Toxicity and lethal effects of herbaceous plant crude extracts against

Background: Tobacco caterpillar, Spodoptera litura, attacks various cultivated plants and causes significant yield losses. In this study, an eco-friendly approach like using botanical insecticides was adopted to examine the toxicological effect of six herbaceous plants (Phyllanthus niruri, Cyperus rotundus, Euphorbia hirta, Boerhavia diffusa, Parthenium hysterophorus and Cynodon dactylon) against S. litura. Results: Six herbaceous plants crude extract showed a definite level of toxicity against S. litura larvae (3rd instar) using the surface diet contamination method. Among tested herbaceous plants crude extract at 5 μg/ml concentration, C. dactylon showed significant high larval mortality (75%) against S. litura larvae compared to P. niruri (39%), C. rotundus (36%), P. hysterophorus (26%), B. diffusa (22%) and E. hirta (22%) treatments. A cent percent S. litura larval mortality was achieved at lower concentration from crude extract of C. dactylon (11.2 μg/ml) compared to E. hirta (18.6 μg/ml), B. diffusa (24.39 μg/ml), P. hysterophorus (31.4 μg/ml) crude extracts. The lethal concentration (LC50) of C. dactylon, P. hysterophorus, C. rotundus, P. niruri, B. diffusa and E. hirta crude extracts was estimated to be 1.45, 4.09, 5.74, 6.75, 10.92 and 13.62 μg/ml, respectively. Conclusion: The study results suggested that C. dactylon crude extract possesses the potential to reduce the S. litura damage on crops as a natural alternative to the chemical insecticides with no toxicity to beneficial species.


Background
Insect pest damage is one of the major reasons for enormous field crop losses. Tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), is a serious polyphagous insect pest that damages around 115 plant species (Atwal & Dhaliwal, 2009). Out of 115 globally recorded host plants of S. litura, 60 are known from India (Garad et al., 1984). During 2008-09, an outbreak of S. litura on soybean in Rajasthan and Maharashtra states of India caused losses up to 64 million and 300 million USD, respectively (Dhaliwal & Koul, 2010). To prevent crop losses, the application of chemical insecticides is the most adopted pest management strategy; however, improper use of insecticides in many nations results in insecticides resistance (Vasantha-Srinivasan et al., 2017). Insecticide pest resistance to different synthetic insecticides is a growing problem, and field-level resistance of S. litura against insecticides was reported by Gandhi et al. (2016) and Senthil-Nathan (2013). Present limitations associated with the insecticides necessitated for searching selective and eco-friendly insecticides.
Botanical insecticides are instrumental in the discovery and development of synthetic chemical products. Thus, continued discovery and development of botanical-based products exemplify the direction for designing new and better pesticides (Senthil-Nathan, 2013). Phyllanthus niruri mostly found in the tropical and subtropical regions has a long history in the traditional medicinal system to prevent or treat various diseases (Bagalkotkar et al., 2006). Cyperus rotundus spread in temperate, tropical and sub-tropical regions of the world (Nagarajan et al., 2015), and its essential oil affects the insect pest behavior (Zandi-Sohani & Ramezani, 2015). Euphorbia hirta extracts showed a broad range of biological properties, including antimicrobial, antifungal, anti-inflammatory, antioxidant, anticancer and antidiabetic activities (Almosnid et al., 2018). Boerhavia diffusa is used in traditional medicine for the treatment of diabetes, stress, dyspepsia, abdominal pain, inflammation, jaundice, enlargement of spleen, heart diseases, bacterial infections and impotence (Nalamolu et al., 2004). Parthenium hysterophorus is used in the treatment of skin diseases, malaria, rheumatism and headache (Raghu et al., 2014). Cynodon dactylon possesses various medicinal properties such as antiviral, antimicrobial (Dhar et al., 1968), antiinflammatory (Biswas & Mukherjee, 2003) and immunomodulatory activity (Santhi & Annapoorani, 2010). Talukder and Miyata (2002) reported plant-derived materials are safe to natural enemies, compatible with biological control agents for Integrated Pest Management (IPM) and non-toxic to the environment. Plant products can be easily produced by farmers in smallscale industries and are less expensive (Talukder & Howse, 1995). Therefore, a century-old practice of protecting crops using plant derivatives, which are known to resist insect attack (Ewete et al., 1996) is considered in this study and planned an in vitro evaluation of dosedependent toxicity of P. niruri, C. rotundus, E. hirta, B. diffusa, P. hysterophorus and C. dactylon crude extract against S. litura larvae.

Collection and extraction of phytochemicals from herbaceous plants
Six different herbaceous plants were collected (entire plant with intact roots) from legumes growing fields (Fig. 1). A weed scientist from the Indian Institute of Pulses Research, Kanpur (Dr. Chaitanya Prasad Nath) identified the herbs up to species viz., P. niruri, C. rotundus, E. hirta, B. diffusa, P. hysterophorus and C. dactylon. Since these are commonly available and well characterized herbs the specimens were not submitted to the repository. In this experiment, heat energy was used to rupture the cell membrane and extract the thermostable bio-compounds from different herbaceous plants. One kilogram (wet weight) of each herbaceous plant was brought to the laboratory in a collection bag; shade dried for a week to remove the moisture from the plant parts. The moisture free herbaceous plants were grinded to powder and stored in PVC bottles at 28 ± 2 °C. The respective herbaceous plant (P. niruri-10 g, C. rotundus-10 g, E. hirta-10 g, B. diffusa-10 g, P. hysterophorus-10 g and C. dactylon-5 g) was weighed in flat bottom round flask along with 50 ml of organic solvent Ethyl Acetate (EtoAc). The contents of the flask were alternatively heated (flasks placed in a hot water bath at 100 ºC) and agitated (flasks placed in an orbital shaker at 150 rpm for 2 h). The heating and agitating step were repeated thrice. The filtrate (organic phase) was collected by passing the contents in the flasks through Whatmanno.1 filter paper. The filtrate was air-dried (24 h) in a glass petri plate, and the remaining residue was suspended in 5-8 ml of Methanol (MeOH). Subsequently, the filtrate was passed through a 0.45µ syringe filter to remove the macroparticles from the crude extract. The crude extract of each herbaceous plant was stored at 8ºC for the bioassay experiment. Aforesaid protocol for extraction of bioactive compounds from herbaceous plants against lepidopteran insect pests was developed and standardized in this study. Crude extract appearance and percent yield (i.e., estimated by comparing mass of crude extract to the mass of fresh material) of individual herbaceous plants are presented in Table 1.

