Patogenisitas Beauveria Bassiana Strain Stgd 7(14)2 dan Stgd 5(14)2 terhadap Wereng Coklat (Nilaparvata Lugens Stål)

Beauveria spp. is one of the entomopathogenic fungi that can be used as biocontrol agents against various insect pests, including brown planthopper (Nilaparvata lugens Stal). This study aimed to test the pathogenicity of Beauveria spp. against N. lugens. Beauveria spp. were isolated from the rice stink bug insects, collected from Situ Gede, Bogor, West Java. Fungal identification was carried out based on the internal transcribed spacer (ITS) rDNA analysis. The pathogenicity assay revealed that Stgd 5(14)1 and Stgd 7(14)2 isolates were virulent against N. lugens, with a mortality of 100%. The LT50 (median lethal time) determination, indicated that Stgd 5(14)2 and Stgd 7(14)2 isolates had low value indicating a faster mortality in brown planthopper insects. Phylogenetic analysis showed that the sequences of Stgd 5(14)2 and Stgd 7(14)2 belong to Beauveria bassiana s.str. This is the preliminary pathogenicity trial of entomopathogenic fungi B. bassiana against brown planthopper and their distribution in rice-growing, Situ Gede area in West Java.


INTRODUCTION
Brown planthopper (BPH) pest (Nilaparvata lugens STAL) damaged more than 130,000 hectares of rice fields (Ditlintan, 2010). A high increase of BPH population led to a decline in rice production, which may have impacts on economic aspects. Pest control of BPH using pesticides has been considered as threats for the environment and human health in the long-term use. The intensive use of chemicals can cause resistance and resurgence of the target-pests, hence it may increase the population level, which is higher than the initial population (Bhisnu et al., 2008). Furthermore, the use of broad spectrum pesticide can kill non-target pests. Therefore, the integrated pest management program to control insect pests, particularly BPH, is necessary (Bednarek et al., 2004;Laba, 2010).
One alternative method of pest control, that is effective for insects and environmentally friendly, is biological control using the formulation of specific microbes, such as fungi, bacteria, and viruses (Bednarek et al., 2004). The search of selective biopesticide against certain insects is increasing, especially the use of natural enemies as a key component of the integrated pest management. The use of biopesticides can inhibit the development of larvae and insect pests, without causing environmental damage (Thungrabeab & Tongma, 2007).
Until now, various efforts to develop BPH control in Indonesia has still been in progress, since no commercial biological agent that is considered really effective against BPH. Therefore, an effort to utilize the potential of Beauveria spp. isolates as potential biocontrol agents against BPH in mass productions scale, is necessary.
The objective of this study is: (i) to determine fungal pathogenicity (LT50) of Beauveria spp., isolated from rice stink bug insects, from Situ Gede (Bogor West Java), against BPH, and (ii) to identify the entomopathogenic fungi using molecular method.
Previously, these isolates were tested for their pathogenicity against rice stink bug insects (Wartono et al., 2016).
Fungal of 15 day colonies on PDA medium were harvested by adding 10 mL of sterile distilled water and 0.1% Triton X100 to the Petri dishes. The conidia then were scrapped from the solid medium with a soft brush (Kim et al., 2014). The calculation of conidial density was done using hemocytometer (Bessey, 1979).
A total of 15 BPH was infested on rice plants and then covered using mylar plastic cylinder. This assay was repeated twice. The inoculation method of fungal spore suspension was carried out using the spray method following the procedure, described by Mahmoud (2009). The distilled water with 0.1% Triton X100 was used as a control treatment. A percentage of BPH mortality due to Beauveria spp. infestation was observed every 24 hours after application.
The experimental data were processed and analyzed by using the Sirichai program v 6.0 at 5% significance level. The determination of the median lethal time (LT 50 ) was done using probit analysis (Chi, 1997). The percentage of mortality was calculated using the following formulation: P = X x 100% Y P : the percentage of mortality X : the number of BPH mortality Y : the total number of BPH ,I WKH PRUWDOLW\ RFFXUUHG RQ FRQWURO " WKH percentage of BPH mortality was then calculated using Abbot formula (Busvine, 1971), as follows: 100 -B AI : percentage of mortality after correction A : percentage of mortality of BPH on treatment B : percentage of BPH deaths on control

Morphological Examination of Stgd 5(14)2 and Stgd 7(14)2 Isolates
The fungal isolates were rejuvenated by culturing 1 2 mycelium disks onto a potato dextrose agar (PDA). The cultures were then incubated for 2 7 days at room temperature (+25 qC). The observation of macroscopic fungal colonies includes colour, colonies form, colonies texture, and shape of the edge of colonies. Microscopic characters of spores, conidia, and hyphae were observed by using compound microscope at 40 1000x magnification (Samson et al., 1988;Glare & Inwood, 1998).

