Endophytic fungi confirmed as entomopathogens of the new invasive pest, the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), infesting maize in South Sumatra, Indonesia

The fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), is a new invasive pest in Indonesia causing financial losses. S. frugiperda larvae hide in the leaf midrib all day. To overcome the hidden larvae, pathogenic endophytic fungi are needed. The objective of this research was to molecularly identify the endophytic fungal species from South Sumatra and determine the most pathogenic species against S. frugiperda larvae. Endophytic fungal identification was based on morphological and molecular characteristics. The molecular identification was based on gene sequential analysis of Intergenic Transcribed Spacer (ITS) 1 and ITS 4. Bioassay of the endophytic fungal species was treated against the 2nd larval instar of S. frugiperda. The results of molecular identification revealed endophytic fungal species consisted of Chaetomium sp. (GaTpeOi, JgTjPr, PiCrPga, and CaCjPga isolates), Aspergillus niger (L.) Van Tieghem (PsgTjPr, JgByU, and JaBuBys), Beauveria bassiana (Balsamo) Vuillemin (JgSPK, JaGiP, JaSpkPGA(2), JgCrJr, and JaTpOi (1) isolates), Curvularia lunata (Wakker) Boed. (JaGiPRB, CMTJP, JaMsBys, JaSpkPga(3), and JgTgSr isolates), Aspergillus flavus Link. (JgPWSR isolate), Penicillium citrinum Thom F 1539 (JaTpOi(2) isolate), and Metarhizium anisopliae (Metschn.) Sorokin (CaTpPGA isolate). All fungal isolates were deposited in the GenBank. The endophytic fungal species of B. bassiana (JgSPK isolate) caused up to (22.67%) mortality, C. lunata (JaSpkPga(3) isolate) caused (17.33%) mortality, and M. anisopliae (CaTpPga isolate) caused (8%) mortality of the pest’s larvae. First report of B. bassiana and C. lunata isolated from maize (Zea mays L.), while M. anisopliae was isolated from red chili (Capsicum annuum L.) as entomopathogenic endophytic fungi against S. frugiperda larvae in Indonesia. The endophytic fungi species of B. bassiana, C. lunata, and M. anisopliae had potentials as entomopathogens of S. frugiperda.

it has spread throughout the world and its first record in Indonesia was in March 2019 in West Sumatra (Sartiami et al. 2020) and in July 2019 in South Sumatra (Hutasoit et al. 2020). Now, it has been spread all over Indonesia. The pest attacks maize (Zea mays L.) and various other plant species by means of its larval stage, eating leaves, stems, flowers, fruits, growing points, and the whole maize plant until it becomes bare (Montezano et al. 2018). Larvae are found on the surface of leaves or maize stalks in the morning and then hide in the leaves' midribs (Gustianingtyas et al. 2021). Such behavior makes it difficult to be controlled.
Laboratory experiments of Herlinda et al. (2020a) showed that the entomopathogenic fungus (EPF), Metarhizium sp., could kill (78%) of S. frugiperda larvae. Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschn.) Sorokin, and M. rileyi (Farlow) Samson also killed more than (80%) S. frugiperda larvae (Ramanujam et al. 2020). Their treatments were applied topically (direct contact) with the larvae exposed. In the field, the presence of hiding larvae in the midribs all the day makes the contacted EPF less effective (Gustianingtyas et al. 2021). To overcome such behavior, EPF found in plant tissues (endophytic fungi) is needed (Ramos et al. 2020). The endophytic fungi systemically colonize plant tissues and associate mutually their host plants (Lira et al. 2020). The results of previous studies have proven that 8 isolates of endophytic EPF could topically kill S. frugiperda larvae (Gustianingtyas et al. 2021); however, the endophytic fungi found in their studies were only isolated from the roots and their molecular identification had not yet been carried out.
In this study, the fungi isolated from leaves, shoots, and roots of maize plants from South Sumatra (Indonesia) were identified morphologically and molecularly and their pathogenicity to S. frugiperda larvae was evaluated.

