Aphid Behavior on Amaranthus hybridus L. (Amaranthaceae) Associated with Ocimum spp. (Lamiaceae) as Repellent Plants

Various plant species contain biocidal and/or semiochemical components. These can be used for managing insect pests, in order to reduce the use of synthetic pesticides and to improve the quality of vegetable crops. This study was conducted to assess the effect of repellent plants Ocimum gratissimum L. and Ocimum basilicum L. on aphids Aphis craccivora Koch, Aphis fabae Scopoli and Myzus persicae Sulzer when they are associated with Amaranthus hybridus L. plants. The results have shown that in the two approaches tested— Ocimum sp. plants surrounded by A. hybridus plants and the dual-choice test—the number of aphids on the A. hybridus plant associated with either O. gratissimum or O. basilicum was significantly less significant compared to the A. hybridus alone. This first study on the association between A. hybridus and Ocimum spp. shows that the Ocimum species might be used as an alternative method for controlling aphids in order to avoid the use of synthetic pesticides on Amaranthus . The ability of Ocimum spp. to repel pests can make it an important companion plant for farmers, because those plants can not only be used to control pests, but they can also be harvested, providing a direct economic return.


Introduction
Aphids are pests that cause economically significant crop losses on many vegetable crops. To control these aphid populations, synthetic pesticides are increasingly used, especially in developing countries [1]. However, there is, nowadays, a growing public concern about the harmful effects of pesticides on humans and non-target organisms, in addition to the resistance of many pests to some commonly used insecticides [2,3]. Together, these problems have motivated the development of environmentally friendly alternative means to manage pests. Biological control using natural enemies is one of these alternatives to control crop pests. However, although this form of control strategy can potentially be successful at a low cost [4], it remains difficult to implement by small-scale farmers in developing countries. In this context, cheap, plant-based solutions can play an important role. Using plants with pesticidal properties to protect crops against pests can have advantageous effects for humans, such as reduced environmental degradation and increased food safety [5]. Indeed, botanical extracts have been reported to be effective on pests and pathogens [6,7]. These plants can be used to protect crops by keeping pests away and then avoiding potential damage [8,9]. The biocidal activity of these plants is generally attributed to volatile organic compounds (VOC) that are emitted and can directly affect herbivorous pests due to their toxic, repellent or dissuasive properties [10,11].
Ocimum spp (Lamiaceae), or basils, are important plants with pesticidal effects, whose biocidal effect has been reported mainly on stored-product pests and malaria vectors [12][13][14]. Some studies indicated that their qualities as repellent or companion plants decreased pest abundance on crops. In a semi-field experiment, O. americanum L. essential oil had a repellent effect on Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae) [15]. Testing O. basilicum as a banker plant in greenhouse tomato crops led to fewer recorded pests [16]. Similarly, near to the tomato plants, Ocimum spp plants (O. gratissimum and O. basilicum) or their essential oils (through diffusers) reduced the oviposition of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) [17]. Intercropping of O. basilicum with Gossypium barbadense L. (Malvaceae) was also found to reduce pest abundance [18]. Introducing O. basilicum in Vicia faba L. (Fabaceae) reduced infestation levels of the bean aphid-Aphis fabae Scopoli (Hemiptera: Aphididae) [19]. A repellent effect was also reported on cabbage pests Phyllotreta sinuata Steph. (Coleoptera: Chrysomelidae), Hellula undalis Fabricius (Lepidoptera: Crambidea), Spodoptera litura Fabricius (Lepidoptera: Noctuidae), Spodoptera littoralis Fabricus (Lepidoptera: Noctuidae) and Plutella xylostella (Lepidoptera: Plutellidae) when this crop was intercropped with Ocimum species [9,20]. Ocimum basilicum reduced pest abundance by 23% on Abelmoschus esculentus L. (Malvaceae) compared to the control, and also reduced the use of synthetic pesticides. In an orchard ecosystem, it was reported that planting Ocimum spp between trees can reduce pest levels and also attract natural enemies, including Coccinellidae, Syrphidae, Chrysopidae and Phytoseiidae [21][22][23]. www.videleaf.com The aim of the present study was to evaluate the repellent effect of O. basilicum and O. gratissimum on the aphids (Hemiptera: Aphididae) Aphis craccivora Koch (Cowpea aphid), A. fabae Scop and Myzus persicae Sulzer (Green peach aphid). The experiments were carried out on Amaranthus hybridus L. (Amaranthaceae), one of the most popular vegetables crops in tropical Africa [24] and highly attacked by aphids [25][26][27]. The Ocimum species are also used as vegetables in several West African countries [28]. The outcome of these experiments shows that Ocimum could be used as a companion plant for pest management.

