International Journal of Food Microbiology 135 (2009) 165–170 Contents lists available at ScienceDirect International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o Efficacy of Lippia alba (Mill.) N.E. Brown essential oil and its monoterpene aldehyde constituents against fungi isolated from some edible legume seeds and aflatoxin B1 production Ravindra Shukla, Ashok Kumar, Priyanka Singh, Nawal Kishore Dubey ⁎ Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India a r t i c l e i n f o Article history: Received 11 February 2009 Received in revised form 30 June 2009 Accepted 1 August 2009 Keywords: Lippia alba Essential oil Geranial Neral Antifungal activity a b s t r a c t The present study deals with evaluation of antifungal properties of Lippia alba essential oil (EO) and two of its monoterpene aldehyde constituents against legume-contaminating fungi. Seventeen different fungal species were isolated from 11 varieties of legumes, and aflatoxigenic isolates of Aspergillus flavus were identified. Hydrodistillation method was used to extract the EO from fresh leaves. The GC and GC–MS analysis of EO revealed the monoterpene aldehydes viz. geranial (22.2%) and neral (14.2%) as the major components. The antifungal activity of EO, geranial and neral was evaluated by contact assay on Czapek'sdox agar. The EO (0.25–1 μL/mL) and its two constituents (1 μL/mL) showed remarkable antifungal effects against all the fungal isolates (growth inhibition range 32.1–100%). Their minimal inhibitory (MIC) and fungicidal (MFC) concentrations for A. flavus were lower than those of the systemic fungicide Bavistin. Aflatoxin B1 (AFB1) production by three isolates of A. flavus was strongly inhibited even at the lower fungistatic concentration of EO and its constituents. There was no adverse effect of treatments on seed germination, and rather, there was enhanced seedling growth in the EO-treated seeds. It is concluded that L. alba EO and two of its constituents could be safely used as effective preservative for food legumes against fungal infections and mycotoxins. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Legumes represent the richest source of proteins and amino acids for human and animal health. The recent trends indicate that legumes share nutraceutical properties which not only contribute to a balanced diet but can also prevent the widely diffused diseases including type II diabetes, cardiovascular diseases, digestive tract diseases and obesity (Duranti, 2006). Fungal contamination in stored seeds and grains is a serious problem in the tropical and sub-tropical countries as the prevailing warm/humid climate is the ideal condition for fungal growth/activity (Weinberg et al., 2008). Their invasion may initiate at any stage from the standing crop through to harvest and post-harvest handlings until they reach the consumer. The infected seeds have decreased nutritional value, loss in germinability, discolouration, increase in free fatty acids (FFA), and more importantly the production of mycotoxins (El-Nagerabi and Elshafie, 2000; Dhingra, et al., 2001; Embaby and Abdel-Galil, 2006). ⁎ Corresponding author. Tel.: +91 9415295765; fax: +91 5422368174. E-mail address: nkdubey2@rediffmail.com (N.K. Dubey). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.08.002 Aflatoxin is one of the most common and hazardous mycotoxins produced by Aspergillus flavus Link ex. Fries. In developing countries, about 4.5 billion people are systematically exposed to excesses of aflatoxin (Williams et al., 2004). Aflatoxicosis induces depressed feed efficiency, abnormal liver chemistry, depressed immune response, carcinogenesis, and even death (Pier, 1992). In plants, aflatoxins inhibit seed germination, seedling growth, root elongation, synthesis of photopigments, proteins, nucleic acids, and some vital enzymes (Jones et al., 1980). Management of fungal contamination of harvested seeds/grains is based on physical (aeration, cooling and rapid drying) and chemical treatments with ammonia, food preservatives or even with pesticides. Since most of these control strategies require expensive chemicals and technical expertise to monitor physical parameters (temperature and pressure), they are not affordable by rural subsistence farmers (Atanda et al., 2007). Also, the widespread and indiscriminate use of chemical preservatives or pesticides has significant drawbacks in not being economical, handling hazards, toxic residues on the grains and more importantly the emergence of resistant food-borne microorganisms (Ishii, 2006). These risks have increased public awareness for safer alternatives to chemical preservatives that are accessible, simple in application, non-toxic to humans and plants, and have sustained broad-spectrum fungitoxicity. Essential oils (EOs) have been classified as GRAS 166 R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170 (Generally Recognized as Safe) (Burt, 2004) and have been recommended as preservatives for food commodities based on their antimicrobial and anti-mycotoxigenic effects (Mishra and Dubey, 1994; Varma and Dubey, 2001; Kumar et al., 2007; Kumar et al., 2008; Rasooli et al., 2008; Razzaghi-Abyaneh et al., 2008; Tatsadjieu et al., 2009). Lippia alba (Miller) N.E. Brown, the aromatic shrub (family: Verbenaceae) is a potential source of EO in India. Ethnopharmacological studies revealed that leaves can be used as an infusion against states of excitement, hypertension, digestive troubles, nausea and cold, to heal wounds locally and as syrup against cough and bronchitis. In addition, the antimicrobial, analgesic, anti-inflammatory and antioxidative potential of EOs have also been ascertained (Oliveira et al., 2006; Hennebelle et al., 2008). In the present study, legume seeds were screened for incidence of mycoflora and the isolation of aflatoxigenic strains of A. flavus. EO of Lippia alba and two of its major components were evaluated for fungitoxicity and anti-aflatoxigenicity against selected fungal isolates. 2. Materials and methods 2.1. Legume seeds and mycoflora analysis Eleven varieties of edible legume seeds viz. peanut (Arachis hypogaea L.), pigeon pea (Cajanus cajan L.), chick pea (Cicer arietinum L.), soya bean (Glycine max L.), lentil (Lens culinaris Medikus), red bean (Phaseolus vulgaris L.), white pea (Pisum sativum L.), moth bean (Vigna aconitifolia (Jacq.) Marechal), black gram (Vigna mungo (L.) Hepper), mung bean Vigna radiata (L.) R. Wilczek and cow pea (Vigna unguiculata (L.) Walp) were purchased locally. The seed mycoflora was examined using the blotter test and the agar plate method as recommended by International Seed Testing AssociationInternational Seed Testing Association, 1999 International Seed Testing Association, 1999. International Rules for Seed Testing. Seed Science and Technology 27 (Suppl.). Seeds were surfacesterilized (1% solution of sodium hypochlorite) and rinsed in three changes of sterile distilled water. Seeds were placed in Petri plates containing blotter pads and potato dextrose agar (PDA) medium incubated for 7 days (28 ± 2 °C). The developing fungal colonies were isolated, identified (Burnett and Hunter, 1999) and routinely maintained on PDA (4 °C). The incidence of fungi was determined based on the occurrence of a particular species in samples of 10 seeds. 