Methods
Purification, identification, and bioassay of fungi were conducted from January to March 2021. The endophytic fungal species were identified based on the molecular analysis carried out at a laboratory accredited according to the ISO 17025 standard. Bioassay of the fungi was carried out in an incubator at controlled and constant temperature and relative humidity, respectively, 25 °C and 97%.

Exploration, isolation, and purification of fungi
Exploration of endophytic fungi was carried out by collecting leaves, shoots, and roots of maize and other crop plants around it such as bananas (Musa spp.), ridged gourd (Luffa acutangula (Roxb.) L.), and red chilies (Capsicum annuum L.). Selection of individual crop plant samples to obtain the endophytic fungi followed the method of Kasambala et al. (2018). Samples of the plant parts were placed into an ice box and then taken to the laboratory. The exploration of endophytic fungi was carried out from the lowlands to the highlands of South Sumatra, Indonesia, namely in Banyuasin District (2.8833°S 104.3831°E), Ogan Ilir District (3.43186°S 104.62727°E), Prabumulih City (3.4328°S 104.2356°E), Pagar Alam City (3°52′43.8″S 103°21′30″E) ( Table 1).
The leaves, shoots, and roots of the sample plants were cleaned and washed aseptically with running tap water. Then, the plant parts were surface sterilized using the method of Elfita et al. (2019) with 70% EtOH (ethyl alcohol) and 1% NaOCl (sodium hypochlorite) and then rinsed 3 times. The last rinse water was used to check whether the microorganisms causing surface contamination were still present and carried out by growing them onto sabouraud dextrose agar (SDA) (Russo et al. 2020). Obtaining the endophytic fungi isolates started with growing the plant parts that sterilized earlier onto a media. The roots were grown on a specific medium, the malt extract agar (MEA), following the method of Silva et al. (2018). The shoots and leaves of the sample plants were grown on SDA media. The plant parts were grown as many as 5 pieces per Petri dish with a length of 5 mm and a diameter of 1-5 mm (depending on the diameter of the leaves, shoots, and roots). Fungi growing from the leaves, shoots, and roots were purified to be one isolate per sample. The isolates were then observed for their microscopic characteristics and continued with the fungal colonization test into the maize seed tissue.

Assess endophytic fungal colonization
Fungal inoculation on maize seeds was carried out to observe the colonization of fungi into the maize seedling tissue and to further ensure that the isolates found were truly endophytic. The 15 seeds of maize were surface sterilized, using 70% EtOH (ethyl alcohol) and 1% NaOCl (sodium hypochlorite) (Elfita et al. 2019). The seeds were immersed in 10 ml of fungal suspension, with a concentration of 1 × 10 6 conidia ml −1 for 6 h, while for the control, the sterilized seeds were only immersed in 10 ml of distilled water. Then, the seeds were grown in a sterile glass bottle (250 ml volume) with a sterile filter paper (Whatman no. 42) at the bottom, which was moistened with 1 ml distilled water and incubated for 10 days. All the treatments in this experiment were repeated 3 times. After the maize seedlings were 10 days old, the stems and leaves were sliced crosswise and longitudinally with a thickness of 0.02 mm each. The slices were observed with a light microscope at 40 × magnification with 0.05% lactofenol trypan blue dye. Mycelia endophytic fungi found in the maize seedling tissue were documented. The fungal isolates that were proven to be endophytic were used for bioassays. Prior to the bioassay, the isolates were observed for microscopic characteristics for morphological identification, followed by molecular identification.

Morphological and molecular identification of fungal species Macroscopic and microscopic observation
Morphological observations were carried out based on the macroscopic characteristics of the endophytic fungi that included colony color and shape, shape, and size of conidia and conidiophores, following the method of Herlinda et al. (2020a).

