Relative toxicity of two natural compounds compared to abamectin against some soybean pests under period rates

This is anOpe Abstract – A sustainable pest management in agro-ecosystems requires parallel assessments of pesticide and natural compounds to control target pests. In the present study, a semi-field experiment was conducted to evaluate the relative toxicity of abamectin (Abamax), humic acid, and Chitosan Nano-Particles (C.N.Ps) against four soybean pests: Tetranychus urticae, Eutetranychus orientalis, Bemisia tabaci, and Phenacoccus solenopsis. The experimental treatments were arranged in a split-plot design with three replicates, where treatments were assigned to main plots and pest populations under different periods in the subplots. The obtained results and graphs demonstrated that there were considerable differences between the total numbers of pests after different periods of treatment. Generally, E. orientalis recorded the minimum pest number at all. Different tested compounds demonstrated a considerable correlation between the densities of the pests. T. urticae exhibited a significant correlation with the other three pests (E. orientalis, B. tabaci, and P. solenopsis). E. orientalis did not correlate with B. tabaci and P. solenopsis. The humic acid recorded the best effect on T. urticae after 3 days with a reduction of 85.45% and E. orientalis after 7 days 65.55%. However, Chitosan Nano-Particles (C.N.Ps) was the best for E. orientalis after 14 days (mortality 74.36%). In contrast, abamectin (Abamax) had a general mean of reduction of 91.17% against T. urticae, whenever, these compounds are promising for controlling T. urticae, E. orientalis, B. tabaci, and P. solenopsis. These results may be a supporting method to overcome some soybean pests. The findings are discussed within the context of integrated management of soybean pests under semi-field conditions.


Introduction
Soybean (Glycine max) is considered an important oilseed crop that has grown in the world (60% of world oilseed production) (US Soybean Export Council, 2019). About 15 000 ha was sown soybean in Egypt, total production was 48 000 tons (FAO, 2018). Numerous kinds of pests attack soybean from seedling to mature stages such as spider mites, aphids, cotton leaf worms, and many other pests. Pests' infestation can lead to yield losses from 20 to 50%. Therefore, the farmers use pesticides to protect their crops (Fikru and Leon, 2003;Massoud et al., 2014).
Polyphagous pests are wide-ranged on many economic and important crops including soybean (Alakhdar et al., 2015). The two-spotted spider mite Tetranychus urticae Koch, and the oriental red mite Eutetranychus orientalis Klein are piercingsucking pests' infest soybean plant. This behavior of feeding leads to the appearance of characteristic yellow-chlorate spots on the leaves. Pale yellow streaks develop along the midrib and veins initially, which later progress to a grayish or silvery appearance of the leaves. In heavier infestations, mites feed and oviposit over the whole surface of the leaf, causing leaf fall and die-back of branches, which may result in defoliated plants (Moghadam et al., 2016). Two insect species also attack soybean and cause a serious economic problem. The whitefly, Bemisia tabaci, can decrease plant health and yield by removing large amounts of plant photosynthetic from the leaves, heavy infestations in young plants can reduce plant growth; later infestations can drop off the number of pods set, and seed size consistency, thus reducing yield and quality. The invasive mealybugs Phenacoccus solenopsis is a highly invasive and polyphagous insect responsible for serious damage to crops and plants. It attacks more than 200 plant species including field crops, vegetables, ornamentals, and weeds, bushes, and trees. (Arif et al., 2009;Fand and Suroshe, 2015).
Pests are controlled using traditional methods such as chemical pesticides. The need to study the correlation between the efficacy of pesticides and other natural alternatives on several target and non-target pests, to reduce their populations, is inescapable. Abamectin belongs to the family known as macrocyclic lactones, which is one of the pesticides applied to control pests. It is often used as an insecticide, acaricide, and nematicide and is found under many commercial names such as Abamax, Vertimec, etc. (Lasota and Dybas, 1990;Alhewairini, 2018). Most of the chemical pesticides are not only expensive but also kill natural enemies. Moreover, the use of these pesticides leads to serious threats such as the resurgence of insects (Kumari et al., 2015), the development of resistance in insects, environmental pollution, and harmful consequences on human health. Many studies have determined the importance of using natural alternatives in integrated pest management programs, one of them is humic acid, which is, a commercial product containing many elements that increase the availability of nutrients and consequently increase plant growth, mineral nutrition, seed germination, seedling growth, root initiation, root growth, shoot development, and the uptake of macro and microelements. Moreover, humic acid considered to induce resistance of plants against some pests (Çelik et al., 2011(Çelik et al., , Xu et al., 2015Ekin, 2019;Alakhdar et al., 2020). Chitosan Nano-Particles (C.N.Ps) is a new natural compound that was derived from chitin (Gan et al., 2005). It shows strong insecticidal and acaricidal activities and may serve as a good alternative for broad-spectrum and highly persistent pesticides. C.N.Ps has a potential for biological control of several pests with a slight effect on some associated natural enemies.
Therefore, the knowledge of the relationship between these pests under different treatments, obtainable through correlation coefficient, was detected to measure only the degree (intensity) and nature (direction) of association (Golkar et al., 2011). The problem gets complicated in selection studies especially when there is a negative interaction between the primary trait of the experiments and the other traits (De Leon et al., 2016) or treatments. Recently, GGE (Genotype plus Genotype by Environment) biplot method was developed by Yan (2014) to use different types of biplot graphs created to discuss the effects of applied treatment on one or all target traits at the same time, allowing the user to assess entire two-way data (Gabriel, 1971). Assessments are usually performed over PC1 and PC2 (the first two principal components) axes calculated from the data of rows and columns from a two-dimensional array produced by the combination of treatment and traits datasets (Akcura and Kokten, 2017).
In Egypt, no reference was found considering the technique of treatment by pest (TP)-biplot graph. Therefore, the objective of the current work was to: 1 Evaluate the efficacy of using natural compounds such as humic acid, and Chitosan Nano-particles, compared to an acaricide Abamectin: Abamax, after different treatment periods against target pests, Tetranychus urticae and Eutetranychus orientalis and non-target pests, Bemisia tabaci, and Phenacoccus solenopsis. 2 Study the interrelationships among pests infestations' using the (TP)-biplot technique.

