International Journal of Sustainable Agriculture 2 (2): 20-25, 2010 ISSN 2079-2107 © IDOSI Publications, 2010

Evaluation of Allelopathic Potential of Neem (Azadirachta indica. A. Juss) Against Seed Germination and Seedling Growth of Different Test Plant Species

Md. Abdus Salam and Hisashi Kato-Noguchi

Department of Applied Biological Science, Faculty of Agriculture, Kagawa University Miki, Kagawa 761-0795, Japan

Abstract: The allelopathic potential of aqueous methanol extract of neem leaves was investigated on seed germination and seedling growth of different plants viz. cress (Lepidum sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), wild buckwheat (Eriogonum compositum Douglas ex Benth.), sand fescue (Festuca myuros L.), timothy (Phleum pratense L.), barnyardgrass (Echinochloa crus-galli [L.] Beauv.) and Echinochloa colonum [L.] Link.). Germination, root and shoot growth of all test plant species were inhibited at concentrations greater than 0.001 g dry weight equivalent extract/mL except timothy and E. colonum. Inhibitory activity was dependent on the extract concentrations and the higher extract concentration had the stronger inhibitory activity. The effectiveness of the extract was much higher on the root growth than the shoot growth of test plants. Comparing in the extract concentrations required for 50% inhibition, germination of sand fesucue was the most sensitive and root and shoot growth of cress was the most sensitive to the extract. These results suggest that neem leaves may contain growth inhibitory substances and may possess allelopathic potential. Therefore, neem leaves may be a possible candidate for the isolation and identification of allelopathic substances and for the development of natural herbicides for sustainable agricultural production.

Key words: Azadirachta indica % Phytotoxicity % Test plant species % Germination % Seedling growth

INTRODUCTION anti-pyretic), gedunin (vasodilator, anti-malaria, anti- fungal), sodium nimbinate (diuretic, spermicide, A variety of crop and weed species have been anti-arthritic), queceretin (anti-protozoal), salannin reported to possess allelopathic activity on the growth of (repellent) and azadirachtin (repellent, anti-feedant, other plant species [1]. Compounds with allelopathic anti-hormonal) [7]. Since neem contains a number of activity are present in many plants and in many organs, useful compounds with multiple uses and adapts including leaves, stems, flowers, fruits and buds [2-4]. to diverse habitats and climatic conditions, the Neem (Azadirachta indica. A. Juss), belongs to the family interest regarding the tree has been increased. Al- Meliaceae, is native to Indo-Pakistan subcontinent [5]. Charchafchi et al. [8] and Ashrafi et al. [9] reported that Since very ancient times neem has been used in extract of neem leaves significantly inhibited the households for giving bath to newly born infants, protect germination percentage and seedling growth of target people from insect bites and cure skin ailments and used plant species used in their research. Although much as traditional medicine for the treatment of various research has been done on neem in different aspects, little diseases [6]. Now-a-days it is widely used in toothpastes, information is available concerning the allelopathic soaps and lotions as well as a biological insecticide. potential of neem on weeds. It was, therefore, of interest These properties have been attributed to hundreds of to determine the allelopathic potential of neem using chemicals present in neem. The most common analyzed aqueous methanol extract of neem leaves by evaluating its compounds are nimbin (anti-inflammatory), nimbidin effect on the seed germination and seedling growth of (anti-bacterial, anti-ulcer, analgesic, anti-arrhythmic, some important test plant species in laboratory bioassay anti-fungal), nimbidol (anti-tubercular, anti-protozoan, condition.

