Extracts of Senna sophera (L.) Roxb ., Syzygium cumuni , and Annona squamosa Linn . as Biopesticides against Army Worm ( Spodoptera exigua ) Larvae

Pests are one of the constraints in agricultural crops, such as the army worm (Spodoptera exigua) , that is considered as one of the most harmful pests in the vegetative phase. In this work, ethanolic leaf extracts of common plants Senna sophera (L.) Roxb ., Syzygium cumuni , and Annona squamosa Linn . in Llanera, Nueva Ecija, Philippines were prepared and the phytochemical constituents were locally found. Direct spraying of the extracts was done to test the effectiveness of the biopesticides. Results showed that the extracts have varying effectivity as biopesticide against Spodoptera exigua and can be related to the chemical constituents present in the extracts. Considering the results from this investigation, the easy availability and effectiveness of these natural pesticides can be developed as alternatives to conventional synthetic pesticides.


INTRODUCTION
The Philippines had experienced an outbreak of onion or beet armyworm, Spodoptera exigua, in 2016. Until now, this remains a pest problem in onion farms in the mainland Luzon that poses a big threat to the agricultural industry of the country 1 . Spodoptera exigua is a polyphagous species that is a known pest to different agricultural products by feeding on leaves and stems, creating holes, and boring holes in fruits. These damages are serious that caused outbreak levels and economic damage in various continents 2 .
To control these pests, farmers and agricultural workers use synthetic pesticides that have negative effects such as pest resistance, resurgence and the emergence of secondary pests 3 . Reports on the resistance of these organisms to various insecticides have been published 4 . Among these insecticides are tebufenozide, metaflumizone, chlorpyrifos, chlorfluazuron, spinosad, chlorpyrifos, trizophos, methomyl, betasiflutrin, siromazin, carbosufan, thiodikarb, and abamectin. Due to these concerns, studies on natural and environment-friendly alternatives such as plant extracts as biopesticides are in urgent need of development. These greener alternatives eliminate the ecological and health threats posed by chemical pesticides 5 .
In this study, extracts of common plants algarrobilla (Senna sophera (L.) Roxb.), java plum (Syzygium cumuni), and sugar apple (Annona squamosa Linn.) found locally in Llanera, Nueva Ecija, Philippines are explored as biopesticides against Spodoptera exigua. Biopesticides are characterized as naturally-derived materials from animals, plants, and microorganisms that are usually biodegradable in short periods of time. Further, plants are excellent sources of biopesticides that can be attributed to the various secondary metabolites that have various biological activities 6 . Other advantages are reduced risk of pest resistance, lower development costs, higher target specificity, lower environmental persistence, and generally improved compatibility with biological controls 7 . Since ancient times, plant extracts as insecticides have been successfully applied to control insects, fungi, and weeds 8 . Phytochemical constituents of the plant extracts were determined qualitatively and the plant extracts were applied via direct spraying to assess its effectiveness as biopesticides against Spodoptera exigua. An efficient and environmentally-friendly biopesticide for controlling and management of this pest is significant due to the migratory dispersal nature of this species that results in pesticide resistance and unwanted consequences 8 . This investigation is a response to the threats imposed by invasive species that resulted in loss of profit to the agricultural sector.

Sample Collection and Preparation of Extracts
Senna sophera (L.) Roxb., Syzygium cumini, and Annona squamosa leaves were collected from trees in Llanera, Nueva Ecija. The samples were washed, dried and powdered. The larvae of Spodoptera exigua were collected from an onion farm in Bongabon, Nueva Ecija.

Fig. 1. Location sites of Llanera and Bongabon in Nueva Ecija, Philippines where the plant and insect specimens were obtained, respectively
The ethanolic extraction of the plant was carried out by soaking 20 g of the powders of leaves in 200 mL of ethanol for each plant and was allowed to stand for 72 h at room temperature. The extracts were filtered through Whatman filter paper No. 1 and were evaporated in a fume hood for two days. They were transferred into sterile bottles and kept in the refrigerator until further used 8 .

