Repellent, Insecticidal, and Oviposition Inhibitory Effects of Nicotiana tabacum, Nicotiana rustica, and Nepeta cataria on the Trialeurodes vaporariorum

INTRODUCTION

Trialeurodes vaporarium (Westwood, 1856) (Homoptera: Aleyrodidae), is a polyphagous pest with great economic importance, high generation number, wide distribution, and global distribution, and is found in most greenhouses, under cover crops, fields, and gardens on many agricultural, horticultural, ornamental and ornamental plants. This pest is very polyphagous (multiple-eating) and feeds on the sap of more than 250 genera and about 600 plant species worldwide with its stinging-sucking mouthparts [1]. The adults and nymphs of this pest feed on the sap of the host plant, but the nymphs feed more intensively and cause more damage. The feeding site on the leaves turns pale, gradually turns yellow, and falls prematurely. In damaged plants, general growth is very slow and the plant becomes weak. This pest completely covers the surface of the plant by secreting honeydew, so that a soot cake grows on the honeydew and the amount of photosynthesis in the plant is reduced. The plants are weakened due to a lack of normal respiration, which reduces the quantity, quality, and marketability of the product. In addition, this pest also causes the transmission of some important plant viruses [2-4].

This pest was first reported in 1856 by Westwood in England and then observed in the United States in 1870, but its origin is in the tropics. Over the past few decades, with the development of greenhouse crops, the prevalence and expansion of the whitefly population as one of the important economic pests has been increasing day by day [5]. Various factors, including a wide host range, multigenerational nature, high reproductive rate, establishment of different stages of this pest on the underside of the leaf, lack of feeding in the puparium stage, abundant honeydew formation on the leaf surface, and resistance to most common chemical pesticides, have led to insufficient effectiveness of pesticides, resulting in excessive use and higher concentrations of pesticides and increased damage to this pest [6]. There are many plant hosts for this pest, the most important of which are field and vegetable plants such as tobacco, tomato, cucumber, eggplant, pepper, and cotton [7-9].

The continuous use of chemical pesticides has created strong selective pressure on the whitefly population, causing resistance, accelerated population growth, and increased fecundity of this pest [10]. The high toxicity of common pesticides for humans, environmental pollution, and the emergence of pest resistance to chemical pesticides have led to many efforts in recent years to introduce low-risk compounds to control plant pests [11]. In the meantime, some plant extracts and essential oils, known as herbal pesticides, have shown good performance as alternatives to chemical pesticides to reduce the adverse effects of chemical compounds [12-14]. The use of plant compounds as pesticides of plant origin is one of the environmentally friendly methods and is an important component of integrated pest management (IPM) for sustainable agriculture and the production of healthy products [15, 16]. Plants have evolved an efficient defense system against most pests, so some plants have become rich sources of compounds with biocidal properties. For example, compounds with repellent, anti-nutritional, growth and reproductive inhibition, anti-ovicidal, ovicidal, and metabolic effects on insect behavior and biology can be mentioned [17-19].

Very limited studies have been conducted on the insecticidal, repellent, and anti-ovicidal effects of some plant extracts and essential oils on Trialeurodes vaporariorum. For example, in 2010, the contact repellent and anti-ovicidal effects of essential oils of Corymbia citriodora Hook, Pogostemon cablin Blanco, and Thymus vulgaris L. were investigated [20]. The repellent and spawning inhibitory effects of aqueous ethanolic and Estonian extracts of several plant extracts against Trialeurodes vaporariorum were investigated. The results showed that the Estonian extracts had no repellent or repellent effects. The aqueous and ethanolic extracts of Raphannus raphanistrum L. and Ambrosia artemisifolia L. had the highest repellent effects with 76% and 72%, respectively, and their repellent effects gradually decreased over time. The ethanolic extract of Piper auritam Kunth had the highest repellent effect with 66%. The highest spawning inhibitory effects were related to the aqueous extract of R. raphanistrum with 76% and P. auritum with 72%, respectively [21].