Corresponding author: Md. Abdus Salam, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0701, Japan. Tel: 81-87-891-1545. 20 Intl. J. Sustain. Agric., 2 (2): 20-25, 2010

MATERIALS AND METHODS colonum). The germinated seeds were counted and the percentage germination was calculated by the reference to Plant Materials: Leaves of neem (Azadirachta indica. A. that of control seedlings. For control treatments, methanol Juss) were collected from Bangladesh Agricultural (0.2 mL) was added to a sheet of filter paper in the Petri University campus, Mymensingh, Bangladesh (lies dish without neem extract (0 g/mL) and evaporated and between 20° 34' and 26° 38' north latitude and between 88° seeds of each test species were sown as described above. 01' and 92° 41' east longitude) in February 2010. The The bioassay experiment was conducted with four leaves were then sun dried and stored in 2°C temperature replications and repeated twice. until extraction. Bioassay for Seedling Growth: Filter paper (No. 2) was Seeds of Test Plants: Seeds of cress (Lepidium sativum placed into the Petri dish and the neem extract was added L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa on it as described above. The filter paper in the Petri L.), wild buckwheat (Eriogonum compositum Douglas dishes was moistened with 0.6 mL of a 0.05% (v/v) ex Benth.) and timothy (Phleum pratense L.) were aqueous solution of Tween 20. Ten seeds of cress, lettuce purchased from Takii (Kyoto, Japan). Seeds of sand or alfalfa, or 10 seedlings of wild buckwheat, sand fescue, fescue (Festuca myuros L.), barnyardgrass and timothy, barnyardgrass or Echinochloa colonum after Echinochloa colonum (L.) Link were purchased from germination in the darkness at 25°C for 24-120 h were Herbiseed (London, UK). Cress, lettuce and alfalfa arranged on the Petri dishes. For control treatments, were chosen as test plants for bioassay because of their methanol (0.2 mL) was added to a sheet of filter paper in known seedling growth behavior and model plant for the Petri dish without neem extract (0 g/mL) as described laboratory bioassay. Wild buckwheat, sand fescue, above. The length of their shoots and roots was measured timothy, barnyardgrass (Echinochloa crus-galli [L.] after 48 h of incubation in the darkness at 25°C. The Beauv.) and Echinochloa colonum were chosen as test bioassay was repeated four times in a completely plants for bioassay due to their existence of weeds in crop randomized design with 10 plants for each determination. field throughout the world. Germination of these seeds The percentage length of seedlings was then determined was tested before use and ranged from 67 to 100%. by reference to the length of control seedlings. The inhibition percentage was calculated as % inhibition = Extraction: Dried leaves (50 g) of neem were cut into small (control length-length with aqueous methanol extract/ pieces (about 1 cm) and extracted with 500 mL of 70% control length) ×100. The concentrations required for

(v/v) aqueous methanol for two days. The extract was 50% inhibition (defined as I50) of the test plant species in filtered through one layer of filter paper (No. 2; Toyo Ltd., the assay were determined by a logistic regression Japan), using a vacuum pump. The residue was re- equation of the concentration-response curves. extracted with 500 mL of methanol for one day and filtered. The two filtrates were then combined and evaporated in Statistical Analysis: Experimental data were subjected to vacuo at 40°C. one-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) to determine Bioassay for Seed Germination: An aliquot of the extract significance differences among mean values at the (final assay concentration was 0.001, 0.003, 0.01, 0.03, 0.1 probability level of 0.05. or 0.3 g dry weight of neem leaves equivalent extract/mL) was evaporated to dryness, dissolved in 0.2 mL of RESULTS methanol and added to a sheet of filter paper (No. 2; Toyo) in a 28 mm Petri dish. Methanol was evaporated in Effects of Aqueous Methanol Extract of Neem Leaves on a draft chamber. The filter paper in the Petri dishes was Seed Germination of Test Plant Species: The inhibitory moistened with 0.6 mL of a 0.05% (v/v) aqueous solution activity of aqueous methanol extract of neem leaves on of Tween 20. Then ten seeds of each test species were the seed germination of cress, lettuce, alfalfa, wild sown on filter paper in Petri dishes and allowed to buckwheat, sand fescue, timothy, barnyardgrass and E. germinate and grow in the darkness at 25°C for two days colonum is shown in Table 1. Germination of cress, wild (cress, lettuce, alfalfa and wild buckwheat), three days buckwheat and sand fescue was significantly reduced by (sand fescue) or five days (timothy, barnyardgrass and E. the extracts at concentrations greater than 0.001 g/mL.

