Insect culture
Whitefly cultures, reared (Gossypium hirsutum on 26 ± 1 °C, 70 ± 10% R.H. and 14:10 h light: dark photoperiod) at the Engineering Research Center of Biological Control, Ministry of Education, South China Agricultural University, were used during this study.
Collection of soil samples and isolation of fungi
Samples of soil for fungal isolation were collected from different provinces of the People’s Republic of China during 2015–2017. The samples were collected from 10 cm below the soil surface and placed in plastic bags. Collected samples were stored at 4 °C. Fungi were isolated by the methods of Imoulan et al. (2011) and Du et al. (2019). Briefly, a mixture of soil sample (3 g) and 30 ml sterilized 0.05% Tween-80 solution in ddH2O was prepared by stirring for 15 min. The prepared suspension (1 ml) was inoculated on PDA plates, followed by incubation at 25 ± 1 °C and 80 ± 5% R.H, 16:8 h (light/dark photoperiod). The PDA plates were observed after 7 days of inoculation, followed by re-inoculation of grown fungi to fresh PDA plates. The inoculation process was repeated until pure fungal culture was obtained. According to the phenotypic characteristics and fungal morphology (Saito and Brownbridge 2016), the cultured fungi were purified until there was only one fungus colony on the PDA plate.
Morphological characterization
Morphological characters of 4 fungal isolates (XI-1, XI-4, XI-5, and J27) were studied through inoculating fungal mycelial plug (1 cm Ø) observed by culturing small fungal mycelia PDA block draped by a cover slip for 10 days (Imoulan et al. 2011; Du et al. 2019). The slides were stained by lactophenol cotton blue and were observed under a phase-contact microscope (×100). Conidial images were taken through Axio Cam HRc camera (Carl Zeiss) having the AxionVision SE64 Release 4.9.1 software.
Fungal growth and conidia production
Expansion rates of fungal colonies and conidia production by different P. lilacinum isolates were observed, following Ali et al. (2009). Fungal mycelial plugs (1 cm Ø) were cultured on PDA plates, followed by incubation at 25 ± 1 °C and 80 ± 5% R.H and 16:8 h (light/dark photoperiod) for 10 days. Colony growth was measured every day, and conidia were scrapped from PDA plates 10 days post inoculation. Conidia were added into 100 ml of 0.05% Tween-80. Muslin cloth was used to filter the conidial suspension. Total conidial yield was calculated by counting the conidia in a hemocytometer under a phase-contrast microscope at × 40 (Ali et al. 2009).
Observation of spores of strains by scanning electron microscopy
Conidia were harvested from a PDA plate, and the conidial suspensions at the 1 × 107 conidia/ml were prepared by using deionized water containing 0.05% Tween-80 solution. Conidial suspensions (8 ml) were inoculated into 100 ml SDA medium (1% peptone, 4% glucose), and the inoculated medium was cultured at 26 °C, 180 rpm for 5 days. The hyphae were obtained through a vacuum suction pump and were fixed with 2.5% glutaraldehyde at 4 °C for 24 h, rinsed with phosphate buffer solution, dehydrated, and dried with alcohol, and the sample was fixed on the sample stage with a conductive adhesive and photographed with a scanning electron microscope (SU8020, Hitachi, Japan).
DNA extraction, PCR amplification, and sequence analysis
Genomic DNA of newly isolated fungi was obtained by using a fungal DNA isolation kit (Ezup, Sangon Biotech, Shanghai, China). The obtained DNA served as a template for amplification of ITS (internal transcribed spacer) fragment through PCR. Reaction mixture (50 μl) was used to perform all PCR reactions. The ITS primers (ITS4F: TCCTCCGCTTATTGATATGC; and ITS5R: GGAAGTAAAAGTCGTAACAAGG) reported by White et al. (1990) were used to amplify ITS regions. The PCR cycling and conditions of Du et al. (2019) were used for amplification of ITS regions. The purity of PCR products was observed through agarose gel electrophoresis, followed by GenGreen (TianGen Biotech, Beijing, China) staining. Pure PCR products were sequenced by Shanghai Majorbio Bio-pharm Technology (Shanghai, China). Geneious version 9.1.4 was used to assemble the sequences, followed by GenBank blast. MEGA version 7.0 was used to compare the sequences whereas Kimura-2-parameter (K2P) was used to build maximum likelihood (ML) tree (Kimura, 1980; Goloboff and Catalano, 2016).
Insect bioassays
Pathogenicity of isolated fungi against B. tabaci
The newly isolated P. lilacinum strains (XI-1, XI-4, XI-5, and J27) grown on PDA were used to prepare conidial suspension (1 × 108 conidia/ml), following Ali et al. (2009). The leaf dip bioassay method of Zhang et al. (2017) was used to carry out the pathogenicity studies. Cotton leaves having 2nd instar B. tabaci nymphs were dipped in the fungal suspension for 30 s. The leaves were air dried and placed in ø 9 cm having a moist filter paper for moisture maintenance. B. tabaci nymphs, dipped in 0.01% Tween 80, served as control. Four cotton leaves having 100 B. tabaci nymphs/leaf were used in each treatment. The whole experimental setup was placed at 25 ± 1 °C and 80 ± 5% R.H and 16:8 h (light/dark photoperiod). The whole experiment was performed thrice (with fresh batch of insects and fresh conidial suspension). Insect mortality was recorded on daily basis until 7 days post treatment.
Pathogenicity of P. lilacinum isolate XI-5 against immature instars of B. tabaci
Different conidial concentrations (1 × 108, 1 × 107, 1 × 106, 1 × 105, and 1 × 104 conidia/ml) of P. lilacinum isolate XI-5 were prepared following Ali et al. (2009). Cotton leaves having B. tabaci nymphs (2nd, 3rd, or 4th instars) were dipped in each conidial concentration for 30 s. The leaves were air dried and placed in ø 9 cm having a moist filter paper for moisture maintenance. B. tabaci nymphs, dipped in 0.01% Tween 80, served as control. Four cotton leaves having 100 B. tabaci nymphs/leaf were used in each treatment. The whole experimental setup was placed at 25 ± 1 °C and 80 ± 5% R.H and 16:8 h (light/dark photoperiod). The whole experiment was performed thrice (with fresh batch of insects and fresh conidial suspension). Insect mortality was recorded on daily basis until 7 days post-treatment.
Transmission electron microscopy
Second instar B. tabaci nymphs treated with P. lilacinum isolate XI-5 (1.0 × 108 conidia/ml) were observed for external as well as internal anatomical changes after different 3, 5, and 7 days post-treatment. The insects’ samples were treated and observed following Du et al. (2019).
Statistical analysis
Data regarding colony expansion rate and conidial yield were analyzed through ANOVA-1, and significant differences among means were calculated through Tukey’s HSD test (P ≤ 0.05). Insect mortality values were percent-transformed, followed by probit analysis (Finney et al. 1971). The SAS 9.2 software was used for all data analysis (SAS et al. 2000).