Antifungal Activity of Aroma Chemicals Against Seed-borne Fungi

Journal of Essential Oil Research: JEOR, Sep/Oct 2004 by Dev, U, Devakumar, C, Mohan, J, Agarwal, P C

Abstract

In vitro inhibition of 16 aroma chemicals was studied against five seed-borne fungal pathogens in the concentration range of 100 to 8000 ppm: Drechslern sorokiniana (Sacc.) Subram. et Jain, Phomompsis sojae Leh., Fusarium solani (Mart.) Sacc., Colletotrichum graminicola (Ces.) Wilson and Macrophomina phaseolina (Tassi) Goid. Minimum inhibitory concentration (MIC) was computed for each chemical against each fungus. All the chemicals had shown dose-dependent inhibitory effect of mycelial growth. Eugenol, methyl eugenol, citral, 2-phenethyl alcohol, 2-phenethyl acetate and 2-phenethyl propionate were found to be highly effective against all the test fungi with MIC values of 270 to 1704 ppm and potentially hold promise in plant disease management and plant quarantine.

Key Word Index

Antifungal activity, seed-borne fungi, Drechslera sorokiniana, Phomompsis sojae, Fusariumsolani, Colletotrichum graminicola, Macrophomina phaseolina, aroma chemicals, eugenol, citral, 2-phenethyl propionate.

Introduction

Colossal crop yield losses occur worldwide, in spite of intensive plant protection efforts. Approximately one third of the losses are attributed to plant pathogenic microorganisms including fungi, bacteria and viruses. Although the application of fungicidal treatment is reported to reduce seed-infection and improve seed germination (1,2), the development of resistance to fungicides in fungal populations is, however, of particular importance (3). To avoid this, new strategies in fungicide use and disease management must be developed and thus the need to identify new chemicals as potent fungicides is imperative. Hence, efforts are being made to find alternatives that are safe and eco-friendly. In this direction, the use of biotic and abiotic agents find their due importance. Extracts/products from medicinal plants and bacterial and fungal strains are note-worthy, and some of the phytochemicals are also known to induce systemic activated resistance in plants against infection (4). Many workers have recommended the use of aroma chemicals in the control of mold infestation during storage (5-8). In the present study, 16 aroma chemicals were evaluated for their antifungal properties against five fungi namely, Colletotrichum graminicola (Ces.) Wils., Drechslera sorokiniana (Sacc.) Subram. et Jain, Fusarium solani (Mart.) Sacc., Macrophominaphaseolina (Tassi) Goid. and Phomopsis sojae Leh. These fungi were reported to be seed-borne on a wide host range (9) and have also been intercepted during the quarantine processing of some new crops and exotic germplasm (10,11).

Experimental

Isolation of seed-borne fungi: The fungal colonies of D. sorokiniana (from the wheat cultivar Arnig), C. graminicola (from sorghum cultivar MSSR001047), M. phaseolina (from sesamum, local cultivar), P. sojae and F. solani (from the soybean cultivar FT 50211) were obtained through blotter test (12). Pure cultures were maintained on potato dextrose agar (PDA).

Test chemicals: Phenyl acetate, methyl anthranilate, benzyl benzoate, phenyl acetaldehyde, 2-pheuctliyl alcohol, 2-phenethyI acetate, 2-phenethyl propionate, eugenol, isoeugenol, citral, 3,7-dimethyloctanol, [alpha]-terpineol and linalyl acetate were obtained from M/s Goldensun, Mumbai, India. Benzyl alcohol and its acetate were procured from laboratory chemical dealers. Methyl eugenol was obtained by methylation of eugenol using methyl iodide in anhydrous acetone containing anhydrous K^sub 2^CO^sub 3^ under refluxing conditions by adopting the procedure of Devakumar et al. (13). The purity of the test chemicals was confirmed to be > 99.5% by gas chromatographic analysis using Hewlett-Packard GC (FID) model 58308 fitted with stainless steel column (G x 1/4) packed with Carbowax 20 m (10%). The injector port and the detector were maintained at 250�C and the column oven temperature at 175�C. Nitrogen was used as the carrier gas with a constant flow rate of 30 mL/min.

Antifungal bioassay: The aroma chemicals were bioassayed by the poisoned food technique (14). Appropriate quantities of each chemical were mixed in autoclaved and adequately cooled PDA to yield mixtures of 8000, 5000, 2500, 2000, 1000 and 500 ppm concentrations. The medium was dispensed into sterilized petriplates. A mycelial disc of 5 mm diameter of the test pathogens taken from 10 day old culture, with the help of a sterilized cork borer was placed at the center of the medium. The mycelial discs on PDA without any test chemical served as control and the plates with 2500 ppm mancozeb served as the fungicide reference. Three replicates were kept for each treatment and incubated for seven days at 20�C. Radial growth of colonies was measured at two points along the diameter of the plate and mean of these two readings was taken as the diameter of the fungal colony. The growth of the colonies in control sets was compared with that of various treatments and the difference was converted into percent inhibition (C-T x 100/C) where C and T are the radial diameters of the colony in control and treatment, respectively. The compounds showing promising activities at 500 ppm were retested at 100, 200, 300 and 400 ppm. Minimum inhibitory concentration (MIC), the minimum dose required to cause percent inhibition of mycelial growth, was computed for each compound using regression equation and FORCAST tool available with Microsoft Excel. The percent inhibition was sine arc converted before computation. ANOVA was carried out to derive least square difference at 5% probability level and Duncan's multiple range test of means (15).

Results and Discussion

The test chemicals (I-XVI) (Figure 1) fall into two broad chemical classes: phenolics and monoterpenoids (Table I). The phenolics can be further classified into simple phenols or their derivatives (C^sub 6^-C^sub 0^) (I), phenyl carbinols (C^sub 6^-C^sub 1^) (II-V), phenylethanoids (C^sub 6^-C^sub 2^) (VI-IX) and phenylpropanoids (C^sub 6^-C^sub 3^) (X-XII). The MIC data expressed in percent (w/w) of the test chemicals against five fungi are given in Table I. It may be mentioned here that mancozeb at 2500 ppm gave 83.75% and 100% inhibition of F. solani and the four other fungi, respectively. Phenyl acetate (I) was found to be only moderately active against the test fungi, with MIC values ranging from 4610 ppm in case of M. phaseolina to 15130 ppm against D. sorokiniana.