Molecular identification of three entomopathogenic fungi infecting the brown plant hopper pest in Indonesia

Abstract

Background

Brown plant hopper (Nilaparvata lugens Stal.) a very damaging pest to rice crops. One of the efforts to control it is the use of entomopathogenic fungi (EPF). Three fungal local isolates found in Indonesia were effective in controlling the brown plant hopper pest. This study aimed to molecularly identify the 3 fungal isolates. Molecular identification is very important to get the exact identity of these fungi. The accuracy of EPF identification will greatly determine the success of control. Molecular identification is based on a partial genetic analysis of the internal transcribed spacer (ITS) locus of ribosomal fungal DNA.

Result

Morphology of the local isolates named J22 and J60 were identified as Paecilomyces sp., while the isolate J34 was identified as Beauveria sp. The results of molecular identification of the isolates J22 and J60 were identified as the fungi Lecanicillium saksenae and Simplicillium sp., while isolate J34 was identified as Myrothecium sp. The results of literature search showed that the 3 fungi have never been previously reported to infect the brown plant hopper.

Conclusion

In Indonesia, 3 types of EPF, namely L. saksenae, Simplicillium sp., and Myrothecium sp., were found having the potential to control the brown plant hopper pest.

Background

Brown planthopper (BPH) Nilaparvata lugens is a major insect pest of rice that causes 20–80% yield loss through direct and indirect damage. The typical damage caused by BPH is drying of plants as if burning (hopperburn) (Balachiranjeevi et al. 2019). BPH can also transmit grassy stunt and ragged stunt viruses (Helina et al. 2019).

The frequency of BPH infestation is increasing frequently in developing Asian countries due to the killing of its natural enemies because of the use of synthetic chemical insecticides (Minarni et al. 2018). Entomopathogenic fungi (EPF) are fungi that can infect and kill insects (Litwin et al. 2020). The EPF that have been widely researched and known to be effective for controlling BPH pests are B. bassiana (Sumikarsih et al. 2019) and Metarhizium sp. (Chinniah et al. 2016). However, in their implementation in the field, the use of EPF to control BPH pests still has many weaknesses. After application in the field, insect pathogens are exposed to various abiotic stresses such as temperature and humidity (Hsia et al. 2014), UV radiation (Shafighi et al. 2014), and edaphic factors (Klingen et al. 2015).

In addition to biotic stress, the effectiveness of EPF in controlling insect pests is influenced by the diversity of varieties or strains or types of them. EPF have large genetic variations among different isolates. The pathogenicity, virulence, enzymatic characteristics, and DNA also varied among different isolates of different insects. The origin of the isolate affects the virulence diversity of the fungus against the host insect, due to the type or race or strain of the fungus (Chen et al. 2017a, b).

The results of previous studies have reported 3 effective fungal isolates to control the brown plant hopper pest. The 3 isolates caused 70–80% mortality within 3.43–4.87 days. The 3 isolates were Pasir Kulon (J22), Cipete (J34), and Papringan (J60). According to morphological characteristics, isolates J22 (Pasir Kulon) and J60 (Papringan) were identified as Paecilomyces sp., while J34 (Cipete) isolate was identified as Beauveria sp. (Minarni et al. 2020).

Accuracy of identification is very important in the use of EPF for insect pest control. Identification based on morphological characters cannot be used to distinguish fungi to the species level so it is necessary to identify them molecularly (Imoulan et al. 2017). This research aimed to precisely identify the 3 previously mentioned EPF isolates that attack the brown plant hoppers.

Methods

Identification process

Fungal isolates J22 (Pasir Kulon), J34 (Cipete), and J60 (Papringan) were identified molecularly based on a partial genetic analysis on the internal transcribed spacer (ITS) locus of ribosomal DNA of fungi. Fungal isolates that will be identified previously were grown in potato dextrose broth (PDB) liquid media. After being incubated for 72 h, the fungal mycelia were harvested, using sterile filter paper and washed with sterile distilled water. The fungal mycelia were crushed in a sterile mortar by a sterile grinder and liquid nitrogen was added. Half a gram of dry fungal biomass was transferred to a 1.5-ml micro-tube containing 600 μl of cetyl trimethylammonium bromide (CTAB) buffer solution. Afterwards, the tube was shaken out and incubated at 65 °C for 30 min, then incubated in ice for 5 min. A mixture of chloroform and isoamyl alcohol with a ratio of 24:1 of 600 μl was added to the tube. The tubes were then centrifuged at 4 °C for 10 min at a speed of 25,000×g. The supernatant was transferred to a new tube and added with 0.1× volume of 2M NaOAc pH 5.2 and 3× volume of ethanol then incubated at − 20 °C for 2 h.

