INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 10–500/ZIP/2011/13–4–517–522 http://www.fspublishers.org Full Length Article Insecticidal Properties of Piper nigrum Fruit Extracts and Essential Oils against Spodoptera litura LOH SIEW FAN, RITA MUHAMAD1, DZOLKHIFLI OMAR AND MAWARDI RAHMANI† Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia †Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 1Corresponding author’s e-mail: [email protected]; [email protected] ABSTRACT Chemical analysis by GC and GC-MS revealed presence of 39 compounds in the essential oil fraction of Piper nigrum fresh fruits. Limonene was the major compound present with 35.06% of total oil followed by beta-pinene (12.95%) and linalool (9.55%). Insecticidal properties of Piper nigrum fruit extracts and essential oils were investigated against tobacco army worm, Spodoptera litura using topical application bioassay on uniform weighted second instar larvae in the laboratory. The hexane extract was most effective in killing the larvae and showed the highest toxicity at 48 h after treatment. Toxicity of extracts decreased in the order of hexane (LD50: 1.8 mg/g) > acetone (LD50: 18.8 mg/g) > chloroform (LD50: NA, the toxicity was very low) > essential oil (no mortality). Insect development and growth index observations showed that the hexane extract had antifeedant properties resulting in severe growth inhibition of Spodoptera litura. 2011 Friends Science Publishers Key Words: Botanical insecticide; Piper nigrum; Spodoptera litura; Topical bioassay INTRODUCTION peepuloidin, piplartin and trichonine contribute in no small measure (Adgeh, 1989; Awoyinka et al., 2006). These Insect pests are a major constraint on crop production, compounds have been demonstrated to be toxic to fruit flies, especially in developing countries. Natural plant extracts adzuki bean weevils, cockroaches and several other insect play an increasingly prominent role as alternatives to species (Su & Hovart, 1981; Gbenwonyo et al., 1993; synthetic pesticides due to the increasing concern on health Awoyinka et al., 2006). Hence, the present study was hazards, environmental pollution and negative effects on conducted to: (i) evaluate the toxicity of P. nigrum extracts non target organisms (Sharma et al., 2006). Many floral against the S. litura larvae and (ii) study the effects of P. volatiles have anti-microbial or anti-herbivore activity nigrum hexane extract on development and antifeedant (DeMoraes et al., 2001; Friedman et al., 2002; Hammer et reaction in Spodoptera litura. al., 2003) and hence act to protect valuable reproductive parts of plants from damage (Dudareva et al., 2004). MATERIALS AND METHODS However, intensive screening is necessary to select compounds with pesticidal properties, but harmless to the Plant materials and preparation of extracts: Fresh and environment and ecosystem. Researches on potential dried fruits of P. nigrum were obtained from Sibu, Sarawak, botanical extracts which are safe with little or no residues East Malaysia. The essential oil fraction was extracted from and naturally derived with minimal technology are urgently fresh fruits by hydrodistillation method (Maisonneune & needed. There are more than 2400 plant species belonging Saint-Ruffine, 1975). The essential oils of P. nigrum was to 189 plant families which are said to be rich sources of stored in glass vial and put in refrigerator (-4º) for further bioactive organic compounds (Rao et al., 2005). Among the experiment. Dried fruits were ground into powder and families of plants investigated to date, one showing soaked in hexane, chloroform and acetone to yield enormous potential is the pepper family, otherwise known sequentially the non-polar, intermediate and polar as Piperaceae (Dodson et al., 2000). The phytochemical compounds. The solvents used were based on the elutropic screening of black pepper fruit shows that it contains 4% series. Three hundred grams of black pepper powder was alkaloids in the berry (Dev & Koul, 1997; Awoyinka et al., soaked in 1 L solvent and incubated for 48 h. The mixture of 2006). Although information on the compounds responsible solvent and plant material was filtered by Whatman No. 1 for the insecticidal activities is scarce, it has been filter paper and the solution was collected. The plant documented that the amide olefinic or alkyl isobutylamides material was soaked in 1 L of hexane for another 48 h. Final compounds such as piperine, piperettine, tricostacine, filtered solution was accumulated and evaporated by rotary To cite this paper: Fan, L.S., R. Muhamad, D. Omar and M. Rahmani, 2011. Insecticidal properties of Piper nigrum fruit extracts and essential oils against Spodoptera litura. Int. J. Agric. Biol., 13: 517–522 FAN et al. / Int. J. Agric. Biol., Vol. 13, No. 4, 2011 evaporator to yield dark viscous extracts. Same extraction μg/g insect that caused 50% mortality) along with 95% methods were repeated by soaking the black pepper powder