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Native Kenyan plants as possible alternatives to methyl bromide in soil fumigation.

Abstract

Methyl bromide (C[H.sub.3]Br) is a biocidal fumigant used widely in crop production and commodity preservation worldwide. C[H.sub.3]Br escapes to the stratosphere and releases bromine atom (Br), which contributes to significant destruction of the ozone ([O.sub.3]). It is therefore necessary to explore alternatives to C[H.sub.3]Br that are environmentally safe and suitable for resource-poor African farmers. We present here the results of a study on the inhibitory activity of crude extracts from Kenyan medicinal plants against three soil pathogens, Fusarium oxysporum, Alternaria passiflorae, and Aspergillus niger. Crude organic extracts of Warburgia ugandensis Sprague, Azadirachta indica A. Juss, Tagetes minuta and Urtica massaica were active against all soil pathogens, while those from U. massaica were not. Chromatographic purification of the crude extract of W. ugandensis provided two pure compounds, muzigadial (1) and muzigadiolide (5). The minimum inhibitory concentration (MIC value) for muzigadial (1) ranged from 5 to 100 ([micro]g/ml. Muzigadiolide (5) was not active. Greenhouse tests of W. ugandensis extracts against F. oxysporium pathogen showed the most effective inhibitory concentration to be at least 5 mg/ml. Quantitative structure-activity relationship (QSAR) models were used to rationalize the variation in biological activities of muzigadial (1), warburganal (2), polygodial (3), ugandensidial (4), muzigadiolide (5), azadirachtin (6), and C[H.sub.3]Br. The models were based on several molecular descriptors including LogP, van der Waals surface area (VD[W.sub.A]), van der Waals volume (VD[W.sub.V]), dipole moment, total energy, polarizability, and differences between the highest occupied molecular orbital and the lowest unoccupied molecular orbital (HOMO-LUMO gap).

[c] 2005 Published by Elsevier GmbH.

Keywords: Native Kenyan plants; Crude extracts; Muzigadial; Muzigadiolide; Soil pathogens; QSAR; Molecular descriptors

Introduction

Methyl bromide (C[H.sub.3]Br) has been used as a fumigant for over 60 years. An important valuable property of C[H.sub.3]Br is the broad spectrum of activity against several pests. The largest single global use is as a soil fumigant (Wang et al., 1997). For example, it is used as a fumigant against pathogens (fungi-, bacteria- and soil-borne viruses), insects, mites, nematodes and rodents (Puckhaber et al., 1998). These pests may be in the soil, in durable or perishable commodities, and in structures and transportation vehicles. The ease of application of C[H.sub.3]Br, along with its reliability and speed of action, has led to its widespread use in agricultural systems producing economically important crops. Although C[H.sub.3]Br is a most useful soil fumigant in specific instances, there are a number of technical and legislative limitations that have led to restrictions on its use.

C[H.sub.3]Br can have adverse effects on a number of commodities; it is phytotoxic and causes taint and odors. Repeated fumigation with C[H.sub.3]Br may result in the production of bromide ion (B[r.sup.-]) residues that accumulate rapidly. Some European countries are concerned about the toxicity of C[H.sub.3]Br in ground water and its ozone-depleting potential. In November 1992, C[H.sub.3]Br was listed as an ozone-depleting substance by the fourth meeting of the parties to the Montreal Protocol on Substances that Deplete the Ozone Layer in Copenhagen (Abritton and Watson, 1992). Since then, plant health services throughout the world have been advocating the phasing out of C[H.sub.3]Br. The technical availability of chemical and non-chemical alternatives to C[H.sub.3]Br has been proposed by the Methyl Bromide Technical Options Committee (Yuen et al., 1991). Non-chemical alternatives include cultural practices, biological control, organic alterations and physical methods (Watson et al., 1992). Chemical alternatives can be either fumigants or non-fumigants. Fumigants include methyl isothiocyanate (MITC), MITC generators, me-tasodium, dazomet and halogenated hydrocarbons (e.g. 1,3-dichloropropene, chloropicrin (trichloronitro-methane) and ethylene dibromide). All these chemicals have major drawbacks: phytotoxicity, skin and eye irritation, sensitization, genotoxicity, and carcinogenicity. Non-fumigant nematicides such as organophosphates or carbamates are neurotoxins (cholinesterase inhibitors) and do not exhibit broad-range disinfestation properties typical of C[H.sub.3]Br. These compounds are therefore not attractive candidates for soil fumigation because they are either decomposed by soil microflora or the soil pests develop resistance easily (Shorter et al., 1995). Encouraged by the recent use of crude plant extracts in combating Striga weeds in Nigerian soils (Rugutt and Berner, 1998), the present study examined the activity of native Kenyan plant extracts against three economically important soil pathogens.

