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. Petri-plates 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 × 106 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 d after application. Data were collected based on the number of the Medfly trapped.
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 (LC50) 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).