Scholarly article on topic 'Chemical composition and antifungal activity of the essential oils of Lippia rehmannii from South Africa'

Chemical composition and antifungal activity of the essential oils of Lippia rehmannii from South Africa Academic research paper on "Biological sciences"

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{Antifungal / Citral / "Essential oils" / "Fruit pathogens" / Lemongrass / " Lippia rehmannii "}

Abstract of research paper on Biological sciences, author of scientific article — J.H. Linde, S. Combrinck, T.J.C. Regnier, S. Virijevic

Abstract Lippia rehmannii H.Pearson (Verbenaceae) is an aromatic bush, indigenous to the northern parts of South Africa. As far as could be ascertained, the essential oil composition has not been previously reported and forms the subject of this investigation. Aerial parts of the shrub were collected from two localities in Gauteng, South Africa, and the isolated essential oils were analysed by gas chromatography. Citral, a mixture of the E- and Z-isomers, was found to be the main constituent of the oils, while borneol, camphor, neryl acetate, isocaryophyllene, p-cymene, β-caryophyllene and β-caryophyllene oxide were other major compounds present. Oil compositions, within and between the two localities, did not differ significantly. The in vitro antifungal activity of L. rehmannii essential oil was compared to that of Cympopogon citratus (lemongrass) and pure citral, against a number of pre- and postharvest fungal food pathogens. At a concentration of 3000µL/L, lemongrass oil and pure citral caused complete growth inhibition of all the pathogens tested. Lippia rehmannii, containing less citral than lemongrass oil, was effective at this concentration against the majority of pathogens, but only partially restricted the growth of Lasiodiplodia theobromae and Botrytis cinerea. This finding suggests that citral may be largely responsible for the observed antifungal activities. Essential oil from L. rehmannii appears to be a good candidate for the in vitro control of Fusarium oxysporum and Rhizoctonia solani and application of these oils in the field should be investigated.

Academic research paper on topic "Chemical composition and antifungal activity of the essential oils of Lippia rehmannii from South Africa"

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South African Journal of Botany 76 (2010) 37 - 42

www.elsevier.com/locate/sajb

Chemical composition and antifungal activity of the essential oils of

Lippia rehmannii from South Africa

J.H. Linde, S. Combrinck *, T.J.C. Regnier, S. Virijevic

Department of Chemistry, Tshwane University of Technology, PO Box 56208, Arcadia, Pretoria 0001, South Africa Received 29 April 2009; received in revised form 11 June 2009; accepted 15 June 2009

Abstract

Lippia rehmannii H.Pearson (Verbenaceae) is an aromatic bush, indigenous to the northern parts of South Africa. As far as could be ascertained, the essential oil composition has not been previously reported and forms the subject of this investigation. Aerial parts of the shrub were collected from two localities in Gauteng, South Africa, and the isolated essential oils were analysed by gas chromatography. Citral, a mixture of the E- and Z-isomers, was found to be the main constituent of the oils, while borneol, camphor, neryl acetate, isocaryophyllene, p-cymene, p.-caryophyllene and p.-caryophyllene oxide were other major compounds present. Oil compositions, within and between the two localities, did not differ significantly. The in vitro antifungal activity of L. rehmannii essential oil was compared to that of Cympopogon citratus (lemongrass) and pure citral, against a number of pre- and postharvest fungal food pathogens. At a concentration of 3000 p,L/L, lemongrass oil and pure citral caused complete growth inhibition of all the pathogens tested. Lippia rehmannii, containing less citral than lemongrass oil, was effective at this concentration against the majority of pathogens, but only partially restricted the growth of Lasiodiplodia theobromae and Botrytis cinerea. This finding suggests that citral may be largely responsible for the observed antifungal activities. Essential oil from L. rehmannii appears to be a good candidate for the in vitro control of Fusarium oxysporum and Rhizoctonia solani and application of these oils in the field should be investigated. © 2009 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Antifungal; Citral; Essential oils; Fruit pathogens; Lemongrass; Lippia rehmannii

