Sampling
Soil samples were collected from corn rhizosphere in 4 regions (districts), namely Bandar Lampung, Pesawaran, Lampung Selatan and Lampung Timur. Three corn fields with a minimum area of 50 m2 were selected in each region. One corn field was selected in each sub-district. Soil samples were collected from 5 corn plant rhizospheres at 10–15 cm of depth, which were diagonally and randomly chosen. As much as 500 g of soil were taken from each plant’s rhizosphere and composited. Totally, 1000 g of the composite soil sample from each field was collected and brought to the laboratory.
Isolation of Entomopathogenic fungi
Isolation of the potential EPF was performed in the Laboratory of Agricultural Biotechnology, Faculty of Agriculture, University of Lampung, Indonesia, using baiting method (Tarasco et al. 2020). The soil samples were sieved by a 600 mesh strainer and moved into plastic trays (35 × 28 × 7 cm). Ten larvae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) were placed on the soil and covered with filter paper. The trays were then incubated in a dark condition at room temperature. Observation was performed for 14 days on the fungus, which emerged on the body surface of T. molitor. The infected T. molitor larvae were sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 30 s. The emerging fungus was taken and cultivated on Potato Dextrose Agar (PDA) medium (Himedia, India) and incubated for 7 days at room temperature.
Pathogenicity test of the obtained entomopathogenic fungi
The 3rd instar larvae of S. litura were used for pathogenicity tests. Ten larvae were placed into sterile plastic petri dish (9 cm of diameter) containing 7 days old fungi, which were obtained from T. molitor. The larvae of S. litura were rolled over to make sure that the body surface of the larvae was completely covered by the mycelium or conidia of the fungi. The larvae were then transferred into plastic jars (14 cm of diameter) containing fresh leaves of Ricinus communis Linnaeus (Malpighiales: Euphorbiaceae) as food. For the control, the healthy Spodoptera larvae were directly placed into a plastic jar. Observation was performed for 14 days on the deaths of S. litura. Percentage of mortality (PM) was calculated using formula [(a/b) × 100%]; a = number of the death of S. litura; b = Total of S. litura observed.
Identification of the entomopathogenic fungi
Identification, conducted to the fungi causing death of Spodoptera larvae, was performed based on the sequence analysis of the Internal Transcribed Spacer (ITS) region.
DNA extraction
DNA extraction was conducted based on the method performed by Swibawa et al. (2020). The conidia of 7 days old of Aspergillus and 21 days old of Beauveria (those were cultured on PDA in sterile plastic petri dish with 9 cm in diameter) were harvested by added with 10 ml of sterile distillate water and carefully grabbed using drigalski. The suspension was then transferred into a 30 ml of centrifuge tube and centrifuged at 14,000 rpm for 10 min, using CF15RXII (Hitachi, Japan). After centrifugation, 1 mL of 70% cold ethanol was directly added into the tube and centrifuged at 14,000 rpm for 10 min. The supernatant was then removed and 1 mL of extraction buffer (0.5 mL Tris HCl, 1 mL SDS 1% + 2.8 mL NaCl, 0.2 mL Mercaptoethanol, 2 mL EDTA, 3.5 mL sterile water) was added to the tube and suspended. The pellet suspension was shifted into a mortar and incubated at – 40 °C for 24 h. After incubation, the frizzed suspension was pounded until completely crushed. In total, 500 µL of pellet suspension was transferred into a 1.5-mL tube. As much as 400 µL of 2% cetyl trimethylammonium bromide (CTAB) was added, gently homogenized and incubated at 65 °C for 1 h, using a water bath (Brookfield TC 550 MX-230, USA). After incubation, 500 μL of Phenol Chloroform Isoamyl Alcohol (PCI) solution (25: 24: 1) was added, hardly homogenized, and centrifuged at 14,000 rpm for 10 min. In total, 600 µL supernatant was conveyed into a new 1.5-mL tube. As much as 600 µL of Chloroform Isoamyl Alcohol (CI) solution (24:1) was added, homogenized, and centrifuged at 14,000 rpm for 10 min. Totally, 400 µL of the supernatant was relocated into a new 1.5-mL tube. As much as 400 µL cold isopropanol was added into the tube, gently homogenized by hand, and incubated at – 40 °C for 20 min. After incubation, the tube was centrifuged at 14,000 rpm for 15 min. The supernatant was discharged, and 500 µL of cool 70% ethanol was added and centrifuged at 14,000 rpm for 5 min. The supernatant was discharged, and the pellet obtained was air-dried at room temperature for 24 h. After air-dried, 50 µL 1 × Tris–HCL EDTA (TE) pH 8.0 (1st Base Malaysia) was added. All centrifugation processes after incubation at − 40 °C were performed using a centrifuge Microspin12 (Biosan, Latvia).
