Isolation and identification of Streptomycessp. AP-123
Streptomyces sp. AP-123 was isolated from Andra Pradesh coast of the Bay of Bengal, India. The 16S rDNA gene (accession number JQ283107) based phylogenetic affiliation was determined by using bioinformatics tools identified Streptomyces sp. AP-123 as Streptomyces sp. with 99% sequence similarity to Streptomyces flavogrecius (Figure 2).
Isolation and identification of polyketide metabolite
Isolation of polyketide metabolite and its identification have already been described in our earlier manuscript [10].
Insect culture collection and monitoring
Larvae of S. litura and H. armigera were collected from the farmers’ field in Kancheepuram district, Tamil Nadu. Insects were cultured by following the methods of Basker et al. [20]. Briefely, the collected H. armigera larvae were reared individually in a plastic container (vials) and fed regularly with lady’s finger, Abelmoschus esculentus L. (Malvaceae) and S. litura larvae were reared on castor leaves and were kept till the larvae became pupae under the laboratory conditions (27 ± 2°C and 74 ± 5% relative humidity). The sterile soil was provided for pupation. After pupation, the pupae were collected from the soil and placed in inside the cage for emergence of adults. Cotton soaked with 10% honey solution (Dabur Honey, India) mixed with a few drops of multi-vitamins (Hi-Media, Mumbai) was provided for adult feeding to increase the fecundity. Potted cowpea plants were kept for H. armigera and groundnut plants were provided for S. litura separately inside the adult emergence cages for egg laying. After hatching, the larvae were collected from the cage and fed with standard artificial diet as recommended by Koul et al. [21] for H. armigera. Castor leaf was provided for S. litura.
Antifeedant activity of the polyketide metabolite
Antifeedant activity of polyketide metabolite was evaluated using leaf disc no-choice method described by Basker et al. [20]. Briefly, fresh young cotton (H. arigera) and castor (S. litura) leaves were collected and cleaned thoroughly with water to remove the dust and other particles and then wiped with cotton to remove the moisture content, after that leaf discs of 4 cm diameter were punched using cork borer. Four different concentrations of the isolated metabolite such as 125, 250, 500 and 1000 ppm were evaluated in this study. The leaves disc were dipped into the metabolite for 15 min. Acetone (Thermo Fisher Scientific India Pvt. Ltd, Mumbai, India) was used as negative control since acetone was used to dissolve the compound and leaf discs dipped in azadirachtin (40.86% purity, obtained from EID-Parry India Ltd., Chennai) was used as positive control. In each plastic petridish (1.5 × 9 cm) wet filter paper was placed to avoid early drying of the leaf discs. Third instar larva of the respective insects was introduced into each petriplates. Progressive consumption of treated and control leaves by the larvae after 24 h was assessed using Leaf Area Meter (Delta-T Devices, Serial No. 15736 F 96, UK). Leaf area eaten by larvae in treatment was corrected from the negative control. Each concentrations were maintained as five replicates with 10 larvae per replicate (total, N = 50). The experiment was performed at laboratory conditions (27 ± 2°C) with 14:10 photoperiod and 75 ± 5% relative humidity. Antifeedant activity was calculated according to the formula of Bentley et al. [22].
Larvicidal activity of the polyketide metabolite
Larvicidal activity was studied using leaf disc no-choice method Basker et al. [20]. Briefly, fresh cotton and castor leaf were obtained from the garden was used in this study. After cleaning the leaves with water leave discs were made and dipped in different concentrations of the compound and assayed as mentioned in antifeedant experiment. After 24 h the larvae were continuously maintained on the untreated fresh cotton and castor leaves for H. armigera and S. litura, respectively. Insect diet was changed every 24 h. Larval mortality was observed and recorded after 96 h of treatment. Five replicates were maintained for each treatment with 10 larvae per replicate (total N = 50). The laboratory conditions were maintained as same as in the antifeedant experiment. Percent mortality was calculated according to Abbott [23].
Pupicidal activity of the polyketide metabolite
The larvae which survived were continuously fed with normal diet as specified in larvicidal activity until they became pupae and adults. Pupicidal activity was calculated by subtracting the number of emerging adults from the total number of pupae.
Larval and pupal durations
The survived larvae in the treatments were reared on fresh untreated leaves and their larval duration after the treatment was recorded. Pupal period was calculated from the day of pupation to the day of adult emergence.
Statistical analysis
The data related to antifeedant, larvicidal and pupicidal activities and larval–pupal durations were analysed by one way Analysis of Variance. Significant differences between treatments were determined using Tukey’s multiple range tests (P ≤ 0.05). Probit analysis was done to calculate median lethal concentration (LC50) and LC90 using SPSS 11.5 version software package [24].