DNA extraction and PCR amplification
The used endophytic fungal DNA extraction method refers to the Swibawa et al. (2020) method. DNA extraction was carried out on fungal conidia. The conidia were harvested from pure cultures of 7-day-old fungus, which was cultured in sterile Petri dishes with a diameter of 9 cm. As much as 10 ml of sterile water was placed into a Petri dish containing fungal cultures and slowly harvested, using a Drigalski. The obtained conidia suspension was transferred to a centrifuge tube (volume 30 ml) and centrifuged using CF15RXII (Hitachi, Japan) for 10 min at a speed of 14,000 rpm. After being centrifuged, 1 ml of 70% ethanol was added to the centrifuge tube and then centrifuged again for 10 min. After that, the supernatant was removed and 1 ml of extraction buffer was added with a composition of 0.5 ml Tris HCl, 1 mL SDS 1% + 2.8 mL NaCl, 0.2 ml Mercaptho Ethanol, 2 ml EDTA, 3.5 ml sterile water, and then homogenized. The suspension was transferred to a sterile mortal and incubated at -40 °C for 24 h. After that, the frozen suspension was ground until it was smooth and melted. A total of 500 µl of suspension was then transferred into a 1.5 ml tube. A total of 400 µl of 2% cetyltrimethylammonium bromide (CTAB) was then added to the tube, homogenized, and then incubated at 65 °C for 1 h using a water bath (Brookfield TC 550 MX-230, USA). After the incubation, 500 µl of Phenol Chloroform Isoamyl alcohol (PCI) (25:24:1) was added, homogenized, and centrifuged (Microspin12; Biosan, Latvia) at 14,000 rpm for 10 min. A total of 600 µl of supernatant was taken and transferred to a new 1.5 ml tube. A total of 600 µl Chloroform Isoamyl Alcohol (CI) (24:1) was added to the tube, homogenized, and centrifuged (Microspin12; Biosan, Latvia) at 14,000 rpm for 10 min. A total of 400 µl of supernatant was then transferred to a new 1.5 ml tube, and 400 µl of cold isopropanol was added, homogenized, and incubated at − 40 °C for 20 min. After the incubation, it was centrifuged (Microspin12; Biosan, Latvia) at 14,000 rpm for 15 min. The supernatant was then discarded. After the supernatant was removed, 500 µl of 70% cold ethanol was added and centrifuged (Microspin12; Biosan, Latvia) at 14,000 rpm for 5 min. The supernatant was then Page 4 of 13 Herlinda et al. Egypt J Biol Pest Control (2021) 31:124 discarded, and the pellets obtained were dried at room temperature for 24 h. After drying, the tube was then 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 the internal transcribed spacer (ITS) using ITS1 and ITS4 primers (White et al. 1990). The PCR was carried out with a total volume of 25 µl consisting of a mixture of Master Mix (Red Mix) (bioline) as much as 12.5 µl, 10 µM of primer ITS 1 (5′TCC GTA GGT GAA CCT TGC GG 3′) and ITS 4 (5′TCC TCC GCT TAT TGA TAT GC 3′) 1 µL each, 1 L of template DNA, and 9.5 µl of sterile water. The DNA amplification stage consisted of 1 initiation cycle at 95 °C for 5 min, followed by 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. The PCR results were then 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 carried out in 1 × TBE buffer solution at 50 V for 70 min. Electrophoresis results were visualized using a DigiDoc UV transilluminator (UVP, USA).

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

Calculation of conidial density and viability
All the isolates of endophytic fungi were counted for their conidial density and viability. The conidial density of the 7-old-day fungi was calculated according to the method of Sumikarsih et al. (2019), using a hemocytometer and observed with a light microscope at 40 × magnification. The viability was observed by growing 1 ml of conidia fungal suspension (1 × 10 6 conidia ml −1 ) onto 2% agar-water medium, and then, the culture was incubated for 1 × 24 and 2 × 24 h under a light microscope at 40 × magnification.

