Aspergillus oryzae and Beauveria bassiana as entomopathogenic fungi of Spodoptera litura Fabricius (Lepidoptera: Noctuidae) infesting corn in Lampung, Indonesia

Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is an important pest causing severe damage to many cultivating plants such as corn worldwide, including Indonesia. This study was performed to obtain and identify entomopathogenic fungi (EPF) of S. litura collected from corn fields in 4 corn producing regions of Lampung, Indonesia, as well as to investigate the damage caused by this pest on corn in Lampung Province. Three corn fields in each region were selected for collecting soil samples. Soil samples were collected from 5 corn plant rhizospheres, at each field in six months of survey. Ten larvae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) were laid on each soil sample as a bait, covered with a filter paper and incubated at room temperature. The emerging fungi from T. molitor cadaver were transferred onto Potato Dextrose Agar (PDA) medium and incubated for 7 days at room temperature. Pathogenicity test was determined against 3rd instar of S. litura larvae. Identification was performed based on the sequence of Internal Transcribed Spacer (ITS) Region. Observations on the corn damage caused by S. litura were conducted at all corn producing areas in Lampung. Twelve fungal isolates were obtained causing 0–75% of mortality of S. litura. Four fungal isolates (NKPT, SKHJ, SDHJ and RAHJ), which caused mortality more than 20%, were further identified. One isolate (NKPT) was confirmed as Beauveria bassiana and the other 3 isolates (SKHJ, SDHJ and RAHJ) were Aspergillus oryzae. S. litura generally caused slight damages to the corn which was found in every observation year performed during 2010–2019. Medium plant damage was observed in 2010–2012 and 2018–2019, severe damage was found in 2011 and crop failure was recorded in 2018. Aspergillus oryzae and B. bassiana were the EPF recorded infecting S. litura in corn in Lampung Province. This was the first report on the isolates of A. oryzae as EPF of S. litura in Indonesia. Slight damages with S. litura were always recorded in every observation year but not for those of medium and severe damages and crop failure.

Page 2 of 12 Fitriana et al. Egypt J Biol Pest Control (2021) 31:127 litura has a high migratory ability and is widely distributed in both tropical and temperate regions. A report in the Philippines confirmed that S. litura caused moderate to severe damages to corn (Gerpacio et al. 2004). The damage caused by this pest on corn in Lampung Province has not been fully revealed.
Recently, the use of insecticides for controlling S. litura has continuously increased, making it possible to become resistant. It has been reported that S. litura is now being resistant to several groups of insecticides resulting in more difficulties for controlling it. The increase in public awareness on the negative impacts on the use of pesticide, especially on human health and environment has led to finding and developing alternative control strategies eco-friendly. One of which is using entomopathogenic fungi (EPF). Application of EPF has no harmful residues and improves the balance of the bionetwork within the ecosystem. Once the EPF has spread in host populations, it provides lifetimes of pest control (Scheepmaker and Butt 2010).
Exploration to obtain biological control agents, including EPF is the first step in the implementation of the biological control techniques. The EPF can be obtained from the infected insects, soils or plants' rhizosphere (Singh et al. 2016). Tesfaye and Seyoum (2010) reported that temperature conditions were one of the factors that influenced pathogenicity of the isolate of EPF. Since the temperature condition varies among regions, indigenus EPF can be one of the alternatives to provide better results since they are more adapted to the local environment (Sayed et al. 2019).
This study was performed to obtain and identify EPF of S. Litura, collected from corn field in 4 corn producing areas in Lampung Province, as well as the potential of EPF against the pest on corn fields in the Province.

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 m 2 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 Page 3 of 12 Fitriana et al. Egypt J Biol Pest Control (2021) 31:127 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).

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

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

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).

Corn damage caused by S. litura in Lampung Province
The  (Fig. 6). The damage caused by S. litura was initially observed in January and continued rising until July. The damage decreased in August and September and again raised in October to December, with the most damage observed in November.
During a year observation, the lowest damage was found in August and September (Fig. 7).