Experimental

Plant collection

All plant materials were collected in Kenya and identified at the Department of Botany, Moi University; voucher specimens have been deposited at the Herbarium there. Leaves of Warburgia ugandensis were collected at Moi University Forestry farm and Nursery in September of 1997. Stem barks were collected in Kerio Valley, Keiyo District in May 1998. Leaves of Azadirachta indica and Urtica massaica were collected in August 1997 in Mombasa and Molo, Nakuru District, respectively. The aerial parts of Tagetes minuta were collected at Chepkoilel Campus, Moi University.

Plant extraction

Wet stem bark (3 kg) of W. ugandensis was extracted using methanol. The resulting crude extract was concentrated in a rotary evaporator in vacuo; water bath temperature was set at 40 [degrees]C. The concentrated crude extract was then partitioned into water and chloroform. Separation of the water-chloroform phases afforded 95 g of crude extract in the chloroform fraction. A portion (15 g) of the concentrated chloroform fraction was filtered (in vacuo) through TLC-grade Merck silica gel using ethyl acetate (EtOAc). The filtrate was concentrated, packed in a pre-packed Merck silica gel column (40-63 mm) and then subjected to flash chromatography. The column was first eluted with neat hexane followed by hexane containing increasing amounts of EtOAc. The fractions eluted were monitored by TLC; the developing solvent was 30% EtOAc in hexane. The compounds on developed TLC plates were visualized either by observation under an ultraviolet lamp or by spraying with concentrated sulfuric acid and then heating at 110 [degrees]C on a hot plate for about 1 min. The crude extract from the bark of W. ugandensis yielded two compounds, muzigadial (1) and warburganal (2). Crude extracts from A. indica, T. minuta, and U. massaica plant materials were obtained following a procedure similar to that described by Rugutt et al. (1999).

Structural elucidation

The IR data of muzigadial (1) and warburganal (2) were obtained from their spectra run using a Shimagzu-IR408 spectrophotometer. Melting point data were obtained on a Reichet Thermovar apparatus. All 1D ([.sup.1.H], [.sup.13.C]) and 2D ([.sup.1.H]-[.sup.1.H] COSY, [.sup.1.H]-[.sup.1.H] NOESY, HMQC, and HMBC) NMR experiments were recorded at 298 K on a Bruker AMX 400 MHz spectrometer using the pulse sequences described by Rugutt et al. (1999). About 0.75 ml of deuterated chloroform (CD[Cl.sub.3]; [[delta].sub.H] 7.24ppm, [[delta].sub.C] 77.0 ppm) was added to 5mg of pure compounds (1-6) contained in 5-mm outer diameter NMR tubes. Chemical shifts are expressed in [delta] (ppm) scale downfield from TMS (internal reference standard). [.sup.1.H]-[.sup.1.H] COSY was performed using the following: acquisition parameters: recycling delay (D1), 1.5s; dummy scans (DS) = 2; number of scans (NS) = 32; D0 increment, 3.0 [micro]s. The [.sup.1.H]-[.sup.1.H] NOESY spectra were recorded using the following parameters: 512H512 data matrix size; time domain (td) = 512 in F1 and 1024 in F2; D1=2s; D0 = 3.0 [micro]s; ns = 96; mixing time ([[tau].sub.m]) = 800 ms. HMQC: 512 x 512 data matrix; td = 512w in F1 and 1024 in F2; D1 = 2s; NS = 48; DS = 4. Pulse sequences used for HMBC: data matrix size; td = 512w in F1 and 1024 in F2; rd = 2 s; ns = 32; DS = 16; D0 = 3.0 [micro]s; the [.sup.3.J.sub.CH] low-pass filter was set to 3.48 ms, and the delay for the evolution of long-range coupling was set to 100 ms.

