Evaluating the repellent effect of four botanicals against two Bactrocera species on mangoes

Insects

The population of both species (B. dorsalis and B. correcta) was reared in the controlled room (25 ± 2 °C, 60 ± 5% relative humidity, and a photoperiod of L:D 14:10 h) at the South China Agricultural University, Guangzhou, China. The temperature was controlled by the air-conditioner (Gree Electric Appliances, Inc. of Zhuhai, Zhuhai, China) and the humidity was maintained by a humidifier (Jaleel et al., 2018a, 2018b). Adult flies were reared in cages (30 × 30 × 30 cm) by providing water-soaked cotton wool in a box (12 × 6.8 × 7 cm), powdered yeast and sugar (2:1) in a petri-dish (6 × 1.5 cm). Larvae were reared on a semi-artificial diet described by Jaleel, Lu & He (2018).

Mangoes

Mango (M. indica L. Hanana Datai Nong Mang, Yellow) fruits were purchased from a local market in Guangzhou, Guangdong Province, China. Fruits were bagged before the stage of ripening (to avoid the attack of wild fruit flies) in fields. To check the field infestation by wild fruit flies, six mangoes were randomly chosen and kept separately in a plastic jar (23.5 × 15.8 × 10 cm) containing a 3-cm layer of soil, either wild flies’ pupae were recovered or not. We did not recover pupae from these fruits. To find out the ripeness or sugar level, the following parameters were measured, total soluble salts (TSS) and the pericarp toughness or firmness of fruits were measured by handheld pocket refractometer pal-1 (ATAGO, PR-101a, Brix 0–45%; Tokyo Tech., Tokyo, Japan) and TMS-Pro texture analyzer (FTC-TV, Rainsville, AL, USA) with probe (one mm diameter) respectively. The hole diameter by the female fly of B. dorsalis has been reported 0.1–0.2 mm on mango fruit. Measurements were taken and recorded at three different locations on mango fruit. Fifteen replications were done (Balagawi et al., 2005; Díaz-Fleischer & Aluja, 2003; Jaleel et al., 2018a; Rattanapun, Amornsak & Clarke, 2009).

Plants

Seeds of S. brevifolium were collected at Halqa 2 Skardu (35.18°N, 75.37°E), Skardu Baltistan, Pakistan. Seeds of A. indica were collected at Multan, Punjab, Pakistan (30.16°N, 71.52°E). Seeds of P. nigrum were purchased from the local market in Guangzhou, China. The fine powder of quercetin (95%) was bought from Sigma–Aldrich Co. (St. Louis, MI, USA).

Preparation of plant extracts solutions

Five hundred grams of seeds of S. brevifolium, P. nigrum and A. indica were dried in the electric oven (DHG-9240A; Shanghai Qi Xin Ke Xue Yiqi, Co., Ltd., Shanghai, China) at 50 °C for 24 h and ground into fine powder in an electronic blender (OPY-908; Zhongshan Opaye Industry Co., Ltd., Zhongshan, China). Two hundred grams were measured and soaked in a conical flask having acetone (97%) with a ratio of 1:2 (w/v). All the filtrates were combined together and allowed to evaporate at the rotary evaporator. Each mixture was stirred by the ultrasound method for 30 min and then placed in the dark for 24 h. Then, the supernatant was filtered with a double layer of Whatman filter paper no. 42. The mentioned procedure was repeated thrice to gain maximumly extractable. To make homogenous and concentrated paste, the filtered solutions were evaporated using a rotary evaporator (RE-52AA; Shandong, Biotechnology Co., Ltd., Taian City, China) under reduced pressure and temperature at 55 °C, then made solvent free in a vacuum desiccator. Then, all extracts were preserved at 4 °C until used (Ilyas, Khan & Qadir, 2017).

Formulation of S. brevifolium, P. nigrum, A. indica and quercetin were prepared using the acetone (20%) and by mixing five drops of tween—80 (0.001%) as emulsifier with five ml of plant extract, then acetone mixture was added up to 100 ml to obtain 5% concentration of each plant extract. The other desired dilutions (2.5% and 1%) were prepared from each stock solution (5%).

Data recording

Treated and untreated mangoes were placed into the cages (45 × 40 × 40 cm) in a free-choice test. In this study, the treated mangoes with one concentration and untreated mango were put in a separate cage. Fifteen mangoes were used for each concentration. Each mango was considered a replication. The experimental design was arranged according to a Completely Randomized Design. The experimental layout has shown in Fig. 1. Twenty gravid female flies of B. dorsalis or B. correcta (18–22 days of age) were released into each cage (treatment). After 24 h, the number of female flies on the surface of treated and untreated mangoes was recorded. Observations were conducted for 10 h; each fruit was observed for 5 min in an hour. Then, after 48 h, the number of female flies on the fruit surface was also recorded using the same method. Then treated and untreated mangoes were removed from the cages, and the number of oviposition punctures/fruit was counted. Then both treated and untreated mangoes were separately kept in plastic jars (12 × 6.0 × 12 cm) having 3 cm soil layer on the bottom for pupation and covered with a muslin cloth. After 10–15 days, the soil was sieved, and pupae/mango of both species were counted.

