Art12_Saha.indd Journal of Applied Botany and Food Quality 85, 207 - 211 (2012) 1Indian Agricultural Research Institute, New Delhi, India 2India Institute of Sugarcane Research, Lucknow, U.P., India Antifungal Acetylinic Thiophenes from Tagetes minuta: Potential Biopesticide Supradip Saha1*, Suresh Walia1, A. Kundu1, B. Kumar1, Deeksha Joshi2 (Received August 11, 2012) * Corresponding author Summary Apart from thiophenes, which possess wide range of biocidal activity, aerial parts of Tagetes sp contain essential oil. Oil com- ponents were reported to have antifungal activity, thus making whole plant of Tagetes very useful for exploiting as natural fungistatic agent. In the present study, Tagetes minuta grown in north western Himalayan condition were evaluated for its potential for use as antifungal agent. Flower essential oil showed minimal antifungal activity. Whereas, leaf essential oil was found signifi cant antifungal activity against three phytopathogenic fungi out of eight tested fungi. ED50 values were 165, 175 and 110 µg mL-1 against Rhizoctonia solani, Sclerotinia sclerotiorum and Sclerotium rolfsii, respectively. Thiophene rich extract of Tagetes minuta was found comparatively lesser active (ED50: 233-484 µg mL-1) than leaf essential oil against the same fungi. The present study shows that essential oil from leaves and thiophene rich extracts from marigold roots have signifi cantly good antifungal activity against a number of soil borne and foliar plant pathogens. The easy availability of these plants makes it an attractive potential candidate for development of natural fungicide. Introduction Plant secondary metabolites and their derivatives have been evaluated as viable alternatives to the persistent and less environmentally friendly synthetic fungicides (DIXON, 2001). Although a large number of phytochemicals are known for their insect control properties, only a few of them are known to impart antifungal activity. The use of pesticides of natural origin is becoming appealing because of the problem of environmental pollution arising from the use of persistent pesticides. To move forward in the discovery of natural plant metabolites, plant extracts were screened for potential candidates with antifungal activity (CROUSE, 1998). Some of the most promising botanicals for use as pesticides have been extracted from species of plants in the families Meliaceae, Rutaceae, Asteraceae, Annonaceae, Labiatae, and Canellaceae (JACOBSON, 1989). Although much of the literature on natural products in agricultural fi eld concerns insect control, a smaller but emerging body of papers has reported the effectiveness of plant extracts and essential oils for controlling microbial pathogens of plants. Marigold (Tagetes sp.), belonging to the Asteraceae family, is a commonly occurring plant all over the world and is well known for a wide range of biological properties. The plant has been credited with having anticancerous and antiageing effects (BLOCK et al., 1992). It contains carotenoids which is used as food colorants and feed additives (TIMBERLAKE and HENRY, 1986). Besides this the plant also has pharmacological properties as it contains fl avonoids (TERSCHUK et al., 1997). The foliar parts of this plant possess essential oils, known for antibacterial and insecticidal properties (PICCAGLIA et al., 1997); and thiophenes which have a marked biocidal activity (HULST et al., 1989). These polyacetylene derivatives, the activity of which depends on photodynamic activation (HUDSON and TOWERS, 1991), act as antibiotics, insecticides, nematicides and fungicides (GOMMERS, 1981; MARES et al., 1990; HUDSON, et al., 1983; ROMAGNOLI et al., 1994, 1998). Further, constituents of essential oil, mainly (Z)- and (E)-ocimenones, along with piperitone, piperitenone, limonene, tagetone and caryophyllene, are the major terpenes present in leaves (VASUDEVAN et al., 1997). In addition, HETHELYI et al. (1986) assumed that the presence of linalool and linalyl acetate characterizes Indian Tagetes patula oil. Due to the high degree of chemodiversity observed within essential oils of Tagetes sp., biological activities also subjected to