X-ray data of muzigadial (1)

Recrystallization of muzigadial (1) in chloroform-hexane (1:3, v/v) afforded colorless crystals, mp 123-126 [degrees]C. A colorless plate was used for data collection on an Enraf-Nonius CAD4 diffractometer equipped with Cu[K.sub.[alpha]] radiation ([lambda] = 1.54184 [Angstrom]), and a graphite monochromator. Crystal data are: monoclinic space group P[2.sub.1], a = 7.441, 6 = 6.224, c = 14.453 [Angstrom], [beta] = 93.20 [degrees] Z = 2. Intensity data were measured by [omega] - 2[theta] scans of variable rate, designed to yield I = 25[sigma](I) for all significant reflections. Two octants of data were collected within the limits 2 < [theta] < 75 [degrees] Data reduction included corrections for background, Lorentz, polarization, and absorption effects. Absorption corrections ([mu] = 8.5 [cm.sup.-1]).

Molecular modeling

Conformational analysis of C[H.sub.3]Br and compounds 1-6 was performed using Alchemy 2000 molecular modeling software (MOPAC 1993 PM3; SciVision, Burlington, MA). Three-dimensional (3D) structures were built using the standard atoms, functional groups, and fragments functions available in the molecule library of the software. The lowest energy conformers of 3D structures were obtained by geometry optimization through MOPAC using PM3 parametrization. To explain the differences in biological activities, molecular descriptors of the lowest energy conformers were determined using QSARIS (SciVision, Burlington, MA). The molecular properties selected for quantitative structure-activity relationship (QSAR) modeling included van der Waals molecular volume (VD[W.sub.V]), van der Waals molecular area (VD[W.sub.A]), dipole moment (D), ovality, LogP, total energy, and the differences in the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) (Table 1).

UMO is the electron-accepting power of the molecule and relates to hydrogen bond donor acidity. HOMO represents the electron-donating power of the molecule and relates to hydrogen bond acceptor basicity. The HOMO-LUMO gap best explained the effect of functional groups on biological activity (Table 1). VD[W.sub.V], [mu], and [alpha] represent molecular bulk, molecular polarity, and molecular hydrophobicity, respectively. The hydrophobic chiral pockets of all the compounds examined in the present study differed in size, ranging from 52 to 230 [[Angstrom].sup.3]. The difference in cavity sizes suggests strongly that the compounds adopt different 3D orientations when interacting with the receptors of soil pathogens.

Inhibitory activities of crude extracts

Diffusion method (Xue et al., 2000) was employed in assessing the antifungal activity of crude extracts against Fusarium oxysporum, Alternaria passifiorae, and Aspergillus niger. Serial concentrations were prepared such that 20 [micro]l of 30% methanol in water contained 1, 10, 20, 50, 100, and 150 mg of the crude extract. Filter paper discs (12 mm in diameter) were treated with a specific volume of test extract solution (prepared in triplicate) and kept in dust-free conditions. The medium (2% malt extract: agar agar) was prepared by adding 20 g of each to 1.01 of distilled water and heating to aid dissolution. The media, distilled water and Petri dishes were sterilized by autoclaving at 120 [degrees]C at 1 bar pressure for 20min. A small amount of sterile distilled water was used to dissolve the test microorganism. This was introduced to the medium after cooling to a temperature just before it solidified and inverted several times to evenly distribute the spores. The medium was then poured into sterile Petri dishes and allowed to solidify. The air-dried discs were then applied on the medium in the Petri dishes and incubated at 37 [degrees]C for 3 days. The clear zone of growth inhibition around the disc was measured and expressed as the inhibition diameter.