Phytochemical screening
Phytochemical analysis for the qualitative detection of active phytochemical constituents was performed on the three leaves (Senna sophera (L.) Roxb., Syzygium cumini, and Annona squamosa) extract based on the reported protocols 9,10,11 . Alkaloids are detected using a Mayer's test, the glycosides using a Keller-Kiliani test, the terpenoids using a Salkowki test, flavonoids using a 2.0% sodium hydroxide solution, tannins using a ferric chloride test, saponins by a permanent foam appearance, and phenols using a ferric chloride solution. Where n = pest population, T = treated, and Co = control.

Biopesticidal activity of the plant extracts
The LC 50 values are calculated using Probit analysis statistical method 15 .

2.
Biopesticidal activity of the plant extracts against Spodoptera exigua (Table 2).   16 . Phytochemicals such as alkaloids, terpenoids, and flavonoids have been reported to interfere with the respiratory system and nervous system of houseflies 17 . Further, alkaloids and flavonoids are compounds known to inhibit the action of acetylcholinesterase enzyme which is an important enzyme in insects and mammals. Inhibition of acetylcholinesterase causes the accumulation of acetylcholine which in turn results in chaos in the impulse delivery system to the muscular system. All these result in muscle spasms and paralysis to insects 17 . Furthermore, the presence of terpenoids, tannins, and saponins in the plant extracts are recommended to be components of biopesticides due to their effects on the insects' nervous or muscle systems, hormonal balance, reproduction, and respiratory system 18 . Table 2 shows the average percent mortality of Spodoptera exigua after the application of the three plant extracts in different concentrations, versus a control group where in a commercially available pesticide was used. It is evident that the biopesticidal activities significantly increase in increasing concentrations of the plant extracts. The biopesticidal activities of Senna sophera (L.) Roxb., Annona squamosa, and Syzygium cumini can be attributed to the presence of the different phytochemicals such as alkaloids, flavonoids, glycosides, saponins, tannins, and terpenoids that have lethal effects on the pests 19 . These results revealed the efficacy of the plant extracts as pesticides against Spodoptera exigua. High concentrations of the plant extracts are comparable with the mortality caused by the commercially available pesticides. These results agreed on the effectiveness of plant extracts as biopesticides that were applied to other species unde the genus Spodoptera 4,20,21,22,23 .

Spodoptera exigua
In terms of toxicity, the LC 50 of the leaf extract of Senna sophera (L.) Roxb. (Fig. 2) obtained from the calculation is 14899.1495ppm or 1.49%.  Table 1, and it revealed the presence of various secondary metabolites of the tested plant extracts. Various phytochemicals such The calculated toxicity is >1%, which indicates it is less toxic. The LC 50 of the leaf extract of Annona squamosa (Fig. 3) obtained from the calculation is 7454.15ppm or 0.75%. The calculated toxicity is <1%, which indicates toxic. And the LC 50 of the leaf extract of Syzygium cumini (Fig. 4) obtained from the calculation is 26320.37ppm or 2.63%. The calculated toxicity is >1%, which indicates less toxic.

CONCLUSION
The extracts obtained from the solvent extraction procedure for three local plants, Senna sophera (L.) Roxb., Annona squamosa, and Syzygium cumini, contained different phytochemicals that can be attributed to the effectiveness on the mortality of Spodoptera exigua. The effectiveness of the plant extracts increases in increasing concentration of the plant extracts. The toxicity assay shows that Annona squamosa is the toxic extract, the other extract Senna sophera (L.) Roxb and Syzygium cumini are less toxic. Future studies on the extraction of other parts of the plants such as fruits, stems, barks, and roots as sources of phytochemicals with potential biopesticidal activities may be conducted. Application of the plant extracts as biopesticides in actual farm set-up may be done to test the activities of the plant extracts and compare the results with the lab-based assays to be able to improve the biopesticidal properties of the plant extracts. Furthermore, this investigation presented the applicability of these local plants as sources of biodegradable and environment-friendly pesticides as alternatives to synthetic chemicals for pest control and management.