Influence of Butylated Hydroxyanisole on the Growth, Hyphal Morphology, and the Biosynthesis of Fumonisins in Fusarium proliferatum

Abstract

Fusarium proliferatum as a common fungus pathogen in foods can produce toxic fumonisins, which can cause animal diseases and increase risks of human cancers. On contrary, butylated hydroxyanisole (BHA) as a synthetic antioxidant offers a clue for preventing growth of fungal species and inhibiting production of mycotoxins. Unfortunately, information of the inhibitory mechanism of BHA on Fusarium species is still limited. In this study, influence of BHA treatment on growth and inhibition of fumonisin production in relation to the expression of the fumonisin biosynthesis-related genes of the F. proliferatum ZYF was investigated, which revealed that BHA had a negative influence on growth and fumonisin production of F. proliferatum. To further elucidate the mechanism of BHA on the growth of F. proliferatum, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the F. proliferatum hyphae. The BHA treatment induced the loss of cytoplasm and cellular constituents, as well as distortion of mycelia, but it did not directly degrade the fumonisin. Furthermore, the BHA treatment markedly inhibited the expressions of FUM1 (a polyketide synthase encoding gene) and FUM8 (an aminotransferase encoding gene) genes, which resulted in the depression of metabolic pathway of F. proliferatum. The transcriptional analyses of the FUM1 and FUM8 genes confirmed a correlation between the fumonisin production and its gene expression. This study provided some insights into mechanisms of production of fumonisin and feasible prevention to reduce fumonisin contamination in favor of human and animal health.

Keywords: Fusarium proliferatum; butylated hydroxyanisole; fumonisin; gene expression; mycelia.

FIGURE 1

Effect of BHA treatment on growth rates of Fusarium proliferatum ZYF incubation in Czapek’s broth (CB) medium (A) and PDA plate (B,C). Values of optical densities were expressed in arbitrary units while diameters of the colonies were expressed as cm. Vertical bars indicated standard errors (n = 3). Experiments were repeated three times with the same results.

FIGURE 2

Scanning electron microscopy (SEM) (A–C) and transmission electron microscopy (TEM) (D–K) images of F. proliferatum ZYF. (A) Mycelia of non-butylated hydroxyanisole (non-BHA)-treated (control) F. proliferatum ZYF after 10 days of culture; (B,C) The BHA-treated F. proliferatum ZYF after 10 days of culture. (D–G) Mycelia of non-BHA-treated (control) F. proliferatum ZYF after 10 days of culture (20, 30, 50, and 150×, respectively). (H–K) Mycelia of the BHA-treated F. proliferatum ZYF after 10 days of culture (20, 30, 50, and 150×, respectively). Arrows referred to the morphologic changes in the hyphae.

FIGURE 3

Effects of BHA on extracellular production of FB1 (A) and FB2 (B) of F. proliferatum ZYF and the FB1 degradation when FB1 was incubated in acetonitrile/water (C) and CB media (D). Vertical bars indicated standard errors of three independent experiments.

FIGURE 4

The expression levels of FUM1(A) and FUM8(B) of F. proliferatum ZYF, and a correlation between fumonisin production and FUM gene expression (C). Induction of FUM genes was monitored by real-time PCR. Data calculated by using the ^ΔΔCT method were expressed as relative units. The data were means of three independent repetitions. Vertical bars indicated standard errors.

FIGURE 5

Influence of BHA on release of cell materials. Data were presented as means ± standard errors (n = 3).