Characterization Antifungal of Microbial Unicellular Phylloplane Chili

ABSTRACT
Biological control agents can be obtained from unicellular microbial exploration. Human
health and sustainable agriculture are the main reasons for using biological control.
Isolates microbes were tested for antagonistic potential against Colletotrichum capsici
the causal pathogen ofed anthracnose diseases. The Mmost potential antagonists was
identified based on the morphology, biochemical and molecular characteristics. That
microbe was also conducted to confirm the antagonist is no’t a pathogen into the plant.
Growth media extract was used as a material for this test. Extracts were filtered from
microbial liquid culture media using a microfilter of 0,0022 µm. Furthermore, extracts
were characterized using a Proteinase K and temperature. The most potential
antagonist was identified as Pseudomonas aeruginosa with the highest inhibition. The
active extract had no effect on Proteinase K and have an influence on the temperature.
P. aeruginosa did no’t hypersensitive and also did no’t have pathogenicity in plants.
Keywords: Colletotrichum capsici, Pseudomonas aeruginosa, biocontrol, biotechnology,
Pseudomonas flourescens
INTRODUCTION
Anthracnose disease is a dangerous chili disease. It causes by the fungal pathogen
Colletotrichum capsici. Anthracnose in chili is characterized by the appearance of small
circular black spots on the skin of the fruit that spread along the long axis so that it
becomes more or less elliptical in shape. The impact can cause crop failure. So it needs
proper control and prevention. The use of chemical pesticides has a negative impact on
humans and the environment. One alternative control with the use of biological agents.
Biological control can be done with unicellular microbe phylloplane.
Phylloplane unicellular microbes are microorganisms that live and live on the surface.
These microbes have various roles including as biological agents to prevent and control
plant pests and diseases. Therefore, it is necessary to conduct research to ascertain the
benefits of its use to control C. capsici.
Chili is an important commodity in Indonesia. However, chili cultivation often fails due to
several factors, one of which is pests and diseases. Anthracnose disease in chili plants
is the most common disease found and almost always occurs in every chili area.
Anthracnose other than result in a decrease in yield can also damage the aesthetic
value of chili. Anthracnose is a dangerous chili disease[1].This disease is caused by the
pathogenic fungus Colletotrichum capsici. Anthracnose disease is characterized by the
appearance of small black spots on the skin of the fruit that spread along the long axis
so that the shape is more or less elliptical. The impact can cause crop failure. So it
needs proper control and prevention. The use of chemical pesticides has a negative
impact on humans and the environment. One of the control alternatives is the use of
biological agents. Biological control can be done with unicellular microbial phylloplane.
Unicellular phylloplane microbes are microorganisms that live and live on surfaces.
Phylloplane microbes are a very diverse and ubiquitous group of microorganisms,
occurring on the surfaces of plant species in major terrestrial and marine habitats.
Hundreds of species of bacteria, mycelial fungi, yeasts, and protozoa have been
detected in the phyllosphere and phylloplane of plants during this time[2]. Many of them
play an important role in the life of the plant, being phytopathogens or pathogen
antagonists, and producing vitamins, phytohormones, antibiotics, or toxins[3]. These
microbes have various roles including as biological agents to prevent and control plant
pests and diseases. Since chitin is an important structural component of the fungal cell
wall, chitindegrading microorganisms can inhibit the development of fungal mycelium.
The opportunity to use chitinolytic enzymes against fungi causing plant diseases was
the first to attract researchers’ attention However, chitinolytic microorganisms inhabiting
the supraterrestrial parts of plants have remained virtually uncharacterized, although
these parts of the plants are also vulnerable to fungal diseases[4].
Studies of the microbial community of the phylloplane of plants are of utmost
importance for understanding interspecies interactions in nature; from the practical point
of view, these studies provide the basis for a variety of biological pest control methods
aimed at increasing the productivity of agriculture, decreasing crop loss caused by
diseases and pests, etc[5]. We focused on study of the exploration, identification of
uncellular phylloplane microbe, and characterization of secondary metabolite under
various conditions may have considerable importance for development of methods for
biological control of phytopathogenic fungi. Therefore, it is necessary to conduct
research to ascertain the benefits of its use to control C. capsici.
MATERIALS AND METHODS

Exploration of Unicelluler Microbe

Chili 50 g was placed into erlenmeyer containing 100 ml of sterile distilled water.
The erlenmeyer was shaken using rotary shaker at 120 rpm for 24 hours. The soaking
water was then diluted into 10 0, 103, 104 and 105. 100 µl of suspension was planted on
YMA media (without antibiotic). The growing uniceluller microbes was then purified and
identification base on their morphological characters.

