A. flavus exposure to A. oryzae volatilome (treatment 1) and the standard VOC gradients (treatment 2)

The effects of A. oryzae volatilome and the varying gradients of standard compounds on A. flavus development were examined using the twin-plate (P1 × P2) experiment (Fig. 5), previously designed and reported by Singh and Lee¹³. The plate 1 (P1—sink of VOCs) with perforated lid and extrinsic filter paper was inoculated with A. flavus and assembled face-to-face with plate 2 (P 2—source of VOCs).

Schematics displaying the design of experiment (DOE) to examine the effects treatment 1 (T1—A. oryzae volatilome) and treatment 2 (T2—1-Octen-3-ol gradients) on A. flavus in twin-plate experiment (P1 × P2). The effects of treatments (T1 and T2) on A. flavus were compared with untreated set serving as the experimental control to normalize the baseline effects of endogenously produced VOCs. All treatments and control sets were examined maintaining the three biological replicates of each.

In treatment 1, we examined the pair-wise Aspergillus interactions through inoculating P2 with A. oryzae. The choice of A. oryzae as the partner strain as well as the source of natural volatilome was inspired by the reports describing the untoward colonization of fermented foods by the toxicogenic strains of A. flavus^24–26. In treatment 2, the selective effects varying concentration gradients of 1-Octen-3-ol on A. flavus were examined using the parallel experimental sets. Briefly, following the 4 day incubation period necessary to acquire competence in A. flavus (P1), the P 2 of twin-plate was infused with varying gradients of standard 1-Octen-3-ol (100 ppm/day, 200 ppm/day, and 400 ppm/day). Another reason rationalizing the timing of standard VOC infusion was the reported onset of C-8 VOCs production in Aspergillus species after 3–4 days of incubation under aerobic conditions⁴⁷. We maintained the experimental controls for treatment 1 and 2 by incubating A. flavus (P1) under the untreated conditions without any VOCs sources in P2 of twin-plate. Herein, we wish to emphasize that the untreated set will serve as the control toward observing the baseline effects of endogenously produced C-8 VOCs on the developmental phenotypes of A. flavus itself. Following the inoculation, all twin-plates were placed flat ensuring P1 (A. flavus) at the top and P2 (A. oryzae in T1 and standard compounds in T2) lay at the bottom, allowing the upward flow of low vapor pressure VOCs from P2 to P1. We maintained three biological replicates for each of the treated (T1 – A. oryzae volatilome and T2 – 1-Octen-3-ol gradients) and untreated sample, all incubated at 30 °C under dark conditions for 7- and 9-days.