Antifungal and Zearalenone Inhibitory Activity of Pediococcus pentosaceus Isolated from Dairy Products on Fusarium graminearum

Abstract

The present study was aimed to evaluate the bio-control efficacy of Pediococcus pentosaceus isolated from traditional fermented dairy products originated from India, against the growth and zearalenone (ZEA) production of Fusarium graminearum. The cell-free supernatants of P. pentosaceus (PPCS) were prepared and chemical profiling was carried out by GC-MS and MALDI-TOF analysis. Chemical profiling of PPCS evidenced that, the presence of phenolic antioxidants, which are responsible for the antifungal activity. Another hand, MALDI-TOF analysis also indicated the presence of antimicrobial peptides. To know the antioxidant potential of PPCS, DPPH free radical scavenging assay was carried out and IC50 value was determined as 32 ± 1.89 μL/mL. The antifungal activity of P. pentosaceus was determined by dual culture overlay technique and zone of inhibition was recorded as 47 ± 2.81%, and antifungal activity of PPCS on F. graminearum was determined by micro-well dilution and scanning electron microscopic techniques. The minimum inhibitory concentration (MIC) of PPCS was determined as 66 ± 2.18 μL/mL in the present study. Also a clear variation in the micromorphology of mycelia treated with MIC value of PPCS compared to untreated control was documented. Further, the mechanism of growth inhibition was revealed by ergosterol analysis and determination of reactive oxygen species (ROS) in PPCS treated samples. The effects of PPCS on mycelial biomass and ZEA production were observed in a dose-dependent manner. The mechanism behind the suppression of ZEA production was studied by reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13), and results showed that there is a dose dependent down-regulation of target gene expression in PPCS treated samples. The results of the present study were collectively proved that, the antifungal and ZEA inhibitory activity of PPCS against F. graminearum and it may find a potential application in agriculture and food industry as a natural bio-controlling agent.

Keywords: F. graminearum; P. pentosaceus; Zearalenone; dual culture overlay technique; ergosterol; minimum inhibitory concentration; reactive oxygen species; reverse transcriptase qPCR analysis.

FIGURE 1

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) identification of peptides from Pediococcus pentosaceus culture supernatant (PPCS).

FIGURE 2

Antioxidant activity of PPCS determined by DPPH free radical scavenging assay. Data were processed using one-way ANOVA following Tukey’s test and p value was significant (<0.05).

FIGURE 3

Antifungal activity of P. pentosaceus on F. graminearum determined by dual culture overlay technique. Data were processed using one-way ANOVA following Tukey’s test and p value was significant (<0.05). Fungal growth at (A) control and (B) P. pentosaceus streaked plate.

FIGURE 4

Dose-dependet antifungal activity of PPCS determined by micro-well dilution method. Data were processed using one-way ANOVA following Tukey’s test and p value was significant (<0.05).

FIGURE 5

Scanning electron microscopic (SEM) observation of mycelia of F. graminearum exposed with P. pentosaceus culture supernatant (PPCS). (A) Control mycelia, (C) control spores and, (B,D) were mycelia and spores, respectively treated with minimum inhibitory concentration (MIC) of P. pentosaceus culture supernatant (PPCS).

FIGURE 6

Dose-dependent effects of P. pentosaceus culture supernatant (PPCS) on ergosterol biosynthesis of F. graminearum determined by (A) UV- spectrophotometry and (B) UHPLC analysis. Data were processed using one-way ANOVA following Tukey’s test and p value was significant (<0.05).

FIGURE 7

Antifungal activity of PPCS on F. graminearum. (A) Effect of PPCS on the generation of reactive oxygen species (ROS). (B) Phase contrast (a–d) and fluorescent images (e–h) of PPCS untreated and treated mycelia. Data were processed using one-way ANOVA following Tukey’s test and p value was significant (<0.05).

FIGURE 8

Effect of PPCS on (A) mycelial biomass, (B) zearalenone (ZEA) synthesis, and (C) expression levels of PKS4 and PKS13 (ZEA metabolic genes). Data were processed using one-way ANOVA following Tukey’s test and p value was significant (<0.05).