1. Introduction

The genus Lippia consists of more than 200 species distributed throughout Africa and South America, many of which are aromatic (Pascual et al., 2001). Infusions of the aerial parts of a variety of these species are widely used in folk medicine for gastrointestinal and respiratory ailments, while the essential oils commonly exhibit antibacterial, antifungal, larvicidal, anesthetic and anti-insecticidal properties (Pascual et al., 2001; Viljoen et al., 2005). The genus Lippia is well known for the intraspecies variation of the essential oil profiles, both within and between wild populations (Viljoen et al., 2005). Five Lippia species are officially recognized as indigenous to

* Corresponding author. Tel.: +27 12 3826126; fax: +27 12 3826286. E-mail address: combrincks@tut.ac.za (S. Combrinck).

South Africa (Retief, 2006). The essential oil compositions of three of these species, L. scaberrima (Combrinck et al., 2006), L.javanica (Viljoen et al., 2005) and L. wilmsii (Terblanche and Kornelius, 1996) are well documented, but no reports on the essential oil composition of Lippia rehmannii could be found. The shrub grows to a height of about 1 m and is frost and drought-resistant (Retief and Herman, 1997). Flowers are white, cream or greenish in colour and the rough leaves have a pleasant and characteristic lemon odour. Consumption of the aerial parts by livestock causes liver damage (Heikel et al., 1960), which is attributed to the presence of icterogenin and rehmannic acids (Barre et al., 1997).

Fungal pathogens are mainly responsible for the postharvest decay of fruit and vegetables (Pathak, 1997) and some are known to produce highly toxic mycotoxins (Phillips, 1984). Fruit, when compared to vegetables, are generally more susceptible to attack by pathogenic fungi due to their low pH, higher moisture content and rich nutrient composition.

0254-6299/$ - see front matter © 2009 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2009.06.011

Lemongrass (Cympopogon citratus) essential oil, characterised by high concentrations of citral (3,7-dimethyl-2,6-octadienal), was shown to exhibit in vitro antifungal activity against Col-letotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus stolonifer and Aspergillus niger (Tsortzakis and Economakis, 2007). A need to replace synthetic fungicides with safer biodegradable alternatives (Wisniewski et al., 2001) has been created by the increasing resistance of pathogens towards commercially used fungicides (Eckert et al., 1994). Essential oils, which are generally recognized as safe (GRAS), have been investigated in the last decade as alternative control measures against many postharvest pathogens (Plaza et al., 2004).

Recent studies demonstrated the use of L. scaberrima essential oil, containing a high concentration of R-carvone, in both the in vitro and in vivo control of two postharvest mango pathogens (Regnier et al., 2008) and Penicillium digitatum on citrus fruit (Du Plooy et al., 2009). These applications of an essential oil in a commercial environment emphasise the importance of plant secondary metabolites in controlling fungal pathogens. The current investigation forms a basis for further studies in this regard.

To evaluate the antifungal activity of the essential oil from L. rehmannii, a variety of common and economically significant fungal pathogens of food, mainly postharvest pathogens of fruit, were selected. Two isolates were obtained from mango fruit, two from citrus, three from avocado fruit and one isolate each from grape, potato and maize. The avocado fruit pathogens, Colletotrichum gloeosporioides, Lasiodiplodia theobromae and Alternaria alternata, are known to be responsible for considerable postharvest losses in production (Sanders et al., 2000). Botrytis cinerea is a well known grape pathogen (De Kock and Holz, 1994), while Rhizoctonia cinerea and Fusar-ium oxysporum, which occurs in soils, have contributed to large scale preharvest decay of potato and maize, respectively (Chutia et al., 2009). Penicillium digitatum (Plaza et al., 2004) and Alternaria citri (Isshiki et al., 2001) are common citrus pathogens that proliferate on fruit under storage. The development of anthracnose and stem-end rot on mango fruit are caused by C. gloeosporioides and L. theobromae, respectively (Regnier et al., 2008).