21 Intl. J. Sustain. Agric., 2 (2): 20-25, 2010

Table 1: Effects of aqueous methanol extract of neem leaves on the germination percentage of test plant species Concentration (g/mL) Cress Lettuce Alfalfa Wild buckwheat Sand fescue Timothy Barnyard grass Echinochloa colonum Control (0) 100.00a 100.00a 100.00a 96.67a 70.00a 76.67a 100.00a 66.67a 0.001 93.33b 100.00a 100.00a 90.00b 40.00b 73.33a 93.33bc 63.33ab 0.003 93.33b 93.33b 93.33b 83.33c 23.33c 73.33a 96.67ab 63.33ab 0.01 60.00c 66.67c 90.00b 66.67d 10.00d 60.00b 90.00c 60.00b 0.03 3.33d 0.00d 70.00c 56.67e 0.00e 33.33c 73.33d 46.67c 0.1 0.00d 0.00d 43.33d 53.33e 0.00e 16.67d 50.0e 36.67d 0.3 0.00d 0.00d 23.33e 6.67f 0.00e 10.00e 20.00f 6.67e Mean with same letters in a column is not significantly different at P<0.05

Table 2: Effects of aqueous methanol extract of neem leaves on the root length (mm) of test plant species Concentration (g/mL) Cress Lettuce Alfalfa Wild buckwheat Sand fescue Timothy Barnyard grass Echinochloa colonum Control (0) 20.52a 8.83a 13.03a 27.23a 13.83a 9.70a 26.73a 18.57a 0.001 12.04b 5.24b 10.26b 24.60b 7.90b 8.54b 19.68b 15.60b 0.003 10.70c 5.24b 8.91c 15.30c 6.26c 7.80c 11.52c 12.37c 0.01 5.45d 2.35c 6.70d 2.80d 2.34d 4.09d 6.52d 6.76d 0.03 2.22e 1.07d 3.13e 1.34de 0.30e 1.52e 2.03e 1.54e 0.1 1.50f 0.00e 0.65f 0.68e 0.00e 0.00f 0.74f 0.38f 0.3 1.19f 0.00e 0.00g 0.28e 0.00e 0.00f 0.00f 0.15f Mean with same letters in a column is not significantly different at P<0.05

Table 3: Effects of aqueous methanol extract of neem leaves on the shoot length (mm) of test plant species Concentration (g/mL) Cress Lettuce Alfalfa Wild buckwheat Sand fescue Timothy Barnyard grass Echinochloa colonum Control (0) 12.60a 2.95a 9.04a 14.50a 9.18a 9.35a 27.56a 16.68a 0.001 8.73b 2.21b 6.07b 13.45b 8.76b 9.77a 27.30a 16.95a 0.003 7.54c 1.52c 5.09c 9.73c 7.93c 9.42a 25.04b 15.79ab 0.01 5.02d 1.00d 3.17d 4.68d 3.73d 8.76b 20.93c 14.87b 0.03 1.91e 0.49e 1.44e 2.74e 1.28e 5.51c 15.38d 8.44c 0.1 1.47f 0.00f 0.19f 1.66f 0.00f 1.16d 6.22e 3.38d 0.3 1.17f 0.00f 0.00g 0.87f 0.00f 0.24e 1.91f 0.76e Mean with same letters in a column is not significantly different at P<0.05

Table 4: I50 values of aqueous methanol extract of neem leaves for germination percentage and root and shoot growth of test plant species.

I 50 values (g dry weight equivalent extract/mL) ------Test plant species Germination Root Shoot Cress 0.0126 0.0019 0.0030 Lettuce 0.0115 0.0034 0.0032 Alfalfa 0.0606 0.0110 0.0042 Wild buckwheat 0.1929 0.0038 0.0051 Sand fescue 0.0018 0.0021 0.0096 Timothy 0.0288 0.0074 0.0438 Banryardgrass 0.1127 0.0026 0.0388 Echinochloa colonum 0.1752 0.0053 0.0489 The values were determined by a logistic regression analysis after bioassay

