KOCH’S POSTULATES

LABORATORY REPORT OF VIROLOGY

Name : Sunu Pertiwi

Student ID : B1B015007
Entourage : II
Group :1
Assistant : Dwi Rizky Nurjanah

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2017
 I. INTRODUCTION

A. Background

Plant disease is a condition when the plant experiences a sustained

physiological dysfunction, resulting in symptoms and signs. This physiological
disorder is caused by biotic factors (bacteria, fungi, viruses and nematodes) as well
as abiotic factors (temperature, moisture, mineral nutrients). One of the ways to
know the biotic factors that cause the disease is an activity based on the Koch’s
Postulate (Semangun, 2004).
Infectious plant diseases (infectious) there are several sequences of sequences
with one another. There are two important sequence events, namely the pathogen life
cycle and the disease cycle. The sequence of events contributes to the development
of pathogens and disease progression. The life cycle of pathogens starts from
growing up to produce reproductive devices. The disease cycle encompasses changes
and changes in pathogens within the plant body and a series of host crop changes as
well as the presence of pathogens (the life cycle of pathogens) within a certain time
span during the growth period of the plant. Important events in the disease cycle
include 'inoculation, penetration, infection (host nutrient utilization), invasion
(extension of attack to other tissues), spread to other sites and pathogen defense
(Pracaya, 2007).
Plants that are infected with the virus usually cause various symptoms in part
or all parts of the plant. These symptoms usually decrease the growth rate of the
plant itself which resulted in the dwarf plant and the plant becomes shorter. Other
symptoms caused by the yaituter virus may line along the lateral leaf bone, there are
patches that are visible when viewed from beneath the leaf surface (Pracaya, 2007).
According to Bollard & Braile (1993), in 1880, Koch used the advancement
of laboratory methods and determined the criteria necessary to prove that specific
microbes were the cause of certain diseases. This criterion is known as Koch's
Postulates:
1. Certain microorganisms are always found associated with infectious diseases.
2. Microorganisms can be isolated and grown as a pure laboratory laboratory.
3. The pure culture when injected to the appropriate plant may cause disease.
4. The microorganism can be isolated from the infected plant
 Plant virus is very various and kind, but there are some characteristics or the
nature of the virus that can be used to group plant viruses. The grouping of plant
viruses is based on the viral genome sequencing, nucleotide sequence homology,
serological relationships, association with vectors, host ranges, pathogenicity, disease
symptoms, and geographic spread. Based on their association with vectors, for
example, viruses that naturally attack Soybean Stunt Virus (SSV), Indonesian
Soybean Swarf Virus (I-SDV), Soybean Mosaic Virus (SMV), Cowpea Mild Mottle
Virus (CPMMV) and only transmissible CPMMV By Bemisia tabacci. Based on the
genome of the virus, viruses with DNA genomes such as Cauliflower mosaic virus
(Akin, 2006).
Plant viruses are in some ways different from viruses attacking animals or
bacteria. These differences, one of which is the mechanism of penetrating the virus
into the host cell. Plant viruses can only enter into plant cells through wounds that
occur mechanically or are caused by vector insects. This is because the plant virus
does not have penetration tools to penetrate the cell wall of plants. Viruses that attack
animals and bacteria can penetrate directly through cell membranes, such as
bacteriophages (bacterial-attacking viruses) have penetrating devices that can
penetrate bacterial cell membranes (Bos, 1983).

B. Objective
The objective of this laboratory activity are:
 To implement the Koch’s postulated in plant disease transmission caused by viral
infection.
 To know the transmission method of plant infecting viruses one to other by using
plant sap.
 II. MATERIAL AND METHOD

A. Material

The tools that we used in this laboratory activity are polybag, mortal and
pestle, transplant plastic, beaker glass, sprayer, millipore + vacum and sterile cotton
bud.
The materials that we used in this laboratory activity are two weeks old of
Leguminoceae (green beans), soil, some pieces of virus-infected leaves, sterile
distilled water, Sartorius membrane filter and carbonundum.
B. Method

I. Extract and sap formulation of virus-infected leaves.

1. A total of 5 green beans leaves are observed and recorded toward its
symptoms.
II. Making SAP solution.
1. Extract the leaves from infected plant by damage the leaves become
smoothly.
2. Filtrate the SAP using Millipore.
III. Treatment.
1. One plant as treatment and one plant as control.
2. The leaves is mechanically injured by using carbonundum.
3. Sterile cotton bud is swabbed into plant sap, then inoculated to the control
green beans.
4. The inoculated leaves is covered using transplant plastic.
5. The green beans is observed for 7 days.
 III. RESULT AND DISCUSSION

Table 1. The observation result of Koch’s postulates of entourage I

Group The Symptoms

1 Chlorosis, necrosis and withered

2 Chlorosis and withered
3 Chlorosis and withered
4 Chlorosis
5 Chlorosis, necrosis and withered

Table 2. The observation result of Koch’s postulates of entourage II

Group The Symptoms

1 Chlorosis and withered

2 Chlorosis and necrosis
3 Chlorosis
4 Chlorosis, necrosis and withered
5 Chlorosis and withered

Table 3. The observation result of Koch’s postulates of entourage III

Group The Symptoms

1 Chlorosis, necrosis and withered

2 Chlorosis
3 Chlorosis and withered
4 Chlorosis, necrosis and withered

Based on the table of observation result the entourage I, group 1 and 5 have
symptoms are chlorosis necrosis and withered, group 2 and 3 have symptoms are
chlorosis and withered and group 4 only chlorosis. Based on the table of observation
result the entourage II, group 4 have symptoms are chlorosis necrosis and withered,
group 1 and 5 have symptoms are chlorosis and withered, group 2 have symptom are
chlorosis and necrosis and group 3 only chlorosis. Based on the table of observation
result the entourage III, group 1 and 4 have symptoms are chlorosis necrosis and
withered, group 3 have symptoms are chlorosis and withered and group 2 only
chlorosis.
 This is in accordance with the statement of Foster et al. (2008), the success of
inoculation is mechanically dependent on viral concentration in sap, inoculum
source, inoculum preparation method, viral resistance to sap, and host plant.
Environmental conditions before and after inoculation, such as light and temperature
also affect the success of inoculation. Leaf infected with the virus generally has a
leaves appearance of a smooth surface, berbercak and not perforated.

Picture 1. The Infected Plant by Koch’s Postulate method

Based on picture of the observation result of Koch’s postulate group 1
entourage II show the symptoms of infected leaves is only chlorosis and withered.
The necrosis is not formed. The control leaves there is not chlorosis, necrosis, mottle
and withered. According Semangun (2004) plant diseases with initial symptoms arise
small patches, round, slightly wet, which then enlarges and the color turns to brown.
Then comes a white mushroom mycelium which eventually forms a black spore
(konidium). The rotten fruit gives off a fermenting odor. Microscopically, the fungus
Aspergillus sp. Hyaline hifa color, simple conidiofor and hyaline disease is caused by
the fungus Aspergillus niger v. Tiegh.
Mottle virus disease on groundnut is caused by Peanut Stripe Virus (PStV).
The disease ia an important constraint in increasing productivity in Indonesia. PStV
is easily transmitted by several species of aphids through non-persistent manner and
infected seeds. The virus have a broad host range, including some leguminous weeds,
which result in the complementary of ecobiology virus-host- vector interrelationship.
The PStV measures have not been effective because of small fields owned by
farmers, limited