PCR amplification
Amplification was conducted with Sensoquest Thermal Cycler Machine (Germany). PCR amplification was performed on Internal Transcribed Spacer (ITS) region, using primer ITS1 (5'TCC GTA GGT GAA CCT TGC GG 3') and ITS4 (5'TCC TCC GCT TAT TGA TAT GC 3') (White et al. 1990). PCR was performed in total volume 25 µL consist of 12.5 µL Master Mix (Red Mix) (bioline), 1 µL of 10 µM of primer ITS 1 and ITS 4, 1 µL DNA template (⁓1 µg/µL) and 9.5 µL sterile distilled water. DNA amplification covering one cycle of the initiation at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 1 min, annealing at 46–52 °C for 1 min, primer extension at 72 °C for 1 min, and one cycle of elongation at 72 °C for 5 min. The PCR results were electrophoresed by 0.5% agarose gel suspended in 20 mL 1× buffer Tris-Boric Acid-EDTA (TBE) (1st Base Malaysia) containing 1 µL Ethidium Bromide (EtBr 10 mg/mL). The electrophoresis was conducted using a 1× TBE buffer at 50 V for 70 min. The results were visualized using DigiDoc UV transilluminator (UVP, USA).
Sequencing and analysis of the result
The PCR product was sent to 1st Base Malaysia for sequencing. The sequencing results were analyzed using Bio Edit program ver. 7.2.6 for windows and submitted to Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to obtain the possible identity. The dendrogram was constructed using Mega 7 program for Windows (Kumar et al. 2016) by neighbor joining method (jukes and cantor model). Reference strains used in this study were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). Detailed information of the reference strains is shown in Additional file 1: Table S1.
Aflatoxin production test
Assessment was performed on the Aspergillus spp. showed capability to cause death of S. litura based on method described by Fente et al. (2001). The 7 days old isolates of Aspergillus spp. were cultivated on yeast extract with supplements (YES) medium (Himedia, India) added to 2% of methyl-ß-cyclodextrin (Sigma Aldrich, USA) and incubated at room temperature. An isolate of A. flavus BIO 3338, an aflatoxigenic fungi collection of Indonesian Culture Collection (InaCC), which was isolated from diseased peanuts, was used as positive control. Observation was performed 5 days after incubation under UV light (356 nm). Aflatoxigenic isolates showed fluorescence but not for non-aflatoxigenic isolates (Fente et al. 2001).
Corn damage caused by Spodoptera litura in Lampung Province
The damage of corn data was obtained from the survey conducted by the Crop and Horticultural Plant Protection Agency of Lampung Province from 2010 to 2019. Survey performed in all corn producing area in Lampung (Bandar lampung, Lampung Selatan, Lampung Tengah, Lampung Timur, Lampung Utara, Lampung Barat, Metro, Pesawaran, Tulang Bawang, Tulang Bawang Barat, Pesisir Barat and Way Kanan, Pringsewu), using method described by Direktorat Perlindungan Tanaman Pangan (2018). The data collected in this study was the damaged area caused by S. litura which were divided into 4 levels namely slight (1 to ≤ 25% of plant damage), medium (> 25 to ≤ 50% of plant damage), severe (> 50 to ≤ 85% of plant damage), and crop failure (> 85% of plant damage).
Weather data in Lampung Province
Rainfall and rainy days were collected from ombrometer collected from 15 sub-districts in Lampung Province, during the years of 2013–2019. Minimum and maximum temperature data were obtained from Statistics of Lampung Province during the year of 2010–2017, which can be freely downloaded from https://lampung.bps.go.id/subject/151/iklim.html#subjekViewTab3.
Statistical analyses
Pathogenicity test was arranged using Completely Randomized Design (CRD) with 3 replicates. The data was analyzed by one-way analysis of variance (ANOVA). If there is a significant difference between the means of two or more isolates, further analysis was carried out using Least Significant Difference (LSD) test. Statistical analysis was performed with R Statistical Software (version 4.1.1; R Foundation for Statistical Computing, Vienna, Austria).