Insect rearing
Test insect, Spodoptera litura, was procured from live insect repository of National Bureau of Agricultural Important Insects, Bengaluru, India, that was reared according to Bapatla et al. (2021). Obtained S. litura pupae were placed in a glass container for adult emergence. Emerged adult moths were released in a glass container (15 cm diameter and 25 cm height) for mating, which was provided with paper sheets for oviposition and cotton swab with 20% honey solution as a source of food to adult moths. From oviposition within 3-4 days, eggs were hatched to neonates that are reared on tender castor leaves for initial 12 days. Subsequently, larvae were individually reared in 12 well insect culture plates on the semi-synthetic diet (chickpea flour-100 g, methyl-p-hydroxybenzoate sodium salt-2 g, 2,4-hexadecenoic acid-1 g, yeast extract powder-10 g, agaragar-12.75 g, L-Ascorbic acid-3.25 g, multivitamin-2 capsules, vitamin E-2 capsules, Streptomycin-0.25 g, 10%, formaldehyde-5 ml, distilled water-780 ml) until pupation (14-16 days) (Babu et al., 2014). Within 10-12 days adults emerged from the pupa and the biological life cycle (35-40 days) was continued. Test insect was reared in an incubator at 28 ± 1 °C temperature, 70 ± 2% relative humidity and 14:10 light and dark period for six generations in the laboratory and, further generations were used in bioassay experiments.

Toxicity of herbaceous plant crude extracts
Oral toxicity of the crude extracts of herbaceous plants was evaluated against third instar larvae of S. litura using the surface contamination method (Micheal et al., 1997). Initially, a semi-synthetic diet was prepared and poured (3 ml) into each well of 12 well insect rearing plates. Later, 100 µl of crude extract from each herbaceous plant was applied to the diet surface (3.8cm 2 ) in each well and swirled the plate to ensure a uniform spread of the extract over the diet. The surface of the diet was allowed to dry in an aseptic condition for three hours to evaporate the solvent residue. Simultaneously, the diet surface was also treated with distilled water (control), MeOH (negative control) and Flubendiamide 39.35SC (positive control) separately. On to the treated surface of the diet, healthy (active), equal-sized (3rd instar) and pre-starved (2 h) test insect larvae were released individually. Each treatment was replicated thrice with ten larvae per replication. The same set of experiments was repeated in three episodes. Larval mortality was taken on every day for three days. Larvae showing moribund symptoms were also considered dead larvae. The number of dead larvae on 72 h after treatment were considered as final larval mortality and presented as percent larval mortality. The experiment was conducted at 28ºC ± 2ºC and 70 ± 2% temperature and relative humidity, respectively.

Lethal toxicity of herbaceous plant extracts
To find out the toxic concentration of each herbaceous plant extract a range-finding test was conducted (at which 20-80% larval mortality can be achieved). For this initially, 5 μg/ml concentration of all herbaceous plant extracts was tested and observed larval mortality. Based  7.27, 11.55, 15.83, 20.11, 24.39 μg/ml), P. hysterophorus (1.5, 5.02, 12.56, 25.12, 31.4 μg/ml) and C. dactylon (0.6, 2.01, 5.04, 10.08, 11.2 μg/ml) extracts against S. litura larvae, respectively. Each treatment was replicated thrice with ten larvae per replication. Larval mortality was taken on every day for three days, and after 72 h final larval mortality was considered.

Statistical analysis
Descriptive statistics showed percent larval mortality data was positively skewed (1.434). Therefore, the data were transformed (log 10 ) to reduce the skewness to 0.903, i.e., within the range (from − 1 to + 1). The transformed data were used for conducting a One way analysis of variance (ANOVA) to observe the significant differences (p ≤ 0.05) between treatments using SPSS software ver.25. Percent larval mortality data in Table 2 were shown in mean ± standard deviation format. The mean of ranks for percent larval mortality attained by crude extracts, water and methanol were compared using Tukey's HSD post hoc test. To estimate the lethal concentrations of crude extracts from herbaceous plants, probit analysis was carried out using SPSS software ver.25 (IBM Corp., 2017).
niruri, E. hirta and C. dactylon was estimated to be 20, 15, 20, 55, 35 and 15 μg/ml by dissolving the semisolid or gum type crude extract with MeOH, respectively. Crude extracts from P. niruri and C. rotundus are olive in color, whereas E. hirta, B. diffusa, P. hysterophorus and C. dactylon extracts were olive green, olive-brown, dark green and bluish green colors, respectively (Table 1).

Conclusion
The crude extracts are easy to prepare and handle in comparison to commercially available chemical insecticides. The present investigation showed the amicable solution for the management of S. litura using C. dactylon crude extract. Further, the development of a stable and consistent product from C. dactylon crude extract may provide a low cost and eco-friendly approach to manage the S. litura.