthcare Life Sciences (Bangalore, India). Supporting plastic materials: 0.18 mm-thick transparency sheets and 2.15 mm-thick transparent acrylic sheets were acquired locally. A double-coated pressure-sensitive adhesive film (PSA; 3M^TM 9731) was used to secure stacked paper and plastic layers. All materials were cut using a 50 W CO₂ laser in a VLS 3.60 laser engraver (Universal Laser Systems, Scottsdale, AZ). All designs were created in AutoCAD (Autodesk, San Rafael, CA).

Chitinase from S. griseus, HRP, cellulase from A. niger, N-acetyl-D-glucosamine and 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium salt were purchased from Sigma-Aldrich (St Louis, MO, USA). One unit of HRP is defined as the amount of enzyme that will form 1.0 mg of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20°C. One unit of chitinase is defined as the amount of enzyme that liberates 1.0 mg of N-acetyl-D-glucosamine from chitin per hour at pH 6.0 at 25°C in a 2-hour assay. One unit of cellulase is defined as the amount of enzyme that liberates 1.0 μmol of glucose from cellulose in 1 hour at pH 5.0 at 37°C. 4-Aminoantipyrine was purchased from Acros Organics (Geel, Belgium), D-glucose monohydrate was purchased from Merck (Darmstadt, Germany) and cellotetraose, chitobiose and chitotetraose were purchased from Dextra (Reading, UK). Cellobiose (purity >98%) was purchased from TCI Europe (Zwijndrecht, Belgium). Whatman filter paper grade 1 was purchased from GE Healthcare Life Sciences (Little Chalfont, UK). E. coli ORIGAMI2 DE3 was purchased from EMD Millipore (Billerica, MA, USA) and pET-SUMO vector was obtained from Invitrogen (Carlsbad, CA, USA).

P. insidiosum strain Pi–S or Basidiobolus ranarum strain KU30017.1 maintained on SD agar were subjected to the preparation of culture filtrate antigen (CFA). Several small agar pieces containing the colony of either microorganism were cultured in SD broth and incubated with shaking (50 rpm) at 37 °C for 10 days. The culture medium was filtrated through a Whatman filter paper grade 1 (GE Healthcare, Chicago, IL, USA) and then a 0.22 μm-pore size Durapore filter membrane (Merck, Darmstadt, Germany) to collect CFA before adding the proteinase inhibitors EDTA and PMSF (at the final concentration of 1 mM each) and concentrating by an Amicon Ultra-15 tube (10 K molecular weight cutoff; Merck, Darmstadt, Germany). Protein concentration was determined using a Qubit Protein Assay kit (Invitrogen, Carlsbad, MA, USA). The obtained P. insidiosum CFA (PiCFA) and B. ranarum CFA (BrCFA) were stored at −80 °C until use.

The powdered arils of M. cochinchinensis were treated with 200 mL liquid nitrogen and stirred until liquid nitrogen was completely evaporated. After evaporation, the aril powders (5 g) were extracted with diethyl ether: CHCl₃ = 1:2 (300 mL) in a 1-L beaker overnight. The suspension was filtered using Whatman filter paper grade 1 (GE healthcare, Chicago, IL, USA). The same procedure was repeated after adding diethyl ether and CHCl₃. The suspension was combined and evaporated under reduced pressure to obtain an oily extract (332.9 mg, 6.65%).

Potato cultivars Agria (Ag), AR-03-3410 (AR), Hansa (Ha), Jazzy (Ja), Luminella (Lu), Melody (Md), Melrose (Mr), Perline (Pe), Piatlina (Pi), Piccolo Star (PS), Postiglione (Po), and Ricciona di Napoli (Ri) were cropped in open field at density of 5 tubers m⁻². Fresh leaves were collected when plants reached the stage of full flowering identified by 2-digit code 69/7N on the BBCH (Biologische Bundesantalt, Bundessortenamt and CHemische Industrie, Germany) scale [18 ] and dried at 70 °C until constant weight. Plant materials were ground to fine powder and extracted by soaking 10 g in 100 mL ethanol (50% vol.) for 24 h at room temperature. The mixtures were filtered (Whatman Grade 1 filter paper, GE Healthcare Life Sciences, Uppsala, Sweden), and the solvent evaporated under vacuum (rotary evaporator at 30 °C, Heldolph, Schwabach, Germany). Dry extracts were dissolved in distilled water at concentration of 200 mg mL⁻¹, and after filter-sterilization (filter 0.22 µm pore size, Merck Millipore Ltd., Darmstadt, Germany), were stored at −20 °C until use.

A total of 120 g of ground M. tenuiflora bark was extracted with 4 L of distilled water under mechanical agitation for 15 h at room temperature. The extract was centrifuged in a sigma 6–16 k centrifuge at 15,000 relative centrifugal field for 15 min (Sigma Laborzentrifugen Gmbh, Osterode am Harz, Germany) and filtered through Whatman grade 1 filter paper (GE Healthcare Life Sciences, Vélizy-Villacoublay, France). Filtrates were adjusted to 120 mL with distilled water and sterilized in an autoclave at 121 °C for 20 min (SMI group UNICOM, Montpellier, France). The final sterile extract was stored at +4 °C until use. Stock filtrates were diluted to experimental concentrations (2 g/L) with distilled water.

This study used two sets of isolates. The first set included the interfertile fungal isolates F. circinatum (CMWF 350; FSP34) and F. temperatum (CMWF 389; Netza9) and their 94 F₁ hybrid progeny (Supplementary File S4, Table S1) that were previously used for GLM construction [26 (link)]. The second set included 19 genetically diverse F. circinatum isolates that were collected from various locations in South Africa, Mexico, California, and Florida during previous studies (Table 1).
Isolates were grown on one of two types of growth media, potato dextrose agar (PDA; 15 g/L; Biolab) and pine-tissue-derived agar medium. The latter was prepared by chopping the above-ground parts of 6-month-old Pinus patula seedlings into small pieces, of which 40 g was added to 1 L of H₂O and autoclaved at 120 °C for 30 min. The extract was filtered through Whatman Grade 1 filter paper (GE Healthcare Life Sciences) to remove the plant debris. The filtrate was mixed with malt extract (20 g/L; BD Difco^TM) and agar (10 g/L; BD Difco^TM), autoclaved, and poured into plates. The medium is referred to as PMEA (for pine-tissue extract containing malt extract agar), and Supplementary File S1 provides an overview of the metabolites it contains, as determined according to Hammerbacher et al. [52 (link)] and Gonzalez-Cabanelas et al. [53 (link)].