Feeding Deterrent Activity of Melia azedarach Linn . and Phytolacca dodecondra ( L ’ Herit ) Plant Extracts Against Cabbage Flea Beetle , Phyllotreta cruciferae ( Goeze )

Aim of the study was to evaluate feeding deterrent activity of cold and hot water extracts of Melia azedarach and Phytolacca dodecandra plant parts against cabbage flea beetle, Phyllotreta cruciferae. Tap water was used for cold water extraction and 100°C boiled water was used for hot water extraction. Five concentrations (w/v) such as 0.625, 1.25, 2.5, 5 and 10 g, respectively of powder/100 mL of water was used to check feeding deterrent activity by leaf discs method. Feeding deterrent activity of plant extracts was monitored for 24, 48, 72 and 96 h, respectively post exposure period with 24 h interval. In addition, mean number of feeding holes were recorded after 96 h post exposure period. The cabbage leaf disc treated with 10 g/100 mL of plant extracts recorded 100% feeding deterrent activity after 24 h exposure period. After 48 h exposure period, except for Melia mature fruit (96.6%) remaining all the extracts recorded 100% feeding deterrent activity at 10 g/100 mL treatment. After 72 h exposure period, at higher concentration (10 g/100 mL), feeding deterrent activity of cold water extract of Phytolacca leaf (96.6%) was on par with hot water extract of Phytolacca leaf (96.6%) and immature fruit (96.6%). After 96 h exposure period, except for hot water extract of Phytolacca leaves (93.3%), remaining all observed 100% feeding deterrent activity at higher concentration. The mean numbers of feeding holes were decreased in increased concentration of the plant extracts. Irrespective of the concentrations, minimum feeding hole was observed at 10 g/100 mL of cold and hot water extract treatment. These two plant extracts proved to be effective by preventing feeding nature of the insects under laboratory. If these plant extracts may provide similar results under field condition it may be useful for controlling flea beetles damage in cabbage crops.


INTRODUCTION
Cabbage, Brassica oleracea is one of the important commercial vegetable crops growing widely throughout Ethiopia.According to Helen (2007) cabbage contains many essential nutrients including dietary fibers, carbohydrates, sugars, fats, protein, thiamine, riboflavin, niacin, panthothenic acid, vitamin B6, vitamin C, vitamin K, calcium, iron, magnesium, phosphorus, potassium and zinc.These nutritional rich vegetable crops are damaged by flea beetle, Phyllotreta spp. in the early stages of development (Palanisamy and Lamb, 1992) and also transmit bacterial and viral diseases (Grubinger, 2006).The economic damage caused by the flea beetles on crop production to be varied based on population densities.The large populations of the beetles quickly devastate seedlings and also feed on the cotyledons and true leaves.The estimated yield losses of about 10% are common in flea beetles abundant fields (Janet and Denise, 2002).Depending on the climate, two to four generations of flea beetles can be produced over the course of one growing season (Metcalf, 1993).
Chemical pesticides are proved to be effective control strategies but due to their negative consequences searching alternatives from the natural resources is gaining importance in recent times.One of the alternative strategies for environmentally safe pest control program is by using botanical pesticides.Botanical pesticides are traditionally used to control various insect pests and diseases considering as broad spectrum solutions to a particular problem.Botanical treatments are particularly relevant for small scale subsistence farmers (Proctor, 1994).Neem, rotenone, pyrethrins, sabadilla and their mixed formulation are recommended for controlling flea beetles (Ellis et al., 1996;Saxena, 1987).The extracts from several species of the family Asteraceae, Euphorbiaceae, Meliaceae, Anacardiaceae was explored well to determine their potential pesticidal effects (Ramos et al., 2010).
Chinaberry berry, Melia azedarach belongs to the family Meliaceae is a close relative of neem tree and their extracts have been studied extensively for their bio-efficacy against various insect pests.The insecticidal potential was reported to be equivalent to that of neem extracts (Champagne et al., 1989;Lee et al., 1991;Chantal and Efat Abou, 2003) particularly berries is extremely poisonous (Fichtl and Adi, 1994).The leaves and bark extracts are commonly used for medicinal purpose in Ethiopia through traditional and scientific practices (Azene, 1993).Several workers have been reported bio-efficacy of fruits, seeds, leaves and barks extracts against aphids and white flies (Abou-Fakher et al., 2001;Zaki, 2008;Zaki et al., 2008;Birhanu et al., 2011;Gebremariam et al., 2012).However, in Ethiopia literature pertaining to the feeding deterrent effects on flea beetle, Phyllotreta cruciferae is limited.
African soapberry, Phytolacca dodecandra belongs to the family Phytolaccaceae is a perennial climbing plants growing rapidly in Ethiopian highland and produce fruits twice in a year from December to February and June to July (Lemma, 1970;Karunamoorthy et al., 2008).In Ethiopia and Zimbabwe, unripe fruits are reported to be used to control bilharzias transmitting snails (Schemelzer and Gurib Fakim, 2008); butanol extract of berries to be toxic to snail and fish (Stobaeus et al., 1990); useful to control mosquito larvae and housefly (Abate and Fesseha, 1994); strongly toxic to aquatic macroinvertebrates (Karunamoorthi et al., 2008) and inhibits egg hatching of Haemonchus contortus (Hermandez-Villegas et al., 2011).In Ethiopia, there is no published work on bio-efficacy of Phytolacca plant extracts against cabbage flea beetle, Phyllotreta cruciferae.Therefore, feeding deterrent activity of cold and hot water extracts of leaves, immature and mature fruits of Melia azedarach and Phytolacca dodecandra were evaluated against cabbage flea beetle, Phyllotreta cruciferae.

