Earias vittella management by utilizing obnoxious weeds extracts of Jammu and Kashmir Himalayas, India

All the three agro-ecological zones (sub-tropical, intermediate and temperate) of the Jammu province were surveyed for potential pesticidal plants. Methanol extracts of all the thirteen plants / plant parts and fractions were assessed for insecticidal activity. Eight treatments (T3 –Boerhavia diffusa stem methanolic extract, T6 –Boerhavia diffusa roots methanolic extract, T7 –Boerhavia diffusa roots hexane fraction, T12 –Arisaema flavum stem + leaves methanol extract, T13 –Arisaema flavum stem + leaves hexane fraction, T14 –Arisaema flavum stem + leaves acetone fraction, T15 –Arisaema flavum roots / tubers methanol extract and T17 –Arisaema flavum roots / tubers acetone fraction) recorded cent percent mortality of first instar larvae, within four hours. All the extracts / fractions evaluated against Earias vitella exhibited contact toxicity, and ovicidal action. The growth indices were also affected. Few extracts / fractions were so toxic that they exhibited 100% control. When larvae were fed on extract / fraction treated food for growth indices studies, in many cases larvae died. These extracts/ fractions have great potential to be developed as botanical pesticide. Active pesticidal compounds from few promising plants may be extracted and further studied for their pesticidal activity. The present studies suggest that the plant extract may be fractionated for better results.

Researchers are looking for alternatives to chemical pesticides, with the increasing safety and health concerns. Besides, pyrethroids and neem products, other plant products need to be explored for their pesticidal potential. Plethora of literature is available on pesticidal activities of several plants extracted with different solvents, but very few have been exploited commercially. Though pesticide consumption in India has declined from 75,033 t in terms of technical grades in 1990-91 to 50,583 t in 2011-12 (CRRI), and biopesticides use has increased 66 times in India in 10 years from 123 t in 1994-95 to 8110 t in 2011-12, cancer cases are on a rise. Use of chemical pesticides is implicated to be one of the foremost reasons for such a rise. The present study is an attempt to reduce, the pesticidal load in the environment and ecological system.
All the three agro-ecological zones (sub-tropical, intermediate and temperate) of Jammu region is very rich in floral diversity that can be explored for pesticidal activity. The zones exhibit plants unique to its climatic conditions. Several obnoxious weeds are widespread in the entire Jammu region. They cover the roadsides, pathways and bunds and are difficult to manage otherwise. Adhatoda vasica (Family -Acanthaceae), locally known as Bryanker, has vasicine and vasicinone as its principal active component, which are known antioxidant and a bronchodialator respectively. Achyranthes aspera (Family -Amaranthaceae), commonly known as Chirchita or Apamarga, locally called Purkanda, contains several important saponins and flavonoids (Dey 2011), which are used in incense sticks and dhoop and also as an insecticide for lice control. Boerrhaavia diffusa (Family -Nyctaginaceae), commonly known as Punarnava and locally called It-sit, is rich in alkaloids, steroids and flavones, terpenoids, essential oils, phenolic compounds, rotenoids and phytosterols, which are responsible for its various pharmacological properties . It is being used as Ayurvedic medicine in India since centuries. Moreover, several new phytochemicals are discovered which again validate its medicinal importance. Its leaves and roots extracts are known to contain rotanoids, boerhavone, boerhavisterol, boerhavinone, etc., that possess anticancerous and antinociceptive activities. Its green stalk contains boerhavin and boerhavic acid.
India is the second largest vegetable producing country in the world, next only to China. These vegetable crops are more prone to insect pests and diseases, probably due to their tenderness and softness as compared to other crops (Uma Shankar and Raju 2012). Okra or Bhindi (Abelmoschus esculentus) is attacked by several insect pests from germination till the harvest of crop. Among them, shoot and fruit borer (Earias vittella) is the most important one. They bore into tender shoots, when the crop is in vegetative phase and later bore into fruits, causing considerable yield losses ranging from 22.79-54.04% (Shivalinaswamy et al. 2002) in India. Presently, farmers use insecticides viz., Malathion or Chlorpyriphos to manage this pest, but as okra needs plucking every alternate day, using such harmful insecticides may lead to several health implications.
Keeping all these facts in mind, research was conducted with the objective to assess the various extracts and fractions of few obnoxious weeds against okra shoot and fruit borer (Earias vittella).
-The weed is abundant in the area, covering roadsides, pasturelands, wastelands and is problematic to the local people. -It possess some medicinal properties based on available literature and information gained from local people.
The collected plant material was shade dried and kept in plastic boxes for further use. All the experiments were conducted at Advanced Centre for Rainfed Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology -Jammu, Bari Brahmana (latitude of 32°39" North and longitude of 74°53″ East at an elevation of 332) in Jammu and Kashmir.