Minimum inhibitory concentrations (MIC values)

MIC values of the pure compounds were determined using the agar macro-dilution method (Xue et al., 2000). Two media, 2% malt extract agar: agar agar and potato dextrose agar (PDA), were used. Malt extract agar:agar agar medium was prepared as described for the diffusion method. PDA medium was prepared by suspending 39 g in 1.01 of distilled water and heating until all of it dissolved. The medium was sterilized by autoclaving at 120 [degrees]C for 20 min. Serial concentrations of the pure compounds, 3, 5, 10, 12, 25, 50 and 100 [micro]g/ml, were prepared in 20% ethanol-water solution, each in triplicate. This was incorporated into a liquefied agar medium (45-50 [degrees]C in a water bath) in sterile screw-capped tubes. The medium was mixed gently by inverting the tubes several times and the contents were poured into an appropriate number of Petri dishes. The plates were then set aside on a horizontal surface and allowed to solidify. One control plate containing the medium without any compound was prepared for each series of dilutions. Each plate was then incubated at 37 [degrees]C in an inverted position for 3 days. The lowest concentration at which no growth was observed visually was determined and indicated as the MIC value.

Greenhouse tests

The pathogen, F. oxysporum was cultured in the laboratory 2 weeks prior to field experimentation. Malt extract agar:agar agar medium (500 ml) was prepared by autoclaving at 120 [degrees]C at 1 bar pressure for 20min. The spores of the pathogen were dissolved in a small amount of sterile distilled water and then introduced into the prepared culture medium. This sterile medium was inverted several times and shaken to distribute the spores evenly, and was then poured into 20 sterile Petri dishes, each containing approximately 25 ml of the culture medium. The dishes were incubated at 37 [degrees]C in an inverted position.

The soils were collected from a fertile field and pasteurized with aerated steam for sterilization before use. The pots were sterilized by cleaning with distilled water, rinsed with sodium hypochlorite and air-dried. For soil infestation, the pathogen (F. oxysporum) spores, already cultured, were dissolved into 360 ml sterile distilled water. A 10-ml portion of Fusarium inoculum stock solution was then diluted to 100 ml to yield a total of 36 of such dilutions. This was poured over the surface of the soil in each pot and allowed to infiltrate the soil. Four pots did not receive this treatment. The soils in the pots were allowed to stand in the greenhouse for 2 weeks; to allow adequate conditioning of pathogen in the soil, distilled water was occasionally added before introducing crude extracts. A total of 10 treatments were prepared. Four replicates of different concentrations of crude extracts (100 ml each) were prepared: 1, 2, 5, 7, 10, 12, and 15mg/ml in 20% ethanol-water mixture, and poured onto soil surface. Four pots had 100 ml of the solvent (20% ethanol-water) poured on the surface of the soil in each pot, with no crude extract. This served as a negative control and helped in establishing whether the solvent had any effect on the pathogen. Four additional pots were treated with the pathogen but with neither the crude extract nor the solvent. These pots also served as negative controls. The four pots with no pathogen inoculum did not receive the crude extract or the solvent treatment. These pots served as positive controls. After this procedure, the soils were monitored for 2 weeks with occasional watering, before planting.

Determination of antifungal infection

Seeds of Lycopersicon esculentum (a certified moneymaker tomato) were planted 2-cm deep into the soil. Five seeds were planted in each pot for 10 different treatments. The completely randomized block design was replicated four times. The pots were arranged randomly at a distance of 60 cm from each other and at a distance of 30 cm from each other within each block. The descriptors for fungal leaf infection (Fusarium wilt) that were monitored were wilted leaves, vascular discoloration of the hypocotyl tissues, branches and leaves exhibiting wilting and chlorosis, necrosis, premature defoliation and eventual plant death. Data indicating the severity of infection were collected for a period of 1 month after seedling emergence. Disease scores indicating leaf infection were taken for days 21, 23, 25, 27, 30, 35, 40, and 50th day after planting and recorded on a 1-9 scale as shown in Table 2.

[FIGURE 1 OMITTED]

Results and discussion

Pathogens continue to be a major problem despite the fact that we live in a world rich in underexploited natural products. Although the use of botanicals in disease management has long been part of traditional practices, the majority of higher plant species are yet to be explored as potential sources of antimicrobial agents. Some farmers in Kenya currently practice organic farming aimed at producing healthy crops by direct incorporation of natural weeds and plants. The present study is based on concepts derived from traditional and organic farming practices. The four native Kenyan plants studied were W. ugandensis, A. indica (Neem tree), T. minuta (Mexican merigold), and U. massaica Mildbr. (Stinging nettle).