MATERIALS AND METHODS
The experiment was conducted in the biology laboratory, College of Natural and Computational Sciences, University of Gondar, Gondar, Ethiopia from February 2013 to May 2013.
Collection and processing of plant materials: Melia azedarach and Phytolacca dodecandra leaves, immature and mature fruits were collected from Atse Tewodros campus, University of Gondar.The parts of the plants were thoroughly washed and dried under shade to avoid denaturation of bioactive principles.After complete drying, plant parts were powdered individually by using electric blender and fine powder was collected by sieving through kitchen strainer.The powdered materials were used to prepare different concentrations of cold and hot water extracts.

Preparation of concentrations:
Cold and hot water extracts of selected plant parts were prepared by mixing 0.625, 1.25, 2.5, 5 and 10 g, respectively of powder individually in 100 mL of water.The tap water was used for cold water extraction and boiled (100°C) water was used for hot water extraction.The known quantity of plant powder was taken in conical flask; added 100 mL of water and allowed for 12 h continuous shaking in a shaker to get complete homogenized solution.After shaking, extracts were filtered through muslin cloth and added 2 mL of soap solution for emulsification purpose.
Flea beetles collection and maintenance: Cabbage flea beetle, Phyllotreta cruciferae were collected from the cabbage field at Hope of Bridge children village farm, near the University of Gondar.The beetles were collected by using insect collection net; transferred to plastic container and brought to the laboratory along with fresh leaves.The beetles were provided fresh cabbage leaves for feeding.The cabbage leaf petiole was tied cotton soaked with water to prevent early drying.These cultures were maintained throughout experimental period.

Feeding deterrent activity of plant extracts:
Feeding deterrent activity of selected concentrations of the plant extracts were tested by applying on both side of the freshly prepared 3 cm cabbage leaf discs.The treated leaf discs were kept in petriplates and introduced 10 healthy flea beetles for each concentration.To prevent early drying of the leaf discs and to maintain moisture content circular Whatmann no 1 filter paper dipped with water was added to petriplate.The leaf discs treated with 2 mL soap solution diluted with 100 mL of water was considered as control.For each concentration experiment was replicated three times.The number of insects on treated leaves and outside were counted continuously for 24, 48, 72 and 96 h, respectively post exposure period and percentage of feeding deterrent activity was calculated.In addition, after 96 h exposure period, numbers of feeding holes for each leaf disc was recorded and mean number of holes were calculated.