Production Test of Antibiosis Compound

Based on the pattern resulting from the antagonistic test[6], it was suggested that
unicellular microbe was capable to produce antibiosis compounds that inhibit C. capsici.
Therefore, the study was then continued to investigate whether the compound was
produced by the unicellular microbe. The unicellular microbe was cultured on Natrium
media following the growth curve of the unicellular microbe curve. The unicellular
microbe was removed from the media using centrifugation, the media were then
sterilized using a 0,22 µm microfilter. Several paper discs were dipped into the medium
for one minute, then placed at the edge of 1-day colony of C. capsici on PDA, and
incubated for 7 days. The percentage of colony inhibition was measured and calculated
using the formula:
r 2−r 1
P= × 100 %
r2
Note: P: the percentage of relative inhibition against the growth of pathogen, r1: the
spoke of pathogen colony which closed to paper discs, and r2: the spoke of pathogen
colony which avoided paper discs.

Biochemistry Identification of Microbe

Biochemical identification is used to identify bacteria. Biochemical tests such as motility

test, nitrate, lysine, omithine, H2S, Glucose, Mannitol, Xylose, ONPG, Indole, Urease,
V-P, Citrate, TDA, Gelatin, Malonate, Inositol, Sorbitol, Rhamonose, Sucrose, Lactose,
Arabinose, Adonitol , Raffinose, Salicin and Arginine using the MicrobactTM NAM
12A/B/E, 24E identification kit. The results of the biochemical test were analyzed using
the Microbact TM Identification Kits software from Oxoid. Bacterial biochemical testing
was carried out at the Microbiology Laboratory, Faculty of Medicine, Universitas
Brawijaya.

Molecular Identification of Microbe

The most potential unicellular microbe were identified their species based on molecular
character (16S ribosomal DNA region) by PCR, using 16S primer. As for the steps of
molecular identification process were as follows:

DNA Isolation
Method of DNA isolation referred to [7] Sambrook and Russell [16]. A total of 2 ml
of rich medium (NA) containing the antibiotic was injected with one colony of
transformed bacteria. Incubate the culture at 27oC with vigorous shaking with 1.5 ml of
culture stored in a microfuge tube. The centrifuge was carried out at maximum speed
for 30 seconds in a microfuge. Save unused parts of the original culture. After
centrifugation is complete, the media is removed by aspiration, allowing the bacterial
pellet to dry as much as possible. Resuspend the microbial pellet in 100 µl cold Alkaline
I lysis solution with strong vortex. Add 200 µl of freshly prepared Alkaline II lysis solution
for each microbial suspension. Close the jar tightly, and mix the contents by rapidly
inverting the jar five times. Keep the jar on ice. Add 150 µl of ice cold Alkaline III lysis
solution. Keep the jar on ice for 3-5 minutes. Microbial lysate centrifugation at maximum
speed for 5 minutes in a microfuge. Precipitate nucleic acid from the supernatant by
adding 2 volumes of ethanol. Collect the precipitated asic nucleic acid by centrifugation
at maximum speed for 5 minutes. Discard the supernatant by gentle aspiration. Add 1
ml of 70% ethanol to the pellet and invert the tube several times. Recover DNA by
centrifugation at maximum speed for 2 minutes. Again, removed all supernatant by
gentle aspiration. Discard any ethanol beads that form on the sides of the tube.
Dissolved the nucleic acid in 50 µl TE (pH 8.0) containing 20 g/ml DNAse-free RNAse
A. Vortex the solution slowly for a few seconds. Stored the DNA solution at -20oC. The
pellets obtained were tested for DNA quality and quantity using Nano-Drop (thermo
Nano-Drop 1000).Inoculate 2 ml of rich medium (NA) containing the appropriate
antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at
27oC with rigorous shaking. Pour 1,5 ml of the culture into a microfuge tube. Centrifuge
at maximum speed for 30 seconds at microfuge. Store the unused portion of the original
culture. When centrifugation is complete, remove the medium by aspiration, leaving the
bacterial pellet as dry as possible. Resuspend the microbe pellet in 100 µl of ice-cold
Alkaline lysis solution I by vigorous vortexing. Add 200 µl of freshly prepared Alkaline
lysis solution II to each microbe suspension. Close the tube tightly, and mix the contents
by inverting the tube rapidly five times. Store the tube on ice. Add 150 µl of ice cold
Alkaline lysis solution III. Store the tube on ice for 3-5 minutes. Centrifuge the microbe
lysate at maximum speed for 5 minutes at microfuge. Precipate nucleic acids from the
supernatant by adding 2 volumes of ethanol. Collect the precipitated nucleid acis by
centrifugation at maximum speed for 5 minutes. Remove the supernatant by gentle
aspiration. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several
times. Recover the DNA by centrifugation at maximum speed for 2 minutes. Again
remove all of the supernatant by gentle aspiration. Remove any beads of ethanol that
form on the sides of tube. Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing
20 µg/ml DNAse free RNAse A. Vortex the solution gently for a few seconds. Store the
DNA solution at -20oC. The obtained pellet was tested of quality and quantity of DNA
using Nano-Drop (thermo Nano-Drop 1000).