2.2. Aflatoxigenic isolates of A. flavus A. flavus isolates from each variety of legume seed were screened for the production of aflatoxin B1 (AFB1) following Sinha et al. (1993). The isolates were cultured separately in 25 mL SMKY broth (sucrose 200 g; MgSO4·7H2O, 0.5 g; KNO3, 0.3 g and yeast extract, 7 g; 1 L distilled water) in 100 mL flask for 10 days. The content of each flask was filtered and extracted with 20 mL chloroform in a separating funnel. The extract was evaporated to dryness on water bath and redissolved in 1 mL chloroform. AFB1 was detected by thin layer chromatography. Fifty micro liter chloroform extract was spotted on TLC plates and developed in the solvent system comprising toluene/ isoamyl alcohol/methanol (90:32:2; v/v/v). The plate was air dried and the intensity of AFB1 observed in UV-transilluminator (360 nm). neral (monoterpene aldehydes) were purchased from Genuine Chemical Co., Mumbai, India. 2.4. Extraction of essential oil Fresh plant leaves (200 g) were collected in the month of June and subjected to hydro-distillation (4 h) using a Clevenger-type apparatus (Kumar et al., 2007). The yield (mL/kg) of EO was averaged over four experiments and calculated based on plant material fresh weight. EO was stored airtight and subjected to gas chromatography-mass spectrometry (GC–MS). 2.5. Oil analysis The EO was analyzed by gas chromatography (PerkinElmer Auto XL GC, MA, USA) equipped with a flame ionization detector and the GC conditions were: EQUITY-5 column (60 m × 0.32 mm× 0.25 µm); H2 was the carrier gas; column Head pressure 10 psi; oven temperature program isotherm 2 min at 70 ºC, 3 ºC/min gradient to 250 ºC, isotherm 10 min; injection temperature, 250 ºC; detector temperature 280 ºC. GC–MS analysis was performed using PerkinElmer Turbomass GC– MS. The GC column was EQUITY-5 (60 m × 0.32 mm × 0.25 µm) fused silica capillary column. The GC conditions were: Injection temperature, 250 ºC; column temperature, isothermal at 70 ºC for 2 min, then programmed to 250 ºC at 37 ºC/min and held at this temperature for 10 min; ion source temperature, 250 ºC. Helium was the carrier gas. The effluent of the GC column was introduced directly into the source of MS and spectra obtained in the EI mode with 70 ev ionization energy. The sector mass analyzer was set to scan from 40 to 500 amu for 2 s. The identification of individual compounds is based on their retention times relative to those of authentic samples and matching spectral peaks available with Wiley, NIST and NBS mass spectral libraries or with the published data (Adams, 2007). 2.6. Antifungal assay The antifungal activity of EO and its two constituents was tested against fungal isolates by contact assay based on hyphal growth inhibition (Chang et al., 2008) using Czapek's-dox agar (CDA) medium (NaNO3, 2 g; K2HPO4, 1 g; MgSO4, 0.5 g; KCl, 0.5 g; FeSO4, 0.01 g; sucrose, 30 g; agar, 15 g; 1 L distilled water, pH 6.8 ± 0.2; Sisco Research Lab., Mumbai). The requisite amount of EO was dissolved in 0.5 mL acetone, and added to 9.5 mL molten CDA in different Petri plates to achieve final concentrations (0.25, 0.50, 0.75 and 1 μL/mL). Geranial and neral were treated at 1 μL/mL. CDA plates containing acetone (0.5 mL) only, served as negative control. In addition, CDA plates treated with Bavistin (50% carbendazim; BASF India Ltd., Mumbai) at 4 mg/mL were used as positive control. A 5 mm disc of test fungus was placed upside down on the center of the plate with fungal species in contact with growth medium. Cultures were incubated in the dark at 28 ± 2 °C (7 days). Antifungal index was calculated as the following— Antifungal index ð%Þ =  1−  Da × 100 Db where Da: the diameter of growth zone in the test plate; Db: the diameter of growth zone in the control plate. 