Impact of Ocimum on the Dispersion of Unwinged Aphids
Three treatments were tested for each aphid species: (1) A. hybridus alone (control), (2) Figure 1). Twenty (wingless) adult aphids were transferred to the central plant (release plant) using a fine brush. The plants located at 8 and 12 cm from the central plant were considered as a potential selected plant by aphids. The trays were individually placed in the 45 × 45 × 45 cm net cages (BugDorm, MegaView Science, Taichung, Taiwan). Twelve days later, the number of aphids on the central and selected plants at 8 cm (n = 4) and 12 cm (n = 4) were counted. Six replicates were assessed per treatment and aphid species. The repellent index (RI) was calculated using the following formula adapted from [29]:

Statistical Analysis
For the dispersal test, the aphid populations recorded on the selected (8 and 12 cm) and central (0 cm [31]) was performed and the -SNK.test‖ function (-agricolae package‖; [32]) was used for means comparison. For the Repellent Index, the function -aov‖ was performed on data. A www.videleaf.com probability level lower than 0.05 was considered statistically significant. All statistical tests were performed using R software version 3.6.3 [33].
Binomial proportion tests (equal distribution hypothesized) were used to compare the number of aphids on the control plant (A. hybridus alone) and the associated treatments (Amaranthus versus Ocimum) during the dual-choice test, according to each modality.

Effect of Ocimum Species on the Abundance of Unwinged Aphids and Repellent Activity
Analysis of the interactions between the factors studied on the aphids shows that only -Treatments‖, -Distance‖, the interaction -Treatments: Distance‖ and -Aphids: Treatments: Distance‖ were statistically significant (p < 0.05) ( Table 1). Synthesis of analysis of the deviance (Anova) for fixed factorsplants, treatments and distanceand their interactions on abundance of each aphid species on amaranth plants is available in the supplementary materials (Table S1). Aphid abundance in each treatment is presented in Figure 2.  The repellent indexes of Ocimum plants on aphids are presented in Figure 3. In the presence of basil plants (Ah + Og and Ah + Ob), the repellent index is positive, and close to one, www.videleaf.com irrespective of the distance from the released plant (central plant) and the aphid species. On the other hand, in the absence of the basil plant (control), the repellent index is negative overall, but varied with aphid species. Statistical analysis showed a significant difference between the control and treatments (Ah + Og and Ah + Ob) irrespective of the distance (p < 0.001). The synthesis of analysis of the Ocimum repellent index on aphids at 8 and 12 cm is available in the supplementary materials (Table S2).

Repellent Activity of Ocimum on Winged Aphids
Two hundred aphids were tested for each modality. In general, more than 75% of the aphids responded (Table 2). Aphids were evenly distributed among A. hybridus when tested alone ( Figure  4). In comparison, the number of aphids observed on A. hybridus plants associated with Ocimum plants (O. gratissimum or O. basilicum) was significantly less significant (p < 0.001) (Figure 4). www.videleaf.com

Discussion
Pesticidal plants are generally an environmentally friendly solution and are more sustainable in integrated pest management programs than synthetic pesticides. Plutellidae [9,17,20,21,35]. Other studies have also demonstrated the repellent effect of Ocimum extracts or essential oils on many storage pests [12,36] and human disease vectors [13,37].
Host plant location by insects for feeding or reproduction is affected by their ability to perceive VOCs emitted by these plants [38]. However, when host plants are located near to nonhost plants, they may be less attractive to insects [39]. Indeed, VOCs from non-host plants can mask or disrupt the chemical environment of the host plants, which would prevent their recognition by pests [34,40]. Thus, it can be assumed that volatiles emitted by Ocimum spp., which is considered as a non-host plant, had changed the chemical environment of the amaranth, and then caused the observed repellent effect. According to some studies, the biocidal activity of plant volatiles was generally attributed to their main compounds [41,42]. Therefore, the repellent activity on aphids could be attributed to the major compounds p-cymene, γ-terpinene, αterpinene, α-thujene E-α-bergamotene, methyl eugenol, E-βocimene linalool, previously identified in the volatiles collected on the tested Ocimum species [17].
For example, the repellent effect of linalool has been shown on Cavariella aegopodii Scopoli, M. persicae and Rhopalosiphum maidis Fitch [42,43]. The two Ocimum species had a similar effect on the three tested aphid species. As they had quite different chemical profiles, according to the relative proportions [17], this could be advantageous in management schemes, as these could be used alternatively to avoid insect habituation.

Conclusions
Our results provide evidence that Ocimum plants have a repellent effect on aphids. This indicates that these plants have potential as natural pesticides. These plants could then be used as an agroecological alternative for aphid control, in order to avoid the use of synthetic pesticides on amaranth or other vegetable crops in West Africa especially. The ability of Ocimum spp. to repel pests and attract natural enemies can make them important companion plants, especially for small-scale farmers. Not only can they be used to control pests, they can also be harvested and commercialized, which may provide a significant economic return for farmers. These benefits can also contribute to the food safety of producers and the consumers. However, additional experiments should be carried out in the field conditions to evaluate the real repellent activity of basil on aphids and how to maximize this effect.