In-vitro Toxicity Evaluation of some Phytoextracts against Mealybug Drosicha mangiferae (Hemiptera: Pseudococcidae) Infesting Citrus Orchards in Pakistan

Citrus is an important fruit crop all over the world including Pakistan. It is being cultivated on approximately 8 million hectares with an annual citrus production of about 122 million metric tonnes (FAO, 2016). Mandarins (Citrus reticulata cv. feutrill’s early and kinnow) and sweet oranges (Citrus sinensis cv. blood red and mosambi) are the most widely cultivated and appraised cultivars of citrus. Pakistan ranks among top citrus producing and exporting countries with an average area and production of about 206,569 hectares and 2.36 million metric tonnes, respectively (GoP, 2017). Sweet oranges, kinnow mandarins, grapefruits and lime are major citrus cultivars of the country. Mandarins (C. reticulata) constitute about 80% of citrus production in Pakistan. The province of Punjab and particularly district Sargodha are the production hubs of citrus in Pakistan sharing approximately 95 and 70% of total citrus production of the country, respectively (Ahmad et al., 2018).

Although Pakistan is famous worldwide for its best production of kinnow mandarins, per unit area production is far behind other citrus producing countries. Incidence of insect pests and diseases is one of the major reasons for citrus decline in Pakistan (Mahmood et al., 2014). Under agro-climatic conditions of district Sargodha (Punjab, Pakistan), canker, slow decline, anthracnose and greening are the major citrus diseases (Anjum and Javaid, 2005), while major insect pests attacking citrus plants include fruit flies (Bactocera dorsalis and B. minax), leafminers (Phyllocnistis citrella), psyllids (Diaphorina citri) and mealybugs (Drosicha mangiferae and Planococcus citri) (Tahir et al., 2015). Among these, mealybugs (Hemiptera: Pseudococcidae) are appearing as regular sucking pests of citrus orchards since last decade. Moreover, a recent survey in district Sargodha has found that among mealybug species infesting citrus plants, Drosicha mangiferae is the most dominant and prevalent species incurring substantial loss to citrus production both in terms of quantity and quality (Tahir et al., 2015).

Table I.- Different botanical extracts evaluated under laboratory conditions against 2nd instar nymphs of mealybug Drosicha mangiferae Green.

D. mangiferae is one of the most economic exotic insect pests of a wide range of agricultural and horticultural crops in South East Asia including Pakistan. Although it is predominantly a pest of mango orchards but for last few decades, it has attained the status of major pest of citrus orchards in the district Sargodha (Afzal et al., 2018). Every year, this mealybug species infests and incurs considerable qualitative and quantitative loss to indigenous citrus crop (Franco et al., 2004; Arshad et al., 2015). These mealybugs are considered as ‘hard-to-kill’ pests because these are difficult to eradicate with synthetic insecticides and have least absorption of pesticides due to presence of waxy mealy powder on dorsal surface of body (Chaudhari, 2012). Usually, there is no effective and operative chemical control option available against D. mangiferae mealybugs infesting different horticultural crops including citrus (Tanwar et al., 2007). Farmers utilize various synthetic insecticides to eradicate D. mangiferae infestations and often use over-dose rates (often double or triple) because of unsatisfactory and partial control of D. mangiferae (Mani and Shivaraju, 2016). Most of these pesticides are highly persistent and cause problems of pesticide residues and environmental contamination (Edwards, 2013).

Therefore, it is imperative to seek out new control tactics for mealybug control on citrus plants which would be more biorational and environment-friendly than conventional synthetic chemicals. In this regard, plant-derived insecticidal compounds appear as one of the biorational pest control potions. For instance, botanical extracts of many plants have been demonstrated to effectively control various phytophagous insect pests including mealybugs (Regnault-Roger, 1997; Prishanthini and Vinobaba, 2014; Lanjar et al., 2015; Badshah et al., 2017; Khan et al., 2019). Keeping in view the above cited situation, the present study was aimed to screen out some indigenous plant extracts and essential oils for their toxicity against D. mangiferae nymphs under laboratory conditions.