The genus Warburgia (Conellaceae) consists of two species distributed in East Africa, W. stuhlmanii Engl, and W. ugandensis. The two species are used widely in the local folk medicine to alleviate toothache, neumatism, general body pains, diarrhea and malaria. In addition, the leaves of W. ugandensis are sometimes used to spice food. The bark of W. ugandensis is commonly known by several different names depending on the local tribe such as 'Apacha' (Luhya), 'Muthiga' (Kikuyu), 'Olosogoni' (Maasai), 'Soget' (Kipsigis), 'Soke' (Tugen) and 'Sogo-maitha' (Luo). The distribution of W. stuhlmanii is limited to the coastal areas (Mombasa), and is known as Mukaa (Swahili). The aqueous methanolic extracts of the barks of W. ugandensis and W. stuhlmanii are active against Gram-positive bacteria, yeast and filamentous fungi. A series of unique sesquiterpentine 1,4-dialdehydes isolated from these plants exhibit broad antibacterial and antifungal activities. Muzigadial (1), warbuganal (2), and polygodial (3) obtained from these plants show similar antibacterial spectra (Fig. 1). These 1,4-dialdehydes not only possess potent antifeedant activity against African armyworms but also taste hot to the human tongue; this parallels their feeding inhibition for animals. These properties are related to the stereochemistry of the aldehyde group at the C-9 position (Jansen et al., 1989). Other compounds that have been isolated from W. stuhlmanii and W. ugandensis include ugandendial (4), muzigadiolide (5), mukaadial, cinnamolide, cinnamolide-3[beta]-01, cinnamolide-3-[beta]-acetate, ugandensolide, and deacetyluganden-solide.

Azadirachtin (6) is hitherto the most interesting constituent of Neem seeds (Azadirachtin indica A Juss; family meliaceae). Compound 6, like all its derivatives, is not only a very potent insect antifeedant but also an insect growth-regulating agent (van der Nat et al., 1991). A. indica is a hardy evergreen tree distributed widely in South Asia, parts of Africa, and other tropical areas. The common name for the "neem" tree in Swahili is "mwarubaini." Neem tree contains a group of compounds called 'triterpenes', more specifically 'limonoids', the most common being azadirachtin (6), salanin, melantiol and nimbin. Nimbin and nimbidin exhibit antiviral activity. Locusts do not touch the leaves of the neem tree; farmers have known this for centuries and use the leaves to protect their grains. Apart from insects, neem extracts affect quite a range of other organisms including nematodes, snails, crustaceans, and fungi. From an African medicinal viewpoint, parts of the neem tree can be used for the treatment of a variety of human ailments, particularly bacterial and fungal diseases. The tree parts are also used as fungicidal, antibacterial, antiviral and antimalarial agents, and for dermatological infections, dental treatments, Chagas disease treatment, pain relief and fever reduction, and birth control. In the field of veterinary medicine, neem extracts are used in controlling insects, bacteria and intestinal worms. In principle, azadirachtin (6) is notable for its chemical complexity and biological activity; it exhibits a wide range of biological activities. The two active halves, "insecticidal" and "antifeedant", of 6 are complementary. However, the present challenge rests in uniting the two fragments (halves). Chemists' efforts to synthesize the two halves have been fruitful. It is worth noting that synthetic analogs retained biological activities of azadirachtin (6). The future of azadirachtin (6) as a potent and environmentally safe insecticide is very bright.

T. minuta is a common weed of the family Compositae (Asteraceae). It is an erect, strong-smelling annual, often very robust but variable in plant habit and very plastic in its response to crowding. Its leaves are pinnate with ellyptic toothed leaflets, heads are creamy yellow, in terminal corymbs with phyllaries 10 mm long. There have been reports on the use of Tagetes species as a nematicide. U. massaica (stinging nettle) belongs to the family Urticaceae. It is a very irritating, painful stinger, often growing in abandoned tracts in montane forest areas. U. massaica is used traditionally for the treatment of fungal diseases. The lectin (UDA) of the rhizome of U. dioica (stinging nettle) possess a remarkable antifungal activity (Boekaert et al., 1989).