Materials and methods
A semi-field experiment was conducted at an experimental farm known as Plant Protection Research Institute, Agricultural Research Center (ARC), Giza, Egypt during the 2nd week of June in the two successive summer seasons of 2019 and 2020 on soybean. Crawford variety was kindly provided by Field Crops Research Institute (FCRI) to study the effect of three compoundsa commercial insecticide (Abamectin: Abamax), an organic compound (humic acid), and a nanomaterials (Chitosan Nano-Particles)after periods (3, 7 and 14 days of spraying) to reduce the populations' density of Tetranychus urticae, Eutetranychus orientalis, Bemisia tabaci, and Phenacoccus solenopsis.

Experiments procedure
Experimental treatments were arranged in a split-plot design with three replications; treatment compounds were assigned to main plots and the pest populations in the subplots. The selected area of about 1.4 kirats (Kirat = 175 m 2 ) was split into 6 plots and controlled each plot consisted of 6 ridges, 70 cm apart, and 4 m long. Three rows of soybean plants, between treatments, were not sprayed as barrier zones to avoid drift spray between treatments. Seeds were sown in the first week of May during the two seasons. Abamectin and humic acid were sprayed with the recommended dose rate, while Chitosan Nano-Particles (C.N.Ps) was applied with as suggested by one of the author's Lc 90 (133.3) ppm of T. urticae (Alakhdar, 2020), one plot was sprayed with water as a control (Tab. 1). Moreover, all other cultural practices were applied as recommended.

The procedure of Bioassay and data recording
After the infestation of pests was confirmed, a pre-spray sample was taken and the treatment was carried out with all tested compounds. Ten leaves were taken from each replicate and the number of each pest, all movable stages of Tetranychus urticae, Eutetranychus orientalis, Bemisia tabaci, and Phenacoccus solenopsis, was counted before treatment and after 3, 7, and 14 days post-treatment by the aid of a stereomicroscope in acarology lab. at PPRI (Alakhdar, 2020). The reduction percentages of the pest's population in all treatments compared to the control was calculated according to the Henderson and Tilton formula (Henderson and Tilton, 1955): Population reduction = Corrected% ¼ 1 À n in Co before treatment Ã n in Tafter treatment n in Co after treatment Ã n in Tbefore treatment Ã 100; where n = insect population, T = treated and Co = control.
The performance of the number of each pest under the tested treatments (Tab. 1) was obtained.

