Spiny bollworm rearing
The SBW larvae used in this study were obtained from the mass rearing culture of bollworms. It had been reared on artificial diet for several generations away from any contamination of insecticides. This artificial diet was described previously by Amer (2015). The experiment was performed under controlled conditions in an incubator at 26 ± 1 °C and 65 ± 5% RH.
Cotton sampling
Different cotton plant samples (leaves, stems and roots) were collected from various areas of Sharkia governorate, Egypt. The collected samples were placed in clean plastic bags and transferred to the laboratory for the isolation steps. The plant material was identified by Dr. Samir Teleb, Lecture of taxonomy of higher plants, Botany Department, Faculty of Sciences. Voucher specimen of this material was deposited in a publicly available herbarium.
Microbiological analysis
Bacterial isolation
Plant samples were washed by tap water, followed by sterile distilled water. Each plant sample was cut into 2 cm long segments, using an aseptic sterile blade under the laminar flow hood and was allowed to dry. The cut surfaces of plant segments were placed in Petri plates containing Nutrient Agar (NA) media (Hi media, India). Each plant segment was inoculated in triplicate. Plates were then incubated at 30 °C for 48 h. Colonies of different pigmentation and morphology were randomly selected from each plate and streaked on fresh NA plates as described above. The pure isolates were preserved in 20% glycerol at − 20 °C for other studies (Girlanda et al. 2001).
Bacterial culture preparations
Bacterial culture prepared by the broth inoculum was taken from plated material and developed in 250 ml flasks, containing 50 ml of the broth. Then the bacterial broth was cultured at 35 °C in an orbital shaker for 24 then 48 h, shaking at 100 rpm.
Characterization of most potent bacterial isolate
Characterization of isolated bacterial colony by light microscope
Developed bacterial colonies were examined daily and the purified bacteria were identified to the species level whenever possible. The identification of bacterial genera and species was carried out by the help of the universally accepted keys for the characterization of the different isolates. Morphology was based on colony shape, height and color of the colony, growth rate and margin characteristics Benson (1998).
Molecular characterization (sequence of 16S rRNA gene of DNA)
Added 200 µl of sample (liquid media that contain bacteria) in micro centrifuge tube and add 95 µl water, 95 µl solid tissue buffer (blue) and 10 µl proteinase K. Mix thoroughly and then incubate the tube at 55 °C for 2 h. Mix thoroughly and centrifugation at 12,000×g for 1 min. Transfer aqueous supernatant to a clean tube (300 ul). Add 600 ul genomic binding buffer and mix thoroughly. Transfer the mixture to a zymo-spin™ IIC-XL column in a collection tube. Centrifuge (≥ 12,000×g) for 1 min. Discard the collection tube with the flow through. Add 400 µl DNA pre-wash buffer to the column in a new collection tube and centrifuge at (12.000×g) for 1 min. Add 700 µl g-DNA wash buffer and centrifuge at (12.000×g) for 1 min. Empty the collection tube. Add 200 µl g-DNA wash buffer and centrifuge at (12.000×g) for 1 min. Discard the collection tube. Add 30 µl elution buffer, incubated for 5 min and then centrifuge at (12.000×g) for 1 min. The primers used were 27F AGAGTTTGATCMTGGCTCAG and 1492R CGGTTACCTTGTTACGACTT. Characterization was done at sigma scientific technical support laboratory Cairo, Egypt.
Bioassay
Pathogenicity effect of 70 bacterial isolates on the newly hatched spiny bollworm larvae
Pathogenicity of 70 isolated bacteria was tested against the newly hatched larvae of SBW as follow: One ml of each selected bacterial isolated broth was distributed on the surface of Petri-dishes (9 cm in diameter) containing 5 g of the artificial diet without antimicrobial agent and left until complete dryness. Twenty newly hatched larvae of SBW were transferred by a soft brush to the surface of treated diet in Petri-dishes then, they were covered by fine and soft paper below the glass cover. Other Petri-dish as a control were prepared containing the same diet but treated with one ml of sterile distilled water and left till dryness and an also twenty larvae were placed on each surface. Treated and control Petri-dishes incubated in an incubator at 26 ± 1 °C and 65 ± 5%RH. The treated and untreated larvae were transferred individually after 24 h from treatment to glass tubes (2 × 7 cm) containing untreated diet (4 g). Tubes were plugged with absorbent cotton and incubated at the above-mentioned conditions. Larval mortality was recorded after two days of treatment.
Pathogenicity of the most affected bacterial isolates on different day-old larvae of SBW
Virulence of the most affected bacterial isolates (no. 12, 18, 36, 66 & 70) against 3, 5, 7 and 10-day old larvae of the SBW were prepared as above. Then, they were fed on treated diet for 24 h. Larval mortality was recorded after 48 and 96 h of treatment (El-Didamony et al. 2016).
Biological effects of B. safensis on some biological aspects of SBW
The latent effects of B. safensis on certain biological aspects of SBW; serial dilutions of B. safensis culture (10–1, 10–2, 10–3, 10−4and 10–5) were studied to provide the most suitable concentration on studying the biological effect. Artificial diet (5 g) without antimicrobial agent was placed in a Petri dish. One ml of B. safensis 10–5 concentration and one ml of water as control were distributed on the surface of diet and left till complete dryness. Twenty-five newly hatched larvae were transferred to the surface of each Petri-dish. Each treatment and control were replicated 4 times. Treated and control Petri-dishes were covered by soft paper below their covers and placed in an incubator at standard conditions of 26 ± 1 °C and 65 ± 5% R H. After 24 h, the alive larvae were transferred to glass tubes (2 × 7 cm) containing untreated diet, kept at the same previous conditions, and followed up daily till pupation. The pupae were transferred to clean tubes until adult emergence. The newly emerged moths of each treatment and control were sexed, placed in glass chimney cages for mating (5 pairs/cage) and replicated 4 times. Inside the cage, a piece of cotton wool previously soaked in 10% sugar solution was suspended to be renewed every 48 h for moths feeding. The cages were examined daily until death of moths. The number of eggs laid by females was counted, placed in clean glass jar and incubated under the same conditions until hatched. Main parameters were estimated such as larvae and pupal durations, weight of full-grown larvae and pupae (one-day old) and pupation percent. The emergence percent and sex ratio were measured as percent of females from the total number of emerged adults. Fecundity (eggs’ number) per female and fertility were calculated. In addition to female longevity as pre-oviposition, oviposition and post-oviposition periods and male longevity were determined.
Statistical analysis
Obtained results were analyzed to compare the mean of treated and control according to Little and Hills (1975) and CoStat computer program Cohort Software. P. O. Box 1149, Berkeley CA 9471 (CoStat program 2005).