vary. However, even though the various properties of the Tagetes plant are well known, less attention has been focused on the studies of biological activity of essential oils from Tagetes species. Therefore, the present work was aimed at developing an antifungal formulation from the total plant extract. Interestingly, synergestic effect on egg hatchability of Globodera rostochinensis was observed when treated with a combination of T. erecta leaf and root extracts (SASANELLI and VITO, 1991). Furthermore, juvenile of M. incognita populations were more suppressed by stem and whole plant rather than root portions (SIDDIQUI and ALAM, 1988). Present study was aimed to evaluate the antifungal activity of the whole plant parts. Thus in vitro antifungal activity was evaluated against eight important plant pathogenic fungi. Antifungal formulation may be developed using essential oil from leaves and thiopene rich extract from roots and shoots. Materials and methods General experimental procedure All chemicals and reagents were procured from Merck® India Ltd. Double-distilled water was used throughout the analysis. GC- MS analysis was carried out on a TRACE-GC (Thermo Finnigan) coupled with fi son MD-800 quadrupole mass detector and DB-17 capillary column (30m × 0.32 mm i.d.; fi lm thickness 0.25 mm). Temperature programming was done from 75-250 °C at 5 °C min-1. Helium was used as the carrier gas at 1 mL min-1 fl ow rate. Mass spectra was recorded over 40-400 amu range at 1 scan s-1 with ionization energy of 70ev and ion source temperature was 250 °C. The split ratio was 1:20. Plant material The plant material (leaves, fl ower and roots) was collected during the month of September from experimental farm of Vivekananda Institute of Hill Agriculture located at Hawalbagh (Altitude: -1220 m a.s.l., Latitude: -29°38’4.8”N, Longitude: -79°37’48.6”E) in North Western Himalayan region. The identifi cation of the plants was confi rmed from Department of Botany, Almora Inter College, Kumaon University, Almora, India. Mancozeb and β-pinene, a constituent of essential oil was used as positive control (procured from Merck®). 208 Supradip Saha, Suresh Walia, A. Kundu, B. Kumar, Deeksha Joshi Test fungi The essential oils / extracts were tested for their antifungal properties against eight plant pathogenic fungi. Pyricularia grisea was isolated from blast infected fi nger millet leaves; Rhizoctonia solani and Fusarium solani were isolated from infected roots of French bean, Sclerotium rolfsii from infected collar portion of French bean, Fusarium oxysporum f.sp. pisi from wilted pea plants, Sclerotinia sclerotiorum from white rot infected pea plants, Fusarium oxy- sporum f.sp. lentis from wilted lentil plants and Alternaria solani from tomato leaves. Extraction of essential oil Leaves (200 g) and fl ower (250 g) of Tagetes sp. were subjected to hydro-distillation separately for 3 h using a Clavenger apparatus to obtain essential oil. Yield of essential oil was 0.3-0.4% and 0.2- 0.3% from leaves and fl ower respectively. The oil was dried with anhydrous sodium sulfate and preserved in a sealed vial at 4 °C until the moment of analysis. Extraction and purifi cation of thiophene Extraction of thiophene from shoots and roots was done by following the modifi ed method of MARGL et al., 2002. Finely chopped air dried shoot and roots (600 g) from Tagetes sp., were extracted with methanol: water (3:1 v/v, 2500 mL) twice at room temperature with the help of mechanical stirrer and fi ltered. Volume of combined extract was reduced in vacuo at 45 °C. The concentrated extract was sequentially partitioned into hexane: chloroform (2:1 v/v). The partitioned organic solvent fractions were concentrated to dryness by rotary evaporation at 35 °C to get dark green coloured thiopene rich concentrate (625 mg). Dark green thiophene rich extract was dissolved in minimum quantity of hexane, fi ltered and concentrated to dryness. Then the residue was dissolved in Et2O and passed through column, preconditioned with Et2O and packed with 60- 120 mesh silica gel. Purifi ed extract was then subjected to GC-MS analysis. Characterization TIC of root and shoot extract was