Sunday, March 23, 2008

Penyakit Tanaman


Plant viruses consist of a nucleoprotein that multiplies only in the living cells of a host. The presence of viruses in host cells often results in disease.

400 or more viruses are known to attack plants (2000 viruses are described for plants, animals, bacteria, etc.). viruses are generally specific, what infects a plant does not cause disease in an animal, and vice versa.

The first record of a disease that was later found to be caused by a plant virus was on tulips in the 17th century in the Netherlands.

First experimental demonstration of the infectious nature of viral disease was recorded by Lawrence, who described the transmission of a disease of jasmine by grafting.

Adolf Mayer (1886) described a disease of tobacco called mosaikkranheit (tobacco mosaic). Disease could be transmitted to healthy plants with sap from diseased plants.

Dmitrii Iwanowski (1892) demonstrated that the agent in tobacco mosaic was filterable. He demonstrated that the causal agent of tobacco mosaic could pass through a filter that retains bacteria.

1898 Martinus Beijerinck - demonstrated that the causal agent was not a microorganism but a contagium vivum fluidum (contagious living fluid). He was the first to use the term virus, which is the Latin word for poison. He concluded that this was not a toxin, because repeated inoculations of diluted infected sap yielded similar amounts of disease as it was passed from one plant to another. If it had been a toxin, it would eventually be diluted away.

Loefler and Frosch (1898) described the first filterable infectious agent in animals - the foot-and-mouth disease virus and Walter Reed (1900) - described the first human virus, yellow fever virus.

In 1929, F. O. Holmes provided a tool by which the virus could be measured by showing that the amount of virus present in a plant sample preparation is proportional to the number of local lesions produced on appropriate host plant leaves rubbed with the contaminated sap.

1935 W. M. Stanley isolated and purified some tiny white crystals from leaves of mosaic-infected tobacco plants. He treated healthy plants with TMV, which had been precipitated out of infected tobacco juice with the help of ammonium sulfate and a technique he had developed. The healthy plants contracted tobacco mosaic disease. Due to the high protein content of the purified virus particles, he concluded that the virus was an autocatalytic protein that could multiply within living cells. Although his conclusions were later proved incorrect, Stanley's work merited him receiving the Nobel Prize. He won the Nobel Prize in chemistry in 1946 for this work.

1937 - Bawden and Pirie demonstrated that virus consists of protein and nucleic acid (RNA).
1939 - Kausche - saw virus particles for the first time with the electron microscope.
1955-1960's Much was learned by various workers, regarding the infectivity of viral (TMV) RNA and the structure and arrangement of viral (TMV) coat protein.

1971 - T. O. Diener discovered viroids, which only consist of nucleic acids. Smaller than viruses, caused potato spindle tuber disease (250-400 bases long of single-stranded circular molecule of infectious RNA). About a dozen other viroids that cause disease in a variety of plants have been isolated. No viroids have ever been found in animals.

1980- Cauliflower mosaic virus, whose genome is a circular double-stranded DNA chromosome, was the first plant virus for which the exact sequence of all its 8,000 base pairs was determined.
In 1982, the complete sequence of the bases in the single-stranded tobacco mosaic virus RNA was determined, as were those of smaller viral RNA and of viroids.

1986 - Use of transgenic plants to obtain resistance against viruses (TMV).

For example, the symptoms of specific plant diseases form the basis for the following disease names: tobacco mosaic, turnip crinkle, barley yellow dwarf, ring spot of watermelon, cucumber mosaic, spotted wilt of tomato.

Some viruses have a broader host range than the name of disease or virus may imply. For example, tobacco mosaic virus (TMV) infects tomato, eggplant, peppers, in addition to tobacco.

noncellular, ultramicroscopic particles, that multiply only in living cells. very, very small! (size measured in nanometers).

most plant viruses consist of protein shells surrounded by a core of positive-stranded nucleic acid (normally ssRNA - nucleotides (guanine, uracil, cytosine, adenine) + 5 carbon sugar called ribose + a phosphate group), but sometimes these viruses contain dsRNA or dsDNA (2 strands of nucleotides with thymine substituted for uracil and deoxyribose instead of ribose).