2.3. Plant material and oil constituents 2.7. Determination of MIC and MFC Lippia alba, growing wild in the premises of Banaras Hindu University, Varanasi was identified by morphological features with the help of Flora of BHU Campus (Dubey, 2004) and the voucher specimen (LHP/Ver-21/2008) was deposited at the Laboratory of Herbal Pesticides, Banaras Hindu University, Varanasi. Geranial and The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for A. flavus (the most prevalent fungus) were determined by broth dilution method as reported earlier (Shukla et al., 2008). Different concentrations of the EO and constituents were dissolved in 0.5 mL acetone and incorporated to 167 R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170 Table 1 Fungi isolated from edible legume seeds. Legumes Common name Fungi isolated Arachis hypogaea L. Cajanus cajan L. Cicer arietinum L. Glycine max L. Lens culinaris Medikus Phaseolus vulgaris L. Pisum sativum L. Vigna aconitifolia (Jacq.) Marechal Vigna mungo (L.) Hepper Vigna radiata (L.) R. Wilczek Vigna unguiculata (L.) Walp Peanut Pigeon pea Chick pea Soya bean Lentil Red bean White pea Moth bean Black gram Mung bean Cow pea ⁎Aspergillus flavus (8), Aspergillus niger (4), Fusarium oxysporum (3) A. flavus (6), Alternaria alternata (5), Aspergillus glaucus (3) A. flavus (10), A. niger (4), A. alternata (4) A. flavus (8), Aspergillus shydowi (5) A. flavus (4), A. alternata (7), Rhizopus stolonifer (4) A. flavus (8), Trichoderma sp. (3) A. flavus (3), Aspergillus terreus (6), Penicillium italicum (2), Fusarium graminearum (2) A. flavus (7), Curvularia lunata (4), Fusarium sp. (2) A. flavus (6), A. niger (5), Rhizoctonia solani (3) ‡ A. flavus (5), A. niger (7), Fusarium nivale (3) † A. flavus (8), Aspergillus fumigatus (3), Cladosporium cladosporioides (3) Aflatoxigenic isolates: ⁎ (NKD-235), ‡ (NKD-116), † (NKD-122). Values in parentheses are incidence of a particular species in samples of 10 seeds. 9.5 mL CD broth tubes containing 106 spores/mL. The tubes were incubated at 30 °C for a week. The lowest concentration that did not permit any visible growth was taken as MIC. Cells from the tubes showing no growth were sub-cultured on treatment-free CDA plates to determine if the inhibition was reversible. MFC is the lowest concentration at which no growth occurred on the plates. 2.8. Efficacy of the essential oil and constituents on aflatoxin B1 synthesis Requisite amount of EO and constituents were dissolved in 0.5 mL acetone, and added to 24.5 mL SMKY to achieve the various concentrations up to MIC. The medium was inoculated with toxigenic isolates of A. flavus to give 106 spores/mL and incubated at 28 ± 2 °C (10 days). The medium was filtered and fungal mat was dried at 80 °C (12 h) to determine the net mycelial dry weight. The filtrate was used for aflatoxin extraction as described above. For quantitative estimations, fluorescent spot of AFB1 on TLC plate was scrapped, dissolved in 5 ml cold methanol, and centrifuged (3000 rpm, 5 min). Optical density of the supernatant recorded at 360 nm and the AFB1 amount calculated according to Sinha et al. (1993): Aflatoxin B1 content ðμg = LÞ = D×M × 1000 E×l where, D = absorbance, M = molecular weight of aflatoxin (312), E = molar extinction coefficient of aflatoxin B1 (21,800) and l = path length (1 cm cell was used). 