PHYSICO-CHEMICAL, VIABILITY EVALUATIONS AND EFFICACY ASSESSMENT OF Bacillus subtilis AGAINTS SOFT ROT DISEASE IN
PHALAENOPSIS
Wakiah Nuryani, Hanudin, Evi Silvia Yusuf, Kurniawan Budiarto Indonesian Ornamental Crops Research Institute (IOCRI)
Jl. Raya Pacet-Ciherang, Pacet, Cianjur (43253), West Java, Indonesia Corresponding author : [email protected]
ABSTRACT
The study of biological agents in controlling plant disease has discovered many potential microbes with numoerous mode of actions.
In the end, these potential microorganism should qualifiedly fulfill several requirements before they are formally stated for commercialization and wider implementation. Evaluations on their physico-chemical characteristics and viabililty after certain storage period were needed to ensure the effectiveness of the product during transportation and commercialization process. The research was aimed to evaluate B. subtilis strains B7 and B30 for their physio-chemical characteristic and viability after 6 months storage and investigate the efficacy the strains against soft root disease of Phalaenopsis. The results showed that both antagonists showed stable perfomances in physico-chemical, viability and afficacy evaluations after 6 months storage. Possitive indication of bacteria existences were demonstrated from the decrase in pH, murky suspensions and foul smelling as indications of fermentative reactions under optimal and minimal nutrients. Both antagonists also had stable viability after storage and effectively inhibited soft rot disease when applied on the infected plants with slightly lesser supression from streptomycin sulphate.
Keywords : Bacillus subtilis, biological control, soft rot, Phalaenopsis, efficacy evaluation.
INTRODUCTION
The uses of biopesticide to control important plant disease in crop production have driven significant attention in the last two decades in Indonesia. The succesful implementation of Integrated Pest Management (IPM) to control leafhoppers in rice during 1980’s, 1
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increased the government confidence to widen the implementation on other important commodities and these resulted in the decrement of synthetic pesticides for more than 300% (Laba, 2010).
The efforts to widen the application of biological agents to control important pests and diseases in crop production have made significant efforts on the research and assessment of pertinent fields not only in food crops, but in industrial, estate and horticultural crops as well, including ornamentals. In Phalaenopsis for example, several reports have indicated the bacterial soft rot was one of the most disastrous diseases that made significant economic losses even up to this moment (Meera, Louis, & Beena, 2016). The general symptom was characterized by soft lesion and decay of the tissues with foul odors. The disease might quickly spread in warmer and humid environment (Nuryani, Yusuf, Hanudin, Djatnika, & Marwoto, 2012).. At least four bacterial pathogens were recognized to be the causal agents of the disease, i.e.
Pectobacterium carotovorum subsp. carotovorum (Samson et al., 2005;
Hanudin & Rahardjo, 2012), Pseudomonas viridiflafa (Hanudin &
Rahardjo, 2011), Dickeya dadantii (Sudarsono, Elina, Giyanto, & Sukma, 2018; Trijoko, Kiswanti, Hanudin, & Subandiyah, 2011; Firgiyanto, Aziz, Sukma, & Giyanto, 2016; Lee, Kim, Lee, Hur, & Koh, 2014), Burkholderia gladioli (Keith, Sewake, & Zee, 2005; Moon, Park, Jeong, Han, & Park, 2017), and P. carotovorum subsp. carotovorum is one of the most 41
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commonly detected in tropical and subtropical nurseries (Trijoko, Kusumandari, & Hartono, 2011).
Growers still relied on chemicals in handling the problem, though such substances were considered costly and dangerous to the environment and human health (Handayati, Hanudin, & Soedjono, 2004). Since there is no resistance variety has been commercially registered, thus the application of antagonistic microbes became the alternative way out to encounter the question addressed. The method was known to be environmental friendly and has been reported successful in reducing the bacterial pathogen attacks in many floriculture crops (Djatnika, 2012).