Third instar female individuals of D. mangiferae were collected from citrus (C. reticulata cv. kinnow mandarin) orchard (32°08’21”N; 72°40’11”E) located in the vicinity of the College of Agriculture, University of Sargodha. Collection was carried out during the 1st fortnight of January upon emergence of early batches of mealybugs and it was ensured that no pesticidal application was made in the orchard against mealybugs yet. These individuals were brought to the laboratory under cool conditions and were reared at 27±2°C and 65±5% relative humidity in plastic cages (90 x 60 cm) up to F2 generation on the young seedlings of C. reticulata. For bioassays, only healthy and active 2nd instar individuals were utilized.

Toxicity potential of essential oils and botanical extracts of eight indigenous plant species (Table I) was assessed under laboratory conditions against 2nd instar nymphs of D. mangiferae. For this purpose, different plant parts as described in Table I were collected from the vicinity of the College of Agriculture, University of Sargodha and washed with clean tap-water and were shade-dried for about a week at room temperature (26°C) and then were powdered using an electric blender. Soxhlet apparatus (Sigma-Aldrich, Germany) was used for the botanical extracts using 1:10 (w/v) methanol as extraction solvent, while essential oils were extracted by hydro-distillation using clevenger-type apparatus. Excess of extraction solvent was removed from the crude plant extracts using a rotary evaporator (Büchi R-3000; Büchi Laboratoriums-Technik, Flawil, Switzerland) set at 41°C. Plant essential oils and extracts were stored in dark colored hermetic glass vials in the refrigerator at 4°C until their utilization in toxicity bioassays.

Standard twig-dip bioassays were conducted to determine the toxicity of botanical extracts and essential oils against 2nd instar nymphs of D. mangiferae. In brief, unsprayed 5 cm long twig-tips of C. reticulata plants were collected washed with clean tap-water and were dried at room temperature (26°C). Their stems were wrapped in moist cotton plug to ensure their freshness for at least three days. Bioassays were laid out according to their label-recommended dose rates according to CRD design with 5 replications per treatment. Twigs were dipped for 5-10 sec in three different concentrations of essential oils (2.0, 1.0 and 0.5% v/v) and botanical extracts (40, 20 and 10% v/v) and after air-drying for 30 min were transferred to Petri plates (diameter 9 cm). Control treatment included distilled water used for the preparation of botanical concentrations. Using camelhair brushes, ten healthy and active 2nd instar mealybug nymphs were released on treated citrus twigs and Petri plates were incubated at 27±2°C and 65±3% relative humidity in an environment chamber set with 16:8 h light–dark photoperiod. Data regarding the mortality of mealybug individuals were recorded at 12, 24, 48 and 72 h post-treatment.

Statistical analysis of data was carried out using SPSS® version 20.0 (IBM Corp., Armonk, NY, USA). Data regarding percent mortality of mealybugs in response to plant essential oils and methanolic extracts insecticides were subjected to factorial analysis of variance (ANOVA) followed by Fischer’s least significant difference (LSD) test at 5% probability level in order to compare the treatments. Prior to ANOVA, mortality data was corrected using Abbott’s formula. Median lethal concentration (LC50) and median lethal time (LT50) values were calculated by probit regression analysis using POLO-PC®.

Results of toxicity bioassay performed with different concentrations of botanical extracts revealed that all botanical extracts caused significant mortality of mealy individuals (F4, 160 = 36.96; p < 0.001) and this mortality response was concentration and time dependent as it augmented along with the concentration of botanical extracts and with different time exposures (Fig. 1). In addition, the interaction of time and concentration had as well a significant impact on the mortality of mealybugs for all botanical extracts (Supplementary Table I).