Isolation of muzigadial (1) and muzigadiolide (5)

A crude organic extract (1.5 g) from W. ugandensis stem bark, purified by flash chromatography column, afforded 1.03 g of muzigadial (1) and 0.25 g of muzigadiolide (5). The spectral (NMR and IR) and melting point data were in agreement with literature reports (Jansen et al., 1989). The structures of compounds 1-6 are shown in Fig. 1. The spectral data for muzigadial (1) and of muzigadiolide (5) are presented below. Muzigadial (1): Needles from EtOAC hexane, melting point, 125 [degrees]C. [.sup.1.H] NMR (400 MHz): [delta] (ppm): 9.64 (1H, s, H-12), 9.44 (1H, H-11), 7.26 (1H, t, H-7), 4.93, 4.76 (2 x 1H, 2xbr [S.sub.1] C[H.sub.2]), 4.08 (1H, s, 9-OH), 2.62 (1H, m, H-5), 1.08 (1H, d, 3-Me), 0.89 (3H, s, 10-Me). IR [v.sub.max][cm.sup.-1] 3460, 2950, 2850, 1715, 1670, 1630. Muzigadiolide (5): Needles from EtOAc hexane, melting point 142 [degrees]C. [.sup.1.H] NMR (400 MHz): [delta] (ppm): 7.18 (1H, dd, H-7), 4.93, 4.75 (2 x 1H, 2 x br s, = C[H.sub.2]), 4.31, 4.26 (2H, 11-C[H.sub.2]), 2.62 (1H, m, H-5), 1.11 (1H, d, Me), 0.74 (3H, s, 10-Me). IR [v.sub.max][cm.sup.-1] 3400-3450, 2800, 1750, 1725, 1680, and 1635.

Antifungal activity of crude extracts

Many soil-borne pathogens are destructive parasites that affect crop production. Fungi are among the soil-borne microorganisms. Some fungal species are parasitic and destructive to host plants. In the present study, the test microorganisms that were used belong to the genera Fusarium, Alternaria, and Aspergillus. Fusarrium is a large genus that contains economically important pathogens causing cereal seedling diseases, root rot, and wilt in several plants. Fusarium species are very successful 'soil inhabitants' and, once established, persist for several years. This renders the soil unfit for production of quality crops. The genus is thus one of the most injurious; several of its species are destructive parasites that invade pant ducts, inhibit water supply and cause a class of Fusarium diseases known as 'wilts'. Fusarium diseases occur in a number of plants, including cereals and grasses, legumes and horticultural crops.

Bioassay data of crude extracts are shown in Table 3. Crude extracts from W. ugandensis, A. indica, and T. minuta inhibited the growth of F. oxysporum, while those from W. ugandensis stem bark inhibited the growth of A. passiflorae. However, a crude extract from U. massaica (leaves) showed no activity against F. oxysporum. As deduced from the diameters of inhibition by the crude extracts, W. ugandensis displayed higher activity than A. indica and T. minuta. T. minuta and A. indica exhibited similar activity patterns against F. oxysporum. There were no definitive relationships between the concentrations of crude extracts and the diameters of inhibition. This may be attributed to limited diffusion; the crude extract and the culture media were both in semi-solid states. Fractions from the crude (leaves and stem bark) extracts of W. ugandensis showed significant activity against F. oxysporum (Table 4). Leaves are renewable resources and are preferred to other parts of plants (such as the stems and roots) as sources of naturally derived fumigants.

Muzigadial (1) was active against all soil pathogens while muzigadiolide (5) was inactive (Table 5). The maximum (optimum) activity against A. niger was at an MIC value of 5 [micro]g/ml. The dramatic differences in activity of compounds 1 and 5 can be attributed to the differences in the dialdehyde functional group that ultimately affects the molecular descriptors (Table 1).

Greenhouse data

The severity of fungal infection, expressed as a function of diameter of inhibition, is shown in Fig. 2.