2. Material and methods

2.1. Plant material and distillation

Aerial parts of L. rehmannii were collected from Zandfontein cemetery (S 25.41'28.3", E 28.04'04.8") and Boschkop (S 25.52'35.6", E 28.27'30.5") in the vicinity of Pretoria, South Africa in March 2007. Voucher specimens were deposited in the herbarium of the South African National Biodiversity Institute, Pretoria (Bosman & Combrinck 21 and Bosman & Combrinck 24, respectively). Seven individual samples were harvested from each locality, in addition to bulk samples of approximately 29 kg each. Individual plants were hydrodistilled in a Clevenger-type apparatus for 3 h, while bulk samples were steam distilled in a custom-built (Tshwane University of

Technology) apparatus connected to a 15 kW boiler (Protherm). The oils obtained were dried with anhydrous Na2SO4 (Merck, AR grade, Darmstadt, Germany). A commercial sample of lemongrass oil (Holistic Emporium, Johannesburg, South Africa) was analysed together with the L. rehmannii oils. Gas chromatographic (GC) analyses were performed using an Agilent 19091 Series GCD system, in splitless mode, equipped with a flame ionization detector (FID) and a mass spectrometer (MS). An Innowax fused silica column (60 mx 0.25 mm id., 0.25 p.m film thickness) was connected simultaneously via splitflow to the detectors. Helium (1.2mL/min) was used as carrier gas. The injector temperature was set at 250 °C and the FID was operated at 300 °C. An initial column oven temperature of 60 °C was elevated to 220 °C at a rate of 4 °C/min and held for 10 min, before ramping to 240 °C at a rate of 1 °C/min. The mass spectrometer conditions were as follows: transfer line temperature at 280 °C, ion source at 230 °C, and the ionization potential at 70 eV. Commercial standards (Sigma Aldrich, Johannesburg, South Africa) were available for the majority of the essential oil constituents and Kovats retention indices were determined for all the sample components. Three MS libraries were used to confirm the identities of the compounds: Wiley GC/MS Library, Ba^er Library of Essential Oil Constituents (not commercially available) and the NIST library of Kovats indices. The relative peak area percentages of the different compounds were calculated based on the FID data.

2.2. Pathogen isolates and toxic medium

Mango (C. gloeosporioides and L. theobromae) and avocado (C. gloeosporioides, L. theobromae and A. alternata) pathogens were isolated from symptomatic fruit. The identification of the pathogens was confirmed by Westfalia Laboratories (Tzaneen, South Africa). Isolates of B. cinerea from grape, F. oxysporum from maize and A. citri from citrus, were obtained from Dr. du Plooy (John Bean Technologies, South Africa), while a potato pathogen (R. cinerea) isolate was generously supplied by Dr. Van der Walt (Department of Microbiology and Plant Pathology, University of Pretoria). Penicillium digitatum, was isolated from symptomatic 'Valencia' oranges, obtained postpackline from Fort Beaufort Packhouse (Eastern Cape, South Africa). All strains were purified and preserved at 24 °C on Malt Extract Agar (MEA; Oxoid, Johannesburg, South Africa). In vitro antifungal assays were done according to the method described by Regnier et al. (2008). The essential oil obtained from the bulk sample collected in the Boschkop locality, mixed with the surfactant, Triton-X 100 (BDH, Wadeville, South Africa), was added to autoclaved MEA to prepare media containing oil concentrations ranging from 10 to 3000 p.L/L. The surfactant was added at a concentration of400 p.L/L agar to ensure miscibility of the liphophilic oil with the hydrophilic agar. A control using only the surfactant mixed with MEA did not show inhibition of the pathogens tested. This procedure was repeated for lemongrass oil and pure citral. After allowing the agar to set, the medium was inoculated with the specific pathogen and incubated at 23 °C for 7 days. This procedure was

Table 1

Essential oil compositions of individual specimens and bulk samples of Lippia rehmannii from Zandfontein (Zandf) and Boschkop (Bosch).