At 12 h post-exposure, maximum mortality of mealybugs was exhibited by 40% extract of A. indica (10%), followed by D. viscosa (6.67%) and P. hysterophorus (6.67%), while N. indicum and G. jasminoides caused minimum mortality (Fig. 1). Extracts of A. indica was more effective with maximum mortality (26.67%) against mealybugs according to observation at 24 h post-treatment, followed by G. jasminoides and D. viscosa (13.33%). In the same way, maximum average mortality of mealybugs was exhibited by A. indica (50%) followed by G. jasminoides (46.67%) and P. hysterophorus (26.67%). Similar trend of mortality was recorded for 48 and 72 h time intervals. Minimum mortality was recorded for 10% extracts varying from zero at 12 h post-exposure for N. indicum, D. viscosa and G. jasminoides to 23.33% for A. indica, followed by G. jasminoides observed at 72 h post-treatment (13.33%; Fig. 1). Nevertheless, factorial analysis and LSD test revealed that, on overall basis, extracts of A. indica and G. jasminoides were the most effective and toxic against 2nd instar D. mangiferae nymphs showing significantly higher mortality than other three extracts (Fig. 1).

Moreover, probit analysis corroborated the same trend of toxicity of botanical extracts against mealybugs. According to probit regression, at 48 h post-exposure, A. indica was the most effective followed by P. hysterophorus with LC50 values of 38.20 and 81.80%, respectively. Similarly, the extract of A. indica showed minimum LC50 value (20.00 %) at 72 h post-treatment, followed by the extract of G. jasminoides, N. indicum and P. hysterophorus (Table II). Similar trend was found in case of median lethal time (LT50) values (Table III). For 20% concentrations, minimum LT50 values were recorded for the extracts of A. indica (68.91 h) and N. indicum (92.65 h), while maximum ones were found for the extracts of P. hysterphorus (124.45 h) and D. viscosa (121.30 h). For 40% extracts, A. indica (47.97 h) and G. jasminoides (71.26 h) were most effective with minimum LT50 values followed by N. indicum and D. viscosa (Table III).

According to the results, all essential oils exhibited a significant mortality of mealy individuals (F2, 120 = 6.42; p = 0.002) and this mortality response was concentration and time dependent as it increased along with the concentration of essential oils and exposure time (Fig. 2). In addition, the interaction of time and concentration had as well a significant impact on the mortality of mealybugs for all essential oils (Supplementary Table II).

At 12 and 24 h time intervals, maximum mortality of mealybug individuals was recorded by 2.00% essential oil of D. alba (6.67 and 23.33%, respectively) and S. aromaticum (6.67 and 16.67%, respectively), while minimum mortality was given by 0.50% concentrations ranged from zero for all essential oils at 12 h to 3.33% for S. aromaticum and C. citratus at 24 h (Fig. 2). In the similar way, maximum average mortality of mealybugs was exhibited by D. alba (30.01%), followed by S. aromaticum (23.33%) and C. citratus (16.67%). Similar trend of mortality was recorded for 72 h time interval.

At 72 h, maximum mortality was exhibited by 2.00% extracts of D. alba 63.33%), followed by S. aromaticum (56.67%) and C. citratus (43.33%). Nevertheless, according to factorial analysis and LSD test, on overall basis, the essential oil of D. alba and S. aromaticum were the most effective and toxic against 2nd instar D. mangiferae nymphs showing significantly higher mortality than C. citratus (Fig. 2).

Table II.- Median lethal concentration (LC50) values of different plant extracts and essential oils bioassayed against 2nd instar nymphs of mealybug Drosicha mangiferae Green.

*Since the significance level is less than 0.15, a heterogeneity factor is used in the calculation of confidence limits.

Table III.- Median lethal time (LT50) values of different plant extracts and essential oils bioassayed against 2nd instar nymphs of mealybug Drosicha mangiferae Green.

*Since the significance level is less than 0.150, a heterogeneity factor is used in the calculation of confidence limits.

Probit analysis showed similar trend of toxicity of plant essential oils against mealybugs as depicted by analysis of variance. According to probit analysis, the essential oil of D. alba was the most effective at 48 h, followed by S. aromaticum with LC50 values of 2.16 and 6.31%, respectively, while the essential oil of C. citratus revealed maximum LC50 value (12.25%). At 72 h post-treatment, essential oil of D. alba and S. aromaticum were more toxic (LC50 = 0.80 and 0.90%, respectively) than oil of C. citratus (LC50 = 1.27%; Table II). Similar trend was found in case of median lethal time (LT50) values (Table III). For 1.00% oil concentrations, minimum LT50 values were found for the essential oils of S. aromaticum (102.47 h), while maximum was recorded for the essential oil of D. alba (115.69 h). For 2.0% essential oil concentrations, D. alba essential oil was most effective with minimum LT50 value of 61.30 h, followed by S. aromaticum (68.58 h) and C. citratus (81.34 h; Table III).