The greenhouse data were subjected to analysis of variance, DMRT, and LSD tests. The differences among the treatments for all the days scored were significant at 1% and 5% levels of significance. All the treatments, except treatment 9 (pathogen plus solvent), differed significantly from the control (treatment 1, pathogen alone). Treatment 2 (1 mg/ml) and treatment 3 (2 mg/ml) were significantly different from other treatments. The diameter inhibition data indicated that crude extract concentration of 5 mg/ml or higher effectively controlled the pathogen, while concentrations of 1 and 2 mg/ml were not effective. Also, the solvent (20% ethanol in water) had no effect on the pathogen, as plants in the pots where only the solvent was applied were equally as infected as those plants in the pots where only the pathogen had been introduced (control).

[FIGURE 2 OMITTED]

Acknowledgments

This research was supported by Rockefeller Foundation Grants (RF 94027 #600 and RF 92-38-419) and the Massachusetts Commonwealth Information Technology Initiative (CITI) Grant (# 201-1017MCL). The authors are grateful for a partial scholarship offered to A.N.N, by Moi University. Special thanks are owed to the technicians and staff of the International Institute of Tropical Agriculture (Ibadan, Nigeria) for reproducing the preliminary research that was performed at Moi University, Kenya.

References

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Boekaert, W.R., van Parijs, I., Leyns, F., Joos, H., Peumans, W.I., 1989. A chitin-binding lectin from stinging nettle rhizomes with antifungal properties. Science 245, 1100-1102.

Jansen, B.M., Kreuger, J.A., de Groot, A., 1989. The conversion of (-)- and (+)-dihydrocarvone into chiral intermediates for the synthesis of (-)-polygodial, (-)-warburganal and (-)-muzigadial. Tetrahedron 45, 1447.

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Rugutt, J.K., Berner, D.K., 1998. Activity of extracts from non-host legumes on the germination of Striga hermonthica seeds. Phytomedicine 5, 293-299.

Rugutt, J.K., Henry, C.W., Franzblau, S.G., Warner, I.M., 1999. NMR and molecular mechanics study of pyrethrins I and II. J. Agric. Food Chem. 47, 3402-3410.

Shorter, J.H., Kolb, C.E., Crill, P.M., Kerwin, R.A., Talbot, R.W., Hines, M.E., Harriss, R.C., 1995. Rapid degradation of atmospheric methyl bromide in soils. Nature 377, 717-719.

van der Nat, J.M., van der Sluis, W.G., van Dijk, H., 1991. Activity-guided isolation and identification of Azadirachta indica bark extract constituents which specifically inhibit chemiluminescence production by activated human polymorphonuclear leukocytes. Planta Med. 57, 65.

Wang, D., Yates, S.R., Gan, J., 1997. Temperature effect on methyl bromide volatilization in soil fumigation. J. Environ. Qual. 26, 1072.

Watson, R.T., Albritton, D.L., Anderson, S.O., Lee-Bapty, S., 1992. Methyl bromide: its atmospheric science, and economics. United Nations Environmental Programme (UNEP), United Nations Headquarters, Ozone Secretariat P.O. Box 30552, Nairobi, Kenya, 1992.

Xue, S., Gan, J., Becker, J.O., 2000. Nematode response to methyl bromide and 1,3-dichloropropnes soil fumigation at different temperatures. Pest Manage. Sci. 56, 737.

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J.K. Rugutt (a,*), A.N. Ngigi (b), K.J. Rugutt (c), P.K. Ndalut (b)

(a) Department of Chemistry, Massachusetts College of Liberal Arts, 375 Church Street, North Adams, MA 0147, USA

(b) Department of Chemistry, Moi University, P.O. Box 1125, Eldoret, Kenya

(c) Department of Education, Illinois State University, Normal, IL 61790-2200, USA

Received 3 September 2001; accepted 6 August 2003

*Corresponding author. Tel.: + 1 413 662 5451; fax: +1413 662 5010.