Compounds

%Relative composition

Zandf specimens

Bosch specimens

1016 a-Pinene 0.09

1059 Camphene 0.27

1103 y8-Pinene 0.05

1116 Sabinene 0.05

1159 a-Phellandrene 0.57

1192 Limonene 0.58

1201 1.8-Cineol 0.63

1231 (Z)-b-Ocimene 0.19

1243 Y-Terpinene 0.19

1269 p-cymene 1.57

1335 6-Methylhept-5-en-2-one 0.29

1453 p -caryophyllene 2.35

1517 Camphor 2.53

1543 Linalool 0.17

1570 Trans-pinocarveol 0.15

1597 Isocaryophyllene 5.21

1615 Caryophyllene 0.12

1683 Neral 25.14

1700 Borneol 3.56

1734 Geranial 43.18

1753 Neryl acetate 1.58

1792 Nerol 0.35

1839 Geraniol 0.60

1995 p-caryophyllene oxide 5.49

Z02 Z03 Z04 Z05 Z06 Z07 Mean

B01 B02 B03 B04 B05 B06 B07 Mean

Bulk samples Zandf Bosch

Monoterpene hydrocarbons Oxygen containing monoterpenes Sesquiterpene hydrocarbons Oxygen-containing sesquiterpenes Other hydrocarbons Total percentage identified

0.39 1.48 0.18 0.11 3.53 1.53 0.75 0.57 0.37 3.31 0.81 2.23 8.66 0.33 0.57

2.96 0.78 21.45 4.59 34.87 2.63 0.95

I.89 2.81

II.45 76.70

5.97 2.81 0.81 97.74

1.53 0.20 0.08 3.01

1.54 0.77 0.33 0.49 2.96 0.48 2.29 8.90 0.16 1.03 2.49 1.86 22.83 2.71 36.83 0.53 0.25 0.23 2.10 10.57 74.24 6.65 2.10 0.48 94.03

0.14 0.61 0.07 0.03 0.72 0.54 0.20 0.04 0.36 0.90 0.92 2.30 3.05 0.31 0.04 3.57 0.13 30.77 2.37 44.37 1.59 0.63 0.67 3.29 3.41 83.99 6.00 3.29 0.92 97.61

0.14 0.22 0.05 0.19 1.66 0.53 1.44 0.56 0.49 1.89 0.57

15.32 2.12 0.09 25.58 1.19 1.32 14.34 1.88 25.00 0.23 0.38 0.29 0.04 3.41

71.33 17.83 0.04 0.57 95.49

0.21±0.14 0.74±0.54 0.10±0.06 0.08±0.06 1.50± 1.29 0.83 ±0.48 0.59±0.46 0.36±0.28 0.38±0.10 1.73 ± 1.05 0.58±0.30 4.19±4.91 4.41 ±3.01 6.82±17.46 3.97±9.53 2.92± 1.43 0.88±0.71 21.82±7.76 3.41 ±1.60 35.34±10.58 1.22±0.88 0.51 ±0.24 0.64±0.59 3.00±1.68 5.94±3.30 78.74±4.68 7.98±4.07 3.00±1.55 0.58±0.27 96.25± 1.41

0.21 0.89 0.11 0.23 0.76 1.03 2.47 0.23 0.17 1.80 0.28 2.19 6.68 0.20 0.14 2.68 0.22 23.14 4.97 37.75 0.86 0.16 0.32 6.07 5.44 76.68 5.09 6.07 0.28 93.57

0.36 1.37 0.16 0.45 1.54 0.91 0.23 0.22 0.30 1.01 0.23 2.12 0.22 0.41 2.50 1.92 0.96 27.80 5.27 38.47 2.80 0.50 1.87 2.74 6.31 80.08 4.99 2.74 0.23 94.35

0.07 0.32 0.05 0.05 0.64 0.43 0.20 0.82 0.44 1.82 0.43 2.28 2.85 0.17 0.55 4.25 0.33 25.57 1.43 41.48 4.46 0.58 0.49 4.98 4.63 77.77 6.87 4.98 0.43 94.68