PCR Amplification
The primers used were 16S rDNA F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and
R (5’-TAC GGC TAC CTT GTT ACG A-3’). The total volume of PCR was 12 µl,
consisted of, 2 µl of dH2O, 1 µl of Forward, 1 µl of Reverse, 5 µl of PCR mix, 3 µl of
DNA template. PCR was run at condition of pre-denaturation at 94 oC for 4 min. After
then, denaturation at 94oC for 1 min, annealing at 50 oC for 1 min and elongation at 72 oC
for 2 min, the steps was repeated 35 cycles. The last was post elongation at 72 oC for 3
min. The PCR product was stored at -20 oC.

Electrophoresis Process
Process of electrophoresis was using 2% agarose gel and the determination of
fragment length of 16s regions was using 1 µl DNA marker 1 kb DNA Ladder. Process
of electrophoresis running at voltage 65 volt for 30 min. Gel of electrophoresis result
was observed by using Dox XR gel (BIO-RAD) connected to the computer to see the
lambent of DNA bands of PCR product. DNA bands at gel of electrophoresis result was
purified using kit Wizard® SV gel and PCR clean-up system (Promega). The last
process in this yeast molecular identification was DNA sequencing by Applied
Biosystem 3100 from 1st Base Company.

Alignment Sequences of 16s Regions

Electropherogram data and text file as a result of sequencing were edited
manually using Bioedit Program. The data of editing result was saved in form of FASTA
that could be matched directly using Basic Local Alignment Search Tool in
www.ncbi.nlm.nih.gov. Yeast species identification was based on the percentage of high
similarity of identical and was supported with the low E-value.

Microbial Growth Curve Calculation

Microbes are grown on artificial media. A total of 2 oses of microbial culture were
placed in 10 ml of liquid media. YMB media for yeast while NB media for bacteria. The
media that has been given microbial isolates is shaken for 1x24 hours. Take 2 ml of the
culture and inoculate it into 100 ml of the new culture medium. Observation at 0 hour
when the inoculant was added. The value of Optical Dentisty (OD) was calculated using
a spectrophotometer with a wavelength of 600. OD observations were carried out every
4 hours with 2 replications until it passed the stationary point of the microbe.

Effect of Extraction Time Active Compound against Antagonistic Ability of

Unicelluler Microbe
Unicellular microbial culture on 100 ml YMB media for yeast and 100 ml NB for
bacteria according to the microbial growth phase. The treatment of bottles and media
was in accordance with the results of the best treatment of pH, temperature and light
tests. Take 1 ml of culture fluid starting at 0, 8, 16, 24, 32, 40 and 48 hours. Then put it
in a microtube to be centrifuged so that it can separate the media from the biomass.
The last step was filtered using a 0.22 µl microfilter. The filter results were tested for
activity against C. capsici using parchment paper.
The Active Compound of P. aeruginosa
In the process of obtaining the active compound, it is necessary to know the right time
to extract based on the growth time of the isolate. Liquid media is one way to grow
microbes. Nutrient liquid media was deliberately made following the optimization of the
treatment of the effects of pH, temperature and light according to the previous test. The
character of the liquid media is that it has a pH of 5 and is placed in the erlenmeyer
tube when the shaker is covered with carbon paper. Once every 8 hours, the microbial
growth media is extracted using a microfilter to obtain the active compound that has the
best inhibition.

Effect of Proteinase K and Temperature Active Compound against Antagonistic

Ability of Unicelluler Microbe
After obtaining the best extraction time based on the highest percentage of
inhibition. The unicellular microbes were re-extracted according to the time treatment by
further testing the effect of adding 500 µl of Proteinase K to 500 µl of the active
compound. This treatment is useful to determine whether the type of active compound
has an effect or not. In addition, the active compound was treated at 100 oC to determine
its effect. The control is without the provision of proteinase K and the effect of
temperature. Then tested using the well method against C. capsici.

Hypersensitivity and Pathogenicity Tests

Bacteria were grown on NA media, and incubated for 24 hours. The bacteria
were harvested with sterile distilled water, then the bacterial suspension was infiltrated
into the underside of the tobacco leaves using a syringe. The control treatment used
sterile distilled water without bacteria. Hypersensitivity reactions will appear in the form
of necrotic wounds after 1-4 days after treatment. In the pathogenicity test, the principle
is almost the same as hypersensitivity, only using chilies that have been sterilized using
a 2% NaOCl solution for 30 seconds.