Weather conditions in Lampung Province
In January, heavy rainfall occurred (274.70 mm) and then decreased in the next months. During July to September, rainfall was lower than 100 mm, with the lowest rainfall observed in August (54.88 mm). The rainfall increased above 100 mm in October (103.64 mm) and increased until December (256.14 mm). The highest rainy days were observed in January (19.39 days) and continue to decrease in the months after. The smallest rainy days occurred in August ( Fig. 1 Percentage of mortality of Spodoptera litura caused by fungal application. The numbers followed by the same letter are not significantly different based on least significant difference test (LSD) analysis at the 5% of significance level. Statistical analysis was performed on the data which were transformed using √x + 0.5. Treatments were arranged using a completely randomized design (CRD). The data are means collected from observation of the mortality of 10 individuals of S. litura applied with entomopathogenic fungi. Assessment was conducted in 3 replicates Page 6 of 12 Fitriana et al. Egypt J Biol Pest Control (2021) 31:127 obtain EPF from soil such as B. bassiana (Sharma et al. 2018). Six out of 12 fungal isolates obtained in this study were confirmed causing mortality to S. litura (3.57-75%). The highest mortality rate was produced by NKPT (75%), followed by SKHJ (60.71%), SDHJ (50%), RAHJ (25%), SDPK (3.57%) and RRPK (3.57%). Some natures of EPF influenced their capability to cause death of herbivorous insects, i.e., spore production and viability (Rosmiati et al. 2018), penetration (Mora et al. 2017), infection (Santos et al. 2018) and enzymes produced by each EPF (Dhawan and Joshi 2017). Different EPF produce several enzymes and toxins. Chitinase was produced by Aspergillus spp., which can degrade chitin of fungal pathogens and pest insects (Purkan and Sayyidah 2016).
Further identification was performed on the 4 isolates which produced mortality more than 20%, i.e., NKPT, SKHJ, SDHJ and RAHJ. Based on the BLAST search result, the NKPT was closely related with the group of B. bassiana; meanwhile SJHJ, SDHJ and RAHJ shared 100% similarity with A. aflatoxiformans, A. flavus and A. oryzae. Based on the phylogenetic tree analysis revealed that NKPT was placed within the group of B. bassiana (Fig. 3); meanwhile, SJHJ, SDHJ and RAHJ were placed within a group of A. aflatoxiformans and A. flavus and A. oryzae (Fig. 4). In the case of NKPT, the isolate was B. bassiana; meanwhile the other 3 isolates should be carefully analyzed on their characters to define their species identity.
The genus of Aspergillus was divided into 4 subgenera (Aspergillus, Circumdati, Fumigati and Nidulantes) and 20 sections. A. oryzae is a representing domesticated group of A. flavus. This species can be differentiated from the other members of A. flavus-clade on their inability to produce aflatoxin (Frisvad et al. 2019).
The three isolates of Aspergillus found in this study (SKHJ, SDHJ and RAHJ) were confirmed on their inability to produce aflatoxin. They did not produce   Peterson (2008), Schneider et al. (2011), Zulkifli and Zakaria (2017) and Frisvad et al. (2019). T = ex type isolate Page 8 of 12 Fitriana et al. Egypt J Biol Pest Control (2021) 31:127 any fluorescence when they were cultured on the YES medium supplemented with 2% of methyl-ß-cyclodextrin. The fluorescence was observed on A. flavus BIO 3338, an aflatoxin producing-isolate, which was used as a positive control. YES medium, which was added by 2% of methyl-ß-cyclodextrin, was reported to be used to differentiate Aspergillus aflatoxin producing-isolate from nonaflatoxin producing-isolate by its capability to produce fluorescence when it was observed under UV light (Fente et al. 2001). Here, it was concluded that SKHJ, SDHJ and RAHJ isolates were A. oryzae. To our knowledge, this is the first report of A. oryzae isolates as an EPF of S. litura in Indonesia. Aspergillus oryzae was established and initially used for food production about 2000 years ago in China (Baker and Bennett 