The general attention and approach to the use of plant compounds for pest control led to the implementation of the current study to evaluate the insecticidal, repellent, and oviposition-inhibiting effects of ethanolic extracts of Nicotiana tabacum, Nicotiana rustica, and Nepeta cataria on adult Trialeurodes vaporarium.

MATERIALS AND METHODS

Collection of the studied plants

After collection, the leaves of Nicotiana tabacum, Nicotiana rustica, and Nepeta cataria plants were first washed superficially and then disinfected with 2% sodium hypochlorite for 5 minutes and then washed three times with distilled water and dried in the shade, away from sunlight and with proper ventilation. Then, the samples were directly powdered in the laboratory and then completely powdered using an electric grinder and passed through a mesh sieve.

Preparation of plant ethanol extract

To prepare the extract, extraction by soaking method was used and ethanol was used as an organic solvent. 100 grams of each plant powder was soaked with 500 ml of 30% ethanol. After stirring under the hood for one hour, the body of the containers was covered with aluminum foil to prevent direct light radiation. The mixture of plant powder and solvent was placed in a shaker at a speed of 100 rpm for 48 hours. The prepared extract was separated from the plant pulp using filter paper and in the next step, it was distilled by a rotary distillation device evaporator at 40 °C and a speed of 100 rpm, then this concentrated extract was placed in an oven at 40 °C to completely remove the remaining solvent and the extracts. The extracts prepared in dark-colored glass jars on which the plant name and extraction date were recorded were stored in a refrigerator at 4 °C until use. These extracts were considered pure extracts and were diluted with distilled water to perform the tests for each extract and used.

Host insect rearing (Trialeurodes vaporarium)

Whitefly rearing was carried out under controlled conditions in an insect breeding room with a temperature of 25 ± 2 °C, a photoperiod of 16 hours of light and 8 hours of darkness, and a relative humidity of 65 ± 5%. For this purpose, pots containing tobacco plants were transferred to wooden cages with dimensions of 80 × 80 × 120 cm, the walls of which were covered with a 10-mesh net. An appropriate number of whiteflies from the tobacco field infected with this pest were collected using a suction aspirator and transferred to the laboratory for identification and breeding. Then, using reliable sources and an identification key, they were identified by an expert and were diagnosed as Trialeurodes vaporarium. Then, 100 of these whiteflies were transferred to each cage. To lay eggs and create the next generations of this pest, adult insects were collected by an aspirator and transferred to pots containing fresh plants, thus providing an appropriate density of whiteflies for subsequent experiments.

Investigation of the insecticidal effect of extracts on Trialeurodes vaporarium adults

Experiments on the contact toxicity effect of extracts on greenhouse whitefly adults were carried out under laboratory conditions. In each experiment, 3 different concentrations (1000, 2000, and 5000 ppm) of each of the plant extracts used were used in 4 replicates. After conducting preliminary experiments, 3 different concentrations of each extract were determined. To conduct the experiments, equal-sized pieces of tobacco leaves were dipped in different concentrations of plant extracts (1000, 2000, and 5000 ppm) using a sampler and after drying on a 1% agar gel surface, they were placed separately in Petri dishes with a diameter of 9 cm and a height of 1 cm. Then, 20 whole whiteflies were transferred to each Petri dish. In the control treatment, only solvent (ethanol) was used. To create ventilation, a round hole with a diameter of 1 cm was made on the lid of each Petri dish and covered with a net to prevent smoke toxicity. The number of dead insects was counted and recorded after 12, 24, and 48 hours and the percentage of lethality was calculated according to the Abbott, 1925 formula. All experiments were carried out in a germinator with a temperature of 25 ± 2 °C, a relative humidity of 65 ± 5%, and a photoperiod of 16 hours of light and 8 hours of darkness [22]. The criterion for identifying dead insects was the absence of reaction in the legs and body of the pest when stimulated by a brush [23].