46.22 1.74 0.65 0.72 3.64 3.50

82.23 6.40 3.64 0.94 96.71

0.05 0.25 0.03 0.06 0.15 0.26 0.06 0.25 0.20 0.63 0.80 2.38 3.67 0.13 0.44 2.34 0.75 30.43 1.12 46.52 2.00 0.54 0.63 2.01 1.87 85.52 5.48 2.01 0.80 95.67

0.16 0.61 0.07 0.03 0.71 0.54 0.21 0.05 0.36 0.88 0.89 2.29 3.07 0.11 0.21 3.59 0.23 29.49 2.61 45.19 0.08 0.59 0.68 3.33 3.40 82.23 6.10 3.33 0.89 95.96

0.16±0.10 0.66±0.38 0.08±0.04 0.12±0.16 0.74±0.41 0.61 ±0.27 0.51 ±0.87 0.24±0.27 0.32±0.10 1.14 ± 0.48 0.64±0.31 2.23±0.09 3.27± 1.88 0.20±0.11 0.60±0.85 3.19±0.88 0.42±0.31 27.50±2.61 2.90± 1.62 42.86±3.64 1.94 ± 1.40 0.52±0.17 0.77±0.51 3.74± 1.37 4.06±1.38 81.07±2.86 5.84±0.64 3.73± 1.26 0.63±0.28 95.35±1.18

0.21 0.74 0.10 0.08 1.50 0.83 0.59 0.36 0.38 1.73 0.58 2.25 3.98 6.82 0.38 2.92 0.38

25.50 3.41 35.37 1.22 0.51 0.64 3.00 5.95 78.43 5.54 3.00 0.58

* Relative retention index (Kovats).

repeated for lemongrass and pure citral (Sigma Aldrich, Johannesburg, South Africa).

2.3. Statistical analyses

The variance amongst averages of the data was evaluated for statistical significance using ANOVA (single factor and two factors without replication). The Least Significant Difference (LSD) test was applied and differences of P < 0.05 were considered significant.

3. Results and discussion

3.1. Essential oil composition and variation

The GC/FID results for 14 individual L. rehmannii oil samples obtained from two localities, along with two bulk samples, are shown in Table 1. Only the 24 most prominent compounds present in the oils, representing between 93.51 and 97.82% of the total oil constituents, are reported. The essential oils were found to contain high concentrations of the geometrical isomers of citral: Z-citral, known as geranial and

E-citral, referred to as neral. Citral was the main constituent of all the specimens, with the exception of one (Specimen Z07), which contained 46.40% linalool, in addition to high levels of citral (17.50% geranial and 9.15% neral). Specimen Z06, containing 15.32% p-caryophyllene, was the only specimen producing a high concentration of the compound, all other samples produced between 2 and 3%. Other major compounds common to all the oils were camphor, borneol, neryl acetate, isocaryophyllene, ^-cymene and p-caryophyllene oxide. Oxygenated monoterpenes were by far the most prominent group of terpenes present. The two bulk samples presented a similar mix of compounds, but the sample harvested from Zandfontein contained a higher percentage of citral (26.78% neral and 42.29% geranial), compared to the sample from Boschkop (25.50% neral and 35.37% geranial). The genus Lippia is well known for chemotypical variability in the essential oil composition (Viljoen et al., 2005) and a more comprehensive study involving specimens from localities, differing in climate and geography, should shed more light on this aspect.

The ANOVA analyses revealed no significant variations between the aromatic profiles of the essential oils obtained from individual specimens growing in the two localities. Specimen

Table 2

Effects in vitro of different concentrations of Lippia rehmanii and lemongrass essential oils, and pure citral on inhibition of mycelial growth of fungal pathogens after 7 days at 23 °C.