2008). As well as in China, this fungus is also extensively employed for food production in Japan, Korea, Thailand and Indonesia (Baker and Bennett 2008). The beneficial aspect of A. oryzae other than its use in food production, especially as a biological control agent, has not been widely reported. The role of A. oryzae as EPF was firstly reported in (Zhang et al. 2015. They described an isolate of A. oryzae, namely XJ-1, showed capability as EPF on locusts. In this study, the 3 isolates of A. oryzae (SKHJ, SDHJ and RAHJ) were found pathogenic to S. litura. Since A. oryzae is not pathogenic to humans, animals or plants (Chuang et al. 2019), it has Generally Regarded as Safe (GRAS) status from Food and Drug Administration (FDA) United States of America (Sewalt et al. 2016). This A. oryzae has a great potential to be developed for biological control agent of herbivorous insects.  et al. Egypt J Biol Pest Control (2021) 31:127 In this study, native corn isolates of EPF belong to B. bassiana (NKPT) and A. oryzae (SKHJ, SDHJ and RAHJ) as pathogen of S. litura infested corn were confirmed. Pathogenicity tests revealed the potential of EPF to be used as a biological control agent against target pest. The EPF found in this study (NKPT, SKHJ, SDHJ and RAHJ) originated from the corn rhizosphere. Here, it will provide more advantages if they were used for controlling S. litura on corn, since it is already well adapted to the corn field environment. However, further study is needed, especially on their optimum capability for controlling S. litura in the field as well as their stability and tolerance to environmental pressure and their effect on non-target organisms including natural enemies.
Application of EPF has been reported to have no or limited adverse effect on some natural enemies (Roy and Pell 2000). For example, application of B. bassiana showed no negative effect on the survival, duration, adult Page 10 of 12 Fitriana et al. Egypt J Biol Pest Control (2021) 31:127 longevity, and fecundity of predator Coccinella undecimpunctata L. and Hippodamia variegate L. (Sayed et al. 2021). The EPF, Metarhizium brunneum, could be potentially applied against Delia radicum with a limited danger to its parasitoid Trybliographa rapae (Rännbäck et al. 2015). On the other hand, some other natural enemies have been reported negatively influenced by EPF application (Abbas 2020). Both strains of B. bassiana (AL1 and ATCC 74040) have also been reported negatively affecting Encarsia formosa, a parasitoid of Trialeurodes vaporariorum (Oreste et al. 2016). Fluctuate damage of corn caused by S. litura was recognized in Lampung Province. The damage was slight (1 to ≤ 25% of plant damage) damage to crop failure (> 85% of plant damage) with slight damage being the most damage observed in each year. The medium (> 25 to ≤ 50% of plant damage) and severe (> 50 to ≤ 85% of plant damage) damage were rarely found in the field. During 2010-2019, the lowest damage area was observed in 2015, with 10 ha of slight plant damage without any medium damage, severe damage and crop failure.
Research performed by Fand et al. (2015) revealed that the development of all the immature stages of S. litura linearly increased until on or about 34-36 °C, but not after this range of temperature. The temperature of 38 °C caused lethal to larval and pupal stages of S. litura and there will be no development to the next stage. Females were unable to lay eggs at low (15 °C) and high (> 35 °C) temperatures. The highest damage of S. litura on cotton was observed when maximum temperature was in the range of 32-35 °C and the minimum temperature was 24-26 °C (Selvaraj et al. 2010). Alteration of temperature negatively influenced feeding performance of 2nd instar of S. litura to yellow cress (Rorippa dubia) (Pham and Hwang 2020). The outbreaks of this pest insect also occurred in heavy rainfall conditions after a long dry spell (Thanki et al. 2003).
The lowest damage of corn in Lampung Province was found in August and September gets together with high temperature and low rainfall.