RESULT AND DISCUSSION

Identification of Unicelluler Microbe

The result of exploration was found 6 isolates of unicelluler microbes but only one
isolate is reported having ability antibiosis mechanism. C3C isolate, macroscopically
microbe colony had white greenish-colored, circle texture, elevation arise, 2-3 mm in
diameters. Solid line was formed less than 1 dai. Microscopic had rod or capsule cell
shape ranged from 1,23-2,70 µm in length and 464,47-741,95 nm in width, hyaline,
single cell.
A B

Figure 1. Colony of C3C isolate A. macroscopically (1) on PDA media age of 1 days
after incubation (dai). B. morphology (2).
Biochemical identification aims to determine the character of bacterial strains in
response to various test media so that they can be compared with strains of the genus
Pseudomonas bacteria that have been studied previously. The test results are shown in
the following table quoted from the books of [8]Schaad et al. [18] and Garrity et al. [7][9].
The result of biochemical test had positive KOH test, gram negative, facultative aerob
and positive flourescens test on King’B media. Another biochemical characteristics
showed positive oxidase, negative motility, negative nitrate, positive lysine, negative
ornithine, positive H2S, negative glucose, negative mannitol, positive xylose, negative
ONPG, negative indole, positive urease, positive gelatin, negative malonate, negative
inositol, positive sorbitol, negative rhaminose, positive sucrose, negative lactose,
positive arabinose, negative adonitol, positive raffinose, negative salicin, and positive
arginine.

Figure 2. Biochemistry of C3C isolate used identification from OXOID kit

The bacterial strains found were not exactly the same as the comparison (P.
aeruginosa and P. fluorescens). However, some characteristics have similarities with P.
aeruginosa such as oxidase test, mannitol, gelatin, arginine, adonitol, inositol, pigment,
growth at 41oC and 4oC. Meanwhile, when compared with P. flourenscens, there were
similarities in the oxidase test, nitrate, gelatin, sucrose, sorbitol, arabinose, adonitol,
arginine, xylose and tobacco HR. Based on biochemical tests using the OXOID
identification kit from MIKROBACTTM and bioinformatics analysis, isolate C3C had a
similarity with P. flourescens-25 by 84.93%. Antibiotics 2,4-diacetylphloroglucinol (Phl)
and phenazine-1-carboxylic acid (PCA) are often produced by P. flourenscens strains.
Therefore, it is necessary to do molecular identification to make sure.
Table 1. Biochemical and physiological characters of C3C isolates

P. fluorescens
Characteristi Isolate P.
biovar I bv. bv. bv. bv.
c C3C aeruginosa
II III IV V
Oksidase + + + + + + +
Motility - TD TD TD TD TD TD
Nitrate - + + + + + -
Lysine + + + +D +D + +D
D D
Ornithine - + + + + +D +D
H2S + TD TD TD TD TD TD
Glukose - + + + + + +
Mannitol - + + + +D + +D
D D
Xylose + - + + + +D +D
ONPG - TD TD TD TD TD TD
Indole - TD TD TD TD TD TD
Urease + - - - - - -
V-P + TD TD TD TD TD TD
Citrate - TD TD TD TD TD TD
TDA + TD TD TD TD TD TD
Gelatin + + + + + + -
Manolate - + + + +D + +D
D D
Inositol - - + + + + +D
Sorbitol + - + + V + V
Rhamnose - - - +D +D - +D
 Sucrose + - + + V + V
Lactose - TD TD TD TD TD TD
Arabinose + - + + V + +D
Adonitol - - + - +D - +D
Raffinose + TD TD TD TD TD TD
Salicin - TD TD TD TD TD TD
Arginine + + + + + + +
Pigmen + (blue- +(blue- TD TD TD - -
green) green) - - - TD TD
Tobacco HR - +D - - - - -
Growth at + + - - - - -
41OC
Growth at - - + + + + +
4OC
Description: +, 80% or more positive strain; +D, 80% or more delayed positive strain; V,
between 21-79% positive strain; -, 80% or more negative strain; TD, not determinate.
Quality and Quantity of DNA
In the molecular identification stage, the first step is to obtain DNA. This stage is
the isolation of DNA by destroying the bacterial cell membrane using a lysis buffer. The
buffer used to destroy the bacterial membrane is SDS. SDS has a function as a
detergent in destroying membrane proteins. In addition, it is necessary to add
cetyltrimethylammonium bromide (CTAB) which aims to separate polysaccharides from
nucleic acids. Under salt conditions, the polysaccharides are bound to a cation
detergent called CTAB. Molecularly identified unicellular microbes were C3C isolates
that had the best inhibition and antibiosis power. The results of DNA isolation were
measured for quantity and quality using Nano-Drop.
From the nano-drop result, the DNA quality of the C3C isolate was good because
the 260/280 nm absorbance ratio was about 1,87. According to Seidmen and Moore
[20], the good quality of DNA ranged from 1.8-2. However, in PCR amplification, DNA
quality was not an absolute requirement. The result of DNA quantity measurement by
using nanodrop showed that C3C isolate DNA concentration was 449,01 nm/µl.
According to Sambrook And Russell [16], DNA concentration that could be used as a
mold during the PCR process should be at least 100nm/µl. The DNA quality
measurement showed that DNA concentration was adequate to be used in the PCR
process.
Table 2. Results of DNA isolation nano drops

