Endophytic fungi from South Sumatra (Indonesia) in seed-treated corn seedlings Affecting development of the fall armyworm, Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) – Egyptian Journal of Biological Pest Control

Exploration of the fungi was performed by collecting infected-host insect cadavers from crops in South Sumatra, Indonesia, from May until June 2021. Purification and isolation of the fungi were carried out from June to July 2021. The morphological identification was carried out in the Laboratory of Entomology, Faculty of Agriculture, Universitas Sriwijaya, in July 2021, and the molecular identification was performed from August to December 2021 at the Laboratory of Agricultural Biotechnology (accredited according to the ISO/IEC 17025 standard), Department of Plant Protection, Faculty of Agriculture, Universitas Lampung, Indonesia. Experimental design used for bioassay was a completely randomized block designs consisted of seven treatments (six fungal isolates and control), and the experiment was repeated three times.

Fungal exploration, isolation, and purification

Fungal exploration from the infected-host cadavers using the method of Ab Majid et al. (2015) by collecting infected-host insects or cadaver infected with the fungi from the fields. The exploration was carried out in Tanjung Pering, Ogan Ilir, South Sumatra (3°13′23″S104°38′27″E), Tanjung Cermin, Pagar Alam, South Sumatra (4°02′23″S103°13″14″E), and Nendagung, Pagar Alam, South Sumatra (3°56′22″S103°12′15″E) (Table 1). The infected insects or cadavers were first surface-sterilized with 70% EtOH (Ethyl alcohol) and 1% NaOCl (Sodium hypochlorite), then rinsed 3 times (Elfita et al. 2019). After that, the sample cadavers were cultured aseptically onto SDA (Sabouraud Dextrose Agar) medium (Russo et al. 2020). The fungal culture was purified to make an isolate per sample. The fungal macroscopic and microscopic characteristics, such as the colonial color and shape, the conidial shape and size, and the conidiophores were observed (Herlinda et al. 2021), and then, molecular identification was performed.

DNA extraction, PCR amplification, and sequencing

DNA was extracted according to the method of Swibawa et al. (2020) and carried out on fungal conidia of 7-day-old fungus. As much as 10 ml of conidia suspension was centrifuged using CF15RXII for 10 min at a speed of 14,000 rpm. Then, 1 ml of 70% ethanol was added to the centrifuge tube and centrifuged again for 10 min. The supernatant was 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. The suspension was incubated at -40 °C for 24 h. The frozen suspension was crushed until pulverized. A total of 500 µl of pellet suspension was put into a 1.5 ml tube, and 400 µl of 2% CTAB (cetyltrimethylammonium bromide) was added, homogenized, and heated at 65 °C for an hour using a water bath (Brookfield TC 550 MX-230, USA). After the incubation, 500 µl of PCI (phenol/chloroform/isoamyl alcohol) (25:24:1) was added, homogenized, and centrifuged at 14,000 rpm for 10 min at 14,000 rpm for 10 min. A total of 600 μl supernatant was transferred to a new 1.5 ml tube, and 600 μl chloroform/isoamyl alcohol (24:1) was added, homogenized, and centrifuged (Microspin12; Biosan, Latvia) again at 14,000 rpm for 10 min. A total of 400 µl of supernatant was then put into to a new 1.5 ml tube, and 400 µl of cold isopropanol was homogenized and incubated at − 40 °C for 20 min. Then, the suspension was centrifuged at 14,000 rpm for 15 min. The supernatant was then discarded, and the pellet was added with 500 µl of 70% cold ethanol and centrifuged at 14,000 rpm for 5 min. The supernatant was then discarded, and the pellets obtained were incubated at room temperature for 24 h to dry. After drying, the pellets were added as much as 50 µl 1 × Tris–HCL EDTA (TE) pH 8.0 (1st Base Malaysia).

PCR amplification was carried out using the Sensoquest Thermal Cycler (Germany) PCR machine on ITS (the Internal Transcribed Spacer) using ITS1 and ITS4 primers (White et al. 1990). The DNA amplification stage consisted of 1 initiation cycle at 95 °C for 5 min, 30 cycles consisting of denaturation at 95 °C for 1 min, primer attachment at 52 °C for 1 min, primer extension at 72 °C for 1 min, and 1 elongation cycle at 72 °C for 5 min. Then, the PCR results were electrophoresed, using 0.5% agarose in 20 ml of 1 × Tris/Boric Acid/EDTA (TBE) buffer (1st Base Malaysia) and added 1 µl of ethidium bromide (EtBr 10 mg/ml). The electrophoresis was under taken in 1 × TBE buffer solution at 50 V for 70 min, and the results were visualized using a DigiDoc UV transilluminator (UVP, USA).