Responding the urgent need from the growers, Indonesian Ornamental Crops Research Institute (IOCRI) has released several bio pesticide products in liquid and powder formulations containing Bacillus substilis and Pseudomonas fluorescens to control the diseases in 2009 (Hanudin, Nawangsih, Marwoto, & Tjahjono, 2013). Consecutive tests conducted on the formulation revealed that 2 isolates B. subtilis, namely B7 and B30 were able to inhibit soft rot development in Phalaenopsis with the percentage suppression of 24.53 and 34.07%, respectively.
Evaluation on liquid formulation at subsequent seasons affirmed that B7
might prevent the disease infection and development up to 34.45%
(Nuryani et al., 2012).
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The bio-bactericide should undergo and succeed several quality evaluations before release for commercialization. The evaluations includes maximum storage period with consistent physico-chemical properties and viability of the active ingredient to ensure the effectivity of the bio-bactericide. The information regarding the results of evaluation would be listed on the package label of product, including the date of test and product expired (Prabayanti, 2010) to protect/guarantee the consumers when using the bio bactericide. Regarding to the said matter, the research was then conducted to evaluate the formulation of B7 and B30 for quality assessment. The evaluation consisted of physico- chemical, viability testing after 6 months storage and effectiveness of the bio bactericide to the target pathogen after storage.
MATERIALS AND METHODS
The research was conducted under laboratory and greenhouse conditions at the IOCRI from January and December 2013. The B.
subtilis formulations strains B7 and B30 were collected from the Biological Control laboratory of IOCRI while the 2 months-old Phalaenopsis plants were obtained from commercial nurseries.
Phsyco-chemical evaluation
The physico-chemical evaluations represented physical and chemical properties B. subtilis isolates i.e. pH, color, and pH and/or color 85
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changes under certain condition. The isolates of B7 and B30 were inoculated into two different media i.e. minimum and optimum nutritive contents. Per litre minimum medium composed of 20 mg bean sprouts and 2 g potato extracts. While per litre optimum medium contained extracts of 20 mg bean sprouts, 10 g carrots, 2 g celery, 2.5 g spinach, 2 g cucumber, 2.5 g cabbage, 2 g squash, 0.5 g cassava starch, 2 g potato and 0.01 % shrimp shell. For about 500 ml of each medium were put 500 ml in elenmeyer flask and then acutoclaved.
After 24 h cooling, the B7 and B30 with the density 109 cfu/ml strains were inoculated into the media based on the arrangement of treatments (Table 1). The inoculated bacteria were then stored under room temperature (25±2 0C) for 6 months. The observation of pH, color and the changes of pH and color were conducted prior and after 3 weeks storage.
Table 1. The treatments of physico-chemical evaluation for B7 and B30 bacterial isolates.
N
o. Code Description of Treatments
1. B7-1 B7 isolate inoculated in minimal nutritive media
2. B7-2 B7 isolate inoculated in optimum nutritive media
3. B30-1 B30 isolate inoculated in minimal nutritive media
4. B30-2 B30 isolate inoculated in optimum nutritive media
5. K7-0 B7 isolates suspended in aquadest (control B7)
6. K30-0 B30 isolate suspended in aquadest 109
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(control B30)
Viability assessment
The suspension cultures of B7 and B30 under optimum, minimal and aquadest were taken every month for viability testing. For about 1 ml suspension were diluted in 9 ml aqudest and gently shaken until homogen. These new suspesions were then serially diluted 5 times with the same aquadest volume. For about 1 µl suspension from the 6th dilution were pippeted and inoculated into 15 ml Nutrient Agar (NA) medium following modified method of Hsu et al. (1994). The viable bacterial colonies were counted using colony counter “Suntex” (model CC-560, 930 801 828) after 48 h incubation.
Efficacy assessment of the stored B. subtilis strains Preparation and maintenace of testing plants
The 2 months-old plants were planted in 10 cm (Φ) pot containing chooped fern bark and spagnum moss (1:1 v/v) as th media. The plant were maintained in a standard procedure under 65% black paranet shading. The irrigation was given twice a day by spraying the plant leaves with water, mainly the abaxial part with a volume of 50 ml/plant.
