Sitophilus zeamais Motschulsky and Sitophilus oryzae (L.) are pests of stored grains capable of surviving in extreme cold and hot temperatures and hence found all over the world (Walgenbach and Burkholder, 1986). Although they are primarily associated with maize and rice, S. zeamais and S. oryzae are capable of developing on all cereal grains and cereal products (Walgenbach and Burkholder, 1986). They are internal feeders that not only cause damage to the grains but also promote secondary insect pest and fungal infestations that further affect the quality and quantity of grains (Gupta et al., 1999). Currently, the control of stored product insects relies heavily on the use of non-natural insecticides and fumigants, which has led to problems such as environmental pollution, pest resurgence, resistance to pesticides and lethal effects on non-target organisms in addition to direct toxicity to the users (Zettler and Arthur, 2000). Plant derived natural products are considered a potential alternative to these toxic and environmentally unsustainable compounds. Because they represent a rich source of bioactive chemicals it was hypothesized that plants native to South Africa could provide alternatives to currently used insect control agents. More than 2,000 plant species have been found to possess insecticidal activity with the most well known botanical pesticides being pyrethrum, neem, rotenone, nicotine and plant essential oils (Philogene et al., 2005; Isman, 2006). Essential oils have been shown to control stored product pests by fumigant activity, contact insecticides and as repellents. Additionally, these bioactive plant secondary metabolites do affect insect growth rate and oviposition (Denloye et al., 2011; Chen et al., 2011; Stefanazzi et al., 2011). The camphor bush, Tarchonanathus campharatus (L.), (family Asteraceae) is a shrub of reaching six meters in height and occurs in a wide range of habitats (van Wyk et al., 1997). The strongly scented tree of T. campharatus has many medicinal applications in traditional healing in South Africa, such as smoking the leaves or drinking infusions or decoctions. The infusions and tinctures of the leaves are used for abdominal pain, headache, toothache, asthma, bronchitis and inflammation and smoke from the fresh or dried plant is inhaled for rheumatism (Hutchings and Van Staden, 1994). In East Africa, the dry leaf infusion is drunk for tapeworm, the leaves are put underarm as perfume and to prevent tiredness and they are used for the control of bedbugs (Anonymous, 2005). The plant shows powerful insect repellent action (Omolo et al., 2004) and wild animals living in the areas where T. camphoratus grows, particularly Cape buffaloes and black rhinoceri, rub themselves against the leaves to deter mosquitoes and flies. The plant also seems to drive away tse-tse fly, a pathogenic agent of trypanosomiasis (Anonymous, 2005). The purpose of this study was to determine the insecticidal activities of the essential oil of the dry leaves of T. camphoratus under laboratory conditions against S. zeamais and S. oryzae. T. camphoratus was selected as a model for this study based on its broad spectrum use in traditional medicine and preliminary reports of its insecticidal activity (Omolo et al., 2004; Anonymous, 2005).

Plant material

 

Fresh materials of T. camphoratus were collected from Sangoyana in the northern part of Kwa-Zulu Natal province, South Africa in March, 2010. The plant was identified by the local people during the time of collection and further identified by Mrs N.R Ntuli in the Department of Botany, University of Zululand. A voucher specimen, (NSKN 1), was deposited at the University of Zululand herbarium.

 

Extraction of the essential oil

 

Air-dried leaves were subjected to hydro-distillation using a Clevenger-type apparatus (British Pharmacopia, 1980). The essential oil was collected 4 h after boiling, weighed and kept at 4°C in sealed glass vials before analysis and bioassay.

 

Determination of the insecticidal activity

 

Rearing of test insects

 

Adults of S. zeamais and S. oryzae were obtained from a colony maintained by the Plant Protection Research Institute, Pretoria. South Africa. These were mass reared on whole maize grains in 5 L glass jars in a controlled chamber, at 28 ± 20°C and 56 to 65% Relative humidity in the Department of Agriculture, University of Zululand. Newly emerged, one week old insects were used in the bioassay (Odeyemi et al., 2008).

 

 

 

The repellent effect of T. camphoratus essential oil against S. zeamais and S. oryzae was studied using a modified area preference method (Tapondjou et al., 2005). The test area consisted of a 9 cm Whatman No.1 filter paper cut into two halves. Different oil concentrations were prepared by diluting 10, 20, 30 and 40 µl of the oil in 1ml hexane and these corresponded to concentrations of 0.314, 0.628, 0.943 and 1.257 µl of oil/cm² of the filter paper respectively. The other half was treated with 0.5 ml hexane alone and this served as a control. Both essential oil treated and hexane treated filter paper halves were air-dried under a fan to evaporate the solvent completely. With the aid of a clear adhesive tape, both halves were later joined together into full discs and placed in 9 cm glass Petri dishes. Twenty one-week old, unsexed adult insects were released at the centre of the rejoined filter paper disc and the Petri dish was covered. Each treatment was replicated four times for each S. zeamais and S. oryzae. The number of insects present on the control and on the treated areas of the filter paper was recorded after 1, 2, 6, 4 and 24 h. Percentage repellency (PR) was calculated as follows (Nerio et al., 2009):

 

PR = ((Nc – Nt)/(Nc + Nt)) x 100

 

Nc was the number of insects on the untreated area after the exposure interval and Nt was the number of insects on the treated area after the exposure interval. The mean number of insects on the treated portion of the filter paper was compared with the number on the untreated portion. Results were presented as the mean of percentage repellency ± the standard error.