2.9. Phytotoxicity assay The phytotoxicity of the EO and compounds in terms of seed germination and seedling growth of chick pea was assayed with respect to control sets following Kordali et al. (2008). Two layers of filter paper were placed on the bottom of each Petri plate (9 cm) and 5 seeds were placed equidistantly on the filter paper moistened with 10 mL of distilled water. Ten microliters of the EO and compounds were dripped on Whatman No. 1 filter paper strip placed on the lid using a micropipette. Petri plates were sealed with parafilm to prevent escaping of volatile compounds and kept at 23 ± 2 °C in a growth chamber. Percent germination of seeds of control and treated sets was recorded .The length of radicle and plumule was monitored at 24, 48, 72, 96, 120 and 144 h interval. 2.10. Statistical analysis Antifungal, antiaflatoxigenic and phytotoxicity experiments were performed in triplicate and data analyzed are mean ± SE subjected to one way ANOVA. Means are separated by Tukey's multiple range tests when ANOVA was significant (p b 0.05) (SPSS 10.0; Chicago, IL, USA). 3. Results Seed samples from eleven legume varieties were tested for the inherent mycoflora wherein a total of 17 different fungal species were isolated (Table 1). Aspergillus (6 species) was the most prevalent followed by Fusarium (4 species). All legumes were found positive for Table 2 Chemical composition of Lippia alba essential oil. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 tr: traces. Compounds Hexenyl acetate Methyl acetyl acetone α-Thujene α-Pinene 1-Hepten-3-ol Methyl heptenone β-Myrcene 3-Octanol 2,3,4 Trimethyl pentane m-Cymene DL-Limonene (Z)- β -ocimene (E)- β -ocimene β-Citronellol Cyclopentylacetone Linalool Perillene E-Chrysanthemal Neolyratol Myrtenal Linalyl acetate β-Citronellol Nerol Nerolidol Neral Geraniol formate Geranial Geranyl acetate, 2,3-epoxyMyrtenyl acetate Geranyl acetate Germacrene-D β-Elemene Methyleugenol E-Caryophyllene γ-Cadinene β-Fernesene α-Humulene E-Caryophyllene γ-Cadinene Germacrene-D α-Copaene Nerolidol (−) Elema-1,3,11(13)-Trien-12-ol 1,5-Diphenylhex-3-ene Phytol Total Percentage 0.07 tr 0.33 1.02 4.46 2.84 7.17 0.11 0.47 0.40 0.25 0.50 0.08 0.05 0.07 1.59 0.06 0.76 0.24 0.75 1.56 0.89 1.17 1.54 14.20 1.86 22.21 1.95 2.46 3.09 tr tr 0.28 6.17 0.58 1.46 0.04 0.24 0.75 1.48 0.75 1.01 4.28 1.42 0.44 91.05 Rt 7.17 8.42 9.25 9.52 10.90 11.20 11.40 11.50 12.32 12.82 13.00 13.22 13.70 13.95 15.57 15.95 16.07 18.40 18.95 20.70 21.82 21.97 22.07 22.27 22.67 23.22 24.00 24.55 26.72 29.22 29.82 29.95 30.20 31.30 31.70 32.60 32.82 33.15 33.72 34.00 35.47 37.22 38.52 41.10 57.40 168 R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170 Table 3 Antifungal activities of Lippia alba essential oil and its components against different fungal isolates. Fungal isolates Antifungal index (%) ‡ ‡ Essential oil Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus Aspergillus niger, Aspergillus shydowi, Aspergillus terreus, Cladosporium cladosporioides Curvularia lunata, Fusarium graminearum Fusarium nivale Fusarium oxysporum, Fusarium sp. Penicillium italicum, Rhizoctonia solani Rhizopus stolonifer Trichoderma spp. ‡ Geranial Neral 0.25 0.5 0.75 1.0 1.0 1.0 59.2 ± 1.9 53.5 ± 2.2 57.0 ± 3.1 63.7 ± 1.7 40.4 ± 3.8 60.0 ± 5.0 51.6 ± 1.6 32.1 ± 1.5 50.7 ± 1.5 63.5 ± 1.7 45.0 ± 0.0 66.1 ± 1.1 77.4 ± 1.1 66.0 ± 3.0 77.0 ± 