Composition and in vitro Fungitoxic Activity of 19 Essential Oils Against Two Post-Harvest Pathogens

Journal of Essential Oil Research: JEOR, Sep/Oct 2004 by Chebli, B, Hmamouchi, M, Achouri, M, Hassani, L M Idrissi

Abstract

The fungitoxic activity against Botrytis cinerea and Phytophthora citrophthora of 19 essential oils distilled from Moroccan medicinal plants is reported. The results of the in vitro trials showed strong activity by the oil of Chrysanthemum viscidehirtum, which completely inhibited the growth of the two fungi at a concentration of 150 ppm. The fungitoxic activity was compared with procymidone for Botrytis cinerea and propamocarbe (HCl) for Phytophthora citrophthora. Gas Chromatographic analysis of the oil from C. viscidehirtum aerial parts showed that it consisted mainly of [beta]-farnesene (25.0%), limonene (21.8%) and oxygenated sesquiterpenes (7.4%). Analysis of Aloysia triphylla oil revealed that its main components were limonene (10.1%), nerol (11.9%), geraniol (15.4%) and spathulenol (13.1%).

Key Word Index

Chrysanthemum viscidehirtum, Asteraceae, Aloysia triphylla, Verbenaceae, essential oil composition, limonene, [beta]-farnesene, nerol, geraniol, spathulenol, antifungal activity, Botrytis cinerea, Phythopthora citrophthora.

Introduction

Phytophthora citrophthora (Smith et Smith) Leonian is the causal agent of the citrus trunk, root rot and the citrus fruit brown rot. Fruits infected with P. citrophthora may not show symptoms when inspected and graded in the packinghouse; therefore, sound fruit can still develop disease in containers during transit and storage. This may particularly be disastrous for citrus fruit export. Botrytis cinerea Pers: Fr (grey mold rot) is a ubiquitous pathogen, which causes severe damage to many fruits, vegetables, and ornamental crops in pre- and post-harvest (1,2). Fruit crops are particularly susceptible to microbial infections in the post harvest period due to their high nutrient and water content in addition to the loss of natural resistance that they had while attached to the tree (3). Failure to control the fungi can result in serious economic loss. Shortened crop production cycle and increased growing pressure to meet the demand of the rapidly expanding market of orchids have caused certain fungi to become more serious pathogens that are often uncontrolled by commercial fungicides (4). Consequently, more frequent applications and higher doses of fungicides have been used. However, this can cause negative effects on the environment and food safety (5). Furthermore, the use of fungicides is more harmful in the post harvest period because of the short time between treatment and consumption. Markets in industrialized countries are desperately looking for new products with less residue in order to comply with the food safety standards. Several studies on the antifungal activity of essential oils have been published (6-13). As part of the rejuvenation of the Moroccan aromatic plants used locally as remedies in folk medicine, we started a program aimed at the evaluation of the fungicidal and the pharmacological properties of the volatile fraction from these plants in the hope of finding new natural biological agents (14-20). Thus, this study evaluates several essential oils from Moroccan medicinal plants for their antifungal activity in vitro against two-post harvest pathogens B. cinerea and P. citrophthora at lower concentrations.

Experimental

Plant collection and essential oil isolation: A number of aromatic plants were collected in different regions of Morocco. They were taxonomically identified at the National Scientific Institute of Rabat (Department of Plant Biology, Laboratory of Botany). A voucher specimen of each sample was deposited in the Herbarium of the Laboratory of Natural Products (Faculty of Medicine and Pharmacy of Rabat). Each plant used for essential oil isolation was separately air-dried and ground. From the powder of each plant a sample of 200 g was subjected to water distillation for 2 h using a Clevengertype apparatus recommended by the French Pharmacopoeia (21 ). The yields (w/w) were determined and reported in Table I. The oils were analyzed using a Hewlett-Packard 5972 MS, fitted with an HP 5890 Series II GC and controlled by a G1034C Chemstation. A sample of 1 �L was injected under the following conditions: DB-1 fused silica capillary column (20 m x 0.20 mm, film thickness 0.2 �m); carrier gas helium (0.6 mL/min); injector temperature 250�C; column temperature 50�-250�C at 3�C/min; MS electronic impact 70 eV The identification of the compounds was achieved by comparing retention times and mass spectra with those of the published standards (22, 23).

Antifungal assay: Potato Dextrose Agar (PDA) (Merck) and V8 medium [PDA was used for Botrytis cinerea, V8 medium (Campbell Soup Co.) a juice mixture of eight vegetables plus agar was used for Phytophthora citrophthora] were autoclaved and cooled in a water bath to 40�C, each oil was added to sterilized water at a concentration of 1,000 ppm and dissolved using an ultrasound homogenizer in ice bath. In order to facilitate the oil dispersion, a surfactant (Tween 80) was used at a concentration of 0.2%. The oil prepared as above was mixed with sterile molten PDA or V8 to obtain final concentrations 0, 50, 150 and 250 ppm. The PDA or V8 containing the oil was then poured into Petri dishes (^sup a^20 mL/ plate), which were then seeded with a 5 mm diameter mycelial plug for each dish from the edge of seven-day-old B. cinerea or P. citrophthora. Plates in three replicates were used for each treatment. All plates were incubated in the dark at 24�C for seven days at which time the growth of the control reached the edge of the plate. Growth inhibition was calculated as the percentage of inhibition of radial growth relative to the control. The effect of the oils was compared to that of the Procymidone a synthetic fungicide belonging to the dicarboximide group for B. cinerea and Previcure (propamocarbe HCl) for P. citrophthora. The concentration of the two fungicides ranged from 0.001 to 10 ppm. Two types of control plates were used. One contained the medium only and the other contained the medium plus 0.2% of Tween 80.