Statistical analysis
Data on individual pests was carried out on the mean values over three replications. At first, the analysis of variance was applied, and then a combined analysis of variance was computed over two seasons according to Sendecor and Cochran (1981). Before running the combined analysis, Levene (1960) test was used to satisfy the assumption of homogeneity of variances. The mean comparison was done using the least significant differences test at a 5% level of probability. The number of pests data was transformed according to (x þ 1) 1/2 and was applied to detect statistical differences among pests numbers. The transformed data analysis can modify the coefficient of variation (C.V. %). Correlations among different pests' data were subjected, according to Sendecor and Cochran (1989), to reveal the relationship among soybean pests. When F was significant (P < 0.05) for the levels of symptoms analysis was performed. The genotype by trait (GGT) biplot, which is an application of the GGE biplot used to study the genotype by trait data (Yan and Rajcan, 2002). The biplot method was employed to display the treatment by trait (TT) two-way data in the biplot graph and denoted as treatment-pest (TP)-biplot graph according to Akcura and Kokten (2017), using GenStat software (18.0 version) by ICARDA.

Efficacy of compounds against different pests
Reduction percentages were calculated for each treatment, showing the effect of sprayed compounds (humic acid, C.N.Ps, and abamectin) against movable stages of different pests (T. urticae, E. orientalis, B. tabaci, and P. solenopsis) after 3, 7, and 14 days of treatment under natural conditions (Fig. 1). All compounds indicated a decrease in the number of mites/insects C.N.Ps were prepared according to Gan et al. (2005), the used concentration was the LC 90 133.3 ppm. recommended for T. urticae (Alakhdar, 2020 Therefore, all three spraying compounds (humic acid, C.N.Ps., and abamectin) had the highest mortality effects against T. urticae after different periods except for C.N.Ps treatment after 3 days. However, B. tabaci had the lowest motility effect for humic acid after 3 and 7 days (Tab. 2). The three spraying compounds (humic acid, C.N.Ps., and abamectin) indicated a reduction in the mean number of pests under study in variance responses. There is a good match between our results and Prabhat and Poehling (2007) who reported heavy reduction percentages on the three nymphal stages of B. tabaci treated with abamectin within 24 h postapplication, while the first instars being most susceptible. Kenneth et al. (2002) indicated that the mortality from abamectin residues was not significantly greater than the control at one day after application, but it was significantly greater than the control after 3, 7, and 14 days of treatment. Few studies recorded the effect of acaricides against E. orientalis, Alhewairini found that the populations of E. orientalis reduced to 76.68 and 78.52 after one-week exposure to the recommended dose (RD) of abamectin under field and laboratory conditions (Márquez et al., 2006;Alhewairini, 2018). The increase of the mean reduction in the population of T. urticae and E. orientalis may due to the specialty of abamectin: Abamax, as acaricide has efficacy on all stages of mites. Whenever its effect on P. solenopsis decreased over time as most of the insecticides used are mixed abamectin with another formula (Rezk et al., 2019).
Particular attention is paid to humic acid, as an organic compound that usually provides plants with a balanced source of nutrients that can influence the composition and physiology of plants. Apart from that, it might have provided some growth-promoting substances, vitamins and these probably have increased the plant resistance to pests or made the plants less palatable to the pest. It emerged superior in minimizing the whitefly 75% compared with other used compounds (Chatterjee et al., 2013). On other the hand, Panda et al. (2005) reviewed the lowest population of sucking pests, jassids, and thrips in chilli. The mechanisms for decreasing pest attacks may be due to the differential availability of mineral nutrients in plants, which might have enhanced the induced resistance development and subsequently helped in escaping suckingpiercing pest infestation. Furthermore, organic treatments reduced the incidence of sucking pest as whitefly and leafhopper that organic amendments comparatively increased the total phenols in the plants and also the activity of the enzymes like polyphenol oxidase and peroxidase, which might be responsible for the reduced pest incidence (Ravi et al., 2006). Consequently, more researches are needed on the mode of action and compatibility of tested compounds with bio-and organic-origin agents .
A similar approach is used for two tetranychid mites, Tetranychus urticae (Koch) and Tetranychus cinnabarinus, and their eggs and immature stages on dry beans (Phaseolus vulgaris L.). It was found that Chitosan Nano-Particles (C.N.Ps) is potent against T. urticae (Alakhdar, 2020). Other studies carried on C.N.Ps to evaluate its insecticidal effect on other pests, and Zhang and Tan (2003) reported that Chitosan exhibited different insecticidal activity to various aphids ranged between 93 and 99% for Hyalopterus pruni (Goffroy) on flowers, while (Rabea et al., 2005) tested the insecticidal activities of Chitosan Nano-Parteciles against larvae of the cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). The same trends were also observed against Aphis gossypii; the mean number of eggs/female of A. gossypi was significantly decreased to 20.9 and 28.9 eggs/female compared with 97.3 and 90.3 of the non-treated controls, under laboratory and semi-field conditions, respectively (Sahab et al., 2015).