Foliar fertilizers were applied regularly with the volume of 30 ml/plant once a week using Growmore (N:P:K = 30 : 10 : 10) in vegetative and 20 : 20 : 20 (N:P:K) at flowering stages.
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Preparation of bacterial pathogen and antagonist B7 and B30 suspension The pathogenic bacterium Pectobacterium carotovorum pv.
carotovorum isolate used was collected from the Laboratory of Micology IOCRI. The antagonist B. subtilis strains B7 and B30 isolates from the varying culture media in Table 1 were monthly taken, separately inoculated from the pathogen isolate in SPA media and incubated for 24 h at 30 ± 2 0C. The grown colonies were collected for about 3 loops, suspended in 10 ml aquadest and vorticed to form homogenous suspension with the density of 1012 cfu/ml. For about 1 ml suspension was diluted into 500 ml Nutrient Broth (NB) medium and gently shaken in 3 rpm for 24 h at 30 0C. The bacteria were then suspended into media treatment (Table 2) until the density reached 109 cfu/ml.
Table 2. The media treatments for efficacy assessment of B7 and B30 antagonistic bacterial isolates.
N
o. Code Description of Treatments
1. B7-1 109 cfu/ml B7 isolate suspended in minimal nutritive media
2. B7-2 109 cfu/ml B7 isolate inoculated in optimum nutritive media
3. B30-1 109 cfu/ml B30 isolate suspended in minimal nutritive media
4. B30-2 109 cfu/ml B30 isolate suspended in optimum nutritive media
5. K7-0 109 cfu/ml B7 isolates suspended in aquadest (control B7)
6. K30-0 109 cfu/ml B30 isolate suspended in aquadest (control B30)
7. K1 Streptomycine sulphate 1.5 WP (2 g/l) (control 1)
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8. K0 Aquadest (control 2)
Inoculation of pathogen and antagonist B7 and B30 applications on the testing plants
The artificial infection of pathogen to the testing plant was conducted using pin pricking method as described by (Hanudin &
Rahardjo, 2011). The pricking was conducted on 2 selected leaves, first and second leaves from the base for each single plant. The leaf adaxial part was 5-diagonally pricked using hypodermic syringe for about 1-2 mm deep depending on the leaf thicknesses. The pricked wounds were then covered with wet cotton that previously dipped in pathogen suspension for 10 minutes. After the cottons containing pathogen were removed, the wounds were then covered by the other cottons that previously dipped in antagonist suspensions as presented Table 2.
Additional antagonists were also sprayed weekly until the plants were flowering.
The observation on the efficacy test included incubation period that was recorded daily up to 7 days after pathogen inoculation.
Disease intensity was observed weekly until 4 months after inoculation.
Degree of antagonist colonization on the leaves and root zones were measured according to Hsu et al. (1994). All the data gathered were analyzed using ANOVA continued by DMRT at 95% level of confidence.
Degree of antagonist suppression against pathogen was calculated using the following formula.
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Where;
DS = Degree of supression
C = Disease intensity of control treatment
T = Disease inensity of antagonist-treated plants
RESULTS AND DISCUSSION Physico-chemical evaluation
Phyco-chemical evaluations are considered as an important stage in quality assessment based on scientific, objective and fair observation on a certain product. The evaluations revealed physical and chemical characteristics (Djunaedy, 2009). The changes in pH and suspension color of B. subtilis B7 and B30 isolates were observed in aquadest, minimum and optimum nutritive media after 3 weeks storage (Table 3).
Foul smelling was also detected on B30 under minimal and optimum and B7 in optimum media. The changes in pH, suspension color and foul odor inferred certain processes of biochemical reaction during storage as indications of biological activity of the bacteria. The decrease of media pH was due to acidic substances released by bacteria as secondary product of metabolism (van Dijl & Hecker, 2013). The acids released from the anaerobic carbohydrate breakdown process are
DS = Σ(C – T)C x
100
% 190
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complex depending the existed biochemical pathways, such as lactic, acetic, propionate and butirate together with CO2 and ethanol (Sansinenea & Ortiz, 2011).