DNA Extraction and PCR Amplification
Genomic DNA of fungal isolates was harvested from 4-day-old isolates on 100 mL of Sabouraud dextrose broth (SDB) medium in Erlenmeyer flask (1 L: 20 g of dextrose, 10 g peptone, and 2 g of yeast extract), and dried in an oven (50 qC) overnight (Trizelia et al., 2012). The dried mycelial fungus was ground in liquid nitrogen using a mortar and pestle (Rogers & Benedich, 1994). The mycelium powder was transferred into an Eppendorf tube containing 700 mL of extraction buffer 1% (2% CTAB: 20 mM EDTA; 0.1 M Tris-HCl; 1.4 M NaCl pH 8.2; 2-mercaptoethanol) and incubated in a shaker water bath for 5 min at 65 qC, then it was vortexed and incubated again for 30 min at 65 qC. About 400 mL solution of chloroform: isoamyl alcohol (24:1v/v) was added and vortexed until the water and the organic layer portion is mixed. The mixture was centrifuged for 10 min at 12,000 rpm (Beckman Coulter Microfuge 22R), and 500 mL the top layer was transferred in new Eppendorf tube and added to 400 mL of chloroform: isoamyl alcohol (24:1v/v). The mixture was centrifuged for 10 min at 10,000 rpm, and the upper layer was moved into the new Eppendorf tube and added with 500 mL cold isopropanol, shaken until mixed, and incubated for 1 h at 20 qC. The mixture was centrifuged for 2 min at 10,000 rpm. The top layer (a mixture of water and alcohol) formed were removed. The bottom layer was air dried for 1 hour. The air-dried pellets were resuspended in 600 mL of TE buffer and stored at 4 qC. DNA was examined by electrophoresis using a 1% agarose gel, which was stained with ethidium bromide (EtBr) (Sambrook & Rusell, 2001;Khosravinia et al., 2007), and the DNA was visualized using ChemiDoc.
The PCR mixture of the current study composed of 1 mL of genomic DNA (20 50 ng/mL), 1 mL of each primer (forward and reverse), 7.5 mL Kappa 2G Fast Ready Mix and 3.5 mL ddH2O. The primer pair of internal transcribed spacer (ITS) 1 (5'-TCC GCGGA GTA GGT GAA CCT-3') and ITS 4 (5'-TCC TCC GCT TAT TGA TATGC-3') (De Beeck et al., 2014) was used in the amplification. PCR reactions were performed using the Esco® PCR machine with the following programs: initial denaturation at 95 qC for 5 min, followed by 30 cycles of 1 min denaturation at 95 qC; 35 s annealing at a temperature of 55 qC; and 30 s elongation at 72 qC. The DNA amplification product was visualized by electrophoresis on a 1.5% agarose gel (White et al., 1990).

RESULT Pathogenicity Assay
Spore suspension density of 15-day-old fungal isolates is showed in Table 1. The highest and the lowest OD was found in Stgd 0213 (OD= 4.44×10 8 ) and Stgd 7(14) 2 (OD= 1.12×10 8 ), respectively. The mean percentage of BPH mortality indicated that Stgd 7(14) 2 and Stgd 8(4) 2 isolates were highly virulent isolates, with an average mortality rate of 100% at the sixth day after the conidial suspension application (Table 1). Isolate Stgd 0213 is a low virulent isolate that showed the lowest percentage of mortality (67.50%). In this study, Stgd 5(14) 2 and Stgd 7(14) 2 isolates showed the ability to reduce BPH population by 50% in a shorter time. The data also showed that an average time required to kill the 50% population (LT 50 ) was after the 3 rd day, while the time required for 90% (LT 90 ) was 5 6 days ( Figure 1).