Mass-rearing of S. frugiperda
The mass-rearing of S. frugiperda was carried out, following the method of Herlinda et al. (2020a). The larvae were collected from the maize plants grown around the campus (3°13′25″S 104°39′51″E) and were reared in the laboratory for more than 5 generations to obtain the larvae with relatively homogeneous genetics and to eliminate the effects of exposure to synthetic insecticides from the fields. In the laboratory, the larvae of S. frugiperda were kept individually in a porous plastic cup (Ø 6.5 cm, height 4.6 cm) because the larvae were cannibals. The larvae were fed daily by maize leaves (measuring 2 cm × 5 cm). Prepupae were transferred to a plastic container (Ø 15 cm, height 25 cm) on which its bottom had a sterile soil (5 cm thick). The plastic container containing the prepupae was placed in a wire mesh cage (30 × 30 × 30 cm 3 ) and inside this cage placed also fresh maize leaves for the adults to lay eggs, which were changed daily. The one-day-old 2 nd instar larvae were used for the bioassay.

Bioassay of endophytic fungi against larvae of S. frugiperda
The bioassay of endophytic fungi followed the method of Zea et al. (2019). The young maize (seedling) already inoculated with the endophytic fungi and not-inoculated (control) aged 10 days old was given to the 2 nd instar larvae of S. frugiperda. Each larva was given one maize seedling whose cotyledons were already removed. The larvae were allowed to feed on the leaves and stems of the young maize for 6 h. in a sterile room (a laminar air flow cabinet), and each replication was tested for 25 individuals of S. frugiperda larvae. Then, the larvae were transferred to a porous plastic cup (Ø 6.5 cm, height 4.6 cm) and fed on healthy non-inoculated leaves measuring 2 cm × 5 cm per day per larvae. The experiment was designed, using completely randomized designs with treatments of 20 isolates, 3 replications per treatment. The dead larvae were recorded daily for 12 days, following the method of Herlinda et al. (2020b). The dead larvae were grown in the agar-water medium to confirm whether the infection was caused by the endophytic fungi or not. The number of dead larvae was used to calculate the mortality.

Data analysis
The differences in the mortality data of S. frugiperda larvae were analyzed using analysis of 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.
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Colonization of endophytic fungi on maize tissues
All fungal isolates, isolated from the leaves, shoots, and roots of the maize, bananas, ridged gourd, and red chilies were found to be 124 isolates. However, after assessing for the fungal colonization in young maize (seedlings) tissue, only 20 isolates of the fungi were confirmed as endophytic fungi (Fig. 1). Evidence of fungal colonization that entered the maize seedlings tissue was that mycelia fungi were seen entering and growing spread throughout the stem and leaf tissue. Mycelia endophytic fungi were able to invade the stem and leaf of the maize tissue, the  mycelia appeared to be branched. The size of mycelia varied daily from 13.51 to 34.25 µm. In the maize tissue, which their seeds were not inoculated by the endophytic fungi (control), the cells in the maize tissue were clean and clear without fungal colonization.