Sampel ng/µl A260 A280 260/280 260/230 Cursor 340

abs. raw
C3C 449,01 8,980 4,794 1,87 0,74 12,173 1,801

Result of Electrophoresis
Electrophoresis results showed the good lambent of single DNA bands for C3C
isolate. Those single bands were 16S rDNA regions fragment amplified with PCR
method. Electrophoresis result showed that 16S rDNA regions amplification was
successfully carried out. The position showed on about 1500 bp. According to
Suryani[10] [24], the result of 16S rDNA was showed about 1500 bp.
M 1kb C3C

1500 bp C3C

Figure 3. Electrophoresis result of PCR product. It was 16s rDNA regions of C3C
isolate.

Alignment Sequences of 16S Regions

Based on results of molecular identification using 16s rDNA regions, the lengths of
C3C isolate was 1393 bp, which the overall 16s sequence similarity of identical 99%
with Pseudomonas aeruginosa strain NA137 [KT005274.1]. The lengths of ITS regions
of Pseudomonas aeruginosa on database was 1401 bp. Microbe which hasve similarity
result in their sequences analysis of the ITS regions about 99-100% was the same
species, while if similarity result their ITS regions was less than 99%, it was different
species[11]. (Sugita et. al. [23]).
Table 3. Sequences Results

Strain/ Blast Max Total Query E Max

Query Score Score Cover value Identit
ID y
C3C Pseudomona 2514 2514 100% 0,0 99%
(lcl| s aeruginosa
16214 strain NA137
9) [KT005274.1]

The results of the BLAST analysis (Table 3) show that the query cover value of
100% indicates the base percentage of the C3C sequence is very compatible with the
homologous strain. The maximum score represents a measure of the statistical
difference in alignment. The maximum value is equal to the total score indicating the
two sequences are the same or very similar. A very low E value (expectation value)
indicates that the two sequences are increasingly similar. The expected value of all
strains appearing in BLAST is zero, meaning that each test strain has a high similarity to
its homologous sequence.

Table 4. Sequence Result of C3C Isolate

 C3C isolate (1393 bp)
GCAGTCGAGCGGATGAGGGAGCTGTGCTCCTGGATTCAGCGGCGGACGGG
TGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACG
GGCGCTAATACCGCATACGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACC
TCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGC
CTACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTG
GAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG
GACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCT
TCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTT
GCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAG
CCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAG
CGCGCGTAGGTGGTTCAGCAAAGTTGGATGTGAAATCCCCGGGGCTCAACC
TGGGAACTGCATCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGA
ATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCG
AAGGCGACCACCTGGACTGATACTGACACTGACGTGCGAAAGCGTGGGGAG
CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGC
CGTTAGGATCCTTGAGATCTTAATGGCGCAGCTAACGCGATAAGTCGACCGC
CTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATCGCCGGGGGCCCG
CACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACC
TGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAAC
TCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGG
GTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGG
TGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGA
CGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCCACAATGG
TCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCG
ATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCT
AGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACAC
ACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTACGCTAGTCTAA
CCGCAA

After finding the sequence results similar to P. aeruginosa, then the phylogenetic
analysis was carried out to compare with the results of biochemical identification. This is
to determine the relationship between the studied bacterial strains with P. aeruginosa
and P. fluorescens species. The comparison nucleotide sequences were accessed from
GenBank on the NCBI site. Based on the results (Table 5) it was found that the C3C
strain was very similar and very compatible with its homologous strain with P.
aeruginosa compared to P. flourescens. P. flourescens did not match the homologous
strain because the cover query value was still very low. Although the expected value of
all strains appearing in BLAST is zero, it means that each test strain has a high
similarity with its homologous sequence. It's just that the percentage value of
identification of P. flourescens is still lower than that of P. aeruginosa.
Table 5. Comparation sequence result with Genbank data

Deskripsi Max Total Query E value Ident Accession

score score cover
Pa1 2514 2514 100% 0.0 99% Query_24713
Pa2 2514 2514 100% 0.0 99% Query_24714
 Pa3 2514 2514 100% 0.0 99% Query_24715
Pa4 2514 2514 100% 0.0 99% Query_24716
Pf1 872 872 46% 0.0 92% Query_24717
Pf2 1103 1103 58% 0.0 92% Query_24718
Pf3 797 797 46% 0.0 90% Query_24719
Description: C3C: Strain C3C; Pa1: P. aeruginosa (KT005274.1); Pa2: P. aeruginosa
(KM659186.1); Pa3: P. aeruginosa (KM948588.1); Pf1: P. flourescens (KF679344.1);
Pf2: P. flourescens (KF864552.1); Pf3: P. flourescens (KR005846.1) ; Bs: B. subtilis
(JX994100.1).

