Biocontrol of <i>Aspergillus</i> Species on Peanut Kernels by Antifungal Diketopiperazine Producing <i>Bacillus cereus</i> Associated with Entomopathogenic Nematode

<div><p>The rhabditid entomopathogenic nematode associated <i>Bacillus cereus</i> and the antifungal compounds produced by this bacterium were evaluated for their activity in reducing postharvest decay of peanut kernels caused by <i>Aspergillus</i> species in <i>in vitro</i> and <i>in vivo</i> tests. The results showed that <i>B. cereus</i> had a significant effect on biocontrol effectiveness in <i>in vitro</i> and <i>in vivo</i> conditions. The antifungal compounds produced by the <i>B. cereus</i> were purified using silica gel column chromatography and their structure was elucidated using extensive spectral analyses. The compounds were identified as diketopiperazines (DKPs) [cyclo-(L-Pro-Gly), cyclo(L-Tyr-L-Tyr), cyclo-(L-Phe-Gly) and cyclo(4-hydroxy-L-Pro-L-Trp)]. The antifungal activities of diketopiperazines were studied against five <i>Aspergillus</i> species and best MIC of 2 µg/ml was recorded against <i>A. flavus</i> by cyclo(4-hydroxy-L-Pro-L-Trp). To investigate the potential application of cyclo(4-hydroxy-L-Pro-L-Trp) to eliminate fungal spoilage in food and feed, peanut kernels was used as a food model system. White mycelia and dark/pale green spores of <i>Aspergillus</i> species were observed in the control peanut kernels after 2 days incubation. However the fungal growth was not observed in peanut kernels treated with cyclo(4-hydroxy-L-Pro-L-Trp). The cyclo(4-hydroxy-L-Pro-L-Trp) was nontoxic to two normal cell lines [fore skin (FS) normal fibroblast and African green monkey kidney (VERO)] up to 200 µg/ml in MTT assay. Thus the cyclo(4-hydroxy-L-Pro-L-Trp) identified in this study may be a promising alternative to chemical preservatives as a potential biopreservative agent which prevent fungal growth in food and feed. To the best of our knowledge, this is the first report demonstrating that the entomopathogenic nematode associated <i>B. cereus</i> and cyclo(4-hydroxy-L-Pro-L-Trp) could be used as a biocontrol agents against postharvest fungal disease caused by <i>Aspergillus</i> species.</p></div

The structure of diketopiperazines.

<p>(<b>A</b>) Cyclo-(L-Pro-Gly), (<b>B</b>) Cyclo(D-Tyr-D-Tyr), (<b>C</b>) Cyclo-(L-Phe-Gly) and (<b>D</b>) Cyclo(4-hydroxy-L-Pro-L-Trp).</p

Microscopic images (Carl Zeiss stereomicroscope, Stemi 200C) of the growth of <i>A. flavus</i> in control and peanut kernels treated with crude ethyl acetate extract and cyclo(4-hydroxy-L-Pro-L-Trp) after 2 days.

<p>(<b>A</b>) Control plates: solvent control-treated with methanol, medium alone- treated with autoclaved modified medium and untreated- peanut kernels without methanol and modified medium (<b>B</b>) crude extract (<b>C</b>) cyclo(4-hydroxy-L-Pro-L-Trp). 1 ×, 2 ×, and 3 × are the 1-fold, 2-fold, and 3-fold MIC concentration of cyclo(4-hydroxy-L-Pro-L-Trp) or crude ethyl acetate extract.</p

HPLC chromatogram of diketopiperazines on a reversed-phase C18 column (LC-20AD).

<p>Samples of 15 µl were injected to a column (250 mm×4.6 mm×5 mm), eluted with 100% methanol. Retention time is 2.778 min. The calculated purity is 96% based on the peak area. (<b>A</b>) Cyclo-(L-Pro-Gly), (<b>B</b>) Cyclo(D-Tyr-D-Tyr), (<b>C</b>) Cyclo-(L-Phe-Gly) and (<b>D</b>) Cyclo(4-hydroxy-L-Pro-L-Trp).</p

Effect of <i>B. cereus</i> and cell free culture filtrate on <i>Aspergillus</i> species.

<p>Values followed by different letters in same row were significantly different according to Duncan’s multiple range test <i>p</i> = 0.05.</p><p>+ full growth up to the edge of the Petri plate.</p><p>Effect of <i>B. cereus</i> and cell free culture filtrate on <i>Aspergillus</i> species.</p

Antifungal activity of diketopiperazines.

<p>–no activity up to 1000 µg/ml.</p><p>Antifungal activity of diketopiperazines.</p

Effect of different concentrations of <i>B. cereus</i> on control of <i>Aspergillus</i> species.

<p>The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan’s multiple range test <i>p</i> = 0.05.</p

Effect of different incubation times of <i>B. cereus</i> on control of <i>Aspergillus</i> species.

<p>The control is treated with sterile distilled water. Values followed by different letters were significantly different according to Duncan’s multiple range test <i>p</i> = 0.05.</p

Details of the pure compounds.

<p>Details of the pure compounds.</p

Microscopic images (Carl Zeiss stereomicroscope, Stemi 200C) of the growth of <i>A. niger</i> in control and peanut kernels treated with crude ethyl acetate extract and cyclo(4-hydroxy-L-Pro-L-Trp) after 2 days.

<p>(<b>A</b>) Control plates: solvent control-treated with methanol, medium alone- treated with autoclaved modified medium and untreated- peanut kernels without methanol and modified medium (<b>B</b>) crude extract (<b>C</b>) Cyclo(4-hydroxy-L-Pro-L-Trp). 1 ×, 2 ×, and 3 × are the 1-fold, 2-fold, and 3-fold MIC concentration of cyclo(4-hydroxy-L-Pro-L-Trp) or crude ethyl acetate extract.</p