Identification results of the endophytic fungal isolates
The colony morphology of 20 isolates of the endophytic fungi showed different colors (Fig. 2) and likewise the shapes of hyphae and conidia of each isolate showed their own characteristics (Fig. 3). The colony isolates of GaTpeOi, JgTjPr, PiCrPga, and CaCjPga grown on SDA media showed similarities; namely, the colony was pale gray, hyphae and mycelia were septate, and the conidia were in the shape of letter D (asymmetric/elliptical) and non-septation. Based on the phylogenetic tree, these 4 isolates were placed within the group of Chaetomium sp. (Fig. 4). The isolates were deposited in the GenBank with the accession number MZ359734 (GaTpeOi), MZ359736 (JgTjPr), MZ359735 (PiCrPga), MZ359737 (CaCjPga) ( Table 1). The colony isolates of PsgTjPr, JgByU, and JaBuBys showed similarities, their colonies were black, the hyphae and mycelia were also black, and the conidia were nonseptate, globose in shape. The 3 isolates were placed within the group of Aspergillus niger (L.) Van Tieghem (Fig. 4). The isolates were deposited in the GenBank with the accession number MZ242060 (PsgTjPr), MZ242059 (JgByU), and MZ242058 (JaBuBys) ( Table 1).
The isolate of JgPWSR had a green colony, green hyphae and mycelia, non-septate globose conidia. The JgPWSR isolate was placed within the group of Aspergillus flavus Link. (Fig. 4). The isolate was deposited in the GenBank with the accession number MZ359829 (JgP-WSR) ( Table 1).
The color of the colony JaTpOi(2) isolate was initially white; as the fungus got older, it turned bluish green with white margin, rounded conidia. The isolate was placed within the group of Penicillium citrinum Thom F 1539 (Fig. 4). The isolate was deposited in the GenBank with the accession number MZ359812 (JaTpOi (2)) ( Table 1).
The isolate of CaTpPGA had a white colony, and as the fungus got older, it turned greenish white to dark green, green hyphae and mycelia, and the conidia are clear, cylindrical, and non-septation. The isolate was placed within the group of M. anisopliae (Fig. 4). The isolate was deposited in the GenBank with the accession number MZ242073 (CaTpPGA) ( There were 7 species from the 20 isolates of the endophytic fungi found in this study. The 4 isolates (GaT-peOi, JgTjPr, PiCrPga, and CaCjPga) were in the group of Chaetomium sp., the three isolates (PsgTjPr, JgByU, and JaBuBys) were in the group of Aspergillus niger, the 5 isolates (JgSPK, JaGiP, JaSpkPGA(2), JgCrJr, and JaTpOi (1)) were placed within the group of B. bassiana, the 5 isolates (JaGiPRB, CMTJP, JaMsBys, JaSpkPga(3), and JgTgSr) were in the group of C. lunata, an isolate (JgPWSR) was in the group of A. flavus, an isolate (JaTpOi(2)) was in the group of P. citrinum, and an isolate (CaTpPGA) was in the group of M. anisopliae.
Page 7 of 13 Herlinda et al. Egypt J Biol Pest Control (2021)  Page 9 of 13 Herlinda et al. Egypt J Biol Pest Control (2021) 31:124 Pathogenicity of the endophytic fungi against S. frugiperda larvae Out of the 7 species of the 20 isolates of the endophytic fungi found in this study, there were 6 isolates that were the most pathogenic against S. frugiperda larvae ( Table 2). The 4 isolates (JgSPK, JaGiP, JgCrJr, and JaT-pOi1) resulted to mortality rates of 22.67, 21.3%, 21.33, and 17.33% consecutively were B. bassiana. In addition, the JaSpkPga(3) isolate of C. lunata caused a fairly high mortality (18.67%) and was non-significantly different than the mortality caused by the isolates of JgSPK, JaGiP, JgCrJr, JaTpOi1. The CaTpPga isolate of M. anisopliae caused (8%) larval mortality and was non-significantly different than the mortality caused by JaSpkPga (3) isolate. The B. bassiana and C. lunata isolates were isolated from maize, while the M. anisopliae isolate was isolated from red chili. The JaSpkPGA(2) isolate of B. bassiana resulting a mortality rate of only 2.67%. Likewise, P. citrinum, Chaetomium sp., A. niger, and A. flavus caused the lowest larval mortality of less than (8%). The larvae of S. frugiperda that died because of feeding on leaves colonized by the endophytic fungi had the characteristics, i.e., dull and dark integument, shriveled body, dry, and odorless. The feces excreted by sick larvae were wetter than those of untreated larvae (control). The larvae that died as a result of treatment with the endophytic fungi when grown in SDA media and their cadavers were not overgrown with mycelia fungi.

