Evaluation of Some Selected Local Phytoextracts Against Wheat Aphid

| Plant-derived insecticides have been focused for biorational pest management because these are relatively quickly biodegradable, cost-effective, less persistent and exhibit low mammalian toxicity than synthetic pesticides. This study assessed the toxicity potential of seven local plant species ( i.e. parthenium Parthenium hysterophorus L., eucalyptus Eucalyptus camaldulensis Dehnh., milkweed Calotropis gigantean (L.) Dryand., bakain Melia azedarach L., neem Azadirachta indica A. Juss., tobacco Nicotiana tabacum L. and thorn apple Datura stramonium L.) against wheat aphid ( Schizaphis graminum Rondani) under laboratory and field conditions. Using foliar spray method, toxicity bioassay was conducted with three different concentrations ( i.e. 250, 500 and 1000 ppm) of methanolic plant extracts under controlled laboratory conditions, while three different concentrations (25, 50 and 75 g/L) of each botanical extract was tested in the field trial. Results showed a significant mortality and suppression of S. graminum individuals at 72 h after treatment in the laboratory and at 10 th day post-treatment in the field. In laboratory bioassay, maximum aphid mortality was caused by the extracts of D. stramonium (92%), A. indica (84%), N. tabacum (78%) and M. azedarach (70%) at 72 h post-treatment at their highest concentration (1000 ppm). Results of field trial showed significant aphid mortality by D . stramonium (78%), A. indica (72%), N. tabacum (68%) and M. azedarach (62%) extracts noted for their highest concentration (75 g/L) at 10 th day after treatment. Based on overall results of the study, the extracts of D. stramonium , A. indica , N. tabacum and M. azedarach are recommended to be considered for biorational management of S. graminum .


Introduction
W heat (Triticum aestivum L.) is the most economical and nutritious staple food around the world (Khan et al., 2009). It is consumed by more than 35% world human population (Khakwani et al., 2012). About 70% of the total population of Pakistan consume wheat grains, therefore it is imperative to improve its yield to feed growing population (FAO, 2019). Pakistan's average wheat yield is much less as compared to other wheat producing countries for which multiple factors are involved including inappropriate sowing schedule, insect pest and weed infestations, use of low yielding varieties and inappropriate use of fertilizers and irrigation water (Khan et al., 2012;Memon et al., 2013;John et al., 2017;Yahya et al., 2017).
Wheat crop in Pakistan is attacked and damaged by several insect pests but among these pests aphids (Hemiptera: Aphididae) are responsible for the foremost economic damage not only by their direct feeding on wheat plants but also by vectoring different plant diseases (Bukvayová et al., 2006;Ali et al., 2015). In Pakistan, the most abundant and prevailing species of wheat aphids include Sitobion avenae Fabricius, Rhopalosiphum padi Linnaeus and Schizaphis graminum Rondani (Shah et al., 2006;Ahmad et al., 2016;Shafique et al., 2016). These aphid species result in 35-40% yield reduction by their direct feeding on plant sap and from 20 to 80% yield reduction indirectly by transmitting some fungal and viral diseases (Carvalho, 2006;Foster et al., 2014). Up to 30% yield loss has been noted in an untreated experimental wheat field by S. graminum infestation (Ahmad et al., 2016).
In Pakistan, different types of synthetic pesticides are being employed for the management of aphid infestations on wheat and other crops (Shafique et al., 2016). Anyhow many species of the aphids got resistant to several of these synthetic insecticides (Foster et al., 2014;Shafique et al., 2016). In case of wheat crop, only a small range of registered synthetic insecticides exist for aphid control which is ultimately resulting in lesser production of wheat (Carvalho, 2006). It is also predicted that ongoing climate changes are also favourable for aphid infestations on wheat crop which could reduce wheat production and impact negatively the world economy (Bezemer et al., 1998;Van der Putten et al., 2010;Simon et al., 2021). All these facts mentioned demand an alternative management strategy against wheat aphids to ensure future food security through sustainable wheat production. Moreover, excessive use of synthetic chemical insecticides has imparted adverse effects on human and animal health as well as on the environment (Duman and Mutlu, 2019;Mamay and Mutlu, 2019;Mutlu et al., 2019;Nazir et al., 2019;Teke and Mutlu, 2021).
Plant-derived pesticides, such as botanical extracts and essential oils, have been a promising biorational strategy for the management of insect pests (Isman, 2020). Botanical pesticides have some advantages over their synthetic counterparts such as these pesticides usually exhibit less mammalian toxicity and environmental persistency and are more environmentfriendly than synthetic insecticides (da Silva et al., 2017;Bedini et al., 2020). Botanical pesticides exert lethal and sublethal effects on insect pests including mortality, repellency, ovipositioning deterrence, ovicidal effects, and reduced fertility and longevity (Isman, 2020). Extracts of many plant species have been evaluated and demonstrated effective against many insect pests including aphids (Nong et al., 2015;Isman, 2020). To this end, this research work was intended to comparatively evaluate the toxicity potential of methanolic extracts of seven indigenous plant species against S. graminum aphids on wheat plants both under laboratory and field conditions.