presented in Fig. 1. Extract contains essential oil component as well as acetylenic thiophenes. First compound in the TIC eluted as dihydrotagetone and it was confi rmed by its mass fragmentation pattern. The compound was eluted at 2.267 minute. Molecular ion peak was detected as m/z 154 [M]+ with the mass fragmentation of m/z 139, 125, 108, 93, 81, 71, 58 (Tab. 1). Second compound eluted in the TIC was confi rmed by its molecular ion peak of m/z 152 and fragments of 137, 109, 95, 81, Fig. 1: TIC of thiophene rich extract. Antifungal thiophenes from Tagetes minuta 209 69, 58 as piperitone. α-terpineol (m/z 154, 136, 121, 111, 93, 83, 71, 58) was eluted at 5.801 minute. Compound at Rt of 6.534 min was remain unidentifi ed. Three thiophenes were identifi ed as 5-(3-buten- 1-ynyl)-2,2’-bithienyl (BBT) (37.57 min), α-terthienyl (47.354 min) and 5-(4-acetoxy-1-butynyl)-2,2’-bithienyl (BBTOAc). Molecular ion peak of these three compounds were m/z 218, 248 and 216 respectively with characteristic fragmentation pattern. Antifungal bioassay The cultures were maintained on PDA slants at 25 °C and were subcultured in petri dishes prior to testing. Weighed quantities of essential oil and extract (extracted under diffused sunlight) to be tested were dissolved in acetone (1 mL) and incorporated into the molten medium (at 45 °C) to reach the desired concentration. PDA containing only acetone served as control while β-pinene at 250 µg mL-1, 500 µg mL-1 and 1000 µg mL-1 served as a positive control. Medium was poured into each sterile petri dish under aseptic conditions and left to settle. Circular discs (5 mm diameter) of the test mycelia mats (punched-in mycelia mats grown in sterile petri dishes) were inoculated centrally and left to grow. In all cases, triplicates were maintained. Radial growth inhibition of test fungi was evaluated. The diameter of fungal growth (mm) was noted at 72 h after inoculation and subsequently every 24 h for a total period of 240 h. These data were subjected to analysis of variance using completely randomised blocks. The percentage inhibition relative to the control, I, was converted to corrected percentage inhibition, IC, using the following formula: IC = [(I − CF)/(100 − CF)] × 100 CF = [(90 − C)/C] × 100 where CF is the correction factor, 90 is the diameter of the petri dish (mm) and C is the diameter of the fungus in the control (mm). Statistical analysis Four replicates were used for each experimental treatment. The effective concentration for 50% inhibition of mycelial growth, ED50 (µg mL-1), was calculated from the IC data using the Basic LD50 program v.1.1 as described by TREVORS, 1986. Percentages were angular transformed (arc sine) for analysis and analysed by complete randomised design. Results Three monoterpenes and three thiophenes were identifi ed from the shoot and root extract of Tagetes minuta. Terpenoids were identifi ed as dihydrotagetone, piperitone and α-terpineol by their mass fragmentation pattern. Out of these three monoterpenes, one is acyclic i.e. dihydrotagetone and other two are cyclic monoterpenes. ROMAGNOLI et al., 2005 reported these compounds to be present in fl ower essential oil along with other monoterpenes. The antifungal activity of fl ower essential oil is not so encouraging (Tab. 2). Percent inhibition of fl ower essential oil at 1000 µg mL-1 was ranged between 8.9-35.1%. Highest activity was found against Fusarium oxysporum pisi and least activity was recorded against Pyricularia grisea. Antifungal activity of leaf essential oil against eight phytopathogenic fungi is presented in Tab. 3. Antifungal activity was comparatively superior in leaf essential oil than fl ower. ED50 value ranged between 110-1016 µg mL-1. Leaf essential oil was most active against Sclerotium rolfsii and least active against Fusarium oxysporum lentis. The in vitro results were classifi ed according to ALIGIANNIS et al., 2001 and DUARTE et al., 2005 who proposed that, when the minimum inhibitory concentration (MIC) is below 500 µg mL-1, the antifungal activity is considered strong, and, if the extracts/compounds display an MIC between 600 and 1500 µg mL-1, the antifungal activity