Investigation of the repellent effect of extracts on Trialeurodes vaporarium adults

The repellent test was performed using the method of Liu and Ho [24]. Thus, in Petri dishes with a diameter of 9 cm and a height of 1 cm, a filter paper with a diameter of 9 cm was divided into two halves. Then, each half of the filter paper was soaked in one ml of a solution of each of the plant extracts with three concentrations of 1000, 2000, and 5000 ppm, and the other half as control was soaked in one ml of solvent (ethanol) and exposed to air for one hour in the room to dry. Then, the two halves of the filter paper were glued together from the bottom with adhesive tape from the cut part and placed inside the Petri dish. Two leaf discs (one treated with the extract and the other with the control) were placed on both sides of a circular piece of moist filter paper 6 cm apart in a Petri dish 9 cm in diameter and 1 cm in height. Also, to eliminate the fumigation effect of the extracts, a circular 2 cm diameter screen was placed on the lid of each Petri dish. Then, 20 adult whiteflies were selected from the greenhouse and placed in the center of the filter paper in each Petri dish. Then, the Petri dishes were kept in a germinator at a temperature of 25 ± 2 °C, a relative humidity of 65 ± 5%, and a photoperiod of 16 hours of light and 8 hours of darkness. After 12, 24, and 48 hours, the number of adult insects on the control and treatment sides were counted and the repellent index was calculated.

Tests to determine the degree of egg-laying inhibition

The effect of ethanolic extracts on the degree of egg-laying inhibition of the greenhouse whitefly was studied on the adult insect of this pest. After immersing filter paper for 10 seconds in concentrations of 1000, 2000, and 5000 ppm of plant extracts and the control treatment with ethanol solvent, the filter papers were then transferred to test containers with a height of 18 cm and a diameter of 8 cm. After 30 minutes and evaporation of the solvent, 20 adult female insects were placed into Petri dishes with a diameter of 9 cm and containing tobacco leaf discs with a diameter of 8 cm. The females were placed on the underside of the leaf discs in the treated and control Petri dishes for 24 hours at a temperature of 25 ± 2 °C and a relative humidity of 65 ± 5%. After this period, every 24 hours for 5 consecutive days, these females were placed on new leaves and the number of eggs laid by them on the underside of the leaves was counted and recorded during this period.

Statistical analysis

This research was conducted as a factorial experiment in a completely randomized design with three factors and four replications. The first factor was plant extract at three levels, the second factor was the concentration at three levels, and the third factor was time at three levels. The data were analyzed by SAS 9.0 software and the LSD (least significant difference) test was utilized to compare the means at the 1% probability level.

RESULTS AND DISCUSSION

Toxicity effect of different concentrations of plant extracts on Trialeurodes vaporarium adult

The results of the analysis of variance of the impact of plant extracts on the percentage of the lethality of Trialeurodes vaporarium at different times of the experiment showed that the effect of plant extracts, concentration, time, the interaction effect of extract × concentration, extract × time, concentration × time, and extract × concentration × time had significant differences at the probability level of one percent (P ≤ 0.01) (Table 1). The average percentage of the lethality of adult insects Trialeurodes vaporarium due to the application of different concentrations of plant extracts after each of the studied times showed a significant difference, and their mean comparison is presented in Table 2. The highest and lowest percentage of the lethality of the extracts after 12 hours were related to Nicotiana rustica extract with a concentration of 5000 ppm at 62.01% and Nepeta cataria extract with a concentration of 1000 ppm at 28.24%, respectively. The highest and lowest percentages of the lethality of the extracts after 24 hours were respectively related to Nicotiana rustica extract with a concentration of 5000 ppm at 79.22% and Nepeta cataria extract with a concentration of 1000 ppm at 33.47%. The highest and lowest percentages of the lethality of the extracts after 48 hours were respectively related to Nicotiana rustica extract with a concentration of 5000 ppm at 95% and Nepeta cataria extract with a concentration of 1000 ppm at 40.05%. In general, in the present study, the highest lethality was observed in the ethanolic extract of Nicotiana rustica, which had 95% lethality at a concentration of 5000 ppm after 48 hours. The lowest lethality was observed in the ethanolic extract of Nepeta cataria with a concentration of 1000 ppm after 12 hours at 28.24%. Based on the findings of the current study, the lethality of the extracts was highest at the highest concentration (5000 ppm) and the longest time (48 hours). In all three plant extracts studied, the rate of adult insect mortality increased with increasing extract concentration (Table 2).

