DOI : http://dx.doi.org/10.21776/ub.jpt.2023.008.2.10
Exploration of Symbiont Bacteria Potentially Degrading Chlorpyrifos Insecticide in the Digestive Tract of Beet Armyworm (Spodoptera exigua) in Kediri Regency
Tita Widjayanti*, Mochammad Syamsul Hadi, Zalfa Meiska Tantri
Departement of Plant Pest and Diseases, Faculty of Agriculture, Universitas Brawijaya
Corresponding author email : [email protected]
submitted 12 September 2023 / accepted 24 October 2023
ABSTRACT
Spodoptera exigua Hubner (Lepidoptera: Noctuidae) is an important pest on shallot crops and cosmopolitan with a fast life cycle. In controlling S. exigua farmers use chemical insecticides such as chlorpyrifos. Intensive application of insecticides causes resistance of S. exigua. Resistance can occur due to several factors, one of which is biological factors. In biological factors, there is a symbiosis between pests and beneficial bacteria called symbiont bacteria. It is suspected that symbiont bacteria symbiotic with S. exigua can degrade chlorpyrifos. The aim of this research is to identify and determine symbiotic bacteria that have the potential to degrade the insecticide chlorpyrifos. The research was conducted from December 2022 to May 2023. The research began with a site survey and sampling in three sub-districts in Kediri Regency, namely Plemahan District, Kepung District, and Badas District.
Furthermore, samples of S. exigua were studied at the Pesticide Toxicology Laboratory, Brawijaya University. The research conducted includes (1) exploration, isolation, and purification of symbiont bacteria in the digestive tract of S. exigua, (2) bioassay test of symbiont bacteria that have the potential to degrade chlorpyrifos by observing the diameter of the clear zone, and (3) characterization of bacteria that have the potential to degrade chlorpyrifos. The results of the exploration of symbiont bacteria in the digestive tract of S. exigua obtained as many as 45 isolates of bacteria capable of growing in NA + chlorpyrifos media. Furthermore, from bioassay testing, 14 isolates of bacteria that have the potential to degrade chlorpyrifos insects. The characterization results showed that bacterial isolates that have the potential to degrade chlorpyrifos insecticides come from the genera Pantoea, Erwinia, and Coryneform.
Keywords : Chlorpyrifos, Insecticides, Spodoptera exigua
INTRODUCTION
Spodoptera exigua Hubner (Lepidoptera: Noctuidae) is an important pest of shallot plants. In Indonesia, S.
exigua is an important pest that causes crop failure in shallot plants (Umboh et al., 2017). The activity of S. exigua in shallot plantings can cause damage to plants which will have an impact on farmers' income. S. exigua larvae that enter the shallot leaves will eat the inside of the leaves and leave the epidermis. High levels of attack cause the leaves to dry out, thereby reducing the quality and quantity of crop yields (Marsadi et al., 2017).
Kediri Regency is one of the shallot centers in East Java (Muzazin, 2022).
There are three sub-districts that have the highest harvest area and production in the Kediri Regency area, including Badas, Pringan, and Kepung. As a center for shallots, insecticide applications in Kediri Regency are carried out intensively so that the pest population does not reach the economic threshold. If not controlled, S.
exigua attacks will reduce shallot production. Based on the statement by Hastuti et al. (2016), S. exigua attacks can cause yield losses ranging from 45 – 57%
and heavy attacks can cause yield losses of up to 100% due to the leaves being eaten by the larvae.
The control of S. exigua that is often carried out by farmers is chemical control using insecticides containing the active ingredient chlorpyrifos. Chlorpyrifos has the molecular formula C9H11Cl3NO3PS and is a type of organophosphate insecticide with a broad spectrum inhibitor of acetylcholinesterase. The active ingredient chlorpyrifos is non-systemic and works when the insecticide comes into contact with the skin, enters the respiratory system due to inhalation, and enters the stomach through ingested plant
parts (Sulaeman et al., 2016). In controlling S. exigua, farmers in Kediri Regency use insecticides with the active ingredient chlorpyrifos intensively. This can cause problems such as pest resistance to insecticides, environmental pollution, death of non-target organisms, and cause residue.
The resistance of S. exigua to chlorpyrifos is thought to be a biological resistance mechanism by symbiotic bacteria in the digestive tract. These symbiotic bacteria can degrade the insecticide chlorpyrifos so that its effectiveness decreases. This is supported by the statement of Hadi et al. (2021), it has been reported that in the digestive tract of Plutella xylostella there are several genera of bacteria that can degrade the active ingredient chlorpyrifos, including Providencia sp., Pseudomonas sp., Serratia sp., Proteus sp., and Aeromonas sp.