Source Pathogens Inhibition (%) at concentration (^L/L) of

10 20 50 100 200 500 1000 2000 3000

Mango Lasiodiplodia theobromae Lippia 0Fi 5Ih 26Gg 33Gf 38He 46Id 51Hc 72Eb 76Ca

Citral 4Ei 14Fh 43Eg 55Cf 63Ce 67Ed 71Ec 81Db 100Aa

Lemongrass 4e 12Fe 45Ed 48Dd 63Cc 66Ec 68Ec 74Eb 100Aa

Colletotrichum gloeosporioides Lippia 0Fh 9Gg 29Gf 30Hf 44Ge 49Hd 64Fc 71Eb 100Aa

Citral 3Eh 7Hg 39Ef 49De 60Dd 69Dc 80Cb 100Aa 100Aa

Lemongrass 4h 12Fg 62Bf 66Be 69Be 72Cd 79Dc 87Cb 100Aa

Avocado Lasiodiplodia theobromae Lippia 0Hi 0Ji 18If 28Ie 34Id 48Hc 83Cb 87Ca 90Ba

Citral 10Fh 19Eg 63Bf 67Be 75 Bd 80Bc 90Bb 100Aa 100Aa

Lemongrass 4Gg 9Gf 48Ce 51Dd 57Dc 58Gc 83Cb 84Cb 100Aa

Colletotrichum gloeosporioides Lippia 0Hi 10Gh 36Fg 46Ef 55Ee 66Ed 73Ec 78Db 100Aa

Citral 3Gh 10Gg 41Ef 64Be 72 Bd 79Bc 89Bb 100Aa 100Aa

Lemongrass 4Ge 10Gh 28Gg 50f 54Ee 61Fd 68Ec 77Db 100Aa

Alternaria alternata Lippia 0Hi 6Hh 25Hg 43Ef 53Ee 61Fd 72Ec 81Cb 100Aa

Citral 5Gh 14Fg 42Ef 57Ce 66Cd 81Bc 87Bb 100Aa 100Aa

Lemongrass 5Ge 13Fh 28Gg 36Ff 53Ee 63Fd 73Ec 87Cb 100Aa

Citrus Penicillium digitatum Lippia 0Fh 0Jh 14Jg 22Jf 33Ie 39Jd 49Ic 64Fb 100Aa

Citral 0Fh 5Ig 12Jf 42Ee 48Fd 66Ec 81Cb 100Aa 100Aa

Lemongrass 0Hg 0Jg 0Lg 13Lf 33Ie 54d 61c 86Cb 100Aa

Alternaria citri Lippia 12Fh 31D 49Df 59Ce 65Cd 71Cc 75Dc 85Cb 100Aa

Citral 0Hh 22Eg 42Ef 55Ce 64Cd 72Cc 77Db 100Aa 100Aa

Lemongrass 10Ff 24Ee 52Cd 56Cd 61Cc 69Db 73Eb 100Aa 100Aa

Grape Botrytis cinerea Lippia 0Hh 0Jh 3Kg 17Kf 31Je 45Id 56Gc 72Eb 79Ca

Citral 0Hh 9Gg 25Hf 64Be 73 Bd 82Bc 92Bb 94Bb 100Aa

Lemongrass 0Hh 9Gg 48Cf 55Ce 63Cd 71Cc 78Db 100Aa 100Aa

Potato Rhizoctonia solani Lippia 63Ab 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa

Citral 19Ec 61Bb 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa

Lemongrass 36Dc 48Cb 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa

Maize Fusarium oxysporum Lippia 69Ab 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa

Citral 57Ba 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa

Lemongrass 45Cc 65Bb 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa 100Aa

Averages (n = 10) within the same column followed by the same upper-case letter do not differ significantly at P < 0.05. Averages (n = 10) within the same row followed by the same lower-case letter do not differ significantly at P < 0.05.

Upper-case and lower-case letters can not be compared to each other and no comparison may be made between letters from different columns or rows.

Z07 was considered an outlier (Q test), on the basis of the linalool content, and was therefore not included in the ANOVA. An inclusion of the specimen would have introduced bias in the comparison of the two populations. The chemical profiles were fairly consistent, particularly those of the specimens harvested from Boschkop. Genetic differences are most probably responsible for the one specimen from Zandfontein producing linalool as a major compound. However, the production of linalool and its oxides have been linked to specific stages of flower development in Clarkia breweri, with an increase in production observed during the flowering stage, and a termination of production once the flowers are pollinated (Cseke et al., 2007).