is considered moderate. Compounds with an MIC above 1600 µg mL-1 are considered weak. As evident from the data, leaf essential oil exhibited strong activity against Rhizoctonia solani, Sclerotinia sclerotiorum and Sclerotium rolfsii. Medium antifungal activity was recorded against other fi ve phytopathogenic fungi. The commercial reference fungicide, mancozeb, was highly effective against all four test fungi. Whereas, essential constituent β-pinene showed ED50 value less than 180 µg mL-1. Antifungal activity of thiophene rich extract from shoot and root is presented in Tab. 4. ED50 value against eight phytopathogenic fungi ranged between 233 to 2227 µg mL-1. The extract was most active against R. solani and least active against P. grisea. Hyphal growth inhibition was maximum in R. solani, S. sclerotiorum and S. rolfsii and the activity was found to be strong. While, moderate antifungal activity was recorded in F. solani, A. solani, F. oxy- sporum pisi and F. oxysporum lentis. Weak antifungal activity was evidenced against P. grisea. Tab. 1: Mass spectra of compounds present in the purifi ed root extract. Compound Rt m/z Mass spectra (m/z, relative intensity) [min] Dihydrotagetone 2.267 154 58(100), 71(41), 81(54), 93(37), 108(54), 125(9), 139(32), 154(31) Piperitone 3.784 152 58 (25), 69(49), 81(100), 95(5), 109(10), 137(6), 152(12) α-terpineol 5.801 154 58(92), 71(100), 83(27), 93(78), 111(78), 121(15), 136(19), 154(18) BBT 37.570 216 69(8), 95(29), 171(29), 216(100), 217(15), 218(12) α-terthienyl 47.354 248 58(5), 69(10), 127(14), 171(9), 203(15), 248(100) BBTOAc 51.054 276 95(6), 171(13), 203(16), 216(100) Tab. 2: Hyphal inhibition of eight major plant pathogens upon application of fl ower essential oil at 1000 µg mL-1 after 3 days. Pathogen Inhibition (%) Rhizoctonia solani 5.7 Fusarium solani 13.8 Sclerotinia sclerotiorum 8.9 Fusarium oxysporum pisi 35.1 Scleroium rolfsii 19.5 Pyricularia grisea 26.4 Fusarium oxysporum lentis 22.9 Alternaria solani 21.9 210 Supradip Saha, Suresh Walia, A. Kundu, B. Kumar, Deeksha Joshi Discussion The marigold plant has been credited with a number of properties including antimicrobial / insecticidal / nematicidal activity (NATA- RAJAN et al., 2006; PICCAGLIA et al., 1997; HULST et al., 1989; ROMAGNOLI et al., 1994). In the present work, a comparative study of the antifungal activity of essential oils / extracts from different parts of the Tagetes plant against eight important plant pathogenic fungi was done. Out of six identifi ed compounds, three were monoterpenic derivative and are component of essential oil. Three compounds namely, dihydrotagetone, piperitone and α-terpineol mostly came from shoot portion of the Tagetes minuta plant. Other three identifi ed compounds were thiophenes and root portion contributed its lion share (DOWNUM, 1983). Antifungal activity of Tagetes sp. published so far is related both to the presence of thiophenic compounds alone and with UV-A irradiation (MARES et al., 1990; ROMAGNOLI et al., 1994). Number of species also reported for different activity including antifungal activity. Reports on exploration of total plant extract of T. minuta for fungistatic activity is not so well documented. Though UHLENBROEK and BIJLOO (1958) found that nematicidal or nematostatic effects of Tagetes were due to α-terthienyl, SASANELLI and VITO (1991) suggested that there was a synergistic effect of leaf and root extract on egg hatchability of G. rostochinensis. Siilar effect was observed by SIDDIQUI and ALAM, 1988, where it was concluded that stem and whole plants was more effective in suppressing juveniles of nematode than root extract. All the different compounds (essential oils and extract) from Tagetes plant as well β-pinene exhibited antifungal activity against the eight pathogens. However, there were signifi cant differences in the antifungal activity of the compounds from different parts of the plant. It was observed that leaf essential oil and root extract at 1000 µg mL-1 showed the highest inhibitory activity against all the eight pathogens which persisted for more than 6 days. Leaf essential oil showed hyphal growth inhibition of 34.1 to 