MATERIALS AND METHODS This research was conducted from December 2022 to May 2023. The first stage was sampling of S. exigua shallot caterpillars which was carried out in the shallot central area of Kediri Regency, East Java which includes Pringan Subdistrict, Badas Subdistrict and Kepung Subdistrict. Meanwhile, the second stage was the exploration of symbiotic bacteria which was carried out at the Pesticide Toxicology Laboratory, Brawijaya University, Malang, East Java.
Research Preparation
Cabbage worm collection was carried out on shallot fields owned by farmers in Pringan Subdistrict, Kepung Subdistrict and Badas Subdistrict (Figure 6). Caterpillar sampling refers to Xia et al.
(2013), namely by taking caterpillars randomly in the field without paying attention to gender. S. exigua sampling was carried out in one day. S. exigua
samples were selected based on the relatively large size of the larvae or larvae that had entered the third instar. S. exigua selection was carried out in the laboratory by placing larvae that have entered the third instar into a treatment box and giving them food in the form of shallot plant leaves that have been applied with insecticide containing the active ingredient chlorpyrifos. Larvae that still survive are taken for further testing.
Observation parameters Sample Isolation
S. exigua larvae from three sub- districts that were resistant to the insecticide chlorpyrifos were dissected and made into a suspension of 10-1 cfu/ml.
The homogeneous suspension was then taken as much as 100 µl and cultured on NA media using the spread plate method.
The isolation results were then incubated for 48 hours. After the isolation results grow, purification will be carried out by growing bacterial colonies on new NA media.
Bioassay Test
Preparation of test isolates is carried out by inserting one cycle of bacterial colonies into Nutrient Broth media which has been placed in an Eppendorf. In each Eppendorf, bacteria from exploration and shaking were added for one day. After one day, put Whatman paper with a diameter of ±0.5 cm in each eppendorf and leave it for 1 hour. After one hour the Whatman paper is taken and then inoculated on the surface of the contaminated media. The observation parameter in the bioassay test is the diameter of the clear zone formed from the interaction between bacteria and media contaminated with insecticide containing the active ingredient chlorpyrifos. The wider diameter of the clear zone formed indicates that the bacteria have greater degradation ability.
Characterization of symbiotic bacteria
Characterization of the symbiont bacteria S. exigua was carried out on bacteria that were successful through bioassay tests. Characterization was carried out morphologically and physiologically. Colony morphological characteristics observed were colony shape, color, edges and elevation (Sabdaningsih et al., 2013). Meanwhile, the physiological characters were observed with reference to the physiological characteristics of the bacteria resulting from selection.
Determination of the genus of each result is based on Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) and Schaad et al., (2001). Tests carried out included gram staining test, 3%
KOH solubility test, spore staining, fermentative oxidative test, fluorescent pigment test on King's B media, catalase test, growth test on YDC media, and growth test on DIM agar media.
Data analysis
Data analysis activities were carried out using Microsoft Excel and IBM SPSS Statistics 25 software. Data obtained from the bioassay test was tabulated and analyzed using Analysis of Variance (ANOVA) at the 5% level with Microsoft Excel software. If the results of data analysis show a real difference then proceed with the DMRT test with a level of 5% using IBM SPSS Statistics 25 software.
RESULTS AND DISCUSSIONS Results of Exploration of Bacteria in the Digestive Tract of S. exigua
The results of the exploration of symbiotic bacteria showed that the number of bacterial isolates was 45 isolates. The bacteria were obtained from a 10-1 dilution level which was then grown on NA media and purified until a single, pure bacterial colony was obtained. The results of exploration of symbiotic bacteria
in the digestive tract of S. exigua are presented in Table 1.
Tabel 1. Exploration results at the S.
exigua sampling location
Location Number of Isolates
Plemahan Subdistrict 18
Kepung Subdistrict 14
Badas Subdistrict 13
Total 45
Symbiotic bacteria found in the host body, in this case S. exigua, are useful in forming defenses. In these conditions, symbiotic bacteria interact with the host to form a mutualistic symbiosis. The bacteria obtained are thought to play a role in helping S. exigua degrade the insecticide chlorpyrifos, causing resistance in S.
exigua (Hillman and Blair, 2016). Apart from that, based on research by Hadi et al.