Insect culture
Collection for mass rearing of wheat aphids (S. graminum) was made from wheat (variety Faisalabad-2008) field (32°08'21''N; 72°40'11''E) near Tehsil Sillanwali, District Sargodha, Punjab, Pakistan. Alate and apterous morphs of S. graminum are usually 1.2 to 2.0 mm long pale green to yellowish green individuals with a median dark green longitudinal line on abdominal notum (Figure 1). This aphid population was reared in the laboratory conditions at 25±2°C of temperature and 55-65% relative humidity under photoperiod of 16:8 L:D hours on the plants of same wheat variety grown in pots at College of Agriculture, University of Sargodha, Sargodha, Punjab, Pakistan. During rearing, old infested plants were replaced with new ones at weekly intervals.

Plants extracts
Seven plant species belonging to five botanical families (Table 1) were selected for the study and their fresh leaves were collected in August 2020 from Sargodha, Punjab, Pakistan. This selection of plant species was made on the basis of their wide availability in district Sargodha and on their literature-based insecticidal properties. For each plant species, one kilogram of healthy leaves was collected, shade-dried and grounded into powder using mortar and pestle in liquid nitrogen. The extraction of plant samples was done following procedure described by Khan et al. (2017). From each sample, five grams of powdered leaves were extracted with 100 mL of 95% methanol by keeping for 2 days in a thermostat water shaker at 35°C temperature. Then, these extracts were cooled down to room temperature (25°C) and were centrifuged for 15 min at 7000 rpm. Then the supernatant was acquired by using Whatman filter paper No. 1 with three repetitions for getting pure supernatant. Later on, this supernatant was passed by a Buchner funnel in the rotary evaporator at 30°C temperature. The process of evaporation of excessive solvent was completed by adjusting the volume of each plant extract to about 100 mL, and got each plant extract in resultant pellet. Each pellet (plant extract) was then mixed in a 1.0% aqueous solution of dimethyl sulfoxide (DMSO) with a final concentration of 50 mg/mL. The final products of all plant extracts were kept at 4°C temperature in air-tight dark glass vials to be used for bioassays.

Laboratory bioassay
The bioassay was carried out against S. graminum under laboratory conditions to assess the efficacy of above mentioned plant extracts. Three different concentrations of each plant extract (i.e. 250, 500 and 1000 ppm) and one control were used as treatment. Seven-day old potted wheat (T. aestivum; variety Faisalabad-2008) plants were used for bioassay. After infesting wheat plants with the test insects, each wheat plant was sprayed with approximately 4 mL of each plant extract concentration using a manual atomizer. Plant extracts were sprayed directly on wheat plants. For the determination of insecticidal efficacy of these plant extracts, data regarding aphid mortality was taken with the help of a 3X magnifying hand lens after 24, 48 and 72 h post-treatment. For all treatments, five replications were maintained. Bioassay was conducted at controlled conditions (i.e. at 25±2°C temperature, 50-60% relative humidity and 16:8 h light: dark photoperiod).

Field experiment
For evaluating the efficacy of these plant extracts against S. graminum, a field trial was conducted at tehsil Sillanwali (Sargodha, Punjab, Pakistan) during the Rabi Season 2020-2021. Three concentrations (i.e. 25, 50 and 75 g/L) of all plant extracts were used along with one control plot. Wheat variety Faisalabad-2008 was sown for the application of these above-mentioned treatments. Plot size was 45 × 100 feet. For each plot, approximately 15 litre solution of each plant extract was sprayed using a Knapsack sprayer which was calibrated before use. Control plots were sprayed with water only. The data for aphid population reduction was noted after 4, 7 and 10 days post-application. For each treatment, three replications/blocks were maintained for each treatment. Uniform agronomic practices were adopted for all experimental plants.

Results and Discussion
Insecticidal effect of plant extracts on S. graminum under laboratory conditions Results of laboratory bioassay revealed that all seven extracts showed a substantial mortality of S. graminum (F = 54.08, p < 0.001;    Table 2).

