Evaluation of the Effect of Different Concentrations of Spirotetramat on the Diaspine Scale Parlatoria ziziphi in Citrus Orchards

: The control of Parlatoria ziziphi (Lucas, 1853) was studied in citrus orchards at Mechraa Belksiri in the Gharb area of Morocco. Three concentrations of spirotetramat T0 = 0 L/Ha as a control experiment, T1 = 0.625 L/Ha, T2 = 0.755 L/Ha, and T3 = 1 L/Ha, were applied on 4 ha of Valencia late orchard (each dose for 1 ha of Valencia late). The effect of spirotetramat was evaluated on the mortalities and survival rates of P. ziziphin during the stages of larvae (ﬁrst instar and second instar) and females (F1, F2, and F3). Results showed that the spirotetramat was effective on larvae and females of P. ziziphi . Among the 11,229 females recorded, 93% were inhibited, while only 7% were intact after the treatment period. Finally, our study highlights that all concentrations tested were effective on the P. ziziphi population; besides, all three concentrations of this product tested were equally effective on larvae and females of P. ziziphi . Thus, during the spread period, spraying a low concentration of this product (0.625 L/Ha) will better control this pest and reduce the environmental impact.


Introduction
In Morocco, the citrus sector is of great importance to socio-economic development, with an estimated production of 2.2 million tons per year [1]. It is mainly produced in Souss, Berkane, Tadla, and Haouz regions, with a total surface area of 126.600 ha [2]. Despite the importance of the citrus sector in the national economy and its particular interest, Agronomy 2021, 11, x FOR PEER REVIEW 3 of 11 Figure 1. Location of studied orchards [23].

Sampling Design
To investigate the impact of spirotetramat on P. ziziphi, an orchard of 'Valencia Late' (Citrus sinensis) was selected and followed [22]. This product systemic (spirotetramat) has been used because of its effectiveness against a wide range of pests, including the diaspine scale insects, and its reduced damage to the environment and natural enemies. The orchard covers 4 ha on Valencia Late trees. The orchard was divided into 4 plots of 1 ha (Figure 2), and each plot was treated by a specific dose of the pesticide: (i) T0 treated only by water (as a control experiment), (ii) T1 = 0.625 L/ha, (iii) T2 = 0.755 L/ha, and (iv) T3 = 1 L/ha. In addition, the Teyme Eolo sprayer (Teyme Tecnologia Agricola, Girona, Spain), with its turbulent nozzle and exit diameter of 12 mm, delivered 1.55 L/min at a pressure of 20 bars. In addition, 2500 L of spray liquid were sprayed by a towed atomizer on each plot (1 ha), at a rate of 6 L of spray for each tree (Figure 2). These 3 concentrations were used based mainly on the quantities of pesticides used by local farmers.
To evaluate the effect of each concentration after treatment (1 to 8 weeks), 200 leaves of Valencia late trees in each plot were collected weekly and randomly from the different directions of the tree (North, East, South, and West), for a total of 20 trees by the plot that belonged to a square block (3 repetitions were performed independently). We left 2 lines between the different plots treated with spirotetramat. Then, we count the mortalities (M) and survival rates (V) of P. ziziphi at two stages: Larvae (L1 and L2) and females (F1, F2, and F3) on each leaf surface.
In the laboratory, the different stages of P. ziziphi on each leaf were determined and counted on both leaf surfaces; we counted the survival rates (V) and mortalities (M) of P. ziziphi at two larval stages (L1 and L2) and three generations of female (F1, F2, and F3) using a binocular microscope.