85.6% while root extract treatment showed inhibition of 21.6 to 77.8% after 6 day against various pathogens. Even at lower concentrations of 500 µg mL-1 and 250 µg mL-1 leaf essential oil resulted in signifi cant reduction in hyphal growth of fi ve of the pathogens both 3 and 6 days after treatment. However, the essential oil from fl owers was not highly potent and exhibited minimal antifungal activity (< 35%) at all three concentrations against the 8 pathogens. Its antifungal activity was particularly low against the fast growing pathogens R. solani and S. sclerotiorum. Marigold (Tagetes sp.) plant is a commonly available plant in all regions of the world. The present study shows that essential oil from leaves and thiophene rich extracts from marigold roots have high antifungal activity against a number of soil borne and foliar plant pathogens. The easy availability of these plants makes them an attractive potential candidate for development of plant based biopesticide. Tab. 4: Antifungal activity thiophene rich extract against eight phytopathogenic fungi. ED50 χ2exp Fiducial limit Regression equation [µg mL−1] (3df, 95%) Rhizoctonia solani 233 3.17 191-284 Y = 1.77+1.37x Sclerotinia sclerotiorum 484 4.37 352-667 Y = 2.36+0.98x Fusarium solani 3656 5.85 1195-11187 Y = 2.42+0.72x Alternaria solani 1045 6.64 634-1722 Y = 2.18+0.93x Pyricularia grisea 2227 2.00 819-6063 Y = 2.82+0.65x Fusarium oxysporum pisi 1131 6.69 611-2094 Y = 2.63+0.78x Fusarium oxysporum lentis 1244 5.56 680-2275 Y = 2.42+0.83x Sclerotium rolfsii 285 4.31 232-350 Y = 1.78+1.31x Mancozeb 32 - - - β-pinene <180 - - - Tab. 3: Antifungal activity of leaf essential oil against eight phytopathogenic fungi. ED50 χ2exp Fiducial limit Regression equation [µg mL−1] (3df, 95%) Rhizoctonia solani 165 2.20 128-213 Y = 2.50+1.13x Sclerotinia sclerotiorum 175 4.32 135-225 Y = 2.59+1.08x Fusarium solani 1776 0.86 642-4915 Y = 3.14+0.57x Alternaria solani 751 4.74 393-1436 Y = 3.29+0.60x Pyricularia grisea 708 5.33 360-1393 Y = 3.10+0.68x Fusarium oxysporum pisi 3716 1.23 890-15520 Y = 3.04+0.55x Fusarium oxysporum lentis 6975 0.46 843-57700 Y = 3.24+0.46x Sclerotium rolfsii 110 2.43 84-144 Y = 2.49+1.22x Mancozeb 32 - - - β-pinene <180 - - - Antifungal thiophenes from Tagetes minuta 211 Acknowledgements The author thanks Dr. K. K. Sharma for assisting in the GC-MS analysis. The authors would also like to thank Mr. Sher Singh for assisting in analysis. References ALIGIANNIS, N., KAPLOTZAKIS, E., MITAKU, S., CHINOU, B. 2001: Com- position and antimicrobial activity of two Origanum sp. J. Agric. Food Chem. 49, 4168-4170. BLOCK, G., PATTERSON, B., SUBAR, A., 1992: Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutr. Cancer 18, 1-29. CROUSE, G.D., 1998: Natural products as leads for new pesticides with reduced risks, in Pesticides: Managing Risks and Optimizing Benefi ts; ACS Symposium Series No. 734, ed. by Ragsdale NN and Seiber JN. American Chemical Society, Washington, DC, 80-95. DIXON, R.A., 2001: Natural products and plant disease resistance. Nature 411, 843-847. DOWNUM, K.R., 1983: Analysis of thiophenes in the Tagetae (Asteraceae) by HPLC. J. Nat. Prod. 46, 98-103. DUARTE, M.C.T., FIGUEIRA, G.M., SARTORATTO, A., REHDER, V.L.G., DELARMELINA, C., 2005: Anti-Candida activity of Brazilian medicinal plants. J. Ethnopharmacol. 97, 305-311. DUNKEL, F.V., JARONSKI, S.T., SEDLAK, C.W., MEILER, S.U., VEO, K.D. 2010: Effects of steam-distilled shoot extract of Tagetes minuta (Asterales: Asteraceae) and entomopathogenic fungi on larval Tetanops myopaeformis. Environ. Entomol. 39, 979-988. GIL, A., GHERSA, C.M., LEICACH, S., 2000: Essential oil yield and composition of Tagetes minuta accessions from Argentina. Biochem. System Ecol. 28, 261-274. GOMMERS, F.J., 1981: Biochemical interactions between nematodes and plants and their relevance to control. Helminthol. Abst., Series B, Plant Nematology 50, 9-24. HETHELYI, E., DANOS, B., TETENYI, P., KOCZKA, I., 1986: GC-MS analysis of the essential oils of four tagetes species and the anti-microbial activity of Tagetes minuta. Flav. Frag. J. 1, 169-173. HULST, A.C., MEYER, M.M.T., BRETELER, H., TRAMPER, J., 1989: Effect of aggregate size in cell cultures of Tagetes patula on thiophene production and cell growth. Appl. Microbiol. Biotechnol. 