 

Statistical analysis

 

Data was analysed using the QED statistics software. Means for percentage repellency for both insects at the four concentrations at a particular time interval were compared using one way ANOVA. The median repellent dose (RD₅₀) was determined from the linear regression equation through regression analysis.

 

Gas chromatography-mass spectrometry analysis

 

The GC-MS analysis was carried out using an Agilent 6890 GC with an Agilent 5973 mass selective detector [MSD, operated in the EI mode (electron energy = 70 eV), scan range = 45-400 amu, and scan rate = 3.99 scans/s], and an Agilent ChemStation data system. The GC was equipped with a fused silica capillary HP-5 MS column of an internal diameter of 0.25 mm, film thickness 0.25 µm and a length of 30 m. The initial temperature of the column was 70°C and was heated to 240°C at a rate of 5°C/min. Helium was used as the carrier gas at a flow rate of 1 ml/min. The split ratio was 1:25. Scan time was 50 min with a scanning range of 35 to 450 amu. A 1%, w/v, solution of the sample in hexane was prepared and 1 µl was injected using a splitless injection technique.

 

Identification of components

 

The identification of the oil constituents was based on their retention indices determined by reference to a homologous series of n-alkanes (C₈-C₃₀), and by comparison of their mass spectral fragmentation patterns with those reported by Joulain and Koening (1998) and Adams (2007) and stored in the MS library [NIST database (G1036A, revision D.01.00)/ChemStation data system (G1701CA version C.00.01.080)]. The percentages of each component are reported as raw percentages based on the total ion current without standardization.

Insecticidal activities

 

The essential oil of T. camphoratus did not show fumigation and contact toxicity against both S. zeamais and S. oryzae at the concentrations used. All the S. zeamais and S. oryzae tested remained alive after the 28 days of exposure. However, the essential oil showed repellent activity against S. zeamais and S. oryzae at the concentrations used. A percent repellence (PR) value of greater than 50% from the four replicates was noted at all concentrations for both S. zeamais and S. oryzae 24 h after treatment (Figures 1 and 2). Repellent action was highly dependent upon oil concentration and exposure time. There were no significant differences between the means of percentage repellence for both insects at the four concentrations at a particular time interval at p< 0.05. The median repellent doses were 0.945 and 0.910 µl/cm² for S. zeamais and S. oryzae respectively. Percent repellence increased with both increasing concentration and time of exposure.

 

 

 

Chemical composition

 

The dry leaves of T. camphoratus yielded 0.23% (w/w) of yellowish green oil with a strong camphor aroma. Twenty seven compounds were identified in the oil accounting for 73.01% of the total oil composition. The oil was dominated by sesquiterpene hydrocarbons (59.18%) of which allo-Aaromadendrene, β-Guaiene, γ-Cadinene, δ-Cadinene, aromadendrene, Beta-caryophyllene and γ-Muurolene were the major components (Table 1). Monoterpene hydrocarbons formed 1.61% of the oil and the percentage composition of the oxygenated monoterpenes and oxygenated sesquiterpenes were 6.26 and 3.19% respectively.

 

The essential oil of the dry leaves of T. camphoratus showed no contact and fumigation toxicity on both S. zeamais and S. oryzae at the concentrations used. Previous studies have shown that the toxicity of essential oils obtained from aromatic plants against storage pests is related to the oil’s main components (Lee et al., 2003). Among the essential oil components monoterpenes have drawn the greatest attention for insecticidal activity against stored product pests (Asgar, 2011). Various monoterpenes like 1,8 cineole, linalool, α-pinene, terpinen-4-ol, and α-terpinene have been reported to show contact and fumigation toxicity to stored product pests (Papachristos et al., 2004; Stamopoulos et al., 2007). These monoterpenes, although present in the essential oil under study were in trace amounts and lack of toxicity of the essential oil may be attributed to the low total concentration of monoterpenes in the oil (Table 1). However, the oil showed good repellent activity against both S. zeamais and S. oryzae. One of the major compounds in the oil, δ-cadinene, has been reported to have repellent activity against some anthropods (Yatagai et al., 2002), and may be responsible for the observed repellent activity of the oil. However, there is a possibility of synergetic action between major and minor components to effect the repellent action of the oil. Biological activity of essential oils has been reported to be affected by interactions among the structural components of the oil where even the minor compounds can have critical function due to coupled effects and additive action between the different chemical classes (Tapondjou et al., 2005).

The essential oil of the dry leaves of T. camphoratus from Kwa-Zulu Natal, South Africa is mainly dominated by sesquiterpene hydrocarbons. The oil is not toxic to S. zeamais and S. oryzae but could be considered a potential in the control of stored product pests as a repellent.

The authors have not declared any conflict of interest.

The authors are grateful to the National Research Fund and University of Zululand, South Africa for the financial support.