Table 1. Analysis of variance of the effect of plant extracts on the percentage of adult insect mortality of Trialeurodes vaporarium at different times of the experiment.

**: Significant at 1% probability level.

Table 2. Comparison of the average effect of plant extracts on the percentage of adult insect mortality of Trialeurodes vaporarium at various times of the experiment.

Means followed by the same letter in each column are not significantly different at a 1% probability level based on the LSD (Least Significant difference).

One of the most important indicators used to compare the insecticidal activity of essential oils and plant extracts is the concentration that causes 50% mortality in the pest. Nicotiana rustica extract had higher contact toxicity against adult Trialeurodes vaporariorum compared to other extracts studied. The highest toxicity was related to Nicotiana rustica extract with LC₅₀ of 10704 ppm and the lowest toxicity was related to Nepeta cataria extract with LC₅₀ of 13989 ppm. A comparison of the 95% confidence limits of the LC₅₀ values ​​showed that there was a significant difference between the LCs calculated for these three extracts (Table 3).

Table 3. Calculated LC₅₀ values ​​of the contact toxicity effect of plant extracts on adult Trialeurodes vaporariorum.

The 95% confidence interval was determined based on the method of Robertson and Preisler (1992).

Repellent effect of different concentrations of plant extracts on Trialeurodes vaporariorum adults

The results of the repellency test showed that all the extracts studied at different concentrations had a repellent effect on Trialeurodes vaporariorum adults, but their repellency rates were different from each other. The highest and lowest repellency rates were observed in Nicotiana tabacum extract after 48 hours at 90% and Nepeta cataria extract after 12 hours at 22.42%. A Chi-square (X²) test was performed for all three extracts between the numbers of insects attracted to the treatment and the control after 48 hours separately. Table 4 shows the percentage of repellency of methanolic extracts of mountain thyme and oregano on greenhouse whitefly adults. The percentage of repellency increased with increasing extract concentration and exposure duration to the extract. Nicotiana tabacum extract had significantly high repellent power and at the highest concentration (5000 ppm) after 48 hours, it had the highest repellent rate (90%), indicating the high potential of this extract for use as a repellent compound against this pest. The lowest repellent effect was related to Nepeta cataria extract, which at the lowest concentration (1000 ppm) after 12 hours, had only a 22.42% repellent effect. Comparison of the repellent effect of plant extracts at different sampling times showed that these extracts were statistically significantly different from each other at the probability level of one percent (P ≤ 0.01).

Table 4. Percent repellent effect of plant extracts used on adults of Trialeurodes vaporariorum.

Means followed by the same letter in each column are not significantly different at a 1% probability level.

The effect of different concentrations of plant extracts on the adult insects Trialeurodes vaporariorum on the inhibition of egg production

The results of the test of the percentage of inhibition of egg production of different concentrations of plant extracts on the adult insects Trialeurodes vaporariorum showed that the highest inhibition effect was reported in Nicotiana rustica extract with a concentration of 5000 ppm at 84.24% and the lowest inhibition effect was reported in catnip extract with a concentration of 1000 ppm at 14.26% (Table 5). The inhibition of egg production increased with increasing concentration.

Table 5. Percentage inhibition of egg production of plant extracts used on the adult insects Trialeurodes vaporariorum.

Means followed by the same letter in each column are not significantly different at a 1% probability level.