5-40% of virus is nucleic acid 60-95% is protein

Protein coats or shells can be different shapes, but are normally rod, filamentous, isometric, quasi-isometric/bacilliform or variants of these structures. See Figure 14-25 on page 501 in handout. For example, Tobacco Mosaic and Barley Stripe Mosaic viruses are rods, while broad bean wilt and maize chlorotic dwarf viruses are isometric or more spherical in shape.

Minimum number of genes in a plant RNA virus could be two: a coat protein and an RNA replicase gene (as is the case with RNA phages). Evidence indicates there are usually 3-5 gene products.

Plant positive-stranded RNA viruses frequently possess divided genomes (refer to Figure 14-4 in handout). In addition, viral genomes are separately encapsulated. Viral genomes consisting of two or three different nucleic acid components, all required for infection are called bipartite, tripartite, or multipartite viruses. More than a single species of genomic RNA. Refer to pages 271-273 in textbook.

Multipartite viruses are potentially at an evolutionary disadvantage. Infectivity dilution curve for Alfalfa mosaic virus (requiring B, M, Tb particles for infectivity) is steeper than for tobacco necrosis virus (single particle). Partition of genome could potentially hinder transmission or infection by a virus.


Kasinis in 1962, described the first satellite viruses. These viruses are serologically unrelated to their helpers and the two genomes exhibit little if any sequence similarity. Satellite viruses are dependent for its replication on the presence of a second, independently replicating virus.
Satellite RNAs have no coat protein of their own and are encapsulated with the help of other viral RNAs.


Mechanical transmission through sap by plants touching one another, through root grafts, and manhandling.

Vegetative propagation and grafting.
Seed, pollen, mites, nematodes, dodder, fungi (carried by zoospores and mycelium) and insects (aphids, leafhoppers, scale insects, thrips, grasshoppers, beetles, whiteflies). For example, cucumber mosaic virus and barley yellow dwarf virus moved by aphids.

Due to the inability to observe plant viruses visually by observing them directly through the light microscope, virologists must resort to the following methods of detecting their presence and in diagnoses.
1. Ability to transmit disease via plant sap by rubbing plant, grafting, dodder or insect transmission.
2. Indexing - indicator plants - sensitive to specific virus and will react a certain way if exposed..
3. Visual inspection with EM.
4. By eliminating possibility that symptoms are not due to other sources (e.g., herbicide, nutritional deficiencies.
5. Serological Tests (ELISA - enzyme-linked immuno sorbent assay). Refer to Figure 14-24; page 499 in the handout.

Indirect (virus + Ab virus + Enzyme conjugated Ab) and direct (double-antibody sandwich technique) (Ab virus + virus + Enzyme-conjugated Ab).
1. Virus or Ab virus added to well and these become attached to walls.
2. Antibody or virus added to well and these attach to their counterpart (i.e., antigen to antibody).
3. Second antibody with enzyme conjugate attaches to first antibody/virus complex.
4. Substrate is catalyzed by enzyme and this causes a color change. ELISA tests are extremely sensitive (small amounts of antisera are needed) results are quantitative, large samples can be run at same time (96 well plates), results can be gathered in a few hours instead of days. ELISAs along with serial dilutions of plant sap and applications of this to the leaves of susceptible hosts (by counting the number of lesions) can be used to quantify the amount of virus present.


Milk inactivates many viruses - use milk to wash tools/hands. "Milk does a plant body good!" Soap and water work well too!

Removing diseased plants, killing and removing potential virus vectors (primarily weeds and insects).

disease-resistant cultivars.
disease or virus free seed, roots or tubers.
cross protection (inoculation with a less-virulent strain of a virus protects the plant from a more virulent strain later when exposed to it).

heat (some viruses are killed at temperatures that will not kill host). For example, dormant propagative organs dipped in hot water (35 C) for few minutes or hours, or by growing plants in greenhouse at 35-40 C for several days, weeks or months may inactivate virus.

Caused by Tobacco Mosaic Virus (TMV) worldwide distribution primarily infects tobacco and tomato, but more than 350 species are susceptible.
tobacco leaves become mottled with light and dark green areas; leave become distorted, puckering or blistering, especially areas of new growth.
stunting of plant growth. in tomato, mottling of leaves occurs and leaflets become long and pointed.