Preparation of plant extracts
Weighed quantity of shade dried plant materials were crushed and kept in a round bottom flask. Methanolic extracts of all the collected plants / plant parts were then prepared by following the standard procedure of refluxing and distillation (Reena et al. 2018). The solvent methanol was added to the shade dried plant material, in a volume just enough to immerse the bits. Refluxing was done by fitting the flask with a water condenser and boiling the set using heating mantle for 6 h. The extract was then filtered out of the flask and was concentrated by distillation process. This refluxing and distillation procedure was repeated thrice for the complete extraction of plant material. The methanolic extracts of all the collected plants / plant parts were then prepared by following the standard procedure of refluxing and distillation.

Fractionation process
These methanolic extracts of these potential plants / plant parts were further subjected to fractionation using hexane first, followed by acetone (based on their increasing polarity). The methanol extract was mixed with silica gel (Column chromatography) in a tray and left for 24 h, after which the mixture was transferred to a glass jar. Hexane was poured to fill threefourth of the jar and distilled to obtain the hexane as residue left at the bottom of distillation flask. This process of adding hexane, leaving for 24 h followed by distillation was repeated thrice. After hexane, the same procedure was followed using acetone to obtain their respective fractions. The quantity of acetone fractions obtained from few plant materials was quite less. So, their bioefficacy studies could not be done. Contact toxicity, ovicidal action and growth inhibitory studies were carried out against Earias vittella, for the below listed extracts and fractions (Table 1).

Rearing of test insect, Earias vittella
Mass culture of E. vitella was established by collecting infested okra fruits carrying E. vittella larvae from the fields, following the procedure mentioned by Reena and Singh 2007a. Larvae were reared till pupation in small plastic trays on okra fruit blocks. Pupae formed were transferred to the rearing cages for adult emergence. Sexing of adults was done by critically observing the caudal region of the abdomen and size. Cotton swab dipped in 15% sucrose solution was provided as food to the adults emerged and muslin was kept as oviposition substrate. Egg laden muslin was replaced daily with a fresh one. Neonate larvae hatched were then provided with okra fruit blocks for further multiplication. The following experiments were conducted under laboratory conditions as and when the eggs, larvae or adults were available in sufficient numbers, in addition to maintaining the general culture of E. vittella sidewise. All the experiments were replicated thrice.

Evaluation of treated surface contact toxicity to first instar larvae
Contact toxicity was assessed against freshly hatched (<12 h old) first instar E. vittella larvae using dry film technique of Gupta and Rawlin (1966). 0.5 ml of different concentrations of extracts and fractions prepared in acetone was taken in rimless test tube (20.0 X 2.0 cm) and evaporated by rotating the tube manually to ensure the formation of a uniform dry film on the inner glass surface. Angle of the tubes was adjusted in such a way that the solution covered three-fourth of the inner surface of each tube. Acetone was taken as control. Ten first instar larvae were released and the tubes were plugged with the help of cotton plug. Larvae were allowed to remain in contact with dry films for four hours and mortality counts were taken thereafter. Moribund larvae were also counted as dead.

