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Effect of plant extracts on feeding activity of cabbage butterfly, Pieris brassicae

The antifeedant activity of various plant extracts were evaluated against cabbage butterfly under laboratory conditions (range of temperature 27-30°C and RH 75-80%). An experiment was conducted to find out antifeedant activity of leaf extracts prepared in cow urine at 2% and 5% concentrations of various plant species viz., Azadiracta indica, Eupatorium triplinerve, Cannabis sativa, Nerium indicum, Parthenium hysterophorus, Melia azedarach, Lantana camara, Annona squamosa, Datura stramonium, against 3rd instar larvae of P.brassicae. These were tested by using ‘no-choice feeding’ bioassay method. The observation on leaf area consumed was recorded on graph sheets and was used for calculations of other parameters viz., Mean leaf area consumed (cm2), Per cent feeding, Per cent Feeding inhibition, Antifeedant activity (%) and Preference index (C-value).


Introduction
Cabbage, Brassica oleracea var. capitata Linn. is an essential cole crop and commercially produce as a leafy vegetable in India. The leafy vegetables, especially Cole crops, make up a major portion of the diet of humans and are sources of phyto-nutriceuticals: vitamins (C, A, B1, B6, B9, and E), minerals, dietary fiber and phytochemicals (Dias, 2012) [4] . The cabbage butterflies, Pieris brassicae, P. canidia and P. rapae have also been found to be major pests of cabbage and cauliflower in India (Bhatia & verma, 1993;Bhutani & Jotwani, 1984;Firake et al., 2012 andLal, 1975) [6,7,12,13] . In one of the studies, it has been documented P. brassicae and Cabbage leaf webber, Crocidolomia binotalis underlay 69% & 28-51% damages respectively to the Cole crops in India (Rai et al., 2014) [20] . In India, it passes winter in the plain and migrates to hilly regions during summer (Gupta, 1984) [6] . This pest damages all plant stages in cabbage crops, i.e., seedlings, vegetative and flowering stages (Ullah et al., 2016) [27] . Young caterpillars of P. brassicae are gregarious leaf feeders (Hasan & Ansari, 2011) [8] . The larvae of P. brassicae feed on all plant parts including leaves, twigs, fruits and seeds of cabbage and cauliflower (Siraj, 1999) [22] . A single larva can consume up to 74 to 80 cm2 of leaf and causes serious damage to the host plant (Younas, et al., 2004) [30] . Extreme infestations of P. brassicae completely destroy the plant foliage and ultimately kill the plant (Hasan & Ansari, 2010) [7] . Plants are rich sources of natural substances that can be utilized in the development of environmentally safe methods for insect control and are able to synthesize a broad range of different chemical compounds called secondary metabolites and many of them are promising new sources of natural pesticides. Secondary plant compounds found in botanical insecticides have been the recent focus of many investigations. Neem (Azadirachta indica, Juss.) has emerged as an excellent alternative to chemical insecticides for the management of insect-pests and was studied by many researchers. The extracts of Datura aborea (Linn.), Nicotiana tobaccum (Linn.) and Zanthoxylum alatum (Roxb.) showed repellent effect and anti-feedant effect on different insect larvae and their lifecycles (Paul and Sohkhlet, 2012) [17] . Numbers of workers uses the plant materials and cow urine for the control of insect pests of field crops (Geetanjaly and Tiwari, 2014). Botanicals are comparatively safer to the parasitoids like Cotesia glomerata which voraciously parasitizes the P.brassicae caterpillar (Ullah et al., 2016) [27] when compared to synthetic insecticides (Yi et al., 2016) [29] . Botanicals are even less toxic to the developing larvae of parasitoids in the host insects exposed to them. In addition, the majority of botanicals contains a diverse mix of active compounds and thus, do not result in pest resistance (Pavela, 2011) [19] . The present investigation was undertaken is to find effective control for larval stages of P. brassiceae by using plant extracts easily available locally, and with user friendly protocols for the farmers.