E-mail address: jrugutt@mcla.mass.edu (J.K. Rugutt).
Table 1. Molecular descriptors of muzigadial (1), warburganal (2),
polygodial (3), ugandensidial (4), muzigadiolide (5), azadirachtin (6),
and methyl bromide (C[H.sub.3]Br)

Compound Energy (a) VD[W.sub.v] (b) VD[W.sub.A] (c) LogP

1 -2612 189 229 2.27
2 -2595 230 266 2.46
3 -2321 226 266 3.55
4 -3412 226 266 3.55
5 -2614 184 205 2.75
6 -8534 651 788 -1.33
C[H.sub.3]Br -507 52 78 0.93

Compound Ovality D (d) LUMO (e) HOMO (f) HOMO-LUMO spcP (g)

1 1.54 7.16 -9.2299 -9.3894 -0.1595 0.0353
2 1.47 11.8 -9.5247 -9.8410 -0.3163 0.0371
3 1.48 10.8 -9.2440 -9.5745 -0.3305 0.0376
4 1.48 10.8 -10.4839 -10.5828 -0.0989 0.0376
5 1.51 8.3 -9.5423 -9.6808 -0.1386 0.0359
6 2.16 4.9 -10.1017 -10.1766 -0.0749 0.0250
C[H.sub.3]Br 1.16 1.46 -7.8298 -10.4228 -2.5930 0.0803

(a) Total steric energy (Kcal/mol).
(b) van der Waals molecular volume ([[Angstrom].sup.3]).
(c) van der Waals molecular area ([[Angstrom].sup.2]).
(d) Dipole moment.
(e) Lowest unoccupied molecular orbital.
(f) Highest occupied molecular orbital.
(g) spc polarizability.

Table 2. Disease scores indicating leaf infection recorded on a 1-9
scale

Scale Disease symptoms

1 No visible disease symptoms
3 Very few wilted leaves (1-3 leaves representing no more than 10%
 of the foliage) combined with limited discoloration of the root
 hypocotyl tissues
5 Approximately 25% of the leaves and the branches exhibit wilting
 and chlorosis
7 Approximately 50% of the leaves and branches exhibit wilting,
 chlorosis, and limited necrosis. Plants are stunted
9 Approximately 75% or more of the leaves and branches exhibit
 wilting, severe stunting, and necrosis, with premature
 defoliation often resulting in plant death

Table 3. Antifungal activity of crude extracts

 Diameter of inhibition
 W. ugandensis W. ugandensis A. indica
 Disc content leaves leaves leaves
Test (mg) diameter (MeOH) (hexane) (MeOH)
microorganism (12mm) extract extract extract

Fusarium sp. 150 23.0 16.5 15.0
 100 16.0 15.5 14.5
 50 15.5 13.0 13.0
 20 14.0 12.5 11.0
 10 12.0 11.5 10.0
 1 10.0 10.0 7.5
Alternaria Stem bark
passiflorae (CH[Cl.sub.3])
 fraction
 150 25.0
 100 23.4 -- --
 50 21.5
 20 19.0
 10 15.0
 1 11.5

 Diameter of inhibition
 T. minuta U. massaica
 Disc content A. indica seeds aerial part leaves
Test (mg) diameter (MeOH) (MeOH) (MeOH)
microorganism (12mm) extract extract extract

Fusarium sp. 150 18.0 16.0 No activity
 100 16.0 15.0 No activity
 50 13.5 13.0 No activity
 20 12.0 12.5 No activity
 10 9.0 10.0 No activity
 1 8.5 9.5 No activity
Alternaria
passiflorae
 150
 100 -- -- --
 50
 20
 10
 1

Table 4. Activity of methanol extract fractions from leaves of W.
ugandensis against F. oxysporum.

Test Disc content Inhibition
microorganism Fractions (mg) diameter (mm)

Fusarium F1 10 No growth
oxysporum inhibition
 1 No growth
 inhibition
 F2 10 10.5
 1 8.0
 F3 10 13.0
 1 10.0
 F4 10 12.5
 1 10.3
 F5 10 No growth
 inhibition and
 other fractions
 below F5 had
 similar results
 1

Table 5. MIC values for muzigadial (1) and muzigadiolide (5) against F.
oxysporum, Alternaria passiflorae, and Aspergillus niger

 MIC ([micro]g/ml)
Soil pathogens Muzigadial (1) Muzigadiolide (5)

Fusarium oxysporum 50 No activity
Alternaria passiflorae > 100 No activity
Aspergillus niger 5 No activity
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Author:Rugutt, J.K.; Ngigi, A.N.; Rugutt, K.J.; Ndalut, P.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Sep 1, 2006
Words:5357
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