D. mangiferae is a damaging pest of different agricultural and horticultural crops including citrus. It has become problematic to control with synthetic insecticides, most probably due to reduced penetration of pesticides and field-acquired resistance (Franco et al., 2004; Sreerag et al., 2016). Therefore, it is imperative to seek out new control tactics for mealybug control on citrus plants which would be more biorational and environment-friendly than conventional synthetic chemicals. The present study was aimed to screen out some plant-based chemicals for their toxicity against D. mangiferae nymphs.

Results revealed a significant effect of botanical extracts and plant essential oils on the mortality of mealybug nymphs as compared to control and this mortality increased along with the treatments (concentration and time). However, no considerable mortality of mealybug nymphs was observed at 12 and 24 h post-treatment for all and even till 48th h for the extracts of D. viscosa (sanatha) and N. indicum (kaner) and for all three essential oils. This delayed toxicity might result from the slow uptake of botanical constituents by the mealybugs as manifested in case of ineffectiveness of chemical insecticides against most of the mealybugs (Mani and Shivaraju, 2016; Sreerag et al., 2016).

Among the tested botanical extracts, A. indica (neem) and G. jasminoides (gardenia) were found to be the most effective ones against 2nd instar D. mangiferae mealybugs. These findings are in conformity with the work of many previous researchers. For instance, seeds and leaves extracts of A. indica have been shown to exhibit excellent insecticidal, repellent and antifeedant properties against a number of insect pest species (Mourier, 1997; Benelli et al., 2017). Likewise, Prishanthini and Vinobaba (2014), Badshah et al. (2017) and Majeed et al. (2018) demonstrated the effectiveness of A. indica extracts against cotton mealybug (Phenococcus solenopsis). Moreover, the methanolic extract of G. jasminoides has been found effective against many sucking pests including Myzus persicae, Tetranychus urticae and Aphis gossypii (Kim et al., 2005; Ahmed and Din, 2009). Similarly, Li and Fang (2010) showed high contact toxicity of petroleum ether extracts of G. jasminoides to 3rd instar nymphs of brown planthopper Nilaparvata lugens causing ≥ 80% mortality.

Among three essential oils tested in this study, D. alba (dhatura) and S. aromaticum (clove bud) were the most effective ones against D. mangiferae nymphs. Many previous researchers have reported the detrimental effects of extracts and essential oils of D. alba on mealybugs (Lanjar et al., 2015), stored grain pests (Ali et al., 2012), aphids (Kuganathan et al., 2008), subterranean termites (Ahmed et al., 2005) and mosquitos (Mehdi et al., 2012). Different alkaloids, particularly tropane alkaloids, are the major bio-constituents of genus Datura plants which are responsible to exert anti-insect effects (Fang, 2009; Monira and Munan, 2012).

Based on the results of this study, it is concluded that although lacking quick knockdown effect, indigenous plant extracts and essential oils can be effectively employed against sucking insect pests such as D. mangiferae. Botanical extracts of A. indica (neem) and G. jasminoides (gardenia) and essential oils of D. alba (dhatura) and S. aromaticum (clove bud) appeared to be the most effective against 2nd instar D. mangiferae individuals with minimum LT50 and LC50 values, hence, are suggested to be incorporated in future biorational management programs for mealybugs, particularly in horticultural and green-house crops. Moreover, the bioactive constituents of these phyto-extracts responsible for the observed mealybug mortality should be focused for their potential characterization in future research.

This laboratory work was financial supported by the research project (No. 6702) funded by the Higher Education Commission of Pakistan under its National Research Program for Universities (NRPU). Authors acknowledge the assistance and help by Prof. Dr. Sohail Ahmed for his valuable comments and proof-reading of the manuscript.

There is supplementary material associated with this article. Access the material online at: http://dx.doi.org/10.17582/journal.pjz/2019.51.5.1815.1822

The authors declare no conflict of interest.