1.0 41.6 ± 1.6 52.0 ± 1.5 73.4 ± 1.6 63.0 ± 1.5 78.3 ± 1.6 69.8 ± 0.9 54.2 ± 2.9 82.9 ± 2.4 73.0 ± 3.6 44.8 ± 2.8 62.0 ± 1.5 74.5 ± 2.3 50.2 ± 2.8 68.5 ± 1.7 80.3 ± 0.3 68.3 ± 1.6 86.8 ± 3.4 91.6 ± 1.6 77.5 ± 3.2 82.5 ± 2.7 67.0 ± 0.7 90.0 ± 1.0 92.7 ± 3.6 67.3 ± 2.3 100 79.3 ± 0.6 59.6 ± 0.3 73.4 ± 3.2 85.3 ± 2.6 70.9 ± 4.3 80.2 ± 4.8 80.6 ± 4.1 74.0 ± 3.0 100 100 89.1 ± 0.6 97.3 ± 2.6 72.5 ± 1.0 100 100 77.4 ± 1.6 100 89.5 ± 1.2 77.6 ± 1.3 96.0 ± 4.0 100 91.6 ± 2.0 100 95.1 ± 2.8 100 100 100 100 100 96.6 ± 1.6 100 100 94.6 ± 2.9 100 100 87.8 ± 1.5 100 100 96.6 ± 3.3 100 100 100 100 100 100 79.4 ± 2.1 68.7 ± 3.1 90.9 ± 0.5 83.1 ± 2.1 71.9 ± 1.5 95.5 ± 4.4 83.5 ± 1.5 58.3 ± 4.4 76.2 ± 1.1 86.7 ± 1.7 82.8 ± 1.4 84.6 ± 3.3 96.7 ± 1.6 95.9 ± 2.1 100 100 73.0 ± 3.7 † Bavistin 24.4 ± 2.8 19.5 ± 0.4 36.7 ± 1.6 40.8 ± 2.8 16.5 ± 2.0 19.7 ± 0.5 46.9 ± 1.8 26.2 ± 1.2 39.9 ± 0.6 56.9 ± 1.7 51.0 ± 2.0 45.2 ± 0.2 59.3 ± 2.9 39.2 ± 2.2 67.0 ± 1.5 76.6 ± 1.6 39.7 ± 2.3 ‡ μL/mL. 4 mg/mL. Values are mean (n = 3) ± SE. † A. flavus. Three AFB1 producing isolates of A. flavus viz. NKD-116, NKD122 and NKD-235 were detected from seeds of Vigna radiata, V. unguiculata and Arachis hypogaea, respectively. The hydro-distillation of L. alba leaves yielded a pale yellow colored oil (yield: 0.8 mL/kg). GC–MS analysis of EO revealed 45 different components, representing 91.05% of the total compounds present. The constituents identified by GC–MS and other parameters are summarised in Table 2. The most abundant constituents were monoterpene aldehydes viz. geranial (22.21%) and neral (14.20%). The other main components of the oil were monoterpene hydrocarbon, β-myrcene (7.17%); sesqueterpene hydrocarbon, E-caryophyllene (6.41%) and 1-hepten-3-ol (4.46%), (−) elema-1,3,11(13)-trien-12-ol (4.28%), geranyl acetate (3.09%), methyl heptenone (2.84%), myrtenyl acetate (2.46%) etc. The EO of L. alba exhibited moderate to high antifungal activity against all the 17 fungal species tested. The antifungal index of the EO increased against each fungus with the rise in concentration from 0.25 to 1 μL/mL onwards (Table 3). Geranial (1 μL/mL), the prime component of the EO caused 100% inhibition of 13 fungi out of 17 tested; whereas, at the same concentration EO and neral arrested 100% mycelial growth of 9 and 2 fungal species, respectively. C. cladosporioides was found to be highly resistant against geranial and neral (1 μL/mL) as well as at lower concentration of EO (0.25 to 0.75 μL/mL) showing least antifungal index amongst the fungi tested. However, A. flavus was found highly resistant against EO at 1 μL/mL. Bavistin, the commercial benzimidazole fungicide was found to be least effective amongst all the treatments with antifungal activity ranging from 16.5 to 76.6%. None of the fungal species was inhibited absolutely at 4 mg/mL of Bavistin. The MICs of EO, geranial and neral were 1.26, 1.03 and 1.58 μL/mL, respectively against A. flavus. MFC of geranial (1.13 μL/mL) was lower than that of EO (1.26 μL/mL) or neral (2.1 μL/mL). All the three MICs and MFCs are significant (p b 0.05) and lower than the positive control (Bavistin, N5 mg/mL). Table 4 Effect of different concentrations of Lippia alba essential oil and its components on mycelial biomass and Aflatoxin B1 production in SMKY medium. Treatments Con. Toxigenic isolates A. flavus (NKD-116) MDW Essential oil Geranial Neral 0.