Combined analysis and mean performance
Variances homogeneity for the studied number of pests in each mite/insect was confirmed according to the Levene (1960) test, which allowed the combined analysis. Accordingly, the mean variability for different treatments on each pest over the two seasons 2019 and 2020 were presented in Tables 3  and 4. Results showed that years affected significantly both T. urticae and E. orientalis. As well, significant differences among the different compound treatments for all pests except E. orientalis were obtained. Each of T. urticae, B. tabaci, and P. solenopsis revealed highly significant differences. Our results are in harmony with Sabbour (2016) and Alakhdar (2020) who reported that spray Chitosan Nano-Particles compounds had a high effect on pests' number on soybean. Concerning periods, highly significant differences were detected for all pests, which demonstrated the existence of a high effect of different periods. The results in this experiment  are in agreement with the results of other researchers such as Ekin (2019) and in terms of the interaction between spray compound treatments and periods, there were highly significant differences for all the traits except E. orientalis. Figure 2 illustrated the effect of tested compounds on the studied pests on soybean over two seasons. Meanwhile, the analysis showed that this data wasn't subjected to normal distribution. Then data of the number of pests trait was transformed according to (x þ 1) 1/2 and reanalyzed for modifying analysis and coefficient of variation (C.V.).

Effect of tested compounds and periods rates
The above results on soybean, the 1st compound (humic acid) recorded the best effect for T. urticae and E. orientalis, meanwhile, 2nd (Chitosan Nano-Particles) was the best for B. tabaci and P. solenopsis. Whenever E. orientalis recorded the minimum number at all. These results agreed with Ekin (2019). Figure 3 represents the significant effects of the interaction between sprayed compounds and periods on the number of mites/insects. Data revealed that the humic compound had effects on T. urticae and E. orientalis mites, Chitosan nanoparticles (C.N.Ps) decreased E. orientalis and abamectin compound had effects on each from T. urticae, E. orientalis, and B. tabaci pests. It could be noticed that the combination in the application of humic acid, C.N.Ps, and abamectin may reduce pests' infestation in soybean plants when applied at this stage of plant growth. This time is accurate to manage pests understudy meanwhile keep their abundance under Economic Threshold (Alakhdar et al., 2015;Czepak et al., 2018;Abd El-Razzik, 2018;Mesbah et al., 2019).

Correlation between pests in the soybean field
Generally, many pests were noted in the soybean field. Adequate knowledge of the relationship that exists between these pests is essential for the identification of infestation in soybeans. The correlation coefficients among all pairs of studied pests of soybean over the two seasons are given in Table 4. The results showed that there was a highly significant positive correlation between T. urticae and each of B. tabaci (0.474 ** ) and P. solenopsis (0.323 ** ). As well as, mealybugs, P. solenopsis had a highly significant positive correlation with the whitefly, B. tabaci. Meanwhile, Eutetranychus (0.302 * ) had only a significant positive association with E. orientalis. On the other hand, E. orientalis exhibited an insignificant correlation with B. tabaci and P. solenopsis. A significant correlation between these pests indicated that the simultaneous infestation of these pests is possible. These findings indicate that the efficiency of spraying compounds for each pest would be accompanied by a high effect of another pest.