TMV is a rod-shaped particle which are 300 nm long by 15-18 nm in diameter. It possesses ssRNA and a protein coat. difficult to inactivate, and can survive for 5 years in dead, dried tissues and many months in living plant tissues. many strains, that vary in virulence from severe to mild symptoms. virus is spread from plant to plant through injuries caused by crop worker, contaminated equipment and chewing insects.

virus overwinters in dead plant tissues and debris, on contaminated equipment, in contaminated soil, greenhouse containers, bedding, tools, and in living hosts, including weeds like horsenettle, Solanum carolinense, and other crop plants (tomato, pepper, and eggplant).

Management of Tobacco Mosaic Disease
use virus-free seed (tomato seed can by treated with acid or bleach)
transplant in noninfested soil
fumigatation with methyl bromide or heated.
no chewing of tobacco or smoking around seedbeds or in greenhouses.
to eliminate spreading of virus wash hand with soap and water or milk.
spraying plants with milk (whole or skim) seems to help reduce
infections. crop rotation with nonhost crops (corn, rice, other cereal grains).
resistant cultivars

Tobacco Mosaic Virus: The Prototype Plant Virus

The stability of the TMV virus particle accounts for its having been the first virus to be identified, purified to homogeneity, and then biochemically and biologically characterized.

Small coat protein subunits (capsomeres) aggregate to form a helical protein coat or capsid (see Fig 2.10 and 2.11 on pages 46 and 47). The virus particle contains an axial channel that is 4 nm wide and the viral RNA lies within a groove in the surrounding protein helix. The nucleic acid core is not in the axial channel, but passes about halfway between the interior channel and the exterior surface of the rod. The overall particle is rod-shaped, narrow, and rigid. The pitch of the helix is 2.3 nm, and each turn contains 16 1/3 coat protein molecules. A full-length virion contains 130 helical turns.
TMV particle is resistant to nucleases and proteolytic enzymes. TMV particles will fall apart in both alkaline and acid solutions. Denaturation is often reversible, as long as temperature and pH are not too extreme. Removal of the denaturant allows the native structure of the viral protein to re-form and near its isoelectric point (pH 4 to 6), the TMV coat protein aggregates to form rod-shaped particles that look exactly like TMV virions.

When virus is subjected to neutral pH with either detergents (e.g., SDS) or 6 M urea or by extraction with phenol then RNA can be extracted in an intact form. When isolated TMV RNA are added to native TMV protein, these form stable "reconstituted" virus, which is more stable (stable from pH 3 to 9) then protein alone (unstable below pH of 4 and above pH 6).

Protein and RNA are more infectious than naked RNA alone (nearly 1000 times the amount of naked RNA is required to cause infection).
Proof that the viral RNA was the sole determinant of tobacco mosaic disease was obtained by a mixed reconstitution of RNA from Holmes ribgrass mosaic virus (RMV) with the protein subunits from TMV. Reconstituted virus caused localized lesions on plants instead of a systemic infection and formed new RMV virus (RMV RNA + protein coat containing histidine and methionine - not found in TMV).

Assembly of Helical Viruses
Aggregates of 33 protein molecules form the double disk. This combines with viral RNA. Attachment of the nucleic acid to the protein aggregate begins at the origin of assembly site (OAS) about 800 nucleotides from the 3' terminus of TMV common strain RNA. Rod growth toward the 5' terminus of the viral RNA is rapid, involving addition of double disks; encapsidation of the 3' terminus proceeds more slowly, through the addition of A protein monomers or small aggregates. Refer to Figure 6.6 in the textbook or to Figure 2.14 on page 50 in handout #2). Cotranslation disassembly - the protein coat is displaced at the 5' end by ribosomes in host cell.

3' end of TMV RNA ends with the sequence -C-C-C-A and can be charged with an amino acid (histidine). This region is non-coding and be folded into a tRNA-like structure preceded by a series of four pseudoknots. Why? Four possibilities exist.
Donating an amino acid during some stage of protein synthesis.
Facilitating translation by disrupting base pairing between the 3' and 5' - terminal regions of the viral RNA
Acting as a recognition site for the viral replicase to initiate negative-strand synthesis
A molecular fossil from the original RNA world where tRNA-like structures tagged RNAs for replication and prevented the uncontrolled loss of nucleotides from third 3' terminus. Subgenomic mRNAs and translational read-through in TMV replication.

