Potato dextrose agar (pda)

Several compounds have been used for the preparation and construction of A. factorovskyi extract and nanoparticles. The 99.9% Ethanol and AgNO₃ were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The Mueller Hinton Agar, Potato Dextrose Agar, Fluconazole, and the antimicrobial susceptibility disks (Tetracycline) were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA).

Triticale grains (Triticum aestivum L. emend. Fiori et Paol) were obtained from local commercial suppliers. Wheat grains with known seed-borne Fusarium infections were obtained from the Human Microbiology Institute (New York, NY, USA). Potato dextrose agar (PDA; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used as a growth medium for seed germination.
Grains in the control group were sterilized according to Htwe et al. (2011) (link), with minor modifications. Briefly, uniformly sized grains were washed in a solution of 4.5% (w/v) NaOCl:water (1:1 ratio) for 20 min. For the experimental groups, uniformly sized grains were treated with different concentrations of three different solutions: 1) 0.1% and 0.05% M451 (a water-based solution of TGV-28 [Figure 1]; Human Microbiology Institute) in combination with 0.01% NaH₂PO₄ (Sigma Aldrich, St. Louis, MO, USA) for 30 min, 2) 0.01% NaH₂PO₄ for 30 min (Sigma Aldrich), and 3) Maxim XL v/v1:10 dilution in water for 12 h according to the manufacturer’s instructions (Syngenta Canada Inc., Guelph, ON, Canada) (Costa et al., 2019 (link); Pfeiffer et al., 2021 (link)). Subsequently, the grains from control and M451-treated groups were washed three times with autoclaved distilled water and then air-dried on filter paper. The grains treated with Maxim XL were left unwashed.

We obtained isolates from 101 patients referred by health providers to the Laboratorio de Micología Médica y Experimental at the Corporación para Investigaciones Biológicas (CIB) in Medellin after obtaining their informed consent. Before we took the samples, we gathered the data on their sex, age, place of residence, and previous use of antifungals. The samples were then obtained from hands and feet fingernails, cornea, sinuses, skin, and secretions by non-invasive procedures warranting the chain of custody at the institutions to avoid cross contamination. Samples were cultured in three different media: Sabouraud agar (Thermo Scientific, USA), potato dextrose agar (Thermo Scientific, USA), and mycosel agar (Fisher Scientific, USA) and 10 inoculation points, at room temperature for one to three weeks. Fusarium was considered the etiological agent based on two criteria: ¹ the growth of the isolate in the media was observed in more than five of the inoculation points and ² (link) the same isolate was obtained in at least two of the three media. Once growth was observed, the macroscopic colonies identified as Fusarium sp. were stored in sterile vials ²⁵ (link).

The samples were removed from the refrigerator at -80 °C and the mixed silage was analyzed microbiologically by plate counting (Olsen and Bakken 1987 (link)). To get the 10^− 1 dilution, first weigh 10 g of the sample, add 90 mL of sterilized distilled water, mix the sample thoroughly in a sterile polyethylene bag, then tap with a sterile homogenizer for 2 min, the 10^− 1-10^− 9 gradient was made by successive dilution with sterilized distilled water at a ratio of 1:9, and the 3 optimal gradients, namely, 10^− 2, 10^− 4, and 10^− 6, were chosen to spread the dilution on the medium, and the number of microorganisms was determined according to the corresponding dilution times. Plate counting method was used to count the microorganisms, and the number of microorganisms was determined by conversion according to the corresponding dilution times (Chen et al. 2012 ). The results were summarized as follows: the number of lactobacilli was determined using BLM medium (De Man Rogosa, Thermo Fisher Scientific). For coliform counts, BLB medium (Blue light broth agar, Thermo Fisher Scientific) was used and incubated for 48 h at 30 °C. For yeast and mold counts, PDA medium (Potato dextrose agar, Thermo Fisher Scientific) was used and incubated at 30 °C for 24 h (Cai et al. 2011 (link)).

A. nidulans strain A26 (Fungal Genetics Stock Center, Kansas City, MO, USA) was used as the parent wild-type strain for all molecular manipulations. Other strains used in this study include A. fumigatus strain Af293 (a generous gift from P. Magee, University of Minnesota, St. Paul, MN, USA), the A. fumigatus Δuge3 mutant [10 (link)], ΔstuA mutant [41 (link)] and clinical isolates of A. flavus and A. niger (obtained from the McGill University Health Center, Montreal, QC, Canada) and A. nidulans isolates from CGD patients (generous gifts from A. Warris and S. Henriett, Radboud University Medical Center, Nijmegen, The Netherlands, and Adrian Zelazny US National Institute of Health, USA). Unless otherwise noted, A. fumigatus strains and A. flavus were maintained on YPD agar (Fisher Scientific), A. niger on potato-dextrose agar (Fisher Scientific), and A. nidulans strains on Aspergillus minimum medium agar [10 (link)] at 37°C. For growth in liquid medium, Brian medium [9 (link)] and phenol red-free RPMI 1640 (Wisent, Inc.) were used as indicated. All growth media for A. nidulans strains were supplemented with biotin (Fisher Scientific).