Biocidal Potential of Indigenous Flora of Soon Valley (Khush- ab, Pakistan) against Helicoverpa armigera Hübner and Spodoptera litura Fabricius (Lepidoptera: Noctuidae) †

Helicoverpa armigera Hübner and Spodoptera litura Fabricius (Lepidoptera: Noctuidae) are destructive pests of agricultural and horticultural crops. Excessive use of synthetic chemicals has created harmful impacts on non-target organisms and environment. This study was aimed to assess the insecticidal potential of acetone extracts of 40 indigenous plant species of Soon valley (Khushab, Punjab, Pakistan) against the 3rd instar larvae of H. armigera and S. litura using leaf-dip bioassay method. Results revealed that maximum mortality of H. armigera larvae was caused by the extracts of Dodonaea viscosa L. (88%), Olea ferruginea Wall. ex Aitch. (69%), Mentha longifolia (L.) Huds. (57%) and Salvia officinalis L. (52.22%), while the extracts of S. officinalis, D. viscosa, O. ferruginea, Sonchus asper (L.) Hill and Nerium indicum Mill. caused significant mortality (i.e. 70 to 90%) of S. litura larvae and exhibited minimum LC50 and LT50 values. These results propose the potential efficiency of indigenous flora against lepidopterous pests and there is a need of further biochemical characterization of these plant extracts.


Introduction
Insect pests have always been an inevitable threat to global food production causing ubiquitously untold damage to different agricultural, horticultural and forest crops. Helicoverpa armigera Hübner and Spodoptera litura Fabricius (Lepidoptera: Noctuidae) are destructive lepidopterous pests [1,2]. Farmers predominantly rely on the synthetic insecticides to combat the infestation of H. armigera and S. litura, which is manifesting many non-target effects such as the development of pesticide resistance, eradication of beneficial fauna including insect parasitoids and predators, and human health hazards [3][4][5]. These ecological drawbacks of synthetic insecticides necessitate looking for novel biorational insect pest management approaches which would be safer and environment-friendly than the synthetic chemical pesticides such as plant-based pesticides [6]. Furthermore, plant-based insecticides usually have low mammalian toxicity and reduced persistency in the environment as compared to conventional synthetic pesticides [7,8].
Native flora of any biogeographical region would contain specific phyto-constitutes potentially effective against indigenous pest species [9]. Soon valley is situated in district Khushab (Punjab, Pakistan) and is highly enriched in flora of ethnomedicinal value [10]. In this study, the insecticidal potential of this local flora (herbs, shrubs and trees) of Soon valley and surrounding salt range of Pakistan was explored against the above mentioned lepidopterous pests.

Collection and Extraction of Plant Materials
Samples of indigenous flora (including trees, shrubs and herbs) were collected from six selected sites (Angah, Dape Shareef, Kenhatti Garden, Khabeki, Khoora and Uchhali) of Soon valley and surrounding Salt Range of district Khushab (Punjab, Pakistan) as detailed in Table 1. Collected plants were identified up to species level and were extracted by the Soxhlet extractor (DH.WHM-12393, Daihan Scientific, South Korea) using pure acetone as extraction solvent using 1:10 plant material: acetone ratio. Pure botanical extract obtained from each plant sample was stored in 50 mL hermetic dark glass vial and was refrigerated until its downstream utilization in the toxicity bioassays.

. Insect Culture
Larvae of H. armigera and S. litura were manually collected from the fields of berseem (Trifolium alexandrinum L.) and sunflower (Helianthus annuus L.), respectively, and were reared on respective plant leaves in plastic jars under controlled lab conditions at 27±3°C temperature, 60±5% relative humidity and 16h L: 8h D photoperiod. Healthy and active 3 rd instar larvae of F3 generation of both insect species were used in all toxicity bioassays.

Insecticidal Bioassays
Leaf-dip bioassay method was used in which leaf discs treated with different plant extract solutions were fixed on 2 mm layer of 1.5% agar in glass 90 mm Petri plates and after release of 10 3 rd instar F3 generation larvae on each leaf disc, these were incubated in a controlled chamber (at 26±2ºC, 60±5% RH and 16:8 L: D photoperiod). Three to six independent replicates were maintained for each treatment. Data regarding mortality of exposed larvae was recorded at 24, 48 and 72 h post-treatment. Moribund insects, which did not show any movement upon touching with camel hair brush, were considered as dead.

Statistical Analysis
Statistical interpretation of data was done using software Statistix 8.1® (Analytical Software, Tallahassee, Florida). Apart from graphical presentation, mortality data were analyzed by factorial analysis of variance (ANOVA) taking botanical solutions and time intervals as factors. Treatment means were compared using honestly significant different (HSD) test at 95% probability level (p ≤ 0.05). Median lethal concentration (LC50) and median lethal time (LT50) were calculated by probit analysis using IBM SPSS® (Version 20) statistics regression software. Prior to probit analysis, mortality was corrected using Abbott's formula [11].

Results
Results of preliminary screening bioassay revealed that some botanical extracts caused significant mortality of 3rd instar larvae of H. armigera (p ≤ 0.05). Maximum larval mortality was observed in case of D. viscosa (88%), O. ferruginea (69%), M. longifolia (58%) and S. officinalis (52%). Second bioassay conducted with 10 most effective botanical extracts recorded from the first bioassay revealed a differential response of S. litura larvae against different plant extracts. According to results, all plant extracts exhibited considerable mortality of S. litura larvae and this mortality response was concentration and exposure time dependent as it increased along with the increase of concentration of botanicals and exposure time (

Conclusions
Based on overall study results, it is concluded that the extracts of S. officinalis, D. viscosa, O. ferruginea, S. asper and N. indicum exhibited significant toxicity potential against both lepidopterous pests. These findings suggest the incorporation of these plants extracts in future management of H. armigera, S. litura and other lepidopterous pests. Nevertheless, the biochemical characterization of these plant extracts in order to find out their bioactive constituents responsible for the observed larval mortality and the laboratory and field evaluation of these plant extracts against natural enemies (insect predators and parasitoids) constitute important future perspectives of this research work. Institutional Review Board Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.