Five open reading frames or ORFs are found in the genome of TMV. Subgenomic mRNAs and translational read-through are two strategies employed by TMV to regulate gene expression.Plant positive-sense RNA viruses have developed several other mechanisms to facilitate and/or regulate the expression of individual genes. 5 strategies of regulating gene expression.


wide host rang, including banana, bean, celery, crucifers, cucumbers, gladiolus, lilies, melons petunias, spinach, squash, tomatoes, and zinnias.

symptoms resemble those of tobacco mosaic and it is difficult to distinguish between the two diseases
Cucumber mosaic virus (CMV) is a polyhedral virus - particles are 30 nm diameter. transmitted mechanically by rubbing and by aphids. overwinters in many weeds and crop plants. Control and Management
resistant cultivars of cucumber, muskmelon, spinach, and tobacco.
reduce aphid populations by eliminate weeds and spraying with aphicides.
erect barriers between cucumbers and inoculum source (i.e. row of sunflowers).
when working with plants wash hands with milk.
aluminum strips between rows reflect UV light, which acts a repellent to aphids.

Barley Yellow dwarf disease infects barley, oats, rye, and wheat.
losses on oats as high as 50 %; 30% on what and barley.
yellowing and dwarfing of leaves, stunting of plants, reduced root system, reduced grain production. leaves may turn red or bronze in color = "red leaf"
BYDV is a polyhedral virus 25 nm in diameter.
not mechanically transmissible.
not seed borne.
does not over winter in plant debris or soil.
survives only in living plant tissue (crop or wild grasses) and bodies of aphids (different species are involved, including the oat-bird cherry aphid and the English grain aphid). Symptoms appear 3-6 weeks after infection by feeding aphids. Management of Barley Yellow Dwarf Disease
avoid planting small grains near large grassy areas - act as source of virus.
insecticides are not that useful.
do not plant oats or barley near end of the normal seeding period.
Subviral Pathogens and Other Virus-like Infectious Agents
Among subviral pathogens, only viroids and prions replicate independently; the prion particle, lacks a genomic nucleic acid.
Satellite viruses and satellite RNAs contain conventional nucleic acid genomes, but their replication is dependent on the presence of a helper virus.
Satellite viruses and RNAs do not exhibit substantial sequence homology with their helper viruses. Satellite Viruses
Satellite viruses were first observed in plants. Their replication is dependent on the presence of a helper virus that provides the replicase, but the satellite virus is not required for helper virus replication. There is little or no sequence similarity between the genomes of satellite viruses and those of their helper viruses. Satellite viruses generally produce their own capsid protein.
Example: The helper virus for satellite tobacco necrosis virus (STNV - a 18 nm particle), is tobacco necrosis virus (TNV - a small 30 nm icosahedral virus). STNV contains a monocistronic mRNA for the synthesis of its 22kDa coat protein. Note that satellite viruses are generally smaller than helper viruses.
STNV is an obligatory parasite of its helper, dependent for its replication on the presence of TNV in the cells it enters.
Specificity of this dependence is illustrated by (1) the inability of plant viruses other than TNV to act as helper and (2) variation in the ability of certain TNV strains to support the replication of different strains of STNV. The presence of STNV greatly suppresses the replication of TNV.
Satellite RNAs

Cucumber mosaic virus (an epiphytotic of lethal necrotic disease among tomato plants in France in 1972). This was unusual because of the severity and atypical nature of the symptoms associated with infections of tomato plants (See description of cucumber mosaic in prior text). Enhanced response of tomatoes to this disease were associated with host used for virus propagation; virus grown in tobacco or tomato yielded an enhanced necrotic response, those propagated in cucurbit hosts yielded a reduce necrotic response. RNA extractions of necrotic tomato plants revealed the presence of variable amounts of a small RNA species in addition to the expected three genomic and one subgenomic RNAs. This 5th RNA species was designated CARNA 5 (i.e., CMV-associated RNA 5 or CMV satRNA), a satellite RNA.

When CARNA 5 is present with CMV, symptoms of the disease are enhanced in tomato plants, while in tabasco pepper the presence of CARNA 5 tends to attenuate the disease symptoms caused by CMV. Other factors including sequence changes in the satellite RNA, use of a different helper virus strain, and changes in environmental conditions appear to greatly enhance or suppress symptoms caused by the helper virus.

