NEMATICIDAL EFFECTS OF LEUCAENA LEUCOCEPHALA AND GLIRICIDIA SEPIUM EXTRACTS ON MELOIDOGYNE INCOGNITA INFECTING OKRA

Two separate but identical greenhouse trials were conducted to investigate the effects of extracts of leaves and roots of Leucaena leucocephala and Gliricidia sepium each at 80,000 mg / kg and 40,000 mg / kg on Meloidogyne incognita on okra. Each potted nine-day old okra seedling was inoculated with 3,000 fresh eggs of M. incognita and 5ml of each of the extracts was added simultaneously around the roots of the seedling. Treatment of okra plant with L. leucocephala and G. sepium extracts resulted in reduced nematode population, reduced galling, reduced nematode reproduction rate and enhanced fruit weight. Chemical analysis revealed that G. sepium leaves contained phenolic compound and carboxylic acid, while the roots showed the presence of aromatic amide, phenolic compound and carboxylic acid. L. leucocephala leaves showed the presence of phenolic compound, aromatic amide and carboxylic acid, while the roots showed the presence of phenolic compound and carboxylic acid. The results of this study suggest that leaf and root extracts of L. leucocephala and G. sepium at the rate of 40,000 mg / kg could be useful in root knot nematode management in vegetable beds.


I n t r o d u c t i o n
Okra, Abelmoschus esculentus (L) Moench is one of the most important vegetables in Nigeria.It is a tropical plant, which grows best in warm climate.It is available all year round, with a peak during summer months.The pods grow rapidly being ready for harvest in about 30-60 days (depending on the variety) when grown from seed.They must be picked about four to five days after flowering, when 4-5 inches or so in length before they mature and toughen.The tender unripe seedpods are used as vegetable, and have a unique texture and sweet flavour.The pods, when cut, exude mucilaginous juice that is used to thicken stews.As a result of the mucilage content, the soups made from okra fruits and young leaves blend well with many solid meals as in many homes.The mucilage is also utilized medicinally (S i g m u n d and G u s t a v , 1991).Okra is a good source of Vitamin C, A and B complex as well as iron and calcium.
Plant parasitic nematodes are important pathogens on most food, vegetable, horticultural and fiber crops and without adequate control will cause loss of yield and quality.Approximate yield losses due to plant parasitic nematodes have been estimated to be $ 100 billion worldwide each year (S a s s e r and F r e c k m a n , 1987).Root-knot nematodes (Meloidogyne species) infect almost all types of plants and may cause considerable damage.During surveys, most vegetable crops including okra were found to be infected with M. incognita (K h a n and K h a n , 1994).Nematode management is complicated and difficult and at present chemical control is employed in many crops to maintain their populations below economic threshold levels (E a p e n et al. 2005).Recently, the control of plant parasitic nematodes by using conventional nematicides has declined internationally because of the inherent toxicity of many existing synthetic pesticides to non-target organisms and their persistence in the environment.There is increasing need to find more acceptable alternatives.The potential for nematicidal activity of indigenous plants and their products has been reported by earlier workers ).There may be many more plants however, not yet tested, which could prove to be effective for the management of plant parasitic nematodes.The present study was therefore designed to investigate nematicidal potential of leaf and root extracts of Leucaena leucocephala and Gliricidia sepium against Meloidogyne incognita on okra.

Materials and Method
Greenhouse trials were conducted to evaluate the effects of extracts of leaves and roots of L. leucocephala and G. sepium on M.incognita infecting okra.

Extraction of Meloidogyne incognita eggs
Pure cultures of M. incognita race 2 were maintained on okra cv.LD-88 in a greenhouse and eggs were extracted from infected okra roots as described in an earlier study (A k i n s a n m i and A d e k u n l e , 2003).

Preparation of plant extracts
Ten-grams pieces each of fresh leaves and roots of gliricidia and leuceana were weighed in duplicates and placed in eight reagent bottles; 100ml of distilled water was added into each reagent bottle.The bottles were then placed in a water bath and heated at 60 0 c for 90 minutes.The extracts were allowed to cool and filtered through Whatman No.1 filter paper.The filterate obtained was 100,000mg/kg (stock extract).Serial dilutions were prepared to produce 80,000mg/kg and 40,000 mg/kg extracts.

