Effectiveness of Secondary Metabolites from Entomopathogenic Fungi for Control Nilaparvata lugens Stål. in the Laboratory Scale

Nilaparvata lugens Stål. is an essential pest in rice plants. This pest attack can reduce crop yields and even crop failure. This research was conducted to obtain secondary metabolites that are effective in controlling brown planthopper (BPH). A randomized block design was used to test the effectiveness of secondary metabolites against BPH. The treatments tested were secondary metabolites produced by eight isolates of fungi consist of three concentrations: 5, 10, and 15%. Water and imidacloprid insecticide were used as control. The eight isolates were: J11 (Aspergillus sp.), J22 (Lecanicillium saksenae.), J34 (Myrothecium sp.), J35 (Beauveria sp.), J41 (Fusarium sp.), J56 (Fusarium sp), J60 (Simplicillium sp.), and J65 (Curvularia sp.). Each treatment was repeated three times. The variables observed were mortality and time of death of BPH. Data were analyzed using the F test and followed by a DMRT if significant differences existed. The results showed that the secondary metabolites of the Lecanicillium saksenae. Myrothecium sp., and Simplicillium sp. fungi effectively controlled BPH pests by 80–100% within 3.22–5.47 days. The fungus L. saksenae, Myrothecium sp., and Simplicillium sp. contain insecticidal compounds, clogging the insect spiraculum, antifeedant, repellant, and antimicrobial.


INTRODUCTION
The brown planthopper (Nilaparvata lugens Stål.) is a major rice pest threatening rice production in Indonesia. Rashid et al. (2016) reported that in the last decade, there was an explosion in the brown planthopper (BPH) population throughout Asia, which resulted in large yield losses. In Thailand, there was a continuous BPH population explosion for ten consecutive growing seasons from 2008 to 2012 and caused a loss of US $ 52 million or the equivalent of approximately 173,000 tonnes. This pest also caused an estimated loss of 1,000,000 tonnes in Vietnam in 2007 and resulted in the government canceling rice exports. According to Bhatt and Tiwari (2015), in Southeast and East Asia, BPH caused yield losses of 30-50 percent. In Indonesia, BPH from October 2016 to August 2017 caused damage to rice crops covering an area of 63,075 hectares and resulted in 20,152 hectares of rice crops experiencing crop failure (Julianto, 2017). The percentage of BPH attacks ranges from 51.6-94.1% in Padang, Indonesia (Syahrawati, 2019).
The frequency of BPH attacks in developing Asian countries continues to increase; this is due to the unwise use of synthetic chemical insecticides, so that natural enemies are killed (Khan et al., 2018;Minarni et al., 2018;Zhu et al., 2018). BPH pests have high genetic plasticity, the use of the same insecticides and continuously can cause BPH resistance to these insecticides (Surahmat et al., 2016;X. Zhang et al., 2016;Y. Zhang et al., 2017;Minarni et al., 2018;Wu et al., 2018;Tian et al., 2019). These problems need to be addressed immediately so that there is no explosion of BPH pests.
The entomopathogenic fungus Hypocreales produces a variety of secondary metabolites. This group of fungi has a very high genome rank and is predicted to have a number of gene clusters that produce unique secondary metabolites. Secondary metabolites have very diverse roles in insect pathogenicity as virulence factors by modulating various interactions between fungi and insect hosts. In addition, secondary metabolites also protect the host carcass from attack by other microbes, play a role in intra and inter-species communication, and reduce biotic stress (L. Zhang et al., 2020).
Secondary metabolites are genetic properties inherent in an organism, which are usually used for the adaptation of fungi to their environment (Hautbergue et al., 2018). The secondary metabolites of entomopathogenic fungi generally have a small molecular size, usually <2,000 MW (molecule weight). The overall production of certain secondary metabolites can be significantly altered by optimizing growth conditions, such as nutrition, temperature, humidity (Mishra et al., 2015;Zaman et al., 2020), and UV radiation (Kaiser et al., 2018, Herlinda et al., 2019. Some entomopathogenic fungi can kill the host even more rapidly by secreting some mycotoxins (such as beauvericin, cyclodepsipeptide, destruxin, and desmethyldestruxin) in the early stages of infestation (Wang et al., 2018).
Based on the previous description, it is necessary to research entomopathogenic fungi' secondary metabolites' effectiveness in BPH control. Results of the literature search show the use of entomopathogenic fungal secondary metabolites to BPH control has not been reported.

Extraction of Secondar y Metabolites of Entomopathogenic Fungi
Fungal secondary metabolites are produced by multiplying the entomopathogenic fungi in PDB (Potato Dextro Broth). Propagation was carried out using five 1 cm diameter fungi cultures and was incubated in a 250 ml Erlenmeyer at room temperature and shaken at 200 rpm for ten days (Kim et al., 2013). Fungi culture was separated between fungal mycelium and its supernatant using a 5,000× g speed centrifuge (Hitachi himac CR 7) for 10 minutes at 4 o C. The supernatant was then filtered with Whatman No 1 filter paper. The supernatant was grown on PDA (Potato Dextro Agar) to ensure no more mycelium was carried (Bandani et al., 2000).
The extraction results were analyzed using GC-MS Shimadzu Type QP-2010 SE, with an SH-Rxi-5Sil MS column, 30 m long, 0.25 mm inside diameter, with an initial column temperature operating conditions of 80 o C and a final temperature of 300 o C, an injector temperature of 128 o C, detector temperature 280 o C, Helium carrier gas, ionizing type EI (Electron Impact), the volume of the sample injected was 0.1 µL. Compound identification was carried out computeraided by Wiley 229, NIST 12, and NIST 62 Library software. Compound analysis using GC-MS will obtain the active ingredient content of the tested fungus. Minarni et al.: Effectiveness of Secondary Metabolites from Entomopathogenic Fungi