The PCR results were sent to 1^st Base Malaysia for a sequencing process. The results of the sequencing were analyzed, using Bio Edit ver. 7.2.6 for windows. The results were submitted to BLAST (the Basic Local Alignment Search Tool) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to obtain the genus or species that had the greatest homology or similarity and molecularly. The phylogeny tree was developed using the Mega 7 for Windows program (Kumar et al. 2016), using the method of UPGMA (jukes and cantor model). The ITS region sequences for several strains used as a reference in this study were obtained from NCBI (https://www.ncbi.nlm.nih.gov/).

Mass-rearing of Spodoptera frugiperda

The mass-rearing of S. frugiperda was performed using the method of Herlinda et al. (2020a, b). The eggs of S. frugiperda were obtained from the Laboratory of Entomology, Faculty of Agriculture, Universitas Sriwijaya. They were reared in laboratory for more than 5 generations at 28–29 °C temperature, and 82–83% RH and the lighting set to photoperiod 12:12 (L:D) hrs. In the laboratory, the larvae of S. frugiperda were maintained individually due to cannibal behaviors and reared using fresh maize leaves. The prepupae and pupae were replaced in a wire mesh cage (30 × 30 × 30 cm³) and inside this cage placed also fresh maize leaves for the adults to lay eggs. Emerged adults were used for bioassays.

Assessing endophytic fungal colonization

Fungal inoculation for maize seeds treated was carried out to assess the ability of the fungal colonization into the maize seedling tissue and to ensure that the fungi used in this experiment were truly endophytic. All the isolates used were grown in SDA medium incubated for 14 days, and then, the SDA fungal culture was transferred to the broth medium (SDB, Sabouraud Dextrose Broth) following the method of Gustianingtyas et al. (2020) and incubated for 7 days on the shaker and 7 days unshaken position. The 45 corn seeds for an isolate were surface-sterilized by using (Russo et al. 2020) method. The seeds were immersed in 10 ml of fungal suspension (1 × 10¹⁰ conidia ml⁻¹) for 24 h, while for the control only 10 ml of sterilized water was treated for the seeds. Then, the seeds were grown in the hydroponic medium, following the method of Novianti et al. (2020) and incubated for 14 days, and this treatment was repeated 3 times for each isolate. The tip leaves of 14-day-old maize seedlings (young maize) were cut of 5 × 5 mm² to be grown onto the SDA medium to detect the mycelia of the endophytic fungi. The leaf materials were first surface-sterilized by using method of (Russo et al. 2020) before grown onto the SDA medium. The leaf material surface-sterilized was carried out by immersion in 70% ethanol, then followed by sodium hypochlorite for 2 min, and rinsed twice in sterile distilled water, and the final rinse water was grown onto SDA and incubated for 10 days. The rest or remaining leaves were used for bioassays as described below.

Bioassay for assessing effect of corn seed treatment on S. frugiperda development

The bioassay for assessing the effect of corn seed treatment on S. frugiperda growth and development followed the method of Russo et al. (2020). The 14-day-old maize seedlings already inoculated with the endophytic fungi as described above were given to be consumed to the first instar neonate larvae of S. frugiperda, while for control treatment, the larvae were provided the non-inoculated young maize and this experiment was repeated three times. The 50 neonate larvae (hatching within 24 h) of first larvae instar were allowed to feed on the treated young maize and untreated ones (control) for 6 h or until the leaves eaten up, and this treatment was replicated three times for each isolate and the control. Then, the larvae were individually kept in a porous plastic cup (Ø 6.5 cm, height 4.6 cm) and were fed on healthy non-inoculated leaves measuring 2 cm × 5 cm per day per larvae and replaced with a fresh new one every day. The treatments of this experiment consisted of the six fungal isolates and the control (water) and used the completely randomized block designs. The variables recorded were development time of each stage (egg, larval, pupal, and adult) and mortality of each stage. The larval and pupal mortality were recorded daily, and the adults emerging were observed every day. The sex of adults emerged was recorded, and the adults were placed in the wire mesh cage for copulation with fresh maize leaves inside to allow egg-laying. Egg collection and 10% honey bee solution replacement for adults were carried out every day. The adult longevity was also observed until the adult death.

Data analysis

The differences in the length of different stages (egg, larval, pupal, and adult), mortality of each stage, adult longevity, eggs laid, and sex ratio of each treatment were analyzed by analysis variance (ANOVA). Tukey’s honestly significant difference (HSD) test (Tukey’s test) was employed to test for the significant differences among the treatments (isolates) at P = 0.05. All data were calculated using software of SAS University Edition 2.7 9.4 M5.