(2021) microorganisms associated with the digestive system of P. xylostella such as Providencia sp., Pseudomonas sp., Serratia sp., Proteus sp., and Aeromonas sp. able to degrade the insecticide chlorpyrifos.
Insecticide Bioassay Test for the Active Ingredient Chlorpyrifos
The results of the chlorpyrifos insecticide bioassay test showed that not all bacteria were able to produce a clear zone (Table 2). There were 14 bacterial isolates capable of degrading the insecticide chlorpyrifos in contaminated media. This shows the influence of the isolate used on the diameter of the clear zone produced during the 96 hour incubation period. The ability of each isolate to degrade the insecticide chlorpyrifos can be seen from the diameter of the clear zone in the test treatment (Table 3).
The highest number of isolates was in Pringan Subdistrict with 18 isolates and the lowest was in Badas Subdistrict with 13 isolates. It is suspected that the diversity of symbiotic bacteria in each sub- district is influenced by the application of
insecticides to the land. According to Hadi et al. (2021) stated that regular application of insecticides will increase the diversity of symbiont bacteria. Especially symbiotic bacteria which can produce insecticide degrading enzymes such as carboxylesterase and esterase.
Table 2. Bioassay test for the insecticide chlorpyrifos with a recommended dose of 1.5 ml/L
Isolate Bioassay test of 1.5 ml/L chlorpyrifos
insecticide
Isolate Bioassay test of 1.5
ml/L chlorpyrifos
insecticide
Kontrol - K5 -
P1 - K6 +
P2 + K7 -
P3 - K8 +
P4 + K9 -
P5 + K10 -
P6 - K11 -
P7 + K12 -
P8 - K13 +
P9 - K14 -
P10 - B1 -
P11 - B2 -
P12 + B3 -
P13 - B4 -
P14 - B5 -
P15 - B6 +
P16 + B7 -
P17 - B8 -
P18 + B9 +
K1 - B10 -
K2 + B11 -
K3 - B12 +
K4 - B13 -
Note: (+) if the bacteria are able to produce a clear zone on contaminated media, while (-) if the bacteria are unable to produce a clear zone.
Symbiotic bacterial isolates in each sub-district have the potential to degrade chlorpyrifos as indicated by the formation of clear zones. The highest number of isolates that were able to produce clear zones were found in Pringan District (P2, P4, P5, P7, P12, P18, and P18). The next highest number of isolates capable of producing clear zones was Kepung District and the lowest was Badas District.
Table 3. Mean diameter of the clear zone in the chlorpyrifos insecticide bioassay test
Isolate
Average Clear Zone Diameter at 24 – 96 hour Observations (cm) 24 Hours
(x̄ ± SD)
48 Hours (x̄ ± SD)
72 Hours (x̄ ± SD)
96 Hours (x̄ ± SD) Kontrol 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 A
P2 0.45 ± 0.06 fg 0.78 ± 0.10 f 1.13 ± 0.13 ef 1.18 ± 0.10 E
P4 0.25 ± 0.06 cd 0.53 ± 0.05 cd 1.03 ± 0.05 de 1.18 ± 0.05 E P5 0.45 ± 0.06 fg 0.75 ± 0.10 ef 1.10 ± 0.12 ef 1.23 ± 0.10 E
P7 0.30 ± 0.08 de 0.45 ± 0.06 bc 0.90 ± 0.08 d 1.15 ± 0.06 E
P12 0.30 ± 0.00 de 0.35 ± 0.06 b 0.68 ± 0.10 c 0.70 ± 0.08 Bc
P16 0.48 ± 0.05 g 0.63 ± 0.10 de 1.08 ± 0.22 ef 1.08 ± 0.22 E
P18 0.45 ± 0.06 fg 0.83 ± 0.15 f 1.45 ± 0.24 g 1.63 ± 0.19 G
K2 0.33 ± 0.05 de 0.55 ± 0.10 cd 0.95 ± 0.10 de 1.23 ± 0.10 E
K6 0.43 ± 0.05 fg 0.85 ± 0.06 f 1.25 ± 0.06 f 1.58 ± 0.10 Fg
K8 0.25 ± 0.06 cd 0.63 ± 0.05 de 0.68 ± 0.10 c 0.85 ± 0.06 Cd
K13 0.38 ± 0.05 ef 0.75 ± 0.06 ef 1.13 ± 0.05 ef 1.45 ± 0.13 F
B6 0.18 ± 0.10 bc 0.43 ± 0.05 bc 0.73 ± 0.10 c 0.90 ± 0.14 D
B9 0.18 ± 0.05 bc 0.33 ± 0.05 b 0.60 ± 0.00 c 0.75 ± 0.10 Cd
B12 0.15 ± 0.06 b 0.33 ± 0.13 b 0.43 ± 0.05 b 0.58 ± 0.05 B
Note: Numbers followed by the same letter in the same column indicate that the results are not significantly different based on the Duncan test, the error level is 5%, SD = Standard Deviation
According to Akbar and Sultan (2016), the clear zone around bacterial colonies indicates phosphate dissolution activity by bacteria. The chlorpyrifos compound is composed of a phosphate group. Thus, it can be seen that the bacterial isolates resulting from exploration have the potential to degrade the insecticide chlorpyrifos. The bioassay results of isolates that form clear zones are presented in Figure 1.