Table 3. pH and suspension color of B. subtilis B7 and B30
before and after 3 weeks storage in aquadest, minimal and optimum media.
Treatment Before storage After 3 weeks storage pH Color pH Change on color and
other characteristic B7-1 (B7 in minimal
nutritive media) 6.5 clear,
yellowish 4.1 murky, whitish B7-2 (B7 in optimum
nutritive media) 6.2 clear,
yellowish 4.4 murky, yellowish, foul smelling
B30-1 (B30 in minimal
nutritive media) 6.5 clear,
yellowish 4.2 murky, yellowish, foul smelling
B30-2 (B30 in optimum
nutritive media) 6.2 clear,
yellowish 4.6 murky, darkish yellow, foul smelling K7-0 (B7 in aquadest
(control B7)
6.2 clear 6.5 Murky
K30-0 (B30 in aquadest
(control B30) 6.5 clear 6.5 Murky
Murky liquid appearance and foul smelling represented the existed bacterial colonies, while the typical odor might be produced from the bio-degradation of protein and lipid and other organic compounds by the bacteria in gaseous forms (Fujita, Matsuoka, & Hirooka, 2007; Fujita, 2009). B. subtilis strains B7 and B30 suspended in steril also changed the appearance of water into murky, yet less foul odor was detected. These conditions inferred that the bacterial colonies still survived after 3 weks storage under lack of nutrient and the less odor reflected less biochemical proccesses within the medium. In prolonged duration, lack of nutrient might contribute to reduced cell size (Chien, Hill, & Levin, 217
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2012) and death of bacteria resulted from nutrient competition (Allocati, Masulli, Di Ilio, & De Laurenzi, 2015).
Viability Assessment
Population dynamic of antagonist bacteria in testing media reflected not only the number of active or living bacterial colonies, but informed the capability of respected strains to expose the potential adaptation mechanism in the provided media (Johnsen, Dubnau, &
Levin, 2009). These inferred that under optimum and minimal media, the population would decrease and/or increase depending on the capability of bacterial colonies and the support of environment conditions including the media in providing any necessary factors for survivals. Under optimal nutrient, both strains B7 and B30 population increased after 1 to 2 months storage and started to decrease here after, though the population after 6 months storage was stilll higher than the initial population before storage (Table 4).
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Table 4. B7 and B30 population dynamic under different media before and after certain storage periods.
Treatment
Bacterial population dynamic (cfu/ml) Before
storage
Storage period (months)
1 2 3 4 5 6
B7-1 (B7 in minimal nutritive media)
(5±3.5)x10
8
(9.45±3.7) x1010
(7.25±2.7) x1010
(9.25±3.5) x109
(5.7±1.7) x109
(3.5±1.1) x109
(1.9±0.3) x108 B7-2 (B7 in
optimum
nutritive media)
(1.45±0.75 )x109
(8.6±3.7)x 1010
(6.7±3.5)x 1010
(5.9±1.9)x 109
(4.7±2.3) x109
(2.9±0.7) x109
(9.7±5.1) x108 B30-1 (B30 in
minimal nutritive media)
(2.5±1.7)x
109 (7.15±1.5)
x1011 (5.5±2.5)x
1011 (7.7±3.5)x
1010 (5.9±2.7)
x1010 (4.7±1.3)
x1010 (4.5±1.4) x109 B30-2 (B30 in
optimum
nutritive media)
(1.7±0.9)x 109
(6.6±2.9)x 1011
(5.6±2.7)x 1011
(4.7±1.9)x 1010
(3.5±0.9) x1010
(2.9±0.7) x1010
(1.9±0.2) x109 K7-0 (B7 in
aquadest (control B7)
(3.4±0.9)x
1010 (7.8±3.7)x
1010 (6.7±3.7)x
1010 (4.9±1.7)x
109 (4.7±1.2)
x109 (3.9±0.8)
x109 (9.8±5.3) x108 K30-0 (B30 in
aquadest (control B30)
(3.0±1.7)x 108
(8.3±5.7)x 1010
(7.2±4.1)x 1010
(9.7±3.9)x 109
(7.7±4.5) x109
(6.7±2.7) x109
(5.7±1.9) x108
Under minimal nutrient, antagonist strain B7 increased in numbers up to 2 months storage (Table 4). The population was then decreased slightly up to 5 months and continued falling down until 38% from the initial population before storage. Strain B30 showed better performance, in that the population was considered higher than initial poulation even after 6 months storage. These conditions indicated that strain B30 were considered more competitive in utilization the limited sources under stress condition and the observable living bacterial colonies of B.