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 A. flavus (NKD-122) AFB1 a 430 ± 2.1 400 ± 4.0ab 374 ± 5.3bc 297 ± 4.3de 217 ± 7.5hi 133 ± 7.0 k 314 ± 3.7d 265 ± 4.7 fg 187 ± 4.5ij 102 ± 4.6 k 0±0l 405 ± 3.2a 356 ± 6.0c 282 ± 11.6ef 239 ± 6.6gh 168 ± 8.7j MDW a 305.8 ± 9.4 192.7 ± 4.8 cd 119.5 ± 1.8f 82.9 ± 4.7 g 0.0 ± 0.0 h 0.0 ± 0.0 h 168.1 ± 4.1de 83.2 ± 5.6 g 0.0 ± 0.0 h 0.0 ± 0.0 h 0.0 ± 0.0 h 237.8 ± 6.0b 197.1 ± 3.6c 146.8 ± 4.4e 83.1 ± 9.3 g 21.6 ± 2.4 h A. flavus (NKD-235) AFB1 a 402 ± 4.3 383 ± 3.6a 303 ± 4.0c 282 ± 2.6c 200 ± 5.7e 94 ± 5.3 g 307 ± 4.6c 238 ± 6.0d 123 ± 12.0f 52 ± 2.3 h 0 ± 0i 391 ± 1.6a 335 ± 5.0b 292 ± 4.0c 221 ± 6.6de 142 ± 3.7f MDW a 143.6 ± 7.7 104.7 ± 3.8b 47.1 ± 4.2c 0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 55.6 ± 4.8c 13.5 ± 6.8d 0.0 ± 0.0d 0.0 ± 0.0d 0.0 ± 0.0d 108.6 ± 6.6b 93.0 ± 5.5b 49.6 ± 2.0c 0.0 ± 0.0d 0.0 ± 0.0d AFB1 a 388 ± 5.7 344 ± 3.7b 324 ± 5.6b 250 ± 4.6c 177 ± 5.3e 98 ± 2.9 fg 271 ± 4.7c 210 ± 4.6d 154 ± 3.3e 84 ± 3.2 g 14 ± 14.6 h 385 ± 5.0a 324 ± 3.7b 253 ± 3.6c 175 ± 4.6e 117 ± 2.4f Con. = Concentration (μL/mL); MDW = Mycelial dry weight (mg); AFB1 = Aflatoxin B1 content (μg/L). Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey's multiple comparison tests. 321.8 ± 4.1a 212.5 ± 7.2b 151.5 ± 6.8c 87.0 ± 5.8d 13.0 ± 6.6 fg 0.0 ± 0.0 g 176.0 ± 4.1c 94.6 ± 6.3d 29.3 ± 8.0ef 0.0 ± 0.0 g 0.0 ± 0.0 g 233.8 ± 9.8b 172.7 ± 5.4c 96.5 ± 2.8d 48.2 ± 3.1e 8.1 ± 4.6 fg R. Shukla et al. / International Journal of Food Microbiology 135 (2009) 165–170 Table 4 summarizes the inhibitory effects of EO and two of its major components on AFB1 production and mycelial dry weight of three toxigenic isolates of A. flavus (NKD-116, NKD-122 and NKD-235). The qualitative TLC of EO, geranial and neral treated cultures showed that these compounds strongly inhibited AFB1 production in a dosedependent manner. Based on the quantitative estimation, AFB1 inhibition was at 0.2 μL/mL and onward concentrations of EO, geranial or neral against all the three isolates. In the negative control of NKD-116, NKD-122 and NKD-235 isolates, AFB1 production was 305.8, 143.6 and 321.8 μg/L, respectively. However, AFB1 production by NKD-116, NKD122 and NKD-235 was inhibited completely at 0.8, 0.6, 1.0 μL/mL of EO and 0.6, 0.6, 0.8 μL/mL of geranial, respectively. On the other hand, only 21.6, 0 and 8.1 μg/L AFB1 were produced by NKD-116, NKD-122 and NKD-235 isolates, respectively at 1 μL/mL dose of neral in SMKY medium. Mycelial dry weight decreased with increasing concentrations of treatments. A 100% germination of seeds was recorded following 48 h in all the four sets, including control. Seedling emergence was significantly (p b 0.05) not different in all the sets until 48 h of incubation, but the size of radicles in EO treated seeds was higher than the control, geranial and neral for the rest of the incubations. The mean length of radicle was a maximum of 133.7 mm in EO treatment compared to 102 mm (neral), 100 mm (control) and 95.7 mm (geranial) at 144 h of exposure. Plumules originated after 96 h, and no significant difference in their length was observed at 144 h in control (23 mm), EO (23.3 mm), geranial (19.3 mm) and neral (21.6 mm) treated seeds according to ANOVA and Tukey's multiple comparison tests. 