30, 18-25. HUDSON, J.B., TOWERS, G.H.N., 1991: Therapeutic potential of plant photosensitisers. Pharmacol. Ther. 49, 181-222. HUDSON, J.B., GRAHAM, E.A., ROSSI, R., CARPITA, A., NERI, D., TOWERS, G.H.N., 1993: Biological activities of terthiophenes and polyynes from the Asteraceae. Planta Med. 59, 447-450. JACOBSON, M., 1989: Botanical pesticides-past present and future. In: Aranson, J.T., Philogene, B.J.R., Morand, P. (eds.), Insecticides of Plant Origin. ACS Symp. Ser. No. 387, 1-10. MARES, D., FASULO, M.P., BRUNI, A., 1990: Ultraviolet-mediated anti- mycotic activity of a-terthienyl on Microsporum cookie. J. Med. Vet. Mycol. 28, 469-477. MARES, D., TOSI, B., POLI, F., ANDREOTTI, E., ROMAGNOLI, C., 2004: Antifungal activity of Tagetes patula extracts on some phytopathogenic fungi: ultrastructural evidence on Pythium ultimum. Microbiol. Res. 159, 295-304. MARES, D., TOSI, B., ROMAGNOLI, C., POLI, F., 2002: Antifungal Activity of Tagetes patula Extracts. Pharm. Biol. 40, 400-404. MARGL, L., TEI, A., GYURJAN, I., WINK, M., 2002: GLC and GLC-MS analysis of thiophene derivatives in plants and in in vitro cultures of Tagetes patula L. (Asteraceae). Z. Naturforsch. C 57, 63-71. MAROTTI, I., MAROTTI, M., PICCAGLIA, R., NASTRI, N., GRANDI, S., DINELLI, G., 2010: Thiophene occurrence in different Tagetes species: agricultural biomasses as sources of biocidal substances. J. Sci. Food Agric. 90, 1210-1217. NATARAJAN, N., CORK, A., BOOMATHI, N., PANDI, R., VELAVAN, S., DHAKSHNAMOORTHY, G., 2006: Cold aqueous extracts of African marigold, Tagetes erecta, for control of tomato root knot nematode, Meloidogyne incognita. Crop Prot. 25, 1210-1213. PICCAGLIA, R., MAROTTI, M., PESENTI, M., MATTARELLI, P., BIAVATI, B., 1997: Chemical composition and antibacterial activity of Tagetes erecta and Tagetes patula essential oils. In: Essential oils: Basic and Applied Research. Proceeding of the 27th International Symposium on Essential Oils, USA, 49-51. ROMAGNOLI, C., BRUNI, R., ANDREOTTI, E., RAI, M.K., VICENTINI, C.B., MARES, D., 2005: Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L. Protoplasma 225, 57-65. ROMAGNOLI, C., MARES, D., FASULO, M.P., BRUNI, A., 1994: Antifungal effects of α-terthienyl from Tagetes patula on fi ve dermatophytes. Phytother. Res. 8, 332-336. ROMAGNOLI, C., MARES, D., SACCHETTI, G., BRUNI, A., 1998: The photo- dynamic effect of 5-(4-hydroxy-1-butinyl)-2,2’-bithienyl on dermato- phytes. Mycol. Res. 102, 1519-1524. SARIN, R., 2004: Insecticidal activity of callus culture of Tagetes erecta. Fitoterapia 75, 62-64. SASANELLI, N., VITO, M.D., 1991: The effect of Tagetes sp. extracts on the hatching of an Italian population of Globodera rostochiensis. Nematol. Mediterranea 19, 135-137. SENATORE, F., NAPOLITANO, F., MOHAMED, M., HARRIS, P.J.C., MNKENI, P.N.S., HENDERSON, J., 2004: Antibacterial activity of Tagetes minuta L. (Asteraceae) essential oil with different chemical composition. Flav. Frag. J. 19, 574-578. SIDDIQUI, M.A., ALAM, M.M., 1988: Toxicity of different plant parts of Tagetes lucida to plant parasitic nematodes. Indian J. Nematol. 18, 181- 185 (1988). TERESCHUK, M.L., RIERA, M.V., CASTRO, G.R., ABDALA, L.R., 1997: Antimicrobial activity of fl avonoids from leaves of Tagetes minuta. J. Ethnopharmacol. 56, 227-232. TIMBERLAKE, C.F., HENRY, B.S., 1986: Plant pigments as natural food colours. Endeavour 10, 31-36. TREVORS, J.T., 1986: A BASIC programme for estimating LD50 value using the IBM-PC. Bull. Environ. Contam. Toxicol. 37, 18. UHLENBROEK, J.H., BIJLOO, J.D., 1958: Investigations on nematicides: I. Isolation and structure of a nematicidal principle occurring in Tagetes roots, Recueil des Travaux Chimiquesdes Pays-Bas 77, 1004-1009. VASUDEVAN, P., KASHYAP, S., SHARMA, S., 1997: Tagetes: a multipurpose plant. Bioresource Technol 62, 29-35. ZYGADLO, J.A., GUZMAN, C.A., GROSSO, N.R., 1994: Antifungal properties of the leaf oils of Tagetes minuta L. and T. fi lifolia Lag. J. Essent. Oil Res. 6, 617-621. Address of the corresponding author: E-mail: s_supradip@yahoo.com