Estimation of ovicidal activity of extracts and fractions
One-day old E. vittella egg-laden (approximately 50 eggs) okra fruit blocks were dipped in various concentrations of extracts as well as fractions and per cent egg hatching was recorded to evaluate the ovicidal action, as mentioned by

Statistical analysis
All the twenty two treatments were replicated thrice in case of contact and ovicidal studies. While, in growth inhibitory studies, fifteen treatments recorded cent percent mortality of larvae and hence could not be evaluated. Rest seven treatments were replicated thrice. The data were subjected to statistical analysis after suitable transformations using One-Way ANOVA in a Completely Randomized Design, SAS Version 9.2. The critical difference was calculated at 5% level of significance. The difference of two means between treatments exceeding Critical Difference (CD) value is significant (Panse and Sukhatme 1978). The differences in mean are denoted as superscripts of different alphabets.

Results
A total of 10 plant materials were collected from the entire Jammu region (Table 1). Methanol extracts of all the ten plants / plant parts and fractions were assessed for insecticidal activity. The plants / plant parts for which sufficient quantities of hexane or acetone fractions were obtained, were assessed for insecticidal activities against E. vitella. List of extracts and fractions that were prepared and assessed is presented in Table 1. For assessment of insecticidal activity more amount of the fraction was needed, so those fractions were left out for which the quantity of fraction obtained was less.

Contact toxicity
The bioefficacy of these extracts and fractions assessed against Okra shoot and fruit borer, E. vitella by dry film technique is depicted in Table 2. Eight treatments (T3, T6, T7, T12, T13, T14, T15 and T17) recorded cent percent mortality of first instar larvae, within four hours. Eleven treatments exhibited >95% mortality in first instar E.vitella larvae, while in control (acetone), it was only 3.33%. Sixteen treatments exhibited >90% mortality of first instar larvae, thus showing proving the efficacy of these extracts and fractions in killing the early stages of this pest. Among the treatments, T1 (Achyranthes aspera stem methanolic extract) recorded significantly lowest mortality percentage (66.67%) followed by its root extracts T2 (73.33%) and T21 (Verbascum thapsus leaves + stem hexane fraction) -76.67%. Mean mortality percentage as depicted in Fig. 1 clearly shows the efficacy of these extracts and fractions, with T3, T6, T7, T12, T13, T14, T15 and T17 exerting almost cent percent mortality and T22 (control) showing the lowest mortality. Hence, all the plant extracts and fractions exhibited high contact action except Achyranthes aspera stem methanolic extract and Verbascum thapsus leaves + stem hexane fraction, that recorded lower mortalities.

Growth inhibitory action
Growth inhibitory activities of these extracts and fractions in relation to food consumption and utilization were also evaluated against E.vittella (Table 4). All the treatments that exhibited significantly higher larval mortality percentage in contact toxicity tests and ovicidal action, exerted complete kill of the fourth instar larvae within 48 h of its exposure. So, in treatments T3, T4, T5, T6, T7, T8, T10, T11, T12, T13, T14, T15,  T16, T17 and T20, larvae did not survive, hence further  growth inhibitory activities could not be recorded. Rest six  treatments (T1, T2, T9, T18, T19 and T21), adversely affected the growth indices (Fig. 3), as compared to control. The consumption index CI was significantly low in T1 (A. aspera stem methanol extract -15.3953), T2 (A. aspera root methanol extract -12.9147), T9 (M. koengii fruits methanol extract -11.8367) and T18 (C. grandis fruits methanol extract -7.9713), as compared to T21 (Control -21.1077). However, the growth rate GR was at par with control (0.8720) in T1, T2 and T9 (0.8707, 0.8777 and 0.8680 respectively), while in T18, T19 and T21 (0.4127, 0.4787 and 0.4537 respectively). On the contrary, the Efficiency of conversion of ingested food ECI as well as Efficiency of conversion of digested food ECD, were significantly reduced in T19 (C. grandis fruits hexane fraction -1.5697 and 1.5863 respectively) and T21 (V. thapsus leaves + stem hexane fraction -1.1507 and 1.1577 respectively), but was slightly enhanced in other treatments T1, T2, T9 and T18. Approximate digestibility AD / Approximate efficiency AE was however at par with control