Genome Structure of Satellite RNAs
Satellite RNAs have been found associated with members of five different plant virus groups. With the exception of TCV (turnip crinkle virus) RNA C, satellite RNAs exhibit only limited sequence homology within the genomes of their respective helper viruses. Satellite RNAs appear to from two size classes - large that are similar in size to the genomes of satellite viruses and much smaller.

Viroids are the smallest known agents of infectious disease - small (246-375) nucleotides), highly structured, single-stranded RNA molecules lacking both a protein capsid and detectable messenger RNA activity.

The first viroid disease to be studied was potato spindle tuber disease. Disease was first recognized and described by Schulz and Fosom in 1923, but it wasn't until Diener demonstrated in 1971, that the fundamental differences between the structure and properties of its causative agent, potato spindle tuber viroid (PSTVd). Vd for viroid.
Viroids exist in vivo as nonencapsidated, low-molecular-weight RNAs;
Infected tissues do not contain virus-like particles;
Only a single species of low-molecular-weight RNA is required for infectivity;
Viroids do not code for any proteins;

Despite their small size, viroids are replicated autonomously in susceptible cells and no helper virus is required.
Single-stranded viroid RNAs are resistance to digestion by ribonucleases and possess a high degree of thermal stability (not easily denatured). The genome of viroids, such as PSTVd are organized into a series of short double helices and small internal loops which form the basis for five domains. Conserved central domain (highly conserved and site where cleavage and ligation to form circular progeny occur), pathogenicity domain (modulate symptom expression), variable domain (greatest sequence variability among otherwise closely related viroids), and two terminal domains (replication and evolution).

Transformation - introduction of a new gene into a plant via some mechanism (i.e., Agrobacterium-mediated gene transfer, bombardment, electroporation, plant virus-mediated gene transfer). Transgenic plants are produce by:
Addition of new genes (to express a new gene product).
Suppression of a gene or a gene product (in the plant - e.g., against ethylene gas production to prevent senescence of fruits or against a potential plant virus - e.g., TMV). Potential benefits of transgenic plants
Genetic engineering can produce plants that are:
able to synthesize oils, starches, and plastics
able to synthesize enzymes for food processing and other uses
more nutritious foods (e.g., plants with a higher protein content, and wider profile of essential amino acids - methionine-rich beans or lysine-rich corn)
able to fix nitrogen for growth
freeze resistant
pest resistant
herbicide resistant
disease resistant. In general, transformed plants that express a viral coat protein are resistant to infection by both that virus and related viruses. Viral coat protein may interfere with viral uncoating of the invading pathogen. Coat protein-mediated resistance now available shows a lot of promise in inferring genetic resistance to various viral pathogens, including TMV. Other promising strategies being investigated include the expression of antisense viral RNAs (which interfere with viral replication) and viral satellite RNAs, and noncoat viral genes (producing high resistance to specific strains).

Potential problems
Allergies to transformed plant products.
Accidental movement of novel genes into wild relatives of cultivated plants.
Consumer resistance to using genetically-modified plant products, especially food.
Ethical and moral considerations. (e.g., releasing transgenic marijuana plants that are poisonous, exploitation of genetic resources for personal gain). Strategies for introducing novel genes into plants

1. Agrobacterium-mediated gene transfer
Agrobacterium tumefaciens is the causal agent for crown gall in wide range of host plants. It enters through wounds and injuries and causes a localized region of uncontrolled cell division (a tumor or gall) on the plant. The bacterial cell contains a Ti or Tumor-inducing plasmid. The Ti plasmid contains genes that call for the production of opines (C and N source for the bacterium) and regulate cytokinin and auxin production in plants (causing hyperplasia - excessive cell division = tumor).
Ti genes removed by using restriction enzymes
Target gene/gene for antibiotic resistance are introduced
Modified Ti plasmid introduced back into A. tumefaciens
Bacteria allowed to infect desired host (usually a tissue culture of the host)
Bacteria incorporates its DNA into host
Antibiotics added to growing callus to (a) eliminate the bacterium and (b) to select against untransformed cells