Statistical analysis:
The data collected for each experiment were subjected to statistical analysis to derive mean and standard deviation.The statistical significant difference for cold and hot water extracts within the plant parts and within the concentrations tested were analyzed by two way Analysis of Variance (ANOVA) by using Microsoft office excel 2007 program.The level of significant difference was compared at 5% level (p<0.05).
After 72 h exposure period, feeding deterrent activity of Melia and Phytolacca plant extracts tested against flea beetles were reported in Table 3.At higher concentration (10 g/100 mL), feeding deterrent activity of cold water extract of Phytolacca leaf (96.6%) was on par with hot water extract of Phytolacca leaf (96.6%) and immature fruit (96.6%).At this concentration, remaining all the extracts tested recorded 100% feeding deterrent activity.At 5 g/100 mL level, 100% feeding deterrent activity was recorded in hot water extract of Melia leaves and the remaining showed ≥80% feeding deterrent activity.At 2.5 g/100 mL treatment, maximum deterrent activity of 86.6% was noted in hot water extract of Melia leaves followed by cold water extract of Melia leaves (83.3%) and hot water extract of Melia mature fruit (83.3%).At 1.25 g/100 mL, maximum deterrent activity of 76.6% was recorded in hot water extract of Melia mature fruit extract.The deterrent activity of cold water extract of Melia immature fruit (73.3%) was on par with cold water extract of Phytolacca leaves (73.3%).At lower concentration (0.625 g/100 mL), maximum feeding deterrent activity was recorded in cold water extract of Melia (66.6%) followed by hot water extract of Melia immature fruit (63.3%) and Phytolacca leaves (60%).The two way ANOVA results confirmed statistically significant (p<0.05)difference within the plant extracts (cold water F = 8.95; hot water F = 10.75) and within the concentrations (cold water F = 101.62;hot water = 120.23).The interaction within the samples of cold water (F = 1.28) and hot water extracts (F = 1-39) were statistically not significant (p>0.05).
After 96 h post exposure period, feeding deterrent activity of plant extracts tested against flea beetle was presented in Table 4.At 10 g/100 mL plant extracts treatment, except for hot water extract of Phytolacca leaves (93.3%), remaining all observed 100% feeding deterrent activity.At 5 g/100 mL treatment, 100% deterrent activity was recorded in cold water extract of Phytolacca leaves and immature fruits.The deterrent activity of hot water extract of Melia immature fruit (96.6%) was on par with hot water extract of Melia leaf (96.6%).At 2.5 g/100 mL level, 90% deterrent activity was observed in hot water extract of Melia leaves.The hot water extract of Melia and Phytolacca mature fruit extract, cold water extract of immature fruit of Melia and Phytolacca was observed 86.6% repellent activity.At 1.25 g/100 mL of plant extracts treatment, all recorded ≥70% deterrent activity.At lower concentration (0.625 g/100 mL), maximum feeding deterrent activity (73.3%) was recorded in cold water extract of Melia leaves.The two way ANOVA indicates that cold water extracts within the plants (F = 4.84); within the concentrations (F = 93.09)tested and interaction among the samples (F = 1.93) were statistically significant (p<0.05).In hot water extracts, within the plants parts tested (F = 2.25) and interaction among the samples were statistically not significant (p>0.05).However, within the concentrations (F = 69.60)results were statistically significant (p<0.05).
After 96 h post exposure period, mean number of feeding holes by flea beetles were counted and presented in Table 5.The flea beetles feeding shows typical "shot hole" appearance on leaves.The mean numbers of feeding holes were decreased in increased concentration of the plant extracts tested.Irrespective of concentrations, minimum feeding hole was observed at 10 g/100 mL of cold and hot water extract.At 5 g/100 mL applied leaf disc, minimum feeding hole was recorded in hot water extract of Phytolacca leaves (2.33) followed by cold water extract of Melia mature fruit (2.66) and immature fruit (7.6).Generally, mean number of feeding holes were increased in decreased concentration of the plant extracts applied leaf discs.The two way ANOVA demonstrates statistically significant differences (p<0.05)within the plant extracts (cold water F = 59.86; hot water F = 9.71) and within the concentrations (cold water F = 456.41;hot water F = 263.99)tested and interaction among the samples (cold water F = 14.55; hot water 8.18).