Testing of Secondary Metabolites to BPH Mortality
Testing of secondary metabolites to BPH mortality was performed using an experimental method with a randomized block design. The treatments tested were secondary metabolites produced by 8 fungal isolates, namely J11 (Aspergillus sp.), J22 (Lecanicillium saksenae.), J34 (Myrothecium sp.), J35 (Beauveria sp.), J41 (Fusarium sp.), J56 (Fusarium sp), J60 (Simplicillium sp.), and J65 (Curvularia sp with three concentration of 5, 10, and 15%. Water and the imidacloprid insecticide (active ingredients 350 g/l) were used as control. Each treatment was repeated 3 times. Each experimental unit used 10 third instar nymphs of BPH. The rice plant used was 21 days after seedlings in a plastic cylinder with a diameter of 5 cm and a height of 20 cm with a leotard cloth. Suspension of secondary metabolites was prepared according to the concentration tested.
The application has been made in contact by spraying the suspension of secondary metabolites, aqua dest, and imidacloprid insecticides on 10 individual third instar nymphs. The distance between the nozzle of the sprayer and the BPH was about 5 cm, the number of sprays was three times. Then the BPH nymphs were transferred into plastic cages containing rice plants. Rice plants were placed in the screenhouse. The experiment was repeated three times; observations were made every 24 hours for seven days. Observed variables: nymph mortality and time of death. BPH's time of death was calculated based on the formula from Susilo et al. (1993): Description: W = Time of death of BPH a = Number of BPH died on the day of infection b = day when the dead BPH n = number of dead BPH at each treatment

Data Analysis
Analysis of secondary metabolites from fungi was done using GCMS Shimadzu Type QP-2010 SE. Data of mortality and time of BPH death were analyzed using ANOVA, and whether real differences were further tested by the Duncan test of 95% accuracy. The analysis of the secondary metabolites content of fungi was carried out on isolates which caused 80% mortality in BPH.

RESULTS AND DISCUSSION
Three fungal isolates were found to be effective in controlling BPH pests in the laboratory, with a mortality of 80-100 percent ( The high mortality of BPH due to the treatment of secondary metabolites of Lecanicillium saksenae, Myrothecium sp., and Simplicillium sp. was suspected because the three fungi produced toxic compounds. The BPH exposed to secondary metabolites of the entomopathogenic fungi showed inactivity, decreased feeding activity, then died drying, and was not overgrown with fungal hyphae. The secondary metabolites of the three fungi were then analyzed for their chemical content using GCMS. The results of GCMS analysis of the three fungi are presented in Tables 2, 3, and 4. The literature search results show that the fungus Lecanicillium saksenae, Myrothecium sp., and Simplicillium sp. contain insecticidal compounds. L. saksenae fungus produces secondary metabolites methyl ester of ricinoleic acid and selinane. Myrothecium sp. produces four secondary metabolites, namely (2.4,4,4,16,16-D6)-3. alpha.,17.beta.-dihydroxy-5.beta.-androstane; (+)-nepetalactone; Alloaroma dendrenoxid-(1) and 2-(4-Bromobenzylidene) cyclohexanone. Meanwhile, the fungus Simplicillium sp. produces secondary metabolites phenylalanine, N, N-bis (trimethylsilyl)-trimethylsilyl ester; papaverine; and octadecanoic acid trimethylsilyl ester.
Based on Table 5, it's known that the more types of secondary metabolites that are insecticidal produced by entomopathogenic fungi, the higher mortality of BPH. Myrothecium sp. produced four toxic compounds, while the Simplicillium sp. and L. saksenae fungi produced three and two toxic compounds. The biological activity of each secondary metabolite produced by each fungus can also be seen in Table 5     The toxin production will differ depending on fungal isolates, culture composition, and pH so that the culture extracts or filtrates from different fungi are thought to contain secondary metabolites or compounds that have different insecticide activity (Sánchez-Pérez et al., 2016). The type and concentration of a compound can vary according to fungal isolates, the composition of the culture medium, and the conditions of propagation (Valencia et al., 2011;Safavi, 2013).
Secondary metabolites of entomopathogenic fungi have the following characteristics: small molecular weight, high stability, not easily damaged, and penetrate the barrier. These characteristics significantly affect the effectiveness of entomopathogenic fungal applications in BPH. Secondary metabolites of L. saksenae), Myrothecium sp., and Simplicillium sp. caused BPH death by 80-100% in the laboratory. These results indicate that the secondary metabolites of entomopathogenic fungi have the potential to be further investigated in the field. If it shows good results, it can be used as an alternative to controlling BPH pests and as a substitute for synthetic chemical insecticides.