Comparison of vetiver root essential oils from cleansed (bacteria- and fungus-free) vs. non-cleansed (normal) vetiver plants

Abstract

‘Karnataka’ and ‘Malaysia’ cultivars of vetiver (Vetiveria zizanioides (L.) Nash, =Chrysopogon zizanioides (L.) Roberty) were subjected to meristem tissue culture in order to produce plants that were bacteria- and fungi-free. Tissue cultured (“cleansed” or phytosanitary) vetiver was grown for five months in sterilized soil contained in pots, and the oil content of plants grown on the medium was compared to that of non-cleansed (normal) vetiver plants grown in unsterilized soil under the same conditions. Statistical analysis of 49 of the major oil components revealed numerous significant differences between tissue culture derived and natural plants for both genotypes.

Although oil yields differed, this may reflect the larger size of the initial plantlets obtained from natural sources. Tissue cultured vs. natural plantlets grown in sterilized soil resulted in the largest number of differences in compounds. The least number of differences of compounds were between tissue cultured vs. natural plantlets grown in non-sterile soil. The thesis that many of the compounds found in vetiver roots originate from endogenous fungi was not supported.

Introduction

Vetiver grass (Vetiveria zizanioides (L.) Nash, syn. Chrysopogon zizanioides (L.) Roberty), the roots of which produce an important essential oil, has been utilized in many parts of the world for soil and water management. Hedges of the non-seeding vetivers provide an effective living dam against erosion (NRC, 1993), and this technique is now in use in more than 100 countries. The origin of the non-seeding vetiver is not known. However, V. zizanioides seems to have originated in the area from India to Vietnam, and its fragrant roots, from which is extracted the essential “Oil of Vetiver”, have been used for centuries for mats and perfumes (NRC, 1993).

Adams and Dafforn (1998) examined 121 accessions of pantropical vetiver and found that 86% appeared to be a single clone (no variation in the DNA examined). That clone was named ‘Sunshine’ (after a collection site in Sunshine, Louisiana, USA). Included in that analysis were plants from Haiti and Reunion that clustered with the ‘Sunshine’ group, indicating that the vetiver cultivars used for commercial essential oil production are ‘Sunshine’ or very similar cultivars. This work was expanded by Adams et al. (1998) to include the closely related genera, Chrysopogon and Sorghum. Based on an overlap of genetic and morphological data, Veldkamp (1999) combined Vetiveria and Chrysopogon under Chrysopogon. Although this has led to the recognition of C. zizanioides (L.) Roberty as a proper classification for V. zizanioides (L.) Nash, in this paper we will continue to use both names for clarity. Analysis of additional collections in Thailand cultivars from Bangkok (Adams et al., 1999) revealed that ‘Sunshine’ and its allied cultivars form the bulk of the vegetatively propagated cultivars in the world. Adams et al. (2003) reported on the growth and oils of 13 distinct DNA types of the ‘Sunshine’ group grown in test plots in Florida, Nepal and Portugal. No single DNA type (cultigen) was found to be superior in all plots. The oil yields (g/g root dry wt.) were highest in Portugal, followed by Nepal, then Florida. However, yields of oil per plant (g/plant) were much higher in Nepal (1.79 g), followed by Florida (1.23 g), then Portugal (0.85 g). The oil composition varied slightly by strains and by plots.

Weyerstahl et al., 1996, Weyerstahl et al., 1997, Weyerstahl et al., 2000a, Weyerstahl et al., 2000b, Weyerstahl et al., 2000c exhaustively examined vetiver oil from Haiti. He stated (Weyerstahl et al., 2000c) that the composition is so complex (most GC peaks contained 2–4 components) that general, routine analyses of vetiver oils are probably not possible. Weyerstahl et al. (2000c) also noted that vetiver oil reminds him of agarwood oil that is obtained from fungus infected trees of Aquilaria, which contain constituents with eremophilane, eudesmane, spirovetivane, guaiane and 2-epi-prezizaane skeletons. These sesquiterpene families are also present in vetiver oil. Vetiver has been reported (Wong, 2003) to contain arbuscular mycorrhizal fungi (AMF). The endomycorrhiza could well be producing biotransformations of the vetiver oil. In addition, Viano et al. (1991) and Bertea and Camusso (2002) reported intracellular bacteria in association with essential oil cells in vetiver root (glands). It is also possible that bacteria could be making biotransformations of the essential oil. Adams et al. (2004) made a preliminary report on ‘cleansed’ vs. ‘normal’ vetiver and found that the single plant arising from tissue culture had considerable amounts of long chain hydrocarbons (C₁₅–C₂₉).

The purpose of this study was to conduct thorough research to compare the essential oils of plants cleansed (i.e., that do not contain internal bacteria or fungi) vs. non-cleansed, wild type plants with their normally associated internal microorganisms. However, as seen in the results, it is very difficult to grow completely sterile plants under ambient (non-sterile) conditions. In addition, these kinds of plants were grown on both sterile and non-sterile soils. To examine these effects, ‘Karnataka’ and ‘Malaysia’ vetivers, obtained from tissue culture (so they had no internal bacteria or fungi) were grown in both sterilized and not sterilized soils along side non-cleansed (normal) plants. The roots were harvested, the oil extracted and analyzed. This paper reports on the comparison of these oils.