# Comparison of Three Biological Control Models of _Pycnoporus__sanguineus_ on Phytopathogenic Fungi
## 2 Materials and Methods
### Model 2: Antifungal Effectiveness Tests of Cinnabarin  
Antifungal activity tests of cinnabarin on the phytopathogens _B. cinerea_ and _F. oxysporum_ were conducted under controlled conditions, where two commercial products were compared using the impregnated disc diffusion method, in which the pathogenic fungi were grown in the center of Petri dishes containing 6 mm diameter paper discs impregnated with the different treatments. These discs were placed one centimeter away from the edge of the Petri dish and arranged at the four cardinal points. For the extraction of the cinnabarin pigment, the growth of _P. sanguineus_ was cultivated in Petri dishes with PDA medium. To facilitate the proper separation of mycelia, a layer of cellophane was placed on the gelatinized culture medium. After thirty days of growth, the mycelia were separated and completely dehydrated in an oven at 45 \({}^{\circ}\)C, with daily measurements taken until a constant weight was achieved. A total of 5.6 g of _P. sanguineus_ dehydrated mycelium was obtained; subsequently, cinnabarin was isolated using extraction techniques. A total of 5.6 g of dehydrated _P. sanguineus_ mycelium was macerated in a 10 mL solution of ethyl acetate to release the pigment, and then adjusted to a volume of 50 mL of solvent, which was allowed to macerate for 2 days for complete the pigment release. To determine the presence and optical characterization of cinnabarin, 0.5 mL of the extract was analyzed using spectrophotometry performed with a spectrometer Cary 100 UV-Vis System, Agilent at 25 \({}^{\circ}\)C and quartz cells with an optical path of 1 cm. UV-vis (C\({}_{4}\)H\({}_{8}\)O\({}_{2}\)): \(\lambda\)max (log \(\varepsilon\)) = 251 (1.09), 427 (0.19), and 446 (0.18) nm (Figure 1a). Additionally, 0.5 milliliters of the extract was analyzed using HPLC performed on a Series 200 Quaternary pump equipped with a Diode Array Detector (DAD) Perkin Elmer using Total Chrom Navigator software (TcNav version 6.2.1). Analytical HPLC analyses were performed using 0-5 min 75% CH\({}_{3}\)CN/H\({}_{2}\)O on a Pecosphera C18 150 \(\times\) 4.6 (5\(\upmu\)m) column at a flow rate of 1.0 mL/min. HPLC (CH\({}_{3}\)CN/H\({}_{2}\)O): the test showed a maximum peak at 1.5 min (Figure 1b). The UV-vis and HPLC values coincide within the previously reported peak area for cinnabarin [22]. Additionally, cinnabarin was determined by studying the \({}^{1}\)H NMR spectra recorded at 500 MHz using a Bruker FT-NMR spectrometer. The chemical shifts (8) are reported in parts per million (ppm), as referenced in the \({}^{1}\)H spectra by the signal of TMS proton resonance: \({}^{1}\)H NMR (500 MHz, CDCl\({}_{3}\)) \(\delta\): 9.79 (1H, brs, 2-NH\({}_{\alpha}\)), 8.50 (1H, brs, 2-NH\({}_{\beta}\)), 7.46-7.04 (4H, m, H-aromatic), and 5.29 (2H, s, H-12). Along the \({}^{1}\)H NMR spectrum of cinnabarin, two broad singlets at 9.79 and 8.50 ppm were assigned to the NH groups. At 7.46-7.04 ppm, signals assigned to four aromatic protons were observed, while at 5.29 ppm, one simple signal appeared to integrate two protons assigned to the methylene. Subsequently, the extract was impregnated into 60 paper discs using a rotavapor at 45 \({}^{\circ}\)C to evaporate the solvent and pigment from the discs simultaneously. Following this, antifungal activity tests were carried out on _B. cinerea_ and _F. oxysporum_ on PDA medium using the impregnated discs. The cinnabarin concentration was 4 mg/mL, which was determined indirectly using an antimicrobial activity technique [12]; the method followed is described in [PERSON] et al. [23]. For the contrast tests, the commercial productsBenomyl 50(r) (with benomyl C14H18N4O3 as the active ingredient) and Captan 50 WP(r) (with captan C9H8 Cl3 NO2S as the active ingredient) were used, along with a control group with discs with sterile water only. The concentrations used for the commercial products were 2 mg/mL for captan and 0.52 mg/mL for benomyl, respectively, according to the indications for their commercial use.