Microscopy Study on the Effect of Essential Oils on Growth and Germination of Aspergillus ssp. in Peanuts

Introduction: Aspergillus flavus infect peanuts and are known to produce carcinogenic aflatoxins. At present, pre and post-harvest afflation management in peanuts is practiced solely through adaptation of suitable agricultural practices such as crop rotation, use of good quality seed and fungicides, and altering the time of planting with adequate irrigation to avoid drought stress. According to the USDA more than $1 billion is spent on infection prevention, which contributes to poor seed quality, low yield, and price reduction and this cost is estimated to exceed $25 million per year.

Purpose: There is no direct action farmers can take to control afflation producing fungi in peanuts. Fungicides have proven to be detrimental to the environment. Increasing interest in antimicrobial agents are being studied as biological control agents against mold in grain, cereals and edible nuts.  Hence, the present study investigates the synergistic effect of essential oils against A. flavus in peanuts.

Methods: Antifungal activity of the essential oils was tested on colony growth and spore germination using modified poisoned food method (Viuda-Martos et al., 2006). Potato dextrose agar (PDA) plates were diffused with combination of two oils and incubated for 7-10 days at 25 °C.  Following incubation, minimum inhibition concentration (MIC) was determined (Hadaceck and Greger, 2000). Changes in conidial morphology and spore germination were observed by light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM).

Results: Incubation time and concentration of oils affected colony growth and spore germination. Colony diameter decreased with increase in concentration and average germ tubes were shorter (2.2 cm) when exposed to 72 h than 48 h (4.4 cm).  A combination of 100 ppm of clove and 500 ppm of cinnamon, respectively, were found to be fungicidal.  The lethal doses 100 and 1000 ppm inhibited about 65% of fungal growth after five days of incubation as compared to control. Disintegration of hyphal wall, disruption of plasma membrane and disorganization of cell organelles leading to large vacuoles in affected cells were observed.  Scanning electron micrographs showed that while length of germ tube varied with exposure time, collapse of spores was visible at higher concentrations.

Significance: Our study revealed that essential oils affect colony growth and spore germination of A. flavus in peanuts. Cinnamon and clove oils showed potential for use as biological control agents. Selected oils may be studied further and integrated in control strategies and aflatoxin reduction in peanuts and other edible nuts.