Analysis of Fusarium graminearum Antifungal Protein’s and Latrodectin-II’s Effect on Growth and Toxigenesis of Aspergillus Fungi with Agrofood Impact ^†

Abstract

Colonization of crops by toxigenic fungi causes important economic losses, which are expected to increase with global change. The presence of mycotoxins in crops affects food security by reducing the edible yield and nutritional values and making cereal consumption unsafe. An efficient method to reduce mycotoxin content is to avoid the appearance of fungi or toxigenesis. This has been traditionally achieved with antifungal chemicals that negatively affect soil and ecosystem health. Current research aims for methods that less harmful for the environment, such as natural proteins or biocontrol. We tested crop-associated fungi against two possible growth-inhibiting proteins: Fusarium graminearum Antifungal Protein (Fg-AFP) and Latrodectin-II (Ltd-II). The first is produced by Fusarium graminearum to compete with other fungi, and the latter is found in Latrodectus hesperus venom and has just recently been correctly purified. In a first assay, we exposed four Aspergillus strains cultured on PDA (potato dextrose agar) against on-surface cellulose discs with 10 µL of different Fg-AFP and Ltd-II concentrations (7 µg/µL, 3.5 µg/µL, 1.4 µg/µL, and 0.7 µg/µL). All tests were carried out in triplicate. Results show that Fg-AFP inhibited development of three of the strains, while Ltd-II did not but may have affected secondary metabolism due to a variation in spore production and pigmentation (no further analysis regarding this event was fulfilled). We subsequently selected Aspergillus niger and A. flavus strains based on their importance in maize crops and sensitivity to the proteins, for a second assay, in which we evenly extended 30 µL of the proteins (1.5 µg/µL) separately on plaques containing PDA. The growth surface was represented over time, the specific growth rate corresponded with the curve slope, and latent period was calculated by obtaining the equation of the line from the slope and the intersection point. An initial diameter of 4 mm was assumed for 2 µL of spore suspension (10⁵ spores/mL) inoculated on the center of the plaque. All tests were carried out in triplicate. Results show that Fg-AFP reduced A. niger and A. flavus growth by 46.3% and 24.3%, respectively, extending fungal latent period by 68.4% and 52.6%, respectively. Ltd-II had no effect on A. flavus, but increased A. niger growth by 18.4%, prolonging the latent period by 67.5%. Toxin production was assessed by thin-layer chromatography under UV light after following a toxin extraction protocol with chloroform and toluene:acetonitrile for quantifying ochratoxin A, and chloroform and methanol for aflatoxin B1 production. Regarding toxigenesis results, ochratoxin A production by A. niger did not vary when it was exposed to the proteins, but aflatoxin B1 synthesis by A. flavus increased with both treatments. Though these proteins have growth-limiting potential, they must be evaluated under wider concentration ranges to assess their effect on toxigenesis and usage as an alternative to harmful chemicals.