Fungal DNA pellets were obtained by centrifugation at 25,000×g at 4 °C for 25 min. The fungal DNA pellets were washed by 500 μl of 70% ethanol, then centrifuged at 25,000×g at 4 °C for 5 min. The fungal DNA pellets were dried in an airtight chamber for 5 min, then dissolved in 0.2× volume of RNAse and 30 μl of sterile TE (TrisHCl 10 mM, pH 7.4, EDTA 1 mM) buffer and then incubated at 37 °C for 10 min and 70 °C for 10 min.

Extraction of fungal DNA was done using Nucleon PhytoPure reagent kit (Amersham LIFE SCIENCE, USA). PCR amplification was at ITS, using ITS Primer 4: 5′-TCC TCC GCT TAT TGA TAT GC-3′ and ITS Primer 5: 5′-GGA AGT AAA AGT CGT AAC AAG G-3′ (White et al. 1990). DNA amplification was carried out by making a volume of 30 μl containing 10.5 μl of alkaline free water, 15 μl 2× PCR mastermix (Promega), 0.75 μl and 10 pmol respectively of primer ITS 4 and ITS 5 and 3 μl (about 250 ng/μl) DNA template. The amplification reaction was carried out in 35 cycles as follows: pre-denaturation at 95 °C for 15 min, denaturation at 95 °C for 30 min, heating (annealing) at 55 °C for 30 s, lengthening at 72 °C for 1.5 min, re-extension at 72 °C for 5 min. and lastly stored at 25 °C for 10 min.

Purification of PCR products was carried out by using Polyethilen Glycol (PEG) precipitation method (Hiraishi et al. 1995) and continued with a sequencing cycle. The results of sequencing cycle were purified again, using the ethanol purification method. Analysis of nitrogen base sequence readings was done using an automated DNA sequencer (ABI PRISM 3130 Genetic Analyzer) (Applied Biosystems). The raw data resulting from the sequencing was then trimmed and assembled, using the BioEdit program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Sequence data that was assembled was then carried out in BLAST with genomic data that was registered at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST) to determine taxon or species that have the greatest homology/similarity and molecularly.

Results

Morphological identification

Fungal isolates, isolated from brown plant hoppers, were infected by EPF. Fungi were purified and cultured on potato dextrose agar (PDA) media. The results of the observation on morphological characteristics, the isolates J22 (Pasir Kulon) and J60 (Papringan) were identified as Paecilomyces sp. while J34 (Cipete) isolate was identified as Beauveria sp. (Minarni et al. 2020). The morphological characters of each EPF isolate (J22, J34 and J60) are presented in (Table 1 and Figs. 1, 2, and 3).

Table 1 Morphological characteristics of entomopathogenic fungi J22, J34, and J60 isolates

Fig. 1

a Colony of 8 days old Pasir Kulon (J22) isolate. b Paecilomyces sp. conidia (Minarni et al. 2020). c Paecilomyces lilacinus conidia (Dong et al. 2016)

Fig. 2

a Colony of 8 days old Cipete (J34) isolate. b Beauveria sp. conidia (Minarni et al. 2020). c Beauveria bassiana conidia (Nuraida and Hasyim 2009)

Fig. 3

a Pure cultures of 8 days old Papringan isolate (J60). b Paecilomyces sp. conidia (Minarni et al. 2020). c Paecilomyces javanicus conidia (Dong et al. 2016)

Molecular identification

The results of the ITS rDNA sequencing of fungal isolates J22, J34, and J60 are as follows:

• 1. ITS rDNA isolate sequence

  • (a) Pasir Kulon_ITS4

1	TCACGTTCAG	AAAGTGGGGT	GTTTTACGGC	GTGGCCACGT	CGGGGTTCCG
51	GTGCGAGGTT	GGATTACTAC	GCAGAGGTCG	CCGCGGACGG	GCCGCCACTC
101	CATTTCGGGG	CCGGCGGTAT	GCTGCCGGTC	CCCAACGCCG	ATTTCCCCAA
151	AGGGAAGTCG	AGGGTTGAAA	TGACGCTCGA	ACAGGCATGC	CCGCCAGAAT
201	GCTGGCGGGC	GCAATGTGCG	TCAAAGATTC	GATGATTCAC	TGAATTCTGC
251	AATTCACATT	ACTTATCGCA	TTTCGCTGCG	TTCTTCATCG	ATGCCAGAAC
301	CAAGAGATCC	GTTGTTGAAA	GTTTTTGATTC	ATTTGTTTTG	CCTTGCGGCG
351	GATTCAGAAG	ATACTCATGA	TACAAAAGAG	TTTGGTGGTC	TCCGGCGGCC
401	GCCTGAGTCC	GGGCCGCGGG	CGGCGCTAGG	CCGTCCGGAC	GCCGGGGCGA
451	GTCCGCCGAA	GCAACATCTT	GGTATGTTCA	CATAAGGGTT	TGGGAGTTGT
501	AAACTCTGTA	ATGATCCCTC	CGCTGGTTCA	CCAACGGAGA	CCTTGTTAC

• (b) Pasir Kulon_ITS5

1	GTTGCTTCGG	CGGACTCGCC	CCGGCGTCCG	GACGGCCTAG	CGCCGCCCGC
51	GGCCCGGACT	CAGGCGGCCG	CCGGAGACCA	CCAAACTCTT	TTGTATCATG
101	AGTATCTTCT	GAATCCGCCG	CAAGGCAAAA	CAAATGAATC	AAAACTTTCA
151	ACAACGGATC	TCTTGGTTCT	GGCATCGATG	AAGAACGCAG	CGAAATGCGA
201	TAAGTAATGT	GAATTGCAGA	ATTCAGTGAA	TCATCGAATC	TTTGAACGCA
251	CATTGCGCCC	GCCAGCATTC	TGGCGGGCAT	GCCTGTTCGA	GCGTCATTTC
301	AACCCTCGAC	TTCCCTTTGG	GGAATTCGGC	GTTGGGGGAC	CGGCAGCATA
351	CCGCCGGCCC	CGAAATGGAG	TGGCGGCCCG	TCCGCGGCGA	CCTCTGCGTA
401	GTAATCCAAC	CTCGCACCGG	AACCCCGACG	TGGCCACGCC	GTAAAACACC
451	CCACTTTCTG	AACGTTGACC	TCGGATCAGG	TAGGAATACC	CGCTGAACTT
501	AA

• (c) Contig-PasirKulon

1	GTAACAAGGT	CTCCGTTGGT	GAACCAGCGG	AGGGATCATT	ACAGAGTTTA
51	CAACTCCCAA	ACCCTTATGT	GAACATACCA	AGATGTTGCT	TCGGCGGACT
101	CGCCCCGGCG	TCCGGACGGC	CTAGCGCCGC	CCGCGGCCCG	GACTCAGGCG
151	GCCGCCGGAG	ACCACCAAAC	TCTTTTGTAT	CATGAGTATC	TTCTGAATCC
201	GCCGCAAGGC	AAAACAAATG	AATCAAAACT	TTCAACAACG	GATCTCTTGG
251	TTCTGGCATC	GATGAAGAAC	GCAGCGAAAT	GCGATAAGTA	ATGTGAATTG
301	CAGAATTCAG	TGAATCATCG	AATCTTTGAA	CGCACATTGC	GCCCGCCAGC
351	ATTCTGGCGG	GCATGCCTGT	TCGAGCGTCA	TTTCAACCCT	CGACTTCCCT
401	TTGGGGAAAT	CGGCGTTGGG	GGACCGGCAG	CATACCGCCG	GCCCCGAAAT
451	GGAGTGGCGG	CCCGTCCGCG	GCGACCTCTG	CGTAGTAATC	CAACCTCGCA
501	CCGGAACCCC	GACGTGGCCA	CGCCGTAAAA	CACCCCACTT	TCTGAACGTT
551	GACCTCGGAT	CAGGTAGGAA	TACCCGCTGA	ACTTAA

• (d) Cipete_ITS4

1	CGGCAGGGGC	TCCGTCCGCT	TCTCCCTATG	CGGAATATCA	CTACTTCCGC
51	AGGGGAGGCC	ACGACGGGTC	CGCCACTAGA	TTTAGGGGCC	GGCCGTCCCT
101	CGCGGGCGCT	GGCCGATCCC	CAACACCACG	CCCTAGGGGC	ATGAGGGTTG
151	AAATGACGCT	CAGACAGGCA	TGCCCGCCAG	AATACTGGCG	GGCGCAATGT
201	GCGTTCAAAG	ATTCGATGAT	TCACTGAATT	CTGCAATTCA	CATTACTTTT
251	CGCATTTCGC	TGCGTTCTTC	ATCGATGCCA	GAACCAAGAG	ATCCGTTGTT
301	GAAAGTTTTT	ATTTATTTGT	AAAAACGACT	CAGAAGATTC	TCAGTAAAAC
351	AAGAGTTAAG	GTCCCCCGGC	GGCCGCCTGG	ATCCGGGGCA	CGCAAGGCGC
401	CCGGGGCGAT	CCGCCGAAGC	AACGATAGGT	ATGTTCACAT	GGGTTTGGGA
451	GTTGTAAACT	CGGTAATGAT	CCCTCCGCTG	GTTCACCAAC	GGA

• (e) Cipete_ITS5

1	TCGTTGCTTC	GGCGGATCGC	CCCGGGCGCC	TTTGCGTGCC	CCGGATCCAG
51	GCGGCCGCCG	GGGGACCTTA	ACTCTTGTTT	TTACTGAGAA	TCTTCTGAGT
101	CGTTTTTACA	AATAAATAAA	AACTTTCAAC	AACGGATCTC	TTGGTTCTGG
151	CATCGATGAA	GAACGCAGCG	AAATGCGAAA	AGTAATGTGA	ATTGCAGAAT
201	TCAGTGAATC	ATCGAATCTT	TGAACGCACA	TTGCGCCCGC	CAGTATTCTG
251	GCGGGCATGC	CTGTCTGAGC	GTCATTTCAA	CCCTCATGCC	CCTAGGGCGT
301	GGTGTTGGGG	ATCGGCCAGC	GCCCGCGAGG	GACGGCCGGC	CCCTAAATCT
351	AGTGGCGGAC	CCGTCGTGGC	CTCCCCTGCG	AAGTAGTGAT	ATTCCGCATA
401	GGAGAGCGAC	GAGCCCCTGC	CGTTAAACCC	CCAACTTTCT	CAGGTTGACC
451	TCAGATCAGG	TAGGAATACC	CGCTGAACTT	A

• (f) Contig-Cipete

1	TCCGTTGGTG	AACCAGCGGA	GGGATCATTA	CCGAGTTTAC	AACTCCCAAA
51	CCCATGTGAA	CATACCTATC	GTTGCTTCGG	CGGATCGCCC	CGGGCGCCTT
101	TGCGTGCCCC	GGATCCAGGC	GGCCGCCGGG	GGACCTTAAC	TCTTGTTTTT
151	ACTGAGAATC	TTCTGAGTCG	TTTTTACAAA	TAAATAAAAA	CTTTCAACAA
201	CGGATCTCTT	GGTTCTGGCA	TCGATGAAGA	ACGCAGCGAA	ATGCGAAAAG
251	TAATGTGAAT	TGCAGAATTC	AGTGAATCAT	CGAATCTTTG	AACGCACATT
301	GCGCCCGCCA	GTATTCTGGC	GGGCATGCCT	GTCTGAGCGT	CATTTCAACC
351	CTCATGCCCC	TAGGGCGTGG	TGTTGGGGAT	CGGCCAGCGC	CCGCGAGGGA
401	CGGCCGGCCC	CTAAATCTAG	TGGCGGACCC	GTCGTGGCCT	CCCCTGCGGA
451	AGTAGTGATA	TTCCGCATAG	GGAGAAGCGG	ACGGAGCCCC	TGCCGTTAAA
501	CCCCCAACTT	TCTCAGGTTG	ACCTCAGATC	AGGTAGGAAT	ACCCGCTGAA
551	CTTAA

• (g) Papringan_ITS4

1	TAGTTGGGTG	TTTTACGGCG	TGGCCGCTTC	GATTTTCCCA	GTGCGAGGTA
51	AGTTACTACG	CAGAGGTCGC	CTCGAAGGGC	CGCCACTGAA	TTTCGGGGGC
101	GGCGTCCCAC	GCCCGGAGGC	GCGGGGCAGT	CTGCCGGTCC	CCAACACCGG
151	GCCGTCTTCC	GAAGAATCGG	GCCCGAGGGT	TGAAATGACG	CTCGAACAGG
201	CATGCCCGCC	AGAATGCTGG	CGGGCGCAAT	GTGCGTTCAA	AGATTCGATG
251	ATTCACTGAA	TTCTGCAATT	CACATTACTT	ATCGCATTTC	GCTGCGTTCT
301	TCATCGATGC	CAGAACCAAG	AGATCCGTTG	TTGAAAGTTT	TGATTCATTT
351	GTTTTTTGCC	TTTCGGCCAC	TCAGATAATG	CTGTAAAAAC	AATAAGAGTT
401	TGATACCCCC	GGCAGCGCCG	GAGCGCCGCC	GAAGCAACAA	GTGGTAAGTT
451	CACATAGGGT	TTGGGAGTTG	AATAAACTCG	ATAATGATCC	CTCCGCTGGT
501	TCACCAACGG	A

• (h) Papringan_ITS5

1	CCACTTGTTG	CTTCGGCGGC	GCTCCGGCGC	TGCCGGGGGT	ATCAAACTCT
51	TATTGTTTTT	ACAGCATTAT	CTGAGTGGCC	GAAAGGCAAA	AAACAAATGA
101	ATCAAAACTT	TCAACAACGG	ATCTCTTGGT	TCTGGCATCG	ATGAAGAACG
151	CAGCGAAATG	CGATAAGTAA	TGTGAATTGC	AGAATTCAGT	GAATCATCGA
201	ATCTTTGAAC	GCACATTGCG	CCCGCCAGCA	TTCTGGCGGG	CATGCCTGTT
251	CGAGCGTCAT	TTCAACCCTC	GGGCCCGATT	CTTCGGAAGA	CGGCCCGGTG
301	TTGGGGACCG	GCAGACTGCC	CCGCGCCTCC	GGGCGTGGGA	CGCCGCCCCC
351	GAAATTCAGT	GGCGGCCCTT	CGAGGCGACC	TCTGCGTAGT	AACTTACCTC
401	GCACTGGGAA	AATCGAAGCG	GCCACGCCGT	AAAACACCCA	ACTATTTTAA
451	GGTTGACCTC	GAATCAGGTA	GGACTACCCG	CTGAACTTAA

• (i) Contig-Papringan

1	TCCGTTGGTG	AACCAGCGGA	GGGATCATTA	TCGAGTTTAT	TCAACTCCCA
51	AACCCTATGT	GAACTTACCA	CTTGTTGCTT	CGCGGGCGCT	CCGGCGCTGC
101	CGGGGGTATC	AAACTCTTAT	TGTTTTTACA	GCATTATCTG	AGTGGCCGAA
151	AGGCAAAAAA	CAAATGAATC	AAAACTTTCA	ACAACGGATC	TCTTGGTTCT
201	GGCATCGATG	AAGAACGCAG	CGAAATGCGA	TAAGTAATGT	GAATTGCAGA
251	ATTCAGTGAA	TCATCGAATC	TTTGAACGCA	CATTGCGCCC	GCCAGCATTC
301	TGGCGGGCAT	GCCTGTTCGA	GCGTCATTTC	AACCCTCGGG	CCCGATTCTT
351	CGGAAGACGG	CCCGGTGTTG	GGGACCGGCA	GACTGCCCCG	CGCCTCCGGG
401	CGTGGGACGC	CGCCCCCGAA	ATTCAGTGGC	GGCCCTTCGA	GGCGACCTCT
451	GCGTAGTAAC	TTACCTCGCA	CTGGGAAAAT	CGAAGCGGCC	ACGCCGTAAA
501	ACACCCAACT	ATTTTAAGGT	TGACCTCGAA	TCAGGTAGGA	CTACCCGCTG
551	AACTTAA

Discussion

Based on the results of the sequences, isolate J22 showed (99.83%) similarity to the L. saksenae strains GFRS14 and L. saksenae isolate Ecu121. Isolate J35 had a similarity with the sequences Myrothecium sp. F129 and Myrothecium sp. 1 TMS-2011 amounted to 98.82 and 98.93%, while isolate J60 had 99.10% similarities to the sequence Simplicillium sp. LCM 845.01 and 98.92% with Simplicillium sp. KYK00024 sequence (Table 2).

Table 2 Results of the nearest fungi taxon BLAST homology ITS1, 5.8S, and ITS2 of rDNA in NCBI (https://www.ncbi.nlm.nih.gov/)

EPF isolates that showed high phylogenetic relationship and had a similarity value of 28S rDNA sequence of more than 99% with the reference species that could be expressed as one species. Ribosomal DNA sequences are used to identify and determine the phylogenetic relationships of organisms to taxa species (Bich et al. 2021). Based on the concept of phylogenetic species, it is stated that an organism is in one species when the difference in DNA sequences is between 0.2 and 1% (Shenoy et al. 2007). According to Henry et al. (2000) isolates, which have a similarity value of 100% can be stated as the same strain and a similarity value of 99% is stated as the same species, while the similarity value of 89–99% belongs to the same genus.

The similarity between 99 and 100% indicated that isolates J22, J34, and J60 each had the same chromosome number, genome size, and gene function as L. saksenae strain GFRS14 and L. saksenae strains isolate Ecu121, Myrothecium sp. F129, and Myrothecium sp. 1 TMS-2011 and Simplicillium sp. LCM 845.01 and Simplicillium sp. KYK00024, respectively.

The identification results based on morphological characters turned out to be different from molecular identification. Accuracy of identification is very important in the use of EPF for insect pest control. Identification based on morphological characters cannot be used as a definite reference. The genera Lecanicillium, Simplicillium, Beauveria, and Isaria have similar morphological characters, so that molecular identification is needed to determine the species certainty of EPF found in Banyumas Regency, Central Java Province, Indonesia. According to Lim et al. (2014) of the genus Lecanicillium, Simplicillium (both previously Verticillium spp.), Beauveria and Isaria belong to family Cordycipitaceae. According to Chen et al. (2016), the genus Myrothecium belongs to family Stachybotryaceae and has a worldwide distribution. Species in this genus were previously classified based on the asexual morphology, especially the characters of conidia and conidiophores. Morphology-based identification alone is imprecise because there are few characters to distinguish between species in the genus and, therefore, molecular sequence data are important in species identification.

Simplicillium sp. is one of the dominant genera of symbiont fungi in unfertilized brown planthopper eggs. The other 3 genera are Microdochium, Fusarium, and Cladosporium (Shentu et al. 2020). One of the species of the genus Simplicillium is S. lanosoniveum. The fungi belong to this genus are known as mycoparasites. However, silkworms (Bombyx mori) inoculated with the fungus isolate S. Lanosoniveum, died during the larval or pupal stage, as shown by the EPF, B. bassiana. The first report on the entomopathogenicity of S. lanosoniveum and demonstrated its potential for use in insect biological control was recorded by Lim et al. (2014). The fungus S. lanosoniveum was able to cause mortality of Hysteroneura setariae ticks on Plum plants by 86.33% (Chen et al. 2017a, b). Chen et al. (2019) found 3 new species, namely Simplicillium cicadellidae, S. formicidae, and S. lepidopterorum. So far, there are limited reports of the fungus Simplicillium sp. being isolated from insects infected with the fungus.

The fungus L. lecanii effectively controlled brown plant hoppers with a density of 10¹⁰ conidia/ml, where the mortality value of (78.33%) and a time of death at 5.81 day after treatment occurred (Khoiroh et al. 2014). L. lecanii can cause more than 50% of brown planthopper mortality within 14 days after treatment (Atta et al. 2020), whereas according to Shaikh and Pandurang (2015), this fungus is less effective in controlling this pest. Sankar and Rani (2018) have found a new Lecanicillium isolate, namely L. saksenae, which can control stink bug (Leptocorisa acuta). This fungus can kill 100% of L. acuta nymphs and imago at 72 h after treatment at conidia densities 10⁷ and 10⁸.

Myrothecium verrucaria has a high activity against extracellular insect cuticles and produces chitinase, proteinase, and lipase (Vidhate et al. 2015).

Based on the literature search, the 3 fungi Simplicillium sp., L. saksenae, and Myrothecium sp. have never been reported to infect brown plant hopper. Data obtained from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/), also showed that these 3 fungi were not obtained from insect pests (Table 2). The results of this study revealed 3 types of new EPF that had the potential to be developed as control agents for brown plant hopper pests.

Conclusion

The results of molecular identification showed that the isolates J22, J34, and J 60 were fungi from L. saksenae, Myrothecium sp., and Simplicillium sp., respectively. The results of literature search showed that these 3 fungi had never been reported to infect brown plant hopper. So that the results of this study can be considered new finding of EPF as biological agents of the control brown plant hopper pests.