Morphological Examination
The observation of macroscopic and microscopic fungal colonies was presented in Figure 2. It showed that macro and microscopic characters of the fungi belong to genus Beauveria (due to their colony morphology are characterised by: (i) white mycelium with white powdery appearance; (ii) conidiophores single; (iii) irregularly grouped or in verticillate clusters tapering to a slender fertile portion that appears zigzag (sympodial) after several conidia are formed on elongating conidiogenous cells.

DISCUSSION
This study showed that there is no variations in pathogenicity of B. bassiana strain Stgd 7(14) 2 and Stgd 5(14) 1 against N. lugens. Both strains were highly virulent because they cause 100% death rate at sixth day after a conidial suspension application. The degree of virulence varies among Beauveria spp. as a consequence of their specific virulence characteristics (Cruz et al., 2006;Carneiro et al., 2008;Safavi et al., 2010). A mortality value of >90 100% indicates that both Stgd 5(14)1 and Stgd 7 (14)2 isolates are potential as biological agents against BPH.
Field observations indicated that the early symptoms of N. lugens mortality was shown by a rigid body and a pale colour body. The mortality of N. lugens can be observed from the first day until the sixth day after the conidial suspension application conducted. Indriyati (2009) reported that the mortality of insects could take place in a short period of time (the tird day to the fifth day. In another study, mortality of N. lugens was obvious at the 6 th or the7 th day after the conidial suspension application (Wahyono, 2013). In this study, a spore germination could possibly be occurred within 24h after inoculation (Lecuona et al., 1997). According to Koswanudin & Wahyono (2014), at the 7 th day after the application, the effectiveness of pathogenicity increased by 80-90%. This study also showed that the 10 8 conidial suspension per mL was sufficient for Beauveria spp. pathogenicity assay, under laboratory and greenhouse condition. A toxin released by Beauveria spp. may further cause insect-tissue damage and the fungal mycelia will grow throughout the body of the insect (Padmini & Padmaja, 2013). In a similar study, Shophiya et al. (2014) reported that the 10 4 10 6 conidial suspension/mL was effective for the pathogenicity evaluation to Lepidoptera ricini. Soetopo & Indrayani (2007) (Yusran et al., 2014). . Phylogenetic tree generated from ITS rDNA sequence showed that Stgd 5(14) 1 and Stgd 7(14) 2 sequences belong to Beauveria bassiana with a strong bootstrap support. The phylo tree was reconstructed using NJ method with 1000 bootstrap replicates The ability of fungal pathogen to infect an insect host is determined by several factors, such as pathogenicity, host insect fitness, and the environment (the availability of water, nutrients, oxygen, pH, and temperature) (Sandhu, 1995;Inglis et al., 2001). In general, the Beauveria infection mechanism on insect may involve a production of beauvericin and other bioactive compounds that can damage the hemolymph function and insect host cell nucleus (Latge et al., 1988;Sandhu et al., 2012). The attached conidia will germinate in 1 2 days and mycelia will grow inside the host body. The infected insect will stop eating so that immunity will decrease, and within a period of 3 5 days, the insects will die with the visible fungal growth of the conidial integument (Deciyanto & Indrayani, 2008).
Beauveria bassiana has been known as the most common causative agent of disease associated with dead and moribund insects in nature (Rehner & Buckley, 2005;Rehner et al., 2011). The close relationship between B. bassiana strain Stgd 5(14) 1 and B. bassiana strain Stgd 7(14) 2 is probably due to the origin of strains. The genetic diversity of Beauveria spp. can be influenced by host range and ecological conditions, as well as climate conditions (Stephen & Rehner, 2005;Kaur & Padmaja, 2008;Trizelia et al., 2012). B. bassiana is the asexual morph of Cordyceps bassiana (Li et al., 2001;Rehner et al., 2011).

CONCLUSION
Beuveria bassiana strain Stgd 5(14) 1 and Stgd 7(14) 2 isolated from rice stink bug insects (Leptocorysa acuta) were virulent against N. lugens pest with the mortality rate of 100%. Phylogenetic analysis based on the ITS rDNA sequence confirmed their identities. Additional research is needed: (i) to determine the effectiveness of both fungal strains to other rice insect pests, and (ii) to conduct further study, by using various different conidial suspension concentration and environmental condition.