Citral is one ofthe main constituents of lemon oil (Chutia et al., 2009), which explains the characteristic odour of L. rehmannii leaves. The compound is also present in high concentrations in lemon balm (Melissa officinalis) oil (Schnitzler et al., 2008). Lemongrass (Cympopogon citrates) generally produces in the region of 41% geranial and 32% neral, thereby yielding total citral content in excess of 70%, with essential oil yields of 1 to 2% on a dry weight basis (Tsortzakis and Economakis, 2007). The results obtained in our laboratory for the commercial lemongrass sample (39% geranial and 34% neral) correlated with the values reported by these authors. Of the fourteen L. rehmannii specimens investigated, six produced oils with citral contents in excess of 70%, while twelve specimens contained more than 50% citral. Lippia rehmannii therefore compares well with lemongrass with regard to citral production, however the essential oil yields were found to be lower (0.22 to 1.2%). Other Lippia species that reportedly produce citral in large amounts include L. alba (Terblanche and Kornelius, 1996) with 12.9% to 36.4% geranial and 9.6 to 28.2% neral and L. citriodora with 26.8 to 38.7% geranial and 21.8 to 24.5% neral (Argyropolou et al., 2007). The chemical profiles of the essential oils of L. rehmannii were found to closely resemble that reported for L alba, originating from South America (Terblanche and Kornelius, 1996).

3.2. Antifungal assay

The in vitro antifungal activities of the essential oils of L. rehmannii and lemongrass, as well as that of pure citral, against the different test pathogens are shown in Table 2. Lemongrass oil and citral at a concentration of 3000 p.L/L, were fungitoxic to all the pathogens tested. Although L. rehmannii essential oil was able to completely inhibit the growth of the majority of fungi at this concentration, it was less effective in controlling the growth of L. theobromae and B. cinerea. The essential oil of L. rehmannii used in the antifungal assays was isolated from plants harvested in Boschkop and contained less citral than lemongrass oil. Citral was, in general, more active than both lemongrass and L. rehmannii essential oils against the fruit pathogens, suggesting that the compound is mainly responsible for the antifungal action of the essential oils. This observation correlates to other reports which highlight the antifungal activity of citral (Rodov et al., 1995). Ben-Yehoshua et al. (1992) found a

correlation between reduced citral concentrations in lemon peels and increased susceptibility to P. digitatum infection.

All three test substances were highly effective against F. oxysporum and R. solani, resulting in total growth inhibition, even at a concentration of 50 p.L/L. However, Lippia rehmannii, at a concentration of only 10 p.L/L, was significantly more effective than lemongrass and citral in controlling the growth of these fungi, with 63 and 69% inhibitions of R. solani and F. oxysporum obtained, respectively. This observation points to a possible synergistic action (Bakkali et al., 2008) of citral with other terpenes present in the oil of L. rehmannii.

4. Conclusion

Lippia rehmannii has very similar morphological attributes to other Lippia spp. indigenous to South Africa, but has a uniquely high content of citral, which gives the species its characteristic citrus odour. Lemongrass and L. rehmannii essential oils show promise as possible candidates for the control of several postharvest fruit pathogens, most probably as a result of their high citral contents. The selection of L. rehmannii specimens with a high citral content for future propagation is feasible. An exciting finding that should be further substantiated by hot-house trials is the excellent in vitro inhibition of R. solani and F. oxysporum, using L. rehmannii oil at concentrations as low as 10 p.L/L. Lippia rehmannii is an excellent substitute for lemongrass as an antifungal agent, since it occurs in the wild and is reasonably drought resistant and well adapted to local climatic conditions.

Acknowledgement

We thank the National Research Foundation (NRF) of South Africa for financial support.

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Edited by J Van Staden