Figure 1. Bioassay results of isolates capable of forming clear zones: (a) control (b) isolate
P1 (c) isolate K6 (d) isolate P18
Based on Table 4, the percentage of degradation of the insecticide chlorpyrifos by symbiotic bacteria increased at each observation time. The highest percentage of chlorpyrifos insecticide degradation was found in isolate P18, namely 72.90%.
Meanwhile, the lowest percentage of degradation was found in isolate B12, namely 48.86%. Symbiotic bacteria that have a high percentage of degradation have potential as bioremediation agents for chlorpyrifos contaminated environments. This is in accordance with the opinion of Puspitasari and Khaeruddin (2016) that bioremediation aims to change dangerous pollutant compounds in polluted environments into harmless compounds by utilizing microorganisms such as bacteria. Bioremediation will produce the final results in the form of carbon dioxide and water. From the data on the percentage of degradation of the insecticide chlorpyrifos, there were 5 isolates that had the best potential in degrading the insecticide chlorpyrifos, namely isolates P18, K6, K13, K2, and P5.
Tabel 4. Percentage of degradation of the insecticide chlorpyrifos by bacteria
Isolat
Persentase Degradasi Insektisida Klorpirifos (%)
24 jam 48 jam
72
jam 96 jam Kontrol 0.00 0.00 0.00 0.00
P2 42.73 56.21 65.08 66.12 P4 29.17 46.59 63.05 66.18 P5 42.73 55.36 64.58 67.05 P7 32.92 42.73 59.91 65.69 P12 33.33 36.67 36.67 53.71 P16 44.09 50.79 63.75 63.75 P18 42.73 57.55 70.43 72.90 K2 35.00 47.50 61.18 67.05 K6 41.36 58.57 67.54 72.37 K8 29.17 50.96 52.75 58.57 K13 38.33 55.49 65.20 70.64 B6 21.73 41.36 54.53 59.71 B9 22.32 35.00 50.00 55.38 B12 19.64 34.28 41.36 48.86
The results of the bioassay test on symbiotic bacterial isolates showed that 14 bacterial isolates had the potential to degrade the insecticide chlorpyrifos, which was indicated by the formation of a clear zone. Based on the statement by Irene et al. (2020), bacterial colonies that are able to form clear zones are thought to be able to degrade hydrocarbons. Bacteria utilize the carbon compounds contained in the insecticide chlorpyrifos as an energy source for cell metabolism.
Based on the results of the identification of symbiotic bacteria, isolates were obtained from the genera Pantoea, Erwinia, and Coryneform. The Pantoea genus has the potential to degrade the insecticide chlorpyrifos. This is supported by the results of research conducted by Wiguna (2019) that bacteria from the genus Pantoea sp. which is capable of degrading the profenofos insecticide which is characterized by the formation of a clear zone with a diameter of up to 1.95 cm at an insecticide concentration level of 0.75 ml/L, 1.86 cm at an insecticide concentration level of 1.5 ml/L and 1.67
cm at an insecticide concentration level of 3 ml/L. Apart from that, Pantoea sp. It has been reported to have been used for industrial purposes as a biodegradation and remediation agent for herbicides and other toxic substances (Hidayat et al., 2018).
Characterization of Symbiotic Bacteria in the Digestive Tract of Spodoptera exigua
Morphological Characterization
Morphological characterization of bacteria can be done by observing the characteristics of a single colony of bacteria that has been grown on Nutrient Agar media. Observations made include shape, elevation, color and edges.
Observation results on 14 bacterial isolates showed that all bacteria had a circular shape. Based on research by Wiguna (2019), bacteria with circular morphological characteristics with convex elevations and entire edges are gram- negative bacteria. The results of the characterization of 14 bacterial isolates are presented in Table 5.