DISCUSSION
Botanicals and their extracts have been used since time immemorial for controlling various agricultural pests in the field and storage by believing multiple mode of action, prevent quick resistance development and may not leave residues.In the present study feeding deterrent activity of various parts of M. azedarach and P. dodecandra plant extracts was varied greatly against flea beetles.Irrespective of concentration, maximum feeding deterrent activity was observed at higher concentration in all the extracts tested.The crude plant extracts may not contain sufficient amount of bioactive principle at lower concentration to prevent the entry of insects in treated leaf disc.Charleston et al. (2005) also reported that feeding deterrent effect of M. azedarach fruit extract against Plutella xylostell larvae was increased at higher doses.Rodriguez and Vendramim (1998) observed repellent action and also malformations in some individuals of Spodoptera frugiperda exposed to M. azedarach extracts.The parts of Melia tree contains some alkaloid and condensed tannins which are able to inhibit development or insect feeding (Mulla and Su, 1999).The present study also agreed with the above reports.In addition, odour of the plant materials on treated leaf disc may be preventing flea beetles to enter on treated environment; as a result most of the time insects are moving around the petriplates thereby percentage of repellent activity was greater at higher concentration.The potential feeding deterrent activity of P. dodecandra was also based on the concentration of plant materials.Nurie et al. (2012) also observed greater immature larval mortality of Culex quinquefasciatus exposed at higher concentration.For example at 1000 ppm concentration of petroleum ether, acetone and benzene extract of P. dodecandra berries were reported 100% larval mortality.The P. dodecandra contains mostly saponin that may be associated with feeding deterrent activity or lemmatoxin, a molluscidal compound isolated from the fruits (Lemma et al., 1972).
The mean number of feeding holes was significantly decreased at higher concentration compared to lower concentration.The bioactive principle in the lower concentration may not be sufficient to deter the feeding rate of the flea beetles.As it is evidenced, at lower concentration most of the insects are entered on treated leaves and started feeding.The part of Melia tree contains limonoids such as meliantriol (Lavie and Jain, 1967), meliacin, meliacarpin (Lee et al., 1991) and meliartenin (Carpinella et al., 2002).Among the limonoids, meliantriol was reported to have strong feeding deterrent properties against desert locust S. gregaria (Kraus et al., 1981) and meliartenin inhibit feeding rate of E. panuelate and S. eridania (Carpinella et al., 2002).The low number of feeding holes at higher concentration may be associated with these limonoids.Minimum feeding holes observed with cold water extract of Phytolacca at higher concentration may be associated with saponin.The detergent nature of the saponin may not allow the insect to feed on treated leaf discs.

CONCLUSION
M. azedarach and P. dodecandra have significant impact on flea beetles by feeding deterrent activities.If these plant extracts provide similar results under field condition it may useful for controlling flea beetles.Because these plants are growing extensively in the study area and it can be easily affordable by small scale farming communities to protect their cabbage crop against flea beetles in an eco-friendly way.

Table 1 :
Percent feeding deterrent activity of plant extracts on P. cruciferae after 24 h exposure period

Table 2 :
Percent feeding deterrent activity of plant extracts on P. cruciferae after 48 h exposure period

Table 3 :
Percent feeding deterrent activity of plant extracts on P. cruciferae after 72 h exposure period

Table 4 :
Percent feeding deterrent activity of plant extracts on P. cruciferae after 96 h exposure period