Germination of lettuce and alfalfa was significantly Effects of Aqueous Methanol Extract of Neem Leaves on reduced at concentrations greater than 0.003 g/mL and the Root Growth of Test Plant Species: Effects of germination of timothy, barnyardgrass and E. colonum aqueous methanol extract of neem leaves on root growth was significantly reduced at concentrations greater than of eight test plant species are shown in Table 2 and 0.01 g/mL. The extract completely inhibited seed Fig. 1. The extract exhibited a marked root growth germination of lettuce and sand fescue at 0.03 g/mL and inhibition which was concentration dependent. Significant the germination of cress at 0.1 g/mL (Table 1). The lowest root growth inhibition was observed at the concentrations

I50 was 0.0018 g/mL for sand fescue and the highest I50 greater than 0.001 g/mL against all the test plant species was 0.1929 g/mL for wild buckwheat (Table 4). (Table 2 and Fig. 1). At concentration of 0.1 g/mL, the root

22 Intl. J. Sustain. Agric., 2 (2): 20-25, 2010

Fig. 1: Effects of aqueous methanol extract of neem leaves on root and shoot growth of cress, lettuce, alfalfa, wild buckwheat, sand fescue, timothy, barnyardgrass and E. colonum. Concentrations of tested samples corresponded to the extract obtained from 0.001, 0.003, 0.01, 0.03, 0.1 and 0.3 g dry weight of neem per mL. Means ± SE from four independent experiments with ten plants for each determination are shown. Asterisk indicates significant difference among treatments in reference to control: *P<0.05,**P<0.01,***P<0.001, NS=non-significant growth of lettuce, sand fescue and timothy was cress, alfalfa, wild buckwheat, timothy, barnyardgrass and completely inhibited and the root growth of cress, alfalfa, E. colonum was inhibited by 88.3, 85.8, 86.7, 86.8, 76.0 and wild buckwheat, barnyardgrass and E. colonum was 73.3%, respectively (Fig. 1). The lowest I50 was 0.0030 inhibited by 93.0%, 94.9%, 97.1%, 97.3% and 98.2%, g/mL for cress and followed by lettuce (0.0032 g/mL). The respectively (Fig. 1). The lowest I50 on root growth was highest I50 (0.0489 g/mL) was observed in E. colonum 0.0019 g/mL for cress and followed by sand fescue (Table 4).

(0.0021 g/mL). The highest I50 was 0.0110 g/mL for alfalfa (Table 4). DISCUSSION

Effects of Aqueous Methanol Extract of Neem Leaves on Aqueous methanol extract of neem leaves inhibited the Shoot Growth of Test Plant Species: Effects of seed germination and root and shoot growth of all test aqueous methanol extract of neem leaves on shoot growth plant species used in this research. The inhibitory activity are shown in Table 3 and Fig. 1. Significant shoot growth was proportional to the extract concentrations and higher inhibition was observed in cress, lettuce, alfalfa, wild concentration had the stronger inhibitory effect. Extract of buckwheat and sand fescue at concentration of 0.001 neem leaves, therefore, had inhibitory effects on a wide g/mL. Stimulatory effect was observed on the shoot range of plant species including dicotyledonous (cress, growth of timothy and E. colonum at same concentration lettuce, alfalfa and wild buckwheat) and (Table 3). At concentrations greater than 0.1 g/mL, the monocotyledonous plants (sand fescue, timothy, shoot growth of lettuce and sand fescue was completely barnyardgrass and E. colonum). The root and shoot inhibited. At same concentration, the shoot growth of growth of test plant species was more suppressed than

23 Intl. J. Sustain. Agric., 2 (2): 20-25, 2010 the seed germination. In addition, the extract of neem ACKNOWLEDGEMENTS leaves had higher root growth inhibition than shoot growth on all the test plant species except in case of wild We are most grateful to Japanese Government for the buckwheat at lowest concentration (0.001 g/mL). The supporting scholarship for the first author of the higher root growth inhibition might be due to the manuscript. permeability of allelopathic substances to root tissue was found to be greater than that to shoot tissue [10] and due REFERENCES to the direct contact between the root and phytotoxic compounds present in extract which in turn might inhibit 1. Rice, E.L., 1984. Allelopathy, 2nd Edn., Academic cell division [11] which is highly active at meristimatic Press, London, pp: 309-316. tissue of the growing root tip. 2. Mahall, B.E. and R.M. Callaway, 1991. Root