subtilis strains B7 and B30 after 6 months in minimal nutritive media have revealed the viability capacities of both antagonists. Similar findings were also reported by Sawant et al., (2016) and Balouiri, Sadiki,
& Ibnsouda, (2016) that different bacterial strains might show similar 252
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fate in responding certain extreme conditions, though the degree of response and persistence of responses might also be different, including the survival and reproductive capacities.
The persistent viability of both B. subtilis strains B7 and B30 after storage were furtherly confirmed when they were suspended in aquadest and stored up to 6 months (Table 4). After 6 months, the population of B7 was 2.9% left, while B30 increased in numbers almost double from the initial before storage. The physico-chemical activities and persistent viabilities of strains B7 and B30 in various media up to 6 months storage indicated that these strains within their carrying subtsances have performed qualified physco-chemical and viability properties as required by formal regulation (Hasyim, Setiawati, &
Lukman, 2015).
Efficacy evaluation of B. subtilis strains on infected plants
In general, the symptom of disease arose when the interaction among three factors, the virulent pathogen, susceptible host and the environment were conducive for the pathogen to grow and develop (Ma et al., 2007). The successful attacks of soft rot P. carotovorum subsp.
carotovorum was presented by the infected plants sprayed with aquadest (Figure 1a) that were characterized by moist and watery decay of the plant leaf after 1 day inoculation (Table 5). The decay symptom was manifestated from the destruction and breakage of the 268
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meristematic and parechyma cell walls, i.e. primary cell wall and inner lamella into simpler compounds that were utilized by the pathogen (Meera et al., 2016). The degradation of cell wall was due to the activity of a set of extracellular pathogen enzymes such as pectinase, cellulase and protease resulting cell wall plasmolisis and death (Joko, Subandi, Kusumandari, Wibowo, & Priyatmojo, 2014).
Figure 1 (a) Initial soft rot infection and (b) enlarged lessions and tissue decay at 3 days after inoculation on Phalaenopsis due to Pectobacterium carotovorum subsp.
carotovorum attacks.
Table 5. Incubation period, disease intensity and percentage of supression of soft rot by B7 and B30 treatment applications.
Treatments Incubation period*)
(Days)
Soft rot disease intensity*)
(% DAI**))
Percentage of supression
1 3 7 (%)
B7-1 (B7 in minimal nutritive media)
1.00 a
0.72 b
12.32 b
27.14 b
68.35 B7-2 (B7 in optimum
nutritive media) 1.00
a 0.99
b 14.62
b 35.41
c 58.71
B30-1 (B30 in minimal
nutritive media) 1.00
a 0.62
b 12.05
b 29.73
b 65.33
B30-2 (B30 in optimum
nutritive media) 1.00
a 1.63
c 15.01
b 33.12
bc 61.38
K7-0 (B7 in aquadest 1.00 1.75 30.07 67.85 20.87
(a) (b)
291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313
(control B7) a c c d K30-0 (B30 in aquadest
(control B30) 1.00
a 1.14
bc 23.67
c 69.74
d 18.67
Streptomycine Sulphate 17.5 b
0.00 a
0.00 a
00.00 a
76.10
Aquadest (Control) 1.00
a 25.75
d 55.75
d 85.75
e -
CV (%) 17.50 15.75 15.75 17.19 -
Remarks *) = Values in the same column followed by different letters, differ significantly under DMRT (P ≤ 5%).
**) DAI = Days after inoculation.
One or several small water-soaked lessions enlarged rapidly to finally rot the whole leaf within 2-3 days after inoculation (Figure 1b).