Callus in tissue culture used to regenerate whole plant 2. Bombardment
DNA-coated particles shot into plant tissue using a microprojectile gun (gun powder charge).
Electrical-acceleration of DNA-coated particles.
Final Product: Population of transformed/untransformed cells. Transformed cells are selected and through tissue culture work a new plant is generated. 3. Electroporation
Protoplasts (cell wall removed with enzymes) or partially-digested apical meristems using enzymes.
Sudden electrical discharge opens up membrane and DNA is allowed to enter.
Final Product: Population of transformed/untransformed cells. Transformed cells are selected and through tissue culture work a new plant is generated. 4. Plant virus-mediated transformation
Tobacco could be infected and the product harvested from altered plants.
‘Geneware' and ‘Pharming'.
The coat protein genes of brome mosaic and tobacco mosaic viruses were replaced by bacterial chloramphenicol acetyltransferase. Such viruses are unable to systemically invade the host plant, and have only limited potential as expression vectors. In the case of TMV, this problem is solved by adding the foreign gene to the viral genome rather than substituting for one of the normal viral genes. Trial runs indicate that expression levels achieved are much lower than those of TMV coat protein. Insertion of such genes at different locations in the viral genome or a satellite-like virus associated with helper viruses may increase these yields.
Mycoviruses and the Biological Control of Chestnut Blight
Mycoviruses are probably widespread in fungi in nature, despite the fact that relatively few have been isolated and characterized, and still fewer have been experimentally transmitted to test fungi and demonstrated to be infectious. A survey of 50 isolates of Rhizoctonia solani from the field, 49 were found to have RNA resembling that of mycoviruses.
Typical mycovirus particles are isometric in shape and have a diameter between 25-48 nanometers. Some mycoviruses are rod-shaped or have some other form. This nucleic acid core usually consists of dsRNA and infrequently contains dsDNA. Some mycoviruses appear to be multipartite. Strains of Penicillium chrysogenum, from which the antibiotic penicillin is commercially produced appears to be infected by a mycovirus. Mycoviruses appear to be transmitted by means of cytoplasm and through spores. Mycoviruses appear to be retained in the cytoplasm of a fungal strain indefinitely.
Many mycoviruses do not cause symptoms in their hosts, despite the fact that cells may harbor large numbers of virus particles. Viral infection in the edible mushroom Agaricus brunnescens causes a degeneration of the mycelium and development of malformed basidiocarps, resulting in a reduction of yield.

Chestnut Blight and Hypovirulence
Introduced in New York City in 1904. Destroyed practically all American chestnut trees throughout natural range in eastern third of the U. S. From Canada to nearly the Gulf of Mexico.
50% of overall value of eastern hardwood timber stands destroyed.
Causal Agent is Cryphonectria parasitica.
Fungus found in North America, Europe and Asia.
Fungus penetrates bark and stems though wounds and grows into inner bark and cambium.
Canker (swollen or sunken) forms and bark becomes covered by pimple-like pycnidia (asexual spores called conidia are contained in an ooze and spread by birds, insects and splashing rain drops) and perithecia (ascospores - shot directly into air).
Cankers eventually girdle the tree killing it. Doesn't kill the root system so shoots continue to come up years later only to become infect and die.
Elimination of American Chestnut (along with habitat destruction and hunting) contributed to the extinction of the passenger pigeon.
In Europe, several strains were found that have reduced virulence (hypovirulence)
Hypovirulent strains contain dsRNA enclosed in a membraneous vesicle. Infected tree can often throw-off an infection and cankers can heal in time if fungus is hypovirulent.
To transmit dsRNA to virulent strains is accomplished by bringing a hypovirulent strain in contact with a virulent strain - hyphal anastomosis or fusion transfers the "virus".
Problem with widespread application of this biocontrol technique is that somatic or vegetative incompatibility often prevents movement of contagious agent from one fungal colony to another.
Only members of the same VCG or anastomosis group can transfer the dsRNA virus.
Sandra Anagnostakis - Connecticut Ag. Expt. Station has spearheaded much of this work.
Michael Milgroom, Cornell Univ. "Population biology of chestnut blight fungus".
Recent efforts to circumvent the problems of vegetative incompatibility and infer hypovirulence to other species of fungi have sought to incorporate the viral genome directly into the genome of the fungus. "European Hypovirulence and construction of transgenic fungal strains for biological control of chestnut blight". D. L. Nuss of the Univ. Of Maryland. This way the viral genome would spread throughout the population through sexual recombination and vegetative compatibility.

This page was assembled by Martin J. Huss, who can be reached at revised on: October 31, 2002. SUmber dari :

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