Discussion
All the extracts / fractions evaluated against E. vitella exhibited high contact toxicity (< 95%) except A. aspera stem methanol fraction and V. thapsus leaves + stem hexane fraction.
Likewise, the methanolic extracts of B. diffusa roots, A. flavum roots and stem + leaves and B. diffusa stem, that recorded less than 10% larval hatching percentage, proved the efficacy of these extracts as ovicides against E. vitella. The growth indices were also affected. Few extracts / fractions were so toxic that they exhibited 100% control. When larvae were fed on extract / fraction treated food for growth indices studies, in many cases larvae died. So, only those extracts / fractions could be further assessed for growth indices, that exhibited lower toxicities in ovicidal and contact toxicity experiments. The plant leaves were reported for the presence of rotenone (Foster and Duke 1990), which is naturally occurring compound commonly used as an insecticide in vegetable gardens (Betarbet et al. 2000). Plants may provide potential alternatives to currently used insect-control agents because they constitute a rich source of bioactive chemicals (Wink 1993). Corroborating our studies, ethyl acetate extract of seeds of A. aspera showed higher insecticidal and growing inhibition activities against Henosepilachna vigintioctopunctata, in a study conducted by Jeyasankar et al. (2014). The more deleterious effect of A. aspera stem hexane extract as compared to that of A. aspera leaves hexane extract against Aedes aegypti  Fig. 3 Pictorial depiction of growth inhibitory activities of extracts and fractions against fourth instar E. vitella larvae. In growth inhibitory studies, fifteen treatments recorded cent percent mortality of larvae and hence could not be evaluated. The triplicated data of rest seven treatments is depicted as Consumption index (CI), Growth Rate (GR), Efficiency of Conversion of Ingested Food (ECI), Approximate Digestibility (AD) and Efficiency of Conversion of Digested Food (ECD) mean ± SEM, p < 0.05, using One-Way ANOVA in a Completely Randomized Design, SAS Version 9.2. All six treatments (T1, T2, T9, T18, T19 and T21), adversely affected the growth indices, as compared to control. observed by Sharma et al. (2018) substantiates our findings. Larvicidal activity of saponin from A. aspera against A. aegypti and Culex quinquefasciatus (Diptera: Culicidae), have been reported by Bagavan et al. (2008). Ecdysone, an insect moulting hormone has been isolated from roots of B. diffusa (Babita et al. 2011). Deshmukh et al. (1982 have also reported insecticidal activity of the hexane and acetone extract of B. diffusa twigs against Culex p. fatigans and Musca domestica nebulo. The methanol extract of V. thapsus leaves have been reported to possess insecticidal activity against red flour beetle (Tribolium casteneum) and rice weevil (Sitophilus oryzae) (Riaz et al. 2013). In accordance with our study, the larvicidal activity of M. koenigii hexane leaf extract have been reported by Arivoli et al. (2015) against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. M. koenigii oil were also reported to posses insecticidal activity against Spilosoma obliqua and Spodoptera litura (Thodsare et al. 2014). Corroborating our study, the repellent and ovicidal activity of Coccinia indica methanol extract against C. quinquefasciatus, A. aegypti and A. stephensi has been reported by Govindarajan (2011). The tubers of Arisaema tortuosum has been reported to posses insecticidal properties (Kaur et al. 2005), supporting our study, where A. flavum extracts (both tuber and stem + leaves) extract proved highly toxic as ovicide and also has high contact and growth inhibitory action. The insecticidal potential of Arisaema jacquemontii incorporated in the artificial diet at different concentrations, showed insecticidal activity, in accordance with our studies. (Kaur et al. 2006).

Conclusion
These potentially active plant extracts (Boerhavia diffusa root and stem, Achyranthes aspera root, Woodfordia fruticosa root, Diplocyclos palmatus fruit, Nicotiana rustica leaves + stem, Arisaema flavum root / tuber and stem + leaves, Coccinia grandis fruit, Vitex negundo leaves and Verbascum thapsus leaves + stem) may be further evaluated under field conditions and utilized for managing this pest. They can also be further developed into botanical pesticides, in similar lines as neem and exploited commercially. Owing to the broad spectrum activity of botanicals in general, they may be used to effectively manage several pest species.