Efficacy of plant extracts against S. graminum under field conditions
Results of the field trial exhibited that all tested plant extracts showed considerable mortality of S. graminum individuals (F = 96.03, p < 0.001; Table  3). Moreover, factorial ANOVA showed a significant impact of all plant extract concentrations (F = 585.02, p < 0.001), time intervals (F = 753.76, p < 0.001) and of their interaction (F = 120.64, p < 0.001) on mean aphid mortality (Table 3) Over the last few decades, cereal aphids (S. avenae, S. graminum and R. padi) have been very common wheat crop pests and pose a major threat to wheat production in Pakistan (Riaz et al., 2022). The use of eco-friendly insecticides is also a part of an integrated management strategy against wheat aphids for the reduction of economic losses (Shahzad et al., 2013). For quick and efficient minimization of yield losses, several synthetic pesticides have been found effective (Shafique et al., 2016). But the chances for accumulation of toxic residual compounds in wheat grain make it a less desirable remedy for the management of wheat aphid, which is our staple food.  Table 3).
The development of biorational control methods such plant extracts have been a vital aspect of plant protection research. The application of botanical extracts and plant essential oils exhibit potential to control aphids (Iqbal et al., 2011;Farooq et al., 2016). The current research work demonstrated in-vitro and in-situ efficacy of seven local plant extracts against S. graminum. Results of the study have revealed that all plant extracts tested might be effective biopesticides for aphid management. Results of this work are corroborated by those of some previous studies demonstrating the efficacy of various plant extracts against wheat aphid S. graminum and some other species of aphid (Khan et al., 2017;Hafeez et al., 2021). In laboratory bioassay, resulted mortality of S. graminum was found time-and dose-dependent as it increased along with the concentrations of the plant extracts and along with the exposure time. Extracts of D. stramonium,A. indica,N. tabacum,M. azedarach,C. gigantean,E. camaldulensis and P. hysterophorus caused 92,84,78,70,66,60 and 54% and 78,72,68,62,54,48 and 40% mortality at 72 h and 10 days post treatment applications in laboratory and field experiments, respectively. These findings validate the conclusions of Roy et al. (2005) who reported maximum mortality (100%) by some botanical insecticides against S. graminum. Plant extracts are found to be a favourable alternative to conventional synthetic insecticides for the effectiveness against a wide range of agricultural insect pests. The results of a current research study are also in accordance with the results of Sohail et al. (2012) for suppressing the population of aphids with an application of plant extracts. Several experimental studies have reported the effectiveness of A. indica formulation against aphids (Tang et al., 2002;Sallam et al., 2009;Melesse and Singh, 2012 (Gahukar, 2000).
However, the results of our findings are also different from some other similar studies. This could be because of the differential response of the targeted insect pest species against a specific plant extract and the varied toxicity of the plant extracts. Earlier studies have shown that plant extracts of many medicinal plant species have different levels of efficacy against different pest species. Likewise, the determination of the toxicity of seven different phytoextracts against four different insect pests revealed that aphids were highly susceptible to all plant extracts which were tested in the study causing 100 % aphid mortality after 24 h of application (Khan et al., 2017).
In another study, plant extracts from Asteraceae family were tested against cabbage aphid Brevicoryne brassicae and were found non-toxic to predatory beetle Coccinella undecimpunctata and C. carnea (Farag and Ismasil, 1999). Similarly, while using plant extracts of nine Ghanaian plants, it was observed that aphid B.
brassicae was effectively controlled with plant extracts as done by the conventional pesticides e.g. emamectin benzoate, and these plant extracts were proven less harmful to ladybird beetles (Amoabeng et al., 2013). According to another research, it is also believed that products based on plants give effective efficacy against insect pests but they also harm some non-target and beneficial insects including natural enemies and pollinators (Czaja et al., 2015;Vivekanandhan et al., 2018). Furthermore, some field studies showed that plant extracts were effective against aphids but were harmless to natural enemies of aphids (Abdel-Rahman et al., 2019).
It is well-known that anti-insect and toxicological properties of plant-based pesticides are due to the existence of some secondary plant metabolites for example alkaloids, flavonoids, terpenoids and quinines (Isman, 2020). According to an estimation, approximately about 9,000 different flavonoid compounds are present in plants. These compounds play several vital functions in plants such as they reduce digestibility, decrease nutritional value and work as toxins or anti-feeding agents for insects (Mierziak et al., 2014). The flavonoids are usually cytotoxic and effect on the growth, development and behaviour of phytophagous insect pests (Simmonds, 2003).

Conclusions and Recommendations
This study in brief has demonstrated the insecticidal efficacy of seven local plant extracts, particularly of A. indica, D. stramonium and N. tabacum against wheat aphid S. graminum causing 54-92% and 40-78% mortality under laboratory and field conditions, respectively. Therefore, in view of the aforementioned concerns of synthetic insecticides, these plant extracts are recommended to be incorporated in future integrated management of wheat aphid S. graminum as biorational pest control tools.