Alignment results were analyzed using the BLAST program (Figure 8), it was found
that the homology level was very high with P. flourescens. There is a gap or dotted line
indicating the occurrence of a high mutation process in the form of insertions or
deletions. The bases that appear indicate identical nucleotides. Although there is still a
gap of 3%, it means that of the 641 bases that are aligned, only 25 are different from the
C3C strain. From the results of this alignment, it is known that the conservative regions
are regions in the sequence that have identical base sequences between species or in
different molecules produced by the same organism. Cross-species conservation shows
that certain nucleotide sequences may have been maintained by evolution even though
speciation is an evolutionary process that occurs even though new species have been
produced[10]. (Suryani, [25]).
Table 6. The similarity matrix of the 16S rDNA sequences

Sekue C3C Pa1 Pa2 Pa3 Pf1 Pf2 Pf3 Bs

n
C3C ID 99,83 99,83 99,83 94,40 94,4 92,46 69,70
0
Pa1 99,83 ID 100 100 94,57 94,5 92,64 69,93
7
Pa2 99,83 100 ID 100 94,57 94,5 92,64 69,93
7
Pa3 99,83 100 100 ID 94,57 94,5 92,64 69,93
7
Pf1 94,40 94,57 94,57 94,57 ID 100 97,68 70,34
Pf2 94,40 94,57 94,57 94,57 100 ID 97,68 70,34
Pf3 92,46 92,64 92,64 92,64 97,68 97,6 ID 68,93
8
Bs 69,70 69,93 69,93 69,93 70,34 70,3 68,93 ID
4
Description: C3C: Strain C3C; Pa1: P. aeruginosa (KT005274.1); Pa2: P. aeruginosa
(KM659186.1); Pa3: P. aeruginosa (KM948588.1); Pf1: P. flourescens (KF679344.1);
Pf2: P. flourescens (KF864552.1); Pf3: P. flourescens (KR005846.1) ; Bs: B. subtilis
(JX994100.1).
Sequence similarity serves as evidence for structural and functional
conservation, as well as evolutionary relationships between sequences. Consequently,
a comparative analysis is the primary means of identifying functional elements. The
genus Bacillus was chosen as an outgroup that has distant relatives to compare its
evolutionary distance. From the results of multiple alignments of all tested sequences, it
can be obtained similarity (similarity matrix), namely a matrix of scores showing the
similarity between two data points. Higher scores are given to sequences that have
higher similarity characters. From the BLAST results, it is known that the C3C strain has
similarities with P. aeruginosa about 99.83% while the similarity with P. flourenscens is
only 94.40% and 92.46%, respectively.
Table 7. Evolution distance matrix between strains
1 2 3 4 5 6 7 8
1 C3C
2 Pa1 0,00
2
3 Pa2 0,00 0,000
2
4 Pa3 0,00 0,000 0,000
2
5 Pf1 0,05 0,054 0,054 0,054
6
6 Pf2 0,05 0,054 0,054 0,054 0,000
6
7 Pf3 0,07 0,074 0,074 0,074 0,023 0,023
5
8 Bs 0,30 0,301 0,301 0,301 0,297 0,297 0,311
3
Description: C3C: Strain C3C; Pa1: P. aeruginosa (KT005274.1); Pa2: P. aeruginosa
(KM659186.1); Pa3: P. aeruginosa (KM948588.1); Pf1: P. flourescens (KF679344.1);
Pf2: P. flourescens (KF864552.1); Pf3: P. flourescens (KR005846.1) ; Bs: B. subtilis
(JX994100.1).
Based on the evolutionary distance matrix, it is known that the C3C strain
belongs to the same cluster as P. aeruginosa with the shortest evolutionary distance of
0.002 against Pa1, Pa2, Pa3. While the distance between C3C and Pf1, Pf2 is 0.056
and Pf3 is 0.075. It is suspected that the C3C strain does not belong to the P.
flourescens group.
Figure 4 Phylogenetic tree of C3C isolate strains with P. aeruginosa and P. flourescens.
From the image of a phylogenetic tree, it is a tree that shows a description of the
relationship of one species to another in the form of a branched diagram that can be
constructed based on similarities and differences in physical or genetic characteristics.
The phylogenetic tree (Figure 4) was made from the Mega6 application, it can be seen
that C3C isolates were included in the Pseudomonas aeruginosa group with the support
of a high biostrap value of 100, while P. flourescens was collected in a separate group.
C3C isolates and P. aeruginosa species form a monophyletic group, which is a
taxonomy containing species derived from a common ancestor[12]. (Holmes, [10]).
Unicellular Microbial Growth
Each microbe has a different growth time so it is necessary to know each growth
process. The method used to determine the growth of unicellular microbes is by
calculating the value of Optical Density or population density. After knowing the
population density in a certain period of time, the lag, log, stationary, and death phases
of these unicellular microbes can be calculated. The calculation results are in the graph.