Physiological and biochemical characterization
Physiological and biochemical characterization was carried out using testing stages including gram staining test, 3% KOH test, oxidative-fermentative test, bacterial growth test on YDC media, catalase test, and endospore staining.
Gram-negative isolates were not tested for catalase and endospore staining.
According to Utama et al. (2018) that catalase testing is generally used to test lactic acid bacteria which are included in the group of gram-positive bacteria.
Meanwhile, endospore staining is only used to test bacteria from the genera Bacillus, Clostridium, Coryneform, and Streptomyces. The results of physiological and biochemical tests are presented in Table 5.
Tabel 5. Results of characterization of symbiotic bacterial in the digestive tract of S. exigua
Keterangan: TU = No Test, Gram Stain (Red) = Gram Negative, Gram Stain (Blue) = Gram Positive, 3% KOH Test (Slimy) = Gram Negative, 3% KOH test (No Mucus) = Gram Positive
Karakerisitik Morfologi Karakterisitik Fisiologi dan Biokimia
Kode
Isolat Bentuk Elevasi Warna Tepi Bentuk Sel
Pewarnaan Gram
Uji KOH
3% Uji Katalase Pengecatan
Endospora Uji OF Uji
YDC Genus
P2 Circular Convex Putih
Keruh Undulate Batang Merah Berlendir TU TU Fermentatif Putih Erwinia P4 Circular Convex Orange Undulate Batang Merah Berlendir TU TU Fermentatif Kuning Pantoea P5 Circular Raised Putih
Keruh Undulate Batang Merah Berlendir TU TU Fermentatif Putih Erwinia
P7 Circular Convex Kuning Entire Batang Merah Berlendir TU TU Fermentatif Putih Erwinia
P12 Circular Convex Putih
Susu Entire Batang Biru Tidak Berlendir
Bergelembung
(Positif) Negatif TU TU Coryneform
P16 Circular Convex Putih
Keruh Erose Batang Merah Berlendir TU TU Fermentatif Putih Erwinia
P18 Circular Convex Orange Entire Batang Merah Berlendir TU TU Fermentatif Kuning Pantoea
K2 Circular Flat Krem Entire Batang Merah Berlendir TU TU Fermentatif Putih Erwinia
K6 Circular Flat Orange Entire Batang Merah Berlendir TU TU Fermentatif Kuning Pantoea
K8 Circular Flat Putih
Keruh Entire Batang Merah Berlendir TU TU Fermentatif Putih Erwinia
K13 Circular Convex Orange Entire Batang Merah Berlendir TU TU Fermentatif Kuning Pantoea
B6 Circular Flat Orange Entire Batang Merah Berlendir TU TU Fermentatif Kuning Pantoea
B9 Circular Flat Kuning Entire Batang Biru Tidak Berlendir
Bergelembung
(Positif) Negatif TU TU Coryneform
B12 Circular Umbonate Kuning Entire Batang Merah Berlendir TU TU Fermentatif Putih Erwinia
DOI : http://dx.doi.org/10.21776/ub.jpt.2023.008.2.10 The gram staining test is a test that aims to determine the gram group of the bacteria being tested. The gram group is divided into two, namely gram negative and gram positive. Based on the results of gram staining on 14 selected bacterial isolates, there were 12 isolates that were classified as gram-negative bacteria, shown by red results when observed under a microscope and 2 isolates of bacteria that were classified as gram-positive bacteria, shown by blue color results when observed (Figure 2).
Figure 2. Gram staining test results: (a) B12 isolate, red bacterial colonies including gram negative (b) P12 isolate, blue bacterial colonies
including gram positive
The 3% KOH test aims to determine the gram group of the bacteria being tested.
Based on testing, it was discovered that as many as 12 bacterial isolates produced mucus when drawn using a loop needle.
Meanwhile, 2 isolates produced mucus when drawn using a loop needle (Figure 3). Based on the statement by Amrulloh et al. (2021), the formation of mucus in gram negatives is caused by bacterial cell walls which are more sensitive and do not have resistance to inhibiting bases. When reacted with KOH, the cell walls of gram-negative bacteria will break and lysis occurs so that the bacterial DNA is not bound.
Figure 3. 3% KOH test results: (a) isolate K13, slimy including gram negative (b) isolate B9, not
slimy including gram positive
The oxidative-fermentative test aims to determine the aerobic or anaerobic properties of the test bacteria on gram- negative bacteria. These results were characterized by a color change in the growth medium from green to yellow, both on media without paraffin oil and on media with added paraffin oil (Figure 4). According to Arfiandi and Tumbol (2020), the fermentative oxidative test is fermentative if the color changes in the test medium from green to yellow, whether the medium has added paraffin oil or not. The color change in the paraffin-covered media indicates that the bacteria are able to utilize carbohydrates under anaerobic conditions through the fermentation process.