Comparing in I50 values, root and shoot of communication among desert shrubs. Proceedings of cress were the most sensitive and root of alfalfa the National Academy of Science of the United State was the less sensitive to the neem extract. In case of of America, 88: 874-876. germination sand fescue was the most sensitive and 3. Inderjit, 1996. Plant phenolics in allelopathy. E. colonum was the least sensitive to the extract. Botanical Rev., 62: 186-202. Therefore, the distinction between dicotyledonous 4. Ashrafi, Z.Y., H.R. Mashhadi and S. Sadeghi, 2007. and monocotyledonous species was not clear in Allelopathic effects of barley (Hordeum vulgare) on root and shoot growth and in seed germination. germination and growth of wild barley (Hordeum There is a complex interrelations between different spontaneum). Pakistan J. Weed Sci. Res., 13: 99-112. characteristics of plant interference for resources and 5. Cheneya, W.R. and O.M. Knudson, 1988. allelopathic interaction [12]. Germination of Azadirachta indica seeds enhanced These bioassays were conducted without addition of by endocarp removal. International Tree Crops J., any nutrient. The necessary nutrients for initial seedling 7: 143-146. growth were provided from seed reserved. Light is also 6. Isman, M.B., O. Koul, A. Luczynski and J. Kaminisky, not essential in the seedling stage. Therefore, the 1990. Insecticidal and antifeedant bioactivities of inhibition by the extract on seed germination and growth neem oils and their relationship to azadirachtin of test plant species might be due to the presence of content. J. Agric. Food Chem., 38: 1406-1411. allelopathic substances in neem extract. The inhibitory 7. Sankaram, A.V.B., 1987. Chemistry, biological activity effect might be occurred through a variety of mechanisms and utilization aspects of some promising neem like reduced mitotic activity in roots and shoots, reduced extractives. In the Proceedings of the Third rate of ion uptake, inhibition of photosynthesis, International Neem Conference, Nairobi, Kenya, respiration and enzyme action [13]. pp: 127-148. 8. Al-Charchafchi, F., I. Al-Nabhani, H. Al-Kharousi, CONCLUSION F. Al-Quraini and A. Al-Hanai, 2007. Effect of aqueous extract of Azadirachta indica (neem) leaves The present research indicates that aqueous on germination and seedling growth of Vigna methanol extract of neem leaves exhibits strong radiata L. Pakistan J. Biol. Sci., 10: 3885-3889. phytotoxicity and possesses the growth inhibitory ability 9. Ashrafi, Z.Y., A. Rahnavard, S. Sadeghi, of weeds including noxious paddy weeds barnyardgrass H.M. Alizade and H.R. Mashhadi, 2008. Study of the and E. colonum. These results suggest that neem could allelopathic potential of extracts of Azadirachta be one of the useful natural resources for developing indica (neem). Online J. Biol. Sci., 8: 57-61. bioherbicide for weed management. Further research to 10. Nishida, N., S. Tamotsu, N. Nagata, C. Saito and identify growth inhibitory substances from aqueous A. Sakai, 2005. Allelopathic effects of volatile methanol extract of neem is underway. The identification monoterpenoids produced by Salvia leucophylla: of these substances might provide chemical basis for the Inhibition of cell proliferation and DNA synthesis in development of bio-herbicides for environmentally the root apical meristem of Brassica campestris friendly sustainable agricultural systems. seedlings. J. Chem. Ecol., 31: 1187-1203.

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11. Rietjens, I.M. and G.M. Alink, 2003. Nutrition and 13. Rice, E.L., 1974. Allelopathy. Academic Press, health-toxic substances in food. Ned Tijdschr New York, pp: 353. Geneeskd, 147: 2365-2370. 12. Furest, E.P. and A.R. Putnam, 1983. Separating the competitive and allelopathic components of interference: theoretical principles. J. Chem. Ecol., 8: 937-944.

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