Average Growth of Psedomonas aeruginosa

2.20 2.29 2.18
2.04 2.04
2.25 1.78 1.62
1.54 1.37
1.75 1.09
1.25 0.89
0.59
0.75 0.08
Colony Density

0.25
Wakt Wakt Wakt Wakt Wakt Wakt Wakt Wakt Wakt Wakt Wakt Wakt Wakt
u0 u4 u 8 u 12 u 16 u 20 u 24 u 28 u 32 u 36 u 40 u 44 u 48
0.08
a 0.59 1.54 1.78 2.04 2.20 2.29 2.18 2.04 1.62 1.37 1.09 0.89
v
e
r
a
g
e Time (Hour)

g
r
o
w
t
h
 Based on the graph of unicellular microbial growth above, it shows that the log or
exponential phase occurs from around the 4th hour to the 20th hour. The stationary
phase occurs around the 20th hour to the 28th hour. [13]Dwijoseputro [6] stated that there
are factors that influence the growth of bacteria, namely the availability of nutrients such
as carbon sources in the media. Hydrocarbons are a source of carbon. Microorganisms
are able to meet their energy needs through oxidation, finding and using as electron
donors[14] (Siregar, [22]). In this study, the carbon source came from nutrients in NA
media in the form of meat extract and peptone.
Data on unicellular microbial growth of Pseudomonas aeruginosa isolates
decreased around the 28th hour to the 48th hour. Microbial cell death is a factor causing
the decrease in the number of isolated cells. This is because there are many cells
whose nutritional needs are not met, thus affecting the availability of nutrients in
insufficient media[13] (Dwijoseputro, [6]). The decrease in the number of Pseudomonas
aeruginosa isolate cells was caused by reduced levels of death (nutrients) in NA media,
therefore many cells died.

Effect of Time Extraction Active Compound against Antagonistic Ability of

Unicelluler Microbe
Time of extraction of the active compound. The first experiment using paper-disc
was found inhibition of C. capsici on PDA media by P. aeruginosa about 4,2-18,6%. It
was suggested that the antibiosis compound(s) was actively produced by P. aeruginosa
under the time of extraction. Treatment extract, the active compounds at hour 0, 8 th and
16th hours couldn’ot suppress the growth of pathogenic fungi C. capsici. While the
percentage of inhibition occurring, starting treatment extracts the 24 th to 48th hours
marked the higher the value of the inhibition. The highest percentage inhibition value
derived from extracts of the active compound with the treatment 48 th hour at 18.6 %.
This is consistent with the statement by [15],Ongena et al. [13], that the establishment
of microbial metabolites begins after 24 hours of incubation time due to amino acids as
nutrient growth has diminished.

20.0
e
18.0
Percentage of Inhibition

16.0
14.0 d
12.0
10.0
c
(%)

8.0 b
6.0
a a a
4.0
2.0
0.0
0 8 16 24 32 40 48
Time Treatment (hour)
Figure 5. Effect of time extraction on the percentage of active compound P. aeruginosa
inhibition against C. capsici after 7 dai
Effect of Proteinase K and Temperature Active Compound against Antagonistic
Ability of Unicelluler Microbe
The active compounds that have the effect of antibiosis may include enzymes and
toxins. As for the other characters, namely the influence of temperature during storage.
Therefore, please note the character possessed by the active compound of P.
aeruginosa. The best time of the extract is based on test previous 48 hours. The
treatment is made by giving the effect of proteinase K and temperature.
From this experiment it can be seen that the active compounds of this type of
enzyme is not because there was no effect of the addition of proteinase K enzyme is a
large protein molecule that analyzing reactions in a living cell which, in any kind of
reaction catalyzsed by a specific enzyme. According[16] Abadi [1], if not the enzyme will
most likely antagonistic microbes secrete a chemical substance in the form of the toxin.
Toxins affect the function of protoplasts, changing the permeability of cell membranes
also function that can damage and kill the host cell.
18

16 b
Percentage of Inhibition (%)

14

12
b
10

4
a
2

0
Control Proteinase K Temperature
Treatment

Figure 6. Effect of proteinase K and temperatur on the percentage of active compound

P. aeruginosa inhibition against C. capsici after 7 dai
Besides the active compound can be affected by temperature due to the
temperature treatment makes the active compound is broken. The active compounds
are very sensitive to the storage location. That is because there is an active compound
that is both volatile and non-volatile. Therefore, the presence of which less appropriate
temperature causes the compound becomes inactive. Storage temperature exceeding
the limit will damage the structure of the molecules of the chemical substance resulting
compound is inactive[16]. (Schaechter [19]).