Assessment of effects of plant extracts on Meloidogyne incognita
Greenhouse studies were conducted in two separate but identical trials.In the first trial, seeds of okra cv-47-4 obtained from National Horticultural Research Institute, Ibadan, Nigeria were sown in 40 five litre plastic pots filled with steamsterilized sandy loam top soil at the rate of one seed per pot.Nine days after planting each of 36 potted seedling was inoculated with 3,000 fresh M. incognita eggs, immediately after egg inoculation extracts of leaves and roots of gliricidia and leucaena at 80,000 mg/kg and 40,000 mg/kg were added to 32 potted seedlings.There were four inoculated control plants to which no extract was added and also four uninoculated control plants.The Experiment was laid out in a Randomized Complete Block Design with ten treatments in four replicates.After eight weeks, the study was terminated.The plants were carefully uprooted, weighed and roots of plants were assessed for galling, nematode egg population on roots of each plant was estimated (A k i n s a n m i and A d e k u n l e , 2003), okra fruits were harvested and weighed and soil nematode population was estimated.
A second trial was conducted with a new set of plastic pots.All procedures and collection of data were as in the first trial without any modifications.

Assessment of root galling
Okra plant in each pot was carefully uprooted eight weeks after planting without damaging their roots.The roots of the plants were assessed for galling using the method described by T a y l o r and S a s s e r (1978) where 0= No galls or egg masses, 1= 1-2 galls or egg masses, 3= 11-30 galls or egg masses, 4= 31-100 galls or egg masses and 5= More than 100 galls or egg masses.

Nematode assay
Soil sample was collected from each of the pots and nematodes were extracted from properly mixed 200 ml soil drawn from the bulk sample collected from each pot.Extraction of nematode was by the method of W h i t e h e a d and H e m m i n g (1965), nematodes were killed by heat and fixed in 4% formaldehyde.Nematodes were counted in Doncaster counting dish under a stereomicroscope (250 x magnifications) and individuals from each sample were further identified under a light microscope (400 x magnification).

Infrared (IR) analysis of leaves and roots of Leucaena leucocephala and Gliricidia sepium.
For Infrared analysis, 0.1g of powders of each of leaves and roots of L. leucocephala and G. sepium were separately used for analysis.Potassium bromide (KBr) disc was prepared by grinding 0.1g of each of the samples with KBr and compressing the whole into transparent disc.The disc was then scanned in a buck scientific 500M IR machine.The IR spectrum of each sample was printed out with the aid of the machine printer (W i l l i a m , 1987).The IR spectrum of each plant material was compared with those of some named compounds in the process of identification of active ingredients.

Statistical analysis
All statistical analysis was performed using the SAS (1985) statistical package while treatment means were partitioned using the Duncan's Multiple Range Test (DMRT) at p= 0.05.