Materials and Methods
Maintenance of Culture Culture of cabbage butterfly, P. brassicae L. The culture of P.brassicae (Linn.) was raised in glass jars on fresh leaves of cabbage, Brassica oleracea L. var. capitata. The nucleus culture of the test insect larvae was collected from the Vegetable Research and Demonstration block, Department of Vegetable Science, College of Horticulture, VCSG UUHF, Bharsar and brought to the laboratory, were reared on fresh cabbage leaves till pupation and healthy pupae were procured for the next generation. The culture was maintained at 27ºC and 70 ± 5% RH. The adults obtained from above culture were released in separate glass jars (18x12 cm2) the walls of which were lined with white paper for egg laying. White paper strips were also kept in the jars for egg laying and muslin cloth and strips were checked daily for egg laying and eggs were removed and placed in separate jars on fresh succulent cabbage leaves for hatching. The neonate larvae were reared on fresh cabbage leaves to maintain the test culture of P.brassicae (Linn.). In adulthood, the culture had been covered with muslin cloth and continuously supplemented with 10% honey solution to adults as a food. To get homogenous population and one generation passed larvae were used for the experiment. Panwar and Chibber (2006).

Selection and collection of plant species for the preparation of extracts
The criteria of selection of plant species, having a bioactive compound was based on literature. Desired plants at the peak of its vegetative growth stage were collected from various locations.

Preparation of plant extracts
The test plants parts were collected from different location, whereas, Cow urine was collected from desi breed cow. The plants collected from various families were brought to the laboratory, washed with dechlorinated water, shade dried under room temperature for 7-9 days and then the plant materials were powdered individually using an electric blender. 10gm of each powdered plant material weight separately by using a top separately balance and dissolve in 90 ml of cow urine to get 1:9 w/v Rani et al. (2009) [26] were sieved using a kitchen strainer. The prepared solution were kept for fermentation for 15 days then the extracts filtered by using muslin cloth after that it was kept in refrigerator at 40 C and working solutions of the desired concentrations were prepared afresh prior to application. To prepare 2 and 5 per cent concentrations of plant extracts in cow urine separately, For 2 per cent dissolve 2 ml of stock solution (Plant extract +Cow urine 1:9w/v) in 98 ml of water. For 5 per cent dissolve 5 ml of stock solution (Plant extract +Cow urine 1:9w/v) in 95 ml of water.

Screening of plant extracts for their antifeedant activity against cabbage butterfly, Pieris brassicae L.
The antifeedant activity of various plant extracts were evaluated against cabbage butterfly under laboratory conditions (range of temperature 27-30°C and RH 75-80%). 'No-choice'feeding bioassay following Singh et al. (1995) [23] technique was used for the determination of antifeedant activity. Cabbage leaves were utilized to treat and feed the larvae during the experimentation. Required concentrations were prepared from the stock solution.
The fresh and soft leaves were plucked, thoroughly washed and dried with the help of filter paper and the leaf discs (area= 5×5 cm²) were cut from them. These leaf discs were later dipped in the extracts for approx. 2 minutes and air dried for a while. These leaf discs were kept in the centre of pre sterilized corning glass Petri-dishes (dia. 9cm) containing an inner lining of moist filter paper. All the treatments were replicated three times along with one control. Pre-starved (3 hours) larvae of uniform size were released in each Petri-dish and were allowed to feed until more than 75 per cent leaf discs were eaten away in control. The data on the mean leaf area consumed was plotted and recorded on graph paper in different concentrations.

Feeding inhibitions (%) at 2 per cent concentration
In Table 4

Preference index at 2 per cent concentration
Overall mean preference index in Table 4.2 indicated that none of the plant extract formulations were found to belong extremely antifeedant category. But the preference indices on treated cabbage leaves discs were significantly less. The minimum preference index was found in T3 (Cow urine+Azadiracta indica) 0.58 and next effective treatments with less preference index found in T7 (Cow urine+ Melia azedarach) 0.75, T4 (Cow urine + Parthenium hysterophorus) 0.85, T8 (Cow urine+ Annona spp.) 0.88.

Discussion
Anti-feedant activity of plant extracts against P. brassicae L.

Leaf area consumed (cm2)
Plants extracts prepared in cow urine have a significant effect on feeding behavior of the P. brassicae. In the present investigations Cow urine+Azadiracta indica and Cow urine+ Melia azedarach was found effective in reducing feeding of the P. brassicae. when treated in cabbage leaves discs.  Meshram et al. (1996) also advocated the antifeedant property of Azadiracta indica against the third instar larvae of P. demoleus.

Conclusion
From the analysis of the present findings it was concluded that plants extracts prepared in cow urine showed promising results in terms of lowering the feeding per cent of P.
brassicae. The studies clearly demonstrated that it is possible to reduce the total load of chemical insecticides on the environment. Hence, isolation of the active ingredients responsible for such antifeedant activity could possibly facilitate in new formulations for effective activity at lower concentrations.
Among the different plants extracts, Cow urine+Azadiracta indica and Cow urine+ Melia azedarach were found to be most effective in feeding behavior, growth and development against cabbage butterfly.