The Active Compound of P. aeruginosa

P. aeruginosa is the microbe that have beneficial properties as an agent of
biological control agents. Besides these microbes useful as PGPR and able to induce
plant resistance to disease. This is reinforced by the statement [17]Saikia et al. that P.
aeruginosa is able to produce the antifungal active compounds in the form of salicylic
acid (SA) could increased plant resistance and inhibits the activity of R. solani, P.
oryzae and H. oryzae. SA was a precursor in the biosynthesis of antibiotics
siderosphore as pyochelin in P. aeruginosa. Besides antibiotics of Pseudomonas 2.4-
diacetylphloroglucinol capable of inducing resistance to plant -related reactions Iavicoli
et al. Pseudomonas capable of producing metabolites in the form of antibiotics such as
DAPG , pyoluteorin , pyrrolnitrin , pyochelin , phenazine , cyanide (Duffy and Defago [5]
[18]; Van Rij et al. [26][19]; [15]Ongena et al.[13]).

A B
Figure 7. Morphology of Collectotrichum. capsici isolate A. Control or without active
compound of Pseudomonas. aeruginosa. B. With active compound of
Psedonomonas. aeruginosa.
Based on the microscopic C. capsici against antifungal effect, it is known inhibited
mycelial growth compared to controls. It is caused by P. aeruginosa exotoxin also have
a grub of active compounds that are excreted by the bacteria. Exotoxin if entry into host
cells and catalyze covalent modification of host cell components that can alter the
physiology of the host cell. According to [20]Barbieri [2], issued exotoxin A bacterium P.
aeruginosa is able to modify the ADP – ribosilation target elongation factor - 2 that can
inhibit protein synthesis and lead to cell death. Antibiotics that have a role in inhibiting
protein synthesis is generally included in a broad spectrum. That is because can bind to
the 30S subunit of the bacterial ribosome, prevent or hinder sticking 50S subunit that is
not fully formed. Additionally, these compounds can distinguish 70S ribosome
prokaryotic and eukaryotic 80S[21] (Hogg [9]).

Hypersensitivity and Pathogenicity Tests

Selected unicellular microbial isolates that have antibiotic power, namely C3C
were tested hypersensitive reaction to know whether the strain is pathogenic or not.
Hypersensitivity test results showed negative C3C isolates, which means they are not
phytopathogenic. These isolates may be phylloplane unicellular microbes which are
also found in chilies. Reaction negative is characterized by the absence of symptoms of
yellow and dry necrosis on injection marks 24-48 hours after inoculation.
Hypersensitivity reactions do not occur
due to improper interaction between microbial isolates with plants, without any
pathogenicity due to their strong defense response from plants.

A B
Figure 8. Hypersensitivity and pathogenicity test of P. aeruginosa. A. Result of
hypersensitivity test; B. Result of pathogenicity test.
Phytopathogenic unicellular microbes induce the formation of oxygen species
active as Hydrogen Peroxide (H2O2), Oxygen (O2) and hydroxyl radicals (OH-) can be
involved initiate of hypersensitivity reactions. Cell membrane permeability plants are
changed through fat peroxidation, resulting in electro lyteleakage and the ion exchange
reaction takes place where K+ out and H+ enter. Impact from an ion exchange reaction
is an increase in the pH of the apoplast fluid during an infection. Reaction
hypersensitivity causes cell death and inhibits growth[22] (Keppler et al.,[11]).
The principle of the pathogenicity test is almost the same as the hypersensitivity
test only using the plant part where the isolate originated. The method is used by
bacterial inoculation into and the surface of chilies. After treatment, the chili is placed in
a sterile box covered with a moist tissue for 7x24 hours to maintain the viability of
unicellular microbes and provide optimal conditions for the pathogenicity process. P.
aureginosa does not cause any symptoms to the chili tested. This can P. aureginosa
may be a saprophytic or endophytic fruit Red chili pepper. This is reinforced by the
statement of Schaad [8] that P. aureginosa is an opportunistic microbial plant and
animal pathogen.

CONCLUSION
The molecular identification suggested that the C3C isolate was P. aeruginosa.
Based on the pattern resulting from the antagonistic test, it was suggested that the
active compound(s) was actively produced by P. aeruginosa on 48th hours incubation of
C. capsici. The active compound was aeffected temperature but did not affect
Proteinase K.

Acknowledgments
Ministry of Education, Culture, Research, and Technology of Indonesia this study was
supported

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