Discussion
The isolates of the endophytic fungi found in this study had an rDNA sequence similarity value of more than 99% to the reference species (BLAST), meaning that the isolates had a high phylogenetic relationship and were in the same species. Bich et al. (2021) stated that ribosomal DNA sequences were used to determine the phylogenetic relationships of organisms to taxa species. Based on the similarity value, it is stated that if the similarity value is 100%, it means that the isolates are the same strain, whereas if the similarity value of 99% indicates that the isolates are the same species, and if the similarity value of the isolates is 89-99%, that means they are the same genus (Henry et al. 2000). An organism is declared the same species when the difference in DNA sequences is between 0.2 and 1% (Shenoy et al. 2007).
The results of identification based on the morphological characters of several species of fungi showed the similarities; for example, the morphology of the colony and the shape of the conidia isolates of B. bassiana were similar to that of P. citrinum, but after the molecular identification, they showed them different species. According to Minarni et al. (2021), proper species identification is very important in determining a species as an entomopathogen.
The fungi in this study proved to be endophytic based on the characteristics of maize stalks and leaves colonized by the fungal mycelia. According to Barra-Bucarei et al. (2020), endophytic fungus (B. bassiana) had a systemic mode of action ability to colonize their host plant (tomato). The endophytic fungi can still be found on roots, stems, and leaves of tomato up to 30 days after inoculation (Carolina et al. 2020). Endophytic B. bassiana colonized 100% of leaves, 80% of stems, and 60% of roots of maize 7 days after foliar spray and caused significant reductions in S. frugiperda growth and reproduction (Russo et al. 2020). The research results of Shikano (2018) showed the endophytic fungi were able to live in the tissue for several months depending on the age of the host plant.
The endophytic fungi species of B. bassiana (JgSPK, JaGiP, JgCrJr, JaTpOi1 isolates), C. lunata (JaSpkPga(3) Fig. 4 Phylogenetic tree based on the sequencing of internal transcribed spacer (ITS) region using UPGMA method (jukes and cantor model) performed with Mega 7 program for windows (Kumar et al., 2016). This research found 20 isolates of entomopathogenic fungi: 4 isolates placed within the group of Chaetomium sp., 3 isolates placed within the group of Aspergillus niger, 5 isolates placed within the group of Beauveria bassiana, 5 isolates placed within the group of Curvularia lunata, 1 isolate placed within the group of Aspergillus flavus, 1 isolate placed within the group of Penicillium citrinum, and 1 isolate placed within the group of Metarhizium anisopliae. A green round symbol = fungal isolates obtained from this research (See figure on next page.) Herlinda et al. Egypt J Biol Pest Control (2021) (2) isolate and all isolates of P. citrinum, Chaetomium sp., A. niger, and A. flavus showed no potential as entomopathogens of S. frugiperda with their resulting mortality rates of less than 8%. In contrast to the research, results of Gustianingtyas et al. (2021) reported that the endophytic fungi species (Aspergillus sp. and Chaetomium sp.) applied topically (contact) caused mortality rates of S. frugiperda larvae as many as 18.67 and 14.67%, respectively, and the mortality rate caused by Beauveria sp. reached 29%. This difference in the rates was due to different ways of application of the fungi; in this study, the larvae of S. frugiperda consumed the plants that had been colonized by the endophytic fungi which were not applied topically.
The larvae of S. frugiperda that were sick and died by feeding on the leaves colonized by the endophytic fungus had different characteristics from the larvae that were sick and died due to the topical suspension of the endophytic fungus. The sick larvae of S. frugiperda in this study had wetter feces, and the cadavers were not overgrown and covered by the fungal mycelia, while the sick larvae caused by contact with suspension of the endophytic fungi according to Gustianingtyas et al. (2021) had dry feces and the cadavers were covered with the fungal mycelia. The dead larvae treated by the endophytic fungi were not overgrown by mycelia fungi because the cadavers were too dry. According to El-Ghany (2015), the success of a fungus in germinating was affected by several external factors, such as humidity (more than 98%). So, if the cadavers were too dry, the fungal conidia could not germinate on the cadavers. Table 2 Mean mortality of Spodoptera frugiperda larvae treated with endophytic fungi (1 × 10 6 conidia ml −1 ), and conidial density and viability of endophytic fungi * = significantly different; values within a column followed by the same letters were not significantly different at P < 0.05 according to Tukey's HSD test