4. Discussion The present investigation revealed that all legume seeds tested, were contaminated with various fungi and A. flavus dominated in almost all the seed samples. These results confirm the earlier observations where Aspergillus spp. were one of the most predominant fungi and aflatoxin producers in some of the stored pulses (ElNagerabi and Elshafie, 2000; Embaby and Abdel-Galil, 2006). The essential oil of L. alba has been standardized on GC–MS data. In the case of L. alba, a number of chemotypes have been reported, abundant in γ-terpinene (Gomes et al., 1993), limonene (Pino et al., 1997), carvone (Matos et al., 1996), linalool (Siani et al., 2002), citralmyrcene (Matos, 1996) or 1,8-cineol-camphor (Dellacassa et al., 1990). In our study, GC–MS data depicted geranial and neral as major components of EO. The chemical composition of L. alba EO is different from earlier reports and a novel biological property of the oil in terms of inhibition of aflatoxin and food borne fungi is being reported. The antifungal activity of EO was also compared with its major components, geranial and neral to conclude, whether, the EO or its components may be recommended as food preservative against food contaminating fungi and aflatoxins. A perusal of the literature reveals the antifungal activity of Lippia berlandieri, L. sidoides and L. rugosa against yeast, dermatophytes and other filamentous fungi (Portillo-Ruiz et al., 2005; Fontenelle, et al., 2007; Tatsadjieu et al., 2008); however, fungitoxocity of L. alba is not well-explored except by Rao et al. (2000) who reported antifungal activity of oil vapours against sugarcane pathogens. Lee et al. (2008) and Park et al. (2007) have also reported bioactivity of geranial and neral against phytopathogens and dermatophytes. To the best of our knowledge, there has not been a relevant study on the effectiveness of L. alba leaf oil and its constituents against aflatoxin production by fungi. In the present study, L. alba EO and two of its components were inhibitory to all the storage fungi although to a varying degree. AFB1 production was significantly inhibited at lower than fungistatic concentrations of oil, geranial or neral. The superiority of EO and its components over the commonly used synthetic fungicide Bavistin (50% carbendazim) at the lowest levels of MICs, further favours their exploitation as the alternative fungitox- 169 icant. Interestingly, there was no adverse effect of treatments on seed germination and, rather, the enhanced seedling growth was recorded in EO treated seeds compared to control, geranial or neral. It collectively suggests that the synergistic effect of constituents in EO may be responsible for the enhanced seed germination. EOs are the potentially useful additives for food preservation to prolong shelf life and improve the quality of stored food products. A few EO-based preservatives are already commercially available. ‘DMC Base Natural’ in this category, comprises 50% EO from rosemary, sage and citrus and 50% glycerol (Mendoza-Yepes et al., 1997). Limitations of employing EOs and their components as preservatives include alteration of organoleptic properties of food commodities. However, recent encapsulation techniques using various surfactants seem to overcome such problems (Gaysinsky et al., 2005). Geranial and neral are the trans- and cis-isomers of citral, respectively and with the characteristic lemon odor. Citral, a widely used natural ingredient, is added to foods, soft drinks and cosmetics as the flavoring and fragrance agent. Hence, there would be no chance of their negative effects on sensory quality, although detailed investigations on organoleptic parameters are needed before final recommendation. A food preservative may be antimicrobial or antioxidant. The antimicrobial and aflatoxin inhibitory activity of L. alba EO is being reported in our study whereas antioxidant activity has been earlier reported (Hennebelle et al., 2008). 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