The wet rot released a foul odor, and furtherly, the disease spread more slowly into the basal shoot and causing root death (Hanudin & Rahardjo, 2012). The development and spreading of the diseases might be triggered in warmer and humid environment (Moh, Massart, Jijakli, &
Lepoivre, 2012) and also influenced by type of orchids, plant resistance and leaf texture (Muharam, Rita, & Hanudin, 2012).
In general, the incubation period occurred in one day except on the plants treated by streptomycine sulphate. The symptom of the disease was observed after 17 days after inoculation in these treatment (Table 5). These indicated that application of the antibiotic might inhibit further infection (Nuryani et al., 2012) as also viewed from the absent of disease incidence up to 7 days after inoculation.
On the other hands, soft rot disease intensities were observed to be varied among the B7 and B30 treatments. Suspended for certain storage periods under minimal and optimal nutritive media, both
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antagonists were able to prevent the development of the disease more effective than those suspended in aquadest. These conditions were observed after 3 up to 7 days after inoculation, where the disease intensities treated by B7 and B30 stored under nutritive media suspension reached only up to 12 to 35.41% compared to those treated B7 and B30 stored under aquadest (23.67 – 69.74%) and control aquadest (55.75 – 85.75%). The condition inferred that numbers of bacterial colonies after certain period storage were not always in line with the effectiveness of the antagonists in supressing the disease infections.
The numbers of bacterial cells suspended in aquadest after 6 month storage were stable for B30 and slightly decrease in B7, while those under minimal and optimal nutrient were dynamic during storage. The number of cells after 6 months storage, especially B7 were higher when suspended in aquadest (Table 4), yet less effective in inhibiting the disease than that when stored under minimal and optimal nutrient (Table 5). These inferred that storing antagonists under the absent of nutrients was predicted affecting the competitive capacity of the antagonists againts the pathogen. Some reports indicated that under aquadest storage, the antagonists suffered nutrient starvation and might resulted in reductive division with alteration of cell morphology.
The protein synthesis might be sharply reduced and associated with the lower expression of proteins necessary for exponential growth. In long term period these conditions led to a rapid loss of cell viability (Abdeljalil 335
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et al., 2016). After 6 months storage under aquadest, the reduction of viability have not been observed from the slight decrease of colony population, however it is predicted that antagonistic capability started to reduce from higher disease intensity on the inoculated plants.
The infected plants treated by antibiotic streptomycine sulphate showed lesser soft rot disease development until flowering time (Table 5). The percentage of supression using the antibiotic reached more than 75%, while those under B. subtilis strains B7 and B30 treatments, the percentage of supression varied in the range of 58.71 – 68.35%.
Based on the results, the utilization of B. subtilis strains B7 and B30 were considered promising since these strains performed stable features during physico-chemical and efficacy evaluations. The substitution and reduction of chemicals and/or antibiotic like streptomycine with bio- bactericide was then should considerably be applied considering many negative effects of long-term pesticide uses in agriculture (Al-Zaidi, Elhag, Al-Otaibi, & Baig, 2011).
CONCLUSIONS
The antagonists B. subtilis strains B7 and B30 performed stable characteristics during physico-chemical, viability and afficacy evaluations after 6 months storage. The change in pH and color of media and smell reflected the acitivity and existence of the bacterial cells after storage. Both antagonists were able to maintain their 358
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viability from the stable cell numbers after 6 months storage under minimal and optimum nutrients. After 6 months storage, the antagonists remained effective in controlling the soft rot infections with the percentage suppression of 63.45%.
ACKNOLEDGEMENT
The authors would like to express their thank and high appreciation to the Director of The Indonesian Agency for Agricultural Research and Development (IAARD), through Center for Horticultural Resaerch and Deveopment (ICHORD), Indonesian Ornamental Crops Research Institute (IOCRI) that financed, gave suggestions, criticisms in the planning and implementation of research. The authors also wish to thank to the following personals; Mr. Saepuloh SP, Mr. Ridwan Daelani, Mr. Asep Samsudin, Mr. Iman Taufiq, and all those who helped and worked during the conduct of the research and report.
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