Statistical analysis

Number of visits, ovipositional punctures, and pupae of both flies to plants were summarized as the percentage of visiting flies (the number of flies visiting treated mangoes divided by the total number of visiting flies on both treated and untreated mangoes), the oviposition punctures on the treated mangoes (the number of oviposition punctures on the treated mangoes divided by the total number of oviposition punctures on both treated and untreated mangoes), and the percentage of pupae developed in the treated mangoes (the number of pupae developed in the treated mangoes divided by the total number of pupae in both treated and untreated mangoes). Data were normally distributed; the percentage data were arcsine square root transformed prior to analysis, if necessary. One-way analysis of variance was used to analyze the number of visits (%), ovipositional punctures (%) and pupae (%). All data analyses were carried out using SPSS version 22.0 (International Business Machines Corp., Armonk, NY, USA).

Fruit characteristics

Characteristics like length (cm), width (cm), thickness (cm), TSS or brix firmness/hardness (N) of mango fruits are shown in Fig. 2.

Visits after 24 h

Female B. dorsalis visits (%) after 24 h were minimum on mangoes treated by the P. nigrum at 5% (F_{3, 20} = 21.30, P < 0.001) and 2.5% (F_{3, 20} = 11.60, P < 0.001) concentrations as compared to the other botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5% and 2.5%) (Fig. 3A). In case of B. correcta, visits (%) after 24 h were less on mangoes treated by the P. nigrum at all concentration for example, 5% (F_{3, 20} = 16.20, P < 0.001), 2.5% (F_{3, 20} = 9.92, P < 0.001), and 1% (F_{3, 20} = 6.74, P = 0.003) as compared to rest of three botanicals (quercetin, S. brevifolium and A. indica) and their concentrations (5%, 2.5% and 1%) (Fig. 3B).

Visits after 48 h

Visits (%) after 48 h done by female B. dorsalis adults were less on the P. nigrum mangoes treated at all concentration (5%; F_{3, 20} = 13.40, P < 0.001, 2.5%; F_{3, 20} = 27.20, P < 0.001 and 1%; F_{3, 20} = 10.40, P < 0.001) as compared to other three botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5%, 2.5% and 1%) (Fig. 4A). In case of B. correcta, visits (%) after 48 h were minimum on the mangoes treated by the P. nigrum at 5% (F_{3, 20} = 2.90, P = 0.045), and 2.5% concentrations (F_{3, 20} = 6.49, P = 0.003) concentrations as compared to rest of three botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5% and 2.5%) (Fig. 4B).

Oviposition punctures

The oviposition punctures made by female flies of B. dorsalis were significantly reduced on mangoes treated by P. nigrum at all concentrations for example, 5% (F_{3, 20} = 16.30, P < 0.001), 2.5% (F_{3, 20} = 11.60, P < 0.001), and 1% (F_{3, 20} = 4.46, P = 0.015) as compared to other three botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5%, 2.5% and 1%) respectively (Fig. 5A). The oviposition punctures (%) done by female flies of B. correcta were minimum on the mangoes treated by P. nigrum at 5% (F_{3, 20} = 3.76, P = 0.027) and 2.5% (F_{3, 20} = 3.03, P = 0.034) concentrations as compared to other three botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5% and 2.5%) (Fig. 5B).

Pupae

The retrieved pupae (%) of B. dorsalis were minimum from mangoes treated by the P. nigrum at all concentrations for example, 5% (F_{3, 20} = 33.30, P < 0.001), 2.5% (F_{3, 20} = 15.20, P < 0.001), and 1% (F_{3, 20} = 9.25, P < 0.001) as compared to other three botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5%, 2.5% and 1 %) (Fig. 5A). Similarly, in the case of B. correcta, the pupae (%) were less from mangoes treated by the P. nigrum mangoes at all concentrations for example, 5% (F_{3, 20} = 6.09, P = 0.004), 2.5% (F_{3, 20} = 39.5, P < 0.001), 1% (F_{3, 20} = 8.83, P < 0.001) as compared to other three botanicals (S. brevifolium, A. indica and quercetin) and their concentrations (5%, 2.5% and 1%) (Fig. 6B). However, extracts of S. brevifolium, P. nigrum, A. indica and quercetin were effective to reduce the visits, ovipositional punctures, and pupae of both species. Among all plants, the P. nigrum was the more active repellent to B. correcta and then B. dorsalis.