confidence intervals. individually by chloroform and acetone. Effect of hexane extract on development of Spodoptera Identification of chemical constituents: Qualitative and litura: Based on the results of the toxicity study, the hexane quantitative data were determined by GC and GC-MS, extract was selected to further evaluate its effects on growth respectively. The identification of the chemical constituents and development of S. litura until adult emergence. The was assigned on the basis of comparison of their mass experiment was conducted by topical bioassay on 2nd instar spectra using Shimadzu NIST/EPA/NIH+ mass spectral larvae as previously described. After application, treated database (225-04793-92), which produces standard bar larvae were placed singly in disposable Petri dishes for graphs for direct comparison with published spectra. observation until adult emergence. Larvae mortality (%), Gas chromatography (GC): The oil was injected onto a larvae head capsule length (mm), larvae weight gain (g), Shimadzu GC-17A system, equipped with an AOC-20i percentage of pupation (%), pupa weight (g), malformed autosampler and a split/splitless injector. The column used pupae (%), adult emergence (%) and sex ratio were was BPX 5, 30 m, 250 µm i.d., 0.25 µm df, coated with 5% recorded. Corrected efficacy (%) of larvae mortality was phenymethylsilane, operated with the following oven calculated using the formula proposed by Abbott (1925), temperature programme: 60ºC, held for 3 min, rising at malformed pupae and pupae mortality were calculated by 20°C/min to 100ºC, rising at 7°C/min to 240ºC, held for 5 Sun-Shepard’s formula (Püntener, 1981) and adult min; injection temperature and volume, 250°C and 1.0 µL, emergence was calculated by Henderson-Tilton’s formula respectively; injection mode, split; split ratio, 28:1; carrier (Henderson & Tilton, 1955). Larval growth index and total gas, helium at 29.6 cm/s linear velocity and inlet pressure developmental growth index were calculated as per Gupta 27.0 kPa; detector temperature, 280°C; column flow rate, and Girah (2001). All data were analyzed using the 0.7 mL/min; sampling rate, 0.50 s. Data was acquired by ANOVA procedure in SAS Statistical package and means means of GC solution software (Shimadzu). were compared by Tukey's Studentized Range (HSD) Test. GC-MS: Shimadzu QP 5050 A mass spectrometer system Antifeedant test: The experiment was conducted using the equipped with a capillary column BPX 5 (30 m x 250 µm, leaf dip method. P. nigrum hexane extract was selected to 0.25 μm film thickness) was used. Helium was used as the test on the 2nd instar of S. litura. The hexane crude extract carrier gas. The MS operating conditions were: ionization (0.3883 g) was dissolved in 1.5 mL xylene (solvent) and 0.1 was induced by an electron impact (EI) at 70 eV, ion source g Triton X-100 (surfactant) as emulsifiable concentrate and 250ºC. The compounds present were identified by finally dissolved in 20 mL distilled water to get a clear conducting a library search using Shimadzu solution. The percentages of formulation were assigned NIST/EPA/NIH+ mass spectral database. The operating based on experimentation including trial and errors. Leaf parameters were identical with those of GC analysis discs, 60 mm diameter, were cut and soaked in the prepared described above. extract, air dried and singly placed into disposable petri Bioassay to evaluate toxicity of extracts: The experiments dishes. Feeding tests were conducted using second instar were conducted using topical application bioassay. larvae of Spodoptera litura. One larva per dish and a total of Armyworm larvae were obtained from laboratory colonies 50 larvae were used per treatment. Leaf disc areas were maintained by Malaysia Agriculture Research and measured using a leaf area meter (LI-3100, LI-COL, USA) Development Institute (MARDI), Serdang. Five doses of at 24 and 48 h after exposure. Antifeedant index (AI) was solvent extracts (ranging from 0 to 40 mg/g) and essential calculated as per Gupta and Girah (2001). oils (ranging from 0 to 40 µL/g) were tested on one hundred larvae per dose with acetone used as a control. A 0.5-µL RESULTS AND DISCUSSION droplet of pesticide-acetone solution or acetone was administered to the thoracic tergum using a micro syringe (5 Chemical constituents of black pepper essential oil: µL, Hamilton Series 600/700 Standard microliter syringe, Distilled oil production from the fresh fruits of P. nigrum Fisher Scientific). After treatment, treated larvae were was 0.8% (v/w), yielding a colourless liquid rich in pungent individually introduced into each Petri dish. Organic fragrance with many volatile components (Table I). Thirty- mustard leaves were applied to consume by the treated nine components were identified. The monoterpene larvae. Mortality was assessed at 24 h and 48 h. Larva was compounds consisted of approximately 64.05% of the total considered dead if it was unable to make a coordinated oil.