Treatment pest biplot (combined data)
Generally, the biplot graphs, according to (Yan et al., 2000;Yan, 2002;Yan and Rajcan, 2002;Yan and Tinker, 2005), can be used to compare genotypes in different environments (GE), genotypes based on multiple traits (GT) or treatment based on multiple traits (TT). The current study depended on the estimation of biplot polygon and vector graphs to study the effects of the used treatments (T) on the studied pests (P) in one graph which is termed as (TP)-biplot graph. This method that uses a combination of treatment and pest datasets is similar to the method of comparing treatments on multiple traits (Yan, 2002;Akcura and Kokten, 2017). The mean values of the effects of three compounds and four dates of accounting numbers of (mite-insects)/each pest (representing 12 factorial treatment combinations) were graphically summarized as shown in the polygon view (Fig. 4). The (TP)-biplot graph gives an overall picture of the interrelationships among factorial treatment and all pests simultaneously.
The treatment Â pest (TP)-biplot model is generated according to Yan and Rajcan (2002). The polygon (whichwon-where) view of the treatment Â pest (TP)-biplot graph is a good tool to interpret the behavior pattern of treatment toward pest provided. Then, the biplot should explain a sufficient amount of the total variation. The principle components (PC) analysis based on (TP)-biplot method together explained that there is about 94.22% of the observed variation for the measured pests on soybean across studied treatments (spraying compounds by period's measurements). The first and second principle components (PC) explained 76.29% and 17.92%, respectively, and the cumulative variance of the first two PCA was found 94.22%. The first two PC ' s reflected more than 60% of the total variation. Therefore, it achieved the goodness of fit for the biplot model.
The polygon-view of the (TP)-biplot graph in Figure 4 indicated which spraying compound by period treatment combinations had the best values for which pests and correlated pests by mega-environment. Mega-environment identification is among the most important objectives of multienvironment statistical trials. The (TP)-biplot showed the variation of the twelve treatments in terms of four pests, treatments as bests ones for single or multiple pests, and grouped the twelve treatments based on pests that make them potential performances. On the right part of the graph (with the Chitosan Nano-Particles compound), the best treatment has obtained for all pests, especially T. urticae. Then, the results of all treatments in the right part of the graph (T11, T12, T9, and T10) revealed that the abamectin effect with the highest insect number for all pests, especially P. solenopsis and B. tabaci. Therefore, this similarity established a strong correlation between P. solenopsis and B. tabaci pests in treatment results (as shown in Fig. 4). The left part of the graph revealed the relatively lowest pest number. Regarding treatments, T2 (humic acid spraying compound after 3 days) and T3 (humic acid spraying compound after 7 days) were the vertex treatment on the left  part of the graph for the E. orientalis mite. Therefore, T2 and T3 (humic acid spraying compound after 3 and 7 days) recorded the best treatments for E. orientalis with a similar effect was obtained. T.urticae, showing the strong correlation between E. orientalis and T. urticae results as shown in (TP)-biplot graph (Fig. 4) and Table 4. Therefore, all treatments on the negative part of the graph recorded the lowest number of pests, then, it was considered as the best treatment and had a good effect for all pests (Yan and Hunt, 2002;Yan and Rajcan, 2002).
The (TP)-biplot graph displayed the relationship among the four pests on soybean. Also, traits with longer vectors are more responsive to the treatment combinations and traits with shorter vectors are less responsive to the treatment combinations as well as those located at the biplot center are not responsive at all (Yan and Hunt, 2002;Yan and Rajcan, 2002;Yan and Frégeau-Reid, 2008). Then, the ideal test trait (pest) effectively discriminates treatments and represents their grouping which can be classified as pests with low treatment discrimination that should be selected as test trait number of insects/mites. In addition to the results of the traditional method of analyzing data, biplot provides more information on the effectiveness of the treatments with the view of identifying the ideal (best) one or pest. Most of the above findings can be verified from the original correlation coefficients.

Conclusion
The present study revealed the reduction of chemical pesticides through judicious application of bio and organic compounds, Chitosan Nano-Particles (C.N.Ps), and humic acid was tested. Abamectin (Abamax) is a recommended pesticide compared to these natural compounds against T. urticae, E. orientalis, B. tabaci, and P. solenopsis on soybean crops. The obtained results demonstrated that there were considerable differences between the total numbers of these pests after different periods. There was a highly significant correlation between these pests indicated that simultaneous infestation of these pests is possible. These findings indicate that the efficiency of spraying these compounds for each pest would be accompanied by a high effect on another pest. Based on the modified treatment-by-pest biplot analysis, it was concluded that the treatments (three spraying compounds by four-period interactions) were identified as effective treatments for pests and these treatments would be considered as key during the selection. Biplot method of treatment Â pest (TP) together established a significant correlation between P. solenopsis and B. tabaci pests in treatment and a significant correlation between E. orientalis and T. urticae results as shown in the (TP)-biplot graph. Treatments on the negative part of the graph recorded the lowest number of pests, then, it was considered as the best treatment and had a good effect on all pests. As similar other results were shown. Then, the biplot graph gave a conclusion for all treatments of all pests. Fig. 4. Polygon-view of (TP)-biplot, showing which treatments had best values for which pests and mega-environment (correlated pests). T1: before spraying humic acid; T2: 3 days after spraying humic acid; T3: 7 days after spraying humic acid; T4: 14 days after spraying humic acid; T5: before spraying C.N.Ps; T6: 3 days after spraying C.N.Ps; T7: 7 days after spraying C.N.Ps; T8: 14 days after spraying C.N.Ps; T9: before spraying abamectin; T10: 3 days after spraying abamectin; T11: 7 days after spraying abamectin; T12: 14 days after spraying abamectin.