Results and Discussion
In Table 1 is presented the effects of L. leucocephala and G. sepium extracts on number of eggs per okra plant infected with M. incognita.Inoculated control plants had the highest number of eggs per plant and this was significantly higher than number of eggs on roots of other plants.Okra plants treated with L. leucocephala root extract at 80,000 mg/kg produced fruits with higher weights than fruits of other plants except those of plants treated with G. sepium leaf and root extracts.(Table 2).
The results presented in Table 3 indicate that there was no significant difference in fresh weights of okra plants infected with M. incognita irrespective of whether or not they were treated with plant extracts.Inoculated control soil supported a significantly higher nematode density than other soils that were treated with plant extracts (Table 4).Reproduction rate of M. incognita was highest in inoculated control plants, while the least reproduction rate was recorded in okra plants treated with gliricidia leaves at 80,000 mg/kg (Table 5).Roots of inoculated control plants were the most galled.There was no significant difference in root galling of plants treated with different plant extracts (Table 6).Infrared spectra of the samples were recorded using KBr pellet technique.The diagnostic peaks of the spectra were interpreted using W i l l i a m (1987) method.G. sepium leaves contained phenolic compounds and carboxylic acid, G. sepium roots showed the presence of aromatic amide, phenolic compounds and carboxylic acid.L. leucocephala leaves showed the presence of phenolic compounds, aromatic amide and carboxylic acid while L. leucocephala roots showed the presence of phenolic compounds and carboxylic acid (Figures 1, 2   Treatment of nematode -infected okra plants with root and leaf extracts of G. sepium and L. leucocephala resulted in a reduction in M. incognita population, reduced root galling which implied reduced pathogenicity of the nematode, reduced nematode reproduction as well as an increase in fruit weight.The extracts of the two plants used in the current study were equally effective at 40,000 mg/kg and 80,000 mg/kg in the management of M. incognita on okra.Similar findings were reported by A k h t a r (1993) on the utilization of plant-origin waste materials for the control of plant-parasitic nematodes.He studied nematicidal properties of several waste materials of plant origin in soil infested with plantparasitic nematodes.Among the materials, press mud, vegetable-and fruitprocessing waste and tobacco wastes were the most effective in reducing the incidence of root knot and development of plant parasitic nematodes on tomato.However amendments to soil with spent tea, wheat straw, paddy husk, paddy straw, sugarcane trash, domestic garbage, dead vegetation and pigeon pea stubble were also found to be beneficial in nematode control.He further asserted that as a consequence of nematode control, plant growth improved, with a few exceptions where higher levels (5% (w/w)) of waste materials were phytotoxic.In the same vein, A d e k u n l e and F a w o l e (2003) reported that application of water extracts of neem leaves, siam weed leaves and roots at 20,000 mg/kg and 40,000 mg/kg or carbofuran at 1.5 kg a.i/ha and 2.5 kg a.i/ha to potted tomato plants, delayed development and consequently reduced population of M. incognita evidenced by a prolonged generation time of the nematode.Same treatments were said to reduce number of nematode egg masses and eggs as well root galling of tomato plants.In another study with plant leaf extracts, L i l a n e et al. (1998) reported that direct exposure of Aphelenchoides saprophilus to leaf extracts of Cassiope tetragona, Empetrum hermaphroditum and Betula pubescens spp.turtuosa in agar cultures resulted in a lower reproduction rate of the nematode.Also, leaf extract treatment in potted soil altered nematode species composition and dominance structure and generally reduced species number and maturity index.Nematode population density was reported to be adversely affected after application of Betula extracts.Our findings are also corroborated by those of A k h t a r and F a r z a n a (1996) who reported that standard extracts of roots and shoots of Datura arborea, D. stramonium, Hyoscyamus albus, H. muticus, Solanum incanum, S. indicum and S. nigrum exhibited 100 % mortality of M. incognita, with less effects of root extracts.They also found that larval hatching and nematode mortality were strongly influenced by the concentration of the extract, plant species and the duration of exposure.
In the current study, addition of plant extracts to the soil enhanced the fruit production of okra.The addition of organic matter to soil for improvement of soil fertility and crop yield is a practice almost as old as agriculture.However beneficial effects of organic materials with respect to the suppression of plant pathogens including nematodes have been recognized only in the recent past (A k h t a r and A l a m , 1993).There is a need for utilizing plant extracts and waste materials for supplementing chemical fertilizers as an additional benefit to their pesticidal properties.The active ingredients identified in the plant extracts may have been contributory to their nematicidal activities as recorded in this study.
(H a s e e b et al. 1981; 1984: P r o t and K o r n p r o b s t , 1983; P r a c e r et al., 1987; A d e k u n l e and F a w o l e , 2003).The nematicidal principles of plant origin in the form of substances such as isothiocyanates, thiophenics, glucosides, alkaloids, phenolics, thianins and fatty acids have been identified (G o m m e r s 1973; F a t o k i and F a w o l e , 2000

Fig. 4 .
Fig. 4. -Infrared spectrum of roots of Gliricidia sepium Each value is a mean of four replicates and two trials.Treatments followed by the same alphabets are not significantly different.(P=0.05)by Duncan`s Multiple Range Test(DMRT).
T a b. 2 -Effect of leucaena leucocephala and Griricidia sepium extracts on weight of fruit per okra plant infected with Meloidogyne incognita T a b. 3 -Effect of Leucaena leucocephala and Griricidia sepium extracts on fresh weight of okra plants infected with Meloidogyne incognita T a b. 5 -Effect of Leucaena leucocephala and Griricidia sepium extracts on nematode reproduction rate on okra plant infected with Meloidogyne incognita Each value is a mean of four replicates and two trials.Treatments followed by the same alphabets are not significantly different.(P=0.05) by Duncan`s Multiple Range Test (DMRT) T a b. 6 -Effect of Leucaena leucocephala and Griricidia sepium extracts on gall index of okra infected with Meloidogyne incognita