Figure 4. The results of the oxidative- fermentative test on isolate P16 were
fermentative
The bacterial growth test on YDC media was carried out to test gram-negative bacteria. The YDC test functions to determine bacteria belonging to the genus Erwinia and Pantoea (Figure 5). Based on
Schaad et al. (2001) if fermentative bacteria grown on YDC media produce white colonies, they belong to the genus Erwinia, whereas if they produce yellow colonies they belong to the genus Pantoea.
Figure 5. YDC test results after 48 hours incubation: (a) isolate K6, genus Pantoea (b)
isolate P2, genus Erwinia
The catalase test is carried out on gram-positive bacteria by dripping hydrogen peroxide (H2O2) solution on a glass object that has been given bacterial suspension (Figure 6). The bubbles produced indicate that the bacteria have the catalase enzyme.
Based on the statement of Khairunnisa et al.
(2018), positive results in the catalase test are indicated by the formation of air bubbles.
Air bubbles can form because bacteria have the enzyme catalase. This enzyme is able to decompose hydrogen peroxide (H2O2) into dihydrogen oxide (2H2O) and oxygen (O2).
Figure 6. Positive results on the catalase test: (a) isolate B9 (b) isolate P12
Endospore staining is carried out on bacteria from the gram-positive group. The purpose of the test is to determine the presence of spores in bacteria. Based on the statement by Purwaningsih and Wulandari (2021), spores are a form of bacteria that defend themselves from environmental conditions that are less supportive of their growth process. Bacteria that are capable of forming spores belong to the Bacillus sp group. and Clostridium sp. Based on the
results of observations, it is known that all bacterial isolates do not have spores (Figure 7). This is indicated by the absence of green color in bacterial colonies and red vegetative cells. This is in accordance with the opinion of Wulandari and Purwaningsih (2020) that the presence of spores in bacteria when stained using 5% malachite green will form a green color. The staining results of spore- bearing bacteria show that the spores will be green while the vegetative cells will be red.
Figure 7. Results of spore staining of test bacterial isolates: (a) P12 isolate (b) B9 isolate (c) bacterial isolate with spores as a comparison
(Wulandari and Purwaningsih, 2020)
Identification of symbiotic bacteria in the digestive tract of S. exigua
The results of the characterization of each of the 14 bacterial isolates were then identified according to morphological, physiological and biochemical characteristics with reference to the identification reference Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) and Schaad et al. (2001) up to the Genus level. Based on the identification results, it was discovered that 7 bacteria were identified as belonging to the Erwinia genus, namely isolates P2, P5, P7, P16, K2, K8, and B12. Furthermore, 5 bacteria were identified as Genus Pantoea, namely isolates P4, P18, K6, K13, and B6.
And as many as 2 bacteria were identified as
the Coryneform Genus, namely isolates P12 and B9.
CONCLUSION
Based on the results of research conducted on symbiotic bacteria in the digestive tract of S. exigua, it can be concluded that:
1. The results of exploration of symbiotic bacteria in the digestive tract of S. exigua showed that there were 45 isolates that were able to grow in contaminated media. A total of 18 isolates were obtained from Pringan subdistrict, 14 isolates from Kepung subdistrict, and 13 isolates from Badas subdistrict.
2. Symbiotic bacteria that have the potential to degrade insecticides can be seen from the clear zone formed. A total of 14 bacterial isolates were able to produce clear zones. These isolates include isolates P2, P4, P5, P7, P12, P16, P18, K2, K6, K8, K13, B6, B9, and B12.
Isolates K6 and P18 had the best degradation ability because they were able to form the widest clear zone, namely isolate K6 at 1.58 cm and isolate P18 at 1.63 cm.
3. Symbiotic bacteria that have the potential to degrade the insecticide chlorpyrifos were identified to genus level.
Identification is carried out through morphological characterization as well as physiological and biochemical characterization. Based on the identification results, three genera of bacteria were obtained that had the potential to degrade the insecticide chlorpyrifos, namely the Erwinia genus, the Pantoea genus, and the Coryneform genus.
ACKNOWLEDGMENT
The author thanks the Faculty of Agriculture, Brawijaya University for
providing support, facilities and funding for this research.
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