Sampling Design
To investigate the impact of spirotetramat on P. ziziphi, an orchard of 'Valencia Late' (Citrus sinensis) was selected and followed [22]. This product systemic (spirotetramat) has been used because of its effectiveness against a wide range of pests, including the diaspine scale insects, and its reduced damage to the environment and natural enemies. The orchard covers 4 ha on Valencia Late trees. The orchard was divided into 4 plots of 1 ha (Figure 2), and each plot was treated by a specific dose of the pesticide: (i) T0 treated only by water (as a control experiment), (ii) T1 = 0.625 L/ha, (iii) T2 = 0.755 L/ha, and (iv) T3 = 1 L/ha. In addition, the Teyme Eolo sprayer (Teyme Tecnologia Agricola, Girona, Spain), with its turbulent nozzle and exit diameter of 12 mm, delivered 1.55 L/min at a pressure of 20 bars. In addition, 2500 L of spray liquid were sprayed by a towed atomizer on each plot (1 ha), at a rate of 6 L of spray for each tree ( Figure 2). These 3 concentrations were used based mainly on the quantities of pesticides used by local farmers.
To evaluate the effect of each concentration after treatment (1 to 8 weeks), 200 leaves of Valencia late trees in each plot were collected weekly and randomly from the different directions of the tree (North, East, South, and West), for a total of 20 trees by the plot that belonged to a square block (3 repetitions were performed independently). We left 2 lines between the different plots treated with spirotetramat. Then, we count the mortalities (M) and survival rates (V) of P. ziziphi at two stages: Larvae (L1 and L2) and females (F1, F2, and F3) on each leaf surface.
In the laboratory, the different stages of P. ziziphi on each leaf were determined and counted on both leaf surfaces; we counted the survival rates (V) and mortalities (M) of P. ziziphi at two larval stages (L1 and L2) and three generations of female (F1, F2, and F3) using a binocular microscope.

Statistics
Statistics of data were done in Minitab software, version 1.1.19, LLC, USA. The results were given as mean ± SD. Moreover, to evaluate the effectiveness of spirotetramat, we calculated the survival (survived specimens/total sampled specimens) and mortality (inhibited specimens/total sampled specimens) of P. ziziphi for both larvae and females [23]. We checked for normality and homogeneity of variance for all variables with the Kolmogorov-Smirnov test. To assess the difference between the survival rates and mortalities between different stages of P. ziziphi, we used the independent t-test, considering the two stages as unrelated variables, while the effect of different doses of the systemic product was tested by the ANOVA One-way test followed by a post hoc Tukey test at p < 0.05. The linear correlation between different larvae and female stages was obtained by the Pearson correlation coefficient at p < 0.05. Principal component analyses were accomplished to elucidate the relationship between the treatment and different stages of P. ziziphi.

Impact on Larvae
Out of 3965 larvae, 83% were inhibited, and only 17% survived after the treatment period. Specifically, during the first instar larval (L1) stage, 620 survived (16%), while 2234 were suppressed (56%). Moreover, in the second instar larval (L2) stage, 53 larvae survived (1%) and 1058 larvae were inhibited (27%). On the other hand, survival and mortality rates were very high during larval stage 1 (Figure 3), while these two parameters were low during larval stage 2. Mortalities and survival rates were uncorrelated during both stages (Table 1).

Statistics
Statistics of data were done in Minitab software, version 1.1.19, LLC, USA. The results were given as mean ± SD. Moreover, to evaluate the effectiveness of spirotetramat, we calculated the survival (survived specimens/total sampled specimens) and mortality (inhibited specimens/total sampled specimens) of P. ziziphi for both larvae and females [23]. We checked for normality and homogeneity of variance for all variables with the Kolmogorov-Smirnov test. To assess the difference between the survival rates and mortalities between different stages of P. ziziphi, we used the independent t-test, considering the two stages as unrelated variables, while the effect of different doses of the systemic product was tested by the ANOVA One-way test followed by a post hoc Tukey test at p < 0.05. The linear correlation between different larvae and female stages was obtained by the Pearson correlation coefficient at p < 0.05. Principal component analyses were accomplished to elucidate the relationship between the treatment and different stages of P. ziziphi.

Impact on Larvae
Out of 3965 larvae, 83% were inhibited, and only 17% survived after the treatment period. Specifically, during the first instar larval (L1) stage, 620 survived (16%), while 2234 were suppressed (56%). Moreover, in the second instar larval (L2) stage, 53 larvae survived (1%) and 1058 larvae were inhibited (27%). On the other hand, survival and mortality rates were very high during larval stage 1 (Figure 3), while these two parameters were low during larval stage 2. Mortalities and survival rates were uncorrelated during both stages (Table 1). ited (L2M) specimens. The T3 concentration was positively correlated with first instar larval Inhibited (L1M) specimens. In contrast, all doses tested were negatively correlated with surviving larvae of the first instar and second instar (L1V and L2V) ( Figure 4). Figure 5 shows that the survival means of the larval stage in the untreated plot increased significantly from week 1 (15.67 AB ± 2.06) to week 8 (22.67 A ± 1.15). On the other hand, the average for surviving larvae remained low for all weeks after treatment with all concentrations of spirotetramat (Table 3).
Regarding the efficacy of the doses on the different larval stages, all concentrations of spirotetramat tested were effective on stage 1 larvae survivors (L1V) with the means of T1 = 10.00 B ± 3.30, T2 = 7.75 B ± 3.17, and 8.08 B ± 2.49, while the T3 dose was very effective on stage 1 dead larvae (L1M) with an average of 41.13 B ± 10.82. Further, all the tested doses (T1, T2, and T3) were effective against stage 2 larvae (Table 2). Furthermore, all doses of this systemic product were positively correlated with second instar larval Inhibited (L2M) specimens. The T3 concentration was positively correlated with first instar larval Inhibited (L1M) specimens. In contrast, all doses tested were negatively correlated with surviving larvae of the first instar and second instar (L1V and L2V) ( Figure 4).   Figure 5 shows that the survival means of the larval stage in the untreated plot increased significantly from week 1 (15.67 AB ± 2.06) to week 8 (22.67 A ± 1.15). On the other hand, the average for surviving larvae remained low for all weeks after treatment with all concentrations of spirotetramat (Table 3).   Means in the same column with different superscripts are significantly different (p < 0.05).  Means in the same column with different superscripts are significantly different (p < 0.05).
(F1V, F2V, and F3V) with averages of T1 = 1.38 B ± 1.17, T2 = 1.13 B ± 0.99, and T3 = 0.67 B ± 0.90. The concentrations T1 = 23.21 A ± 11.33 and T2 = 24.00 A ± 6.08 were effective on stage 1 dead females (F1M). What is more, all doses of this systemic product tested were effective on dead females of stage 2 (F2M) with the averages T1 = 43.42 B ± 14.27, T2 = 49.92 B ± 11.55, and T3 = 47.38 B ± 16.05, while spirotetramat showed no effect on stage 3 females (F3M) ( Table 4). In addition, the doses of this systemic product were positively correlated with inhibited females of all stages (F1M, F2M, and F3M), while all doses tested were negatively correlated with surviving females (F1V, F2V, and F3V) (Figure 4). Figure 7 shows that female survival in the untreated plot decreased significantly from week 1 (46.00 A ± 5.56) to week 8 (28.00 B ± 2.00 14.33 AB ± 2.45). While the mean survival of females in the 0.625 L/ha treated plot was very high in the first week after treatment (21.00 A ± 2.31), the means then decreased significantly over the last 7 weeks after treatment to (14.33 AB ± 2.45) in week 8 ( Table 5). The survival averages of the females remained low for all 8 weeks after treatment with the concentrations 0.755 L/he and 1 L/he (Table 5). In terms of comparisons between stages, the means of surviving females were higher in F3, followed by F2 and then F1. Similarly, inhibited females were more numerous in F3, followed by F2 and F1. On the other hand, survival averages were variable for all periods. Similarly, the means of females eliminated from the three stages were significantly different during the entire follow-up period (Table 4).  The three doses were all effective on the survival rates of females in all three stages (F1V, F2V, and F3V) with averages of T1 = 1.38 B ± 1.17, T2 = 1.13 B ± 0.99, and T3 = 0.67 B ± 0.90. The concentrations T1 = 23.21 A ± 11.33 and T2 = 24.00 A ± 6.08 were effective on stage 1 dead females (F1M). What is more, all doses of this systemic product tested were effective on dead females of stage 2 (F2M) with the averages T1 = 43.42 B ± 14.27, T2 = 49.92 B ± 11.55, and T3 = 47.38 B ± 16.05, while spirotetramat showed no effect on stage 3 females (F3M) ( Table 4). In addition, the doses of this systemic product were positively correlated with inhibited females of all stages (F1M, F2M, and F3M), while all doses tested were negatively correlated with surviving females (F1V, F2V, and F3V) (Figure 4). Figure 7 shows that female survival in the untreated plot decreased significantly from week 1 (46.00 A ± 5.56) to week 8 (28.00 B ± 2.00 14.33 AB ± 2.45). While the mean survival of females in the 0.625 L/ha treated plot was very high in the first week after treatment (21.00 A ± 2.31), the means then decreased significantly over the last 7 weeks after treatment to (14.33 AB ± 2.45) in week 8 ( Table 5). The survival averages of the females remained low for all 8 weeks after treatment with the concentrations 0.755 L/he and 1 L/he (Table 5).   Means that do not share a letter are significantly different.

Discussion
Based on our nationwide survey, this is the first research on the control of P. ziziphi using a systemic product, spirotetramat. Our main objective was to provide detailed and extensive data on the efficacy of this systemic product in controlling this pest. These results are the first and only data supplied in the control of P. ziziphi in citrus in North Africa, which is of great importance for further comparative research and the adoption of less environmentally damaging and more effective doses to control the pest.
Our results showed that in the larval stage, inhibition was nearly 83%, while in fe-

Discussion
Based on our nationwide survey, this is the first research on the control of P. ziziphi using a systemic product, spirotetramat. Our main objective was to provide detailed and extensive data on the efficacy of this systemic product in controlling this pest. These results are the first and only data supplied in the control of P. ziziphi in citrus in North Africa, which is of great importance for further comparative research and the adoption of less environmentally damaging and more effective doses to control the pest.
Our results showed that in the larval stage, inhibition was nearly 83%, while in females, the inhibition rate was more than 92%. All three doses tested were effective on the survival rates of females in all three stages (F1V, F2V, and F3V) during the whole follow-up period, and the same results were reported by [24], where spraying spirotetramate on citrus fruit reduced the survival rate and fecundity of the mealybug (Lepidosaphes beckii) to 100% Agronomy 2021, 11, 1840 9 of 11 172 days after treatment. In addition, spirotetramat showed no effect on the mortality rates of stage 3 females (F3M). This may be due to other unknown factors that contributed to the increased mortality in the untreated plot. In Egypt, the same results were reported on navel orange trees, Citrus sinensis, which reported high mortality due to unknown factors [7]. All doses of spirotetramat tested were effective on stage 1 larvae, while the T3 concentration was very effective on stage 2 larvae. According to the findings of Nauen [25], spirotetramat causes death in larvae deposited by adult female aphids after 24 h of its application. At the larval stages, the efficacy of the doses used was variable. Regarding the mortality rate, the T3 concentration (1 L/he) was effective against larva 1, while all three doses tested were effective on the survival rate of larva 1, while on larva stage 2, all concentrations showed the same efficacy. Our objective was to search for lower concentrations of pesticides that have effective control against P. ziziphi. This will reduce pesticide use and avoid pesticide resistance [26][27][28]. Therefore, for easy marketing, the remaining residues are suggested to be less on treated fruits [29,30].
Among the total population of 15,194 treated with this systemic product, 90.59% were inhibited, and only 9.41% were intact after treatment. Similar results were reported in Morocco and Algeria where spirotetramat controlled Parlatoria pergandii with encouraging results in the citrus orchard [23,31]. All doses of spirotetramat used showed a significant effect on larvae and females of P. Ziziphi. All the concentrations of spirotetramat used were effective on the population of Parlatoria blanchardi of date palm. Among all concentrations tested, we noticed that the survival rate remained low for 8 weeks after the application of the treatment. According to [24], spirotetramat spraying on citrus fruit resulted in 100% lower survival and fecundity of Lepidosaphes beckii for 172 days after treatment. Therefore, the choice of the right concentration remains a necessity in order to preserve the agroecosystem. In general, large amounts of pesticides control pests but negatively influence natural enemies and the environment [32]. In contrast, the combination of the effect of parasitism (natural enemies) and low doses of pesticides would provide effective and environmentally friendly pest control [23].

Conclusions
Our findings highlight two key messages: (1) The data indicated that spirotetramat was effective on larvae and females of P. ziziphi and (2) all doses tested were effective on the P. ziziphi population. Since spirotetramat is an effective and widely used pesticide, further studies on its effect on other natural enemy populations and the residues remaining on fruits are needed to establish appropriate management strategies.