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
a Pure cultures of 8 days old Papringan isolate (J60). bPaecilomyces sp. conidia (Minarni et al. 2020). cPaecilomyces javanicus conidia (Dong et al. 2016)
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.
Availability of data and materials
All data are available in the article and the materials used in this work are of high quality and grade.
Abbreviations
BPH:
Brown plant hopper
BLAST:
Basic Local Alignment Search Tool
CTAB:
Cetyl trimethylammonium bromide
DNA:
Deoxyribonucleic acid
ITS:
Internal transcribed spacer
PCR:
Polymerase chain reaction
PDA:
Potato dextrose agar
PDB:
Potato dextrose broth
PEG:
Polyethilen Glycol
UV:
Ultraviolet
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The author would like to thank the Head of the Laboratory of Plant Protection, Faculty of Agriculture, Jenderal Soedirman Purwokerto University, The Biology Laboratory of Indonesian Academy of Sciences, and all those who have helped research and write this scientific article.
Funding
The author would like to thank the Directorate of Research and Community Service of the Ministry of Education and Culture of the Republic of Indonesia for funding this research through the Doctoral Dissertation Research Grant in 2020.
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Authors and Affiliations
Departement of Agrotechnology, Faculty of Agriculture, Universitas Jenderal Soedirman, Purwokerto, Indonesia
Endang Warih Minarni, Loekas Soesanto, Agus Suyanto & Rostaman
EWM performed the experiments on bioassay and analyzed the data. The manuscript was prepared by EWM, LS, AS, and R. All the authors read and approved the manuscript.
The authors declare that they have no competing interests.
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Minarni, E.W., Soesanto, L., Suyanto, A. et al. Molecular identification of three entomopathogenic fungi infecting the brown plant hopper pest in Indonesia.
Egypt J Biol Pest Control31, 62 (2021). https://doi.org/10.1186/s41938-021-00412-7