Spinosad combined with entomopathogenic nematode for biocontrol of the Mediterranean fruit fly (Ceratitis capitata [Wiedemann]) on citrus

Cultivation of citrus (Sapindales: Rutaceae) crops is continuously expanding in Egypt given the favorable ingredients of citriculture. Notwithstanding the Egyptian rank as the world's largest orange exporter, the Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is one of the major pests that considerably reduces the quality of citrus crops. Contrary to hazardous organophosphate insecticides that are commonly used to control the Medfly, biologically-based C. capitata control tactics were tried herein. The effect of spinosad as a bacterial fermentation product and the nematode Steinernema riobrave as biological insecticides applied singly or in combination on laboratory and field strains of Medfly were investigated. A significant difference in LC50 values was observed between laboratory strain (4.78 PPM) and field strain (8.12 PPM) of C. capitata larvae exposed to spinosad. A 1.7 fold decrease in susceptibility of field strain was recorded after treatment with spinosad. In a field experiment, a reduction in Medfly population by 80, 37, and 92% for spinosad, S. riobrave, and spinosad + nematode treatments was recorded, respectively. Utilization of spinosad-S. riobrave combination in citrus fields, as a novel alternative for unhealthy chemical insecticides to control C. capitata in Egypt can be suggested. Use of this combination should be incorporated into a holistic management package that can be economically feasible and environmentally sustainable for Egyptian agriculture.


Background
Citrus production suffers from the infestation by many insect pests of which the Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) ranks high. It is one of the most globally damaging pests of horticulture in the fruit fly group. Several specific control methods have been developed and applied successfully in many countries against C. capitata (Abd-Elgawad 2021). Common techniques are partial or cover-spraying of chemical insecticides with or without lures, hygiene, the Sterile Insect Technique (SIT), and the Bait Application Technique (BAT). They are recommended for control and suppression of C. capitata populations in fruit groves including citrus. Females of Tephritidae flies need certain amino acids as nutrition for developing their eggs and so they are attracted by the relevant baits. Buminal (5, 10 and 15% concentrations) was superior in attracting the Medfly (Amin 2003) as BAT is a widely used approach for general control of fruit flies.
Page 2 of 5 Abdel-Razek and Abd-Elgawad Egypt J Biol Pest Control (2021) 31:112 Development of chemical insecticides' resistance in pest and vector populations, the damage caused to non-target organisms, and the realization of other environmental hazards of these chemicals have led to an increasing interest in biological control measures (Trdan et al. 2020). Entomopathogenic nematodes (EPNs) are safe biocontrol agents that have been isolated and used successfully in the control of many insect pests in Egypt (Abd-Elgawad 2020) and elsewhere (Koppenhöfer et al. 2020). Moreover, chances that ease incorporating them into holistic management systems of various pests and pathogens should be seized. For example, using EPNs with certain chemical insecticides have proved to increase their effectiveness against pests (Koppenhöfer et al. 2020). Also, developing novel (compatible) techniques or leveraging synergies between EPNs and other pest management tactics to offer economically feasible application could be sought (Abd-Elgawad 2019). Thus, utilizing EPNs + spinosad, a bacterial fermentation product, is hypothesized in the present study to increase their biocontrol efficacy against C. capitata populations. As a natural substance, spinosad is toxic to fruit flies either by contact or following ingestion as it contains 2 chemicals: spinosyn A and spinosyn D (Ekesi et al. 2016).
This study examined the following points: (1) basic susceptibility of Medfly to spinosad, (2) susceptibility of Medfly to the EPN Steinernema riobrave, and (3) synergistic effects between spinosad and S. riobrave.

Spinosad
Spinosad was obtained from Dow-Agro-sciences as Tracer 24% ® formulation. Spinosad has already been organically certified (Racke 2007) and is being used against many insect pests, especially different fruit fly species (Abd-Elgawad 2021).

The entomopathogenic nematode, Steinernema riobrave
This highly pathogenic species is maintained in the microbial control laboratory, Giza, Egypt. It was originally isolated from the Rio Grande Valley of Texas, USA, and possesses several promising features (Grewal et al. 2005); its effective host range runs across multiple insect orders. It has ability to exploit aspects of both ambusher and cruiser means of finding hosts and tolerate soil temperatures at approx. 35 °C with persistence even under semi-arid conditions. Its small size provides high yields of EPN-infective juveniles (IJs) whether using in vivo or in vitro production methods.

Laboratory tests
Two different C. capitata strains were used, a laboratory strain (L) and a field strain (F), collected from Mashtoul El-Souk Center, Sharkia Governorate, Egypt. The (L) strain was obtained from the standard laboratory culture, Egypt. Medfly was reared on a standard laboratory media; a formulated diet contained 4.83% Nutrifly, 15% corn cob fractions, 8% corn flour, 8.33% sugar, 0.23% sodium benzoate, 0.11% niacin, 0.13% citric acid, and 63.37% water (Hernandez et al. 2010) under constant conditions (25 ± 3 °C and 70-80% RH with a 16:8 (light: dark) cycle. Bioassay of the biological activity of the spinosad (Tracer 24% ® ) was determined, using treated media bioassay. The first instar larvae of C. capitata were used for both C. capitata strains. Spinosad doses used in both bioassays were 0 (only water), 2, 4, 8, 16, 32, 64, 125, 250, 500 and 1000 part per million (ppm). All sterile 9-cm-diam. Petriplates of treated diet with spinosad were first air-dried for 1/2 h. Control plates were prepared by diets treated with only distilled water. Afterwards, 5 first instar C. capitata larvae of each strain were separately placed in single plates and allowed to feed on the treated media for 24 h. Percentages of mortalities were measured after 24 h. The experiment was repeated 3 times in a completely randomized design.

Field experiment
Three rows each contains 12 'Succari' orange trees of 15 years old were divided into 4 blocks (3 trees each) in an organic farm at Mashtoul El-Souk Center, Sharkia governorate, Egypt. Treatments' blocks were separated by a minimum buffer area of 25 m. Treatments were carried out in a randomized complete block design via applying the following 4 treatments just before sunset in January 2020: (1) Spinosad as cover spray treatment at the rate of 110 ppm in 5 l water tree −1 , (2) S. riobrave as cover spray at the rate of 3 × 10 6 IJs in 5 l water tree −1 . The spray (EPN suspension) is assumed to fall either on the fruits to protect them from depositing eggs by the insect females and kill the hatched larvae or drain into the ground beneath the tree canopy to kill the emerging insect adults, (3) S. riobrave-IJs + spinosad in 5 l water tree −1 , and (4) untreated control group (sprayed with water only). Treatments were applied as a foliar and fruit spray, using a back-back sprayer system with a nozzle attached to spray the whole tree. Three plastic International Yellow Pheromone traps with Concept's Medfly Biolure ® (ammonium acetate, putrescine, trimethylamine) were placed in the middle of each replicate for the 4 treatments, 5 m between adjacent trees and 35 m between adjacent blocks to attract male flies in addition to the predominant female via attractive integrated pest management (IPM) trap with Biolure ® as food bait (Ekesi et al. 2016). Traps were placed just after treatment applications, checked and removed 7

Statistical analysis
Data were analyzed using probit analysis models in the Stat program (Finney 1964;Brown et al. 2001). Mortality rates were corrected by using the Abbott formula (Abbott, 1925). The significant differences between spinosad concentrations expected to kill 50% of C. capitata larvae or median lethal concentration (LC 50 ) values based on overlap of 95% confidence intervals were recorded. Data were analyzed using one-way analysis of variance (ANOVA) and followed by the least significant difference (LSD) test as a comparison of the mortality means. Dose-response chart of the dosage were plotted, using percentage mortality rates in Microsoft Excel spreadsheets. A randomized complete block design consisting of 4 treatments, with 3 replicates, each with 3 trees was used. Their field data were analyzed via two-way ANOVA and means separated using Tukey's (P = 0.05).

Laboratory bioassays
The C. capitata larvae displayed a concentrationdependent response to spinosad. A 100% mortality of the C. capitata field strain was detected after using 250 and 500 ppm but all doses induced mortality of the Laboratory strain (P < 0.05). An average of approximately 125 ppm of spinosad caused 70 and 62% mortality of laboratory and field strains, respectively (Table 1). According to LC 50 values (Table 2), a significant difference was observed between laboratory strain (4.78 ppm) and field strain (8.12 ppm), 24 h. after treatment. Therefore, the laboratory strain was more susceptible to spinosad than the field one ( Table 2). Obtained results suggested that even common confidence profiles set in this test would not overlap based on the 95% confidence interval (CI). The former strain demonstrated 4.96-4.59 CI at 95% probability level (slope = 2.85 ± 0.001) compared to 9.25-6.99 CI at 95% probability level (slope = 8.09 ± 0.39) for the latter strain whose susceptibility decreased to 1.7 fold.

Field experiment
There was a significant (P ≤ 0.05) difference between the control and any of the 3 other treatments for the captured Medfly (Table 3). Means of the captured Medfly adults showed that the least number was recorded at the combined treatment of spinosad and nematode followed by spinosad alone, and then nematode alone in an ascending order. The highest numbers of the captured Medfly were recorded in the untreated check.   These results showed a reduction in Medfly populations by 80, 37 and 92%, for spinosad, nematode, and spinosad + nematode treatments, respectively, compared to the control treatments.

Discussion
In the laboratory study, the LC 50 values concerning C. capitata 1 st instar larvae after 24-h exposure to the biological compound, containing concentrate formulation of spinosad, was quite different between the 2 insect strains. Based on these LC 50 values of spinosad, the field strain was less susceptible than the laboratory strain possibly due to drop in fitness of this latter as a common phenomenon in laboratory-reared organisms based on excessive culturing (Yu et al. 2010). As the laboratory strain has been in culture quite longer than the field strain, it is conceivable that some of the higher mortality recorded in the laboratory strain is due to trait deterioration in the laboratory strain. This goes in parallel to spinosad LC 50 of C. capitata in Tunisia where 22 ppm and 127.95 ppm for laboratory and field population, respectively, were recorded (Guerfali et al. 2020). Reasonably, the field strain may simply have innate fitness factors. Nevertheless, since the field strain was approximately 1.7 fold more insensitive to spinosad than the laboratory strain, the difference might also be due to the differential susceptibility of strains and intensive insecticide selection pressure in that area of collection. Notwithstanding the above-mentioned innate fitness in field populations of insect pests, a variety of biotic and abiotic factors can contribute to the apparent difference and fitness of field population relative to laboratory one (Yu et al. 2010). Moreover, Stark et al. (2004) found that spinosad was remarkably similar in toxicity to all 3 economically important fruit fly (Diptera: Tephritidae) species: C. capitata, the melon fly Bactrocera cucurbitae (Coquillett), and the oriental fruit fly B. dorsalis (Hendel).
Obtained data proved that spinosad had not only significant effectiveness in the control of C. capitata but also its combination with S. riobrave could improve the EPN efficacy as biocontrol agent. Therefore, in order to minimize the negative effects of the chemicals on the environment and natural enemies in the management of pests, the natural insecticide spinosad could also be rotated with other pesticides or in low rates simultaneous with different insecticidal categories to avoid development of insect resistance in such programs. On the other hand, the present experiment showed a strong efficacy of the spinosad (i.e. 100% mortality) on C. capitata when applied at high rates, resulting in complete control in the laboratory bioassays. However, it is expected that the resistance to the spinosad might be developed if it is applied exclusively, i.e., without other biocontrol agent such as entomopathogenic nematodes. Moreover, field spraying of 'Succari' orange trees with a combination of spinosad-nematode material compared to either spinosad alone or S. riobrave alone and control provided acceptable control of the pest in commercial citrus cultivations. Likewise, comparable results of spinosad have been observed for wild Medflies in an eradication plan in Guatemala and Mexico (Rendon et al. 2000). Such plans should be fully exploited especially in the new citruscultivated areas as a recently published study (Dritsoulas et al. 2021) confirmed that reclaimed desert habitats of Egyptian citrus favor entomopathogenic nematode and microarthropod abundance. On the contrary, an exclusive use of spinosad may possibly generate the insect resistance (Guerfali et al. 2020). This might be indirectly shown herein by the higher resistance in field strain than laboratory strain as well. Therefore, spinosad may be used with a precaution that its long-term use can be contingent.

Conclusions
Obtained results indicated that spinosad + S. riobrave together may be used as an effective alternative tool for control of the Medfly in Egypt. As a novel alternative for synthetic chemical insecticides to control C. capitata in Egypt, this tactic should be applied on a wider scale to be incorporated into an integrated pest management programs to achieve economically feasible and environmentally sustainable agricultural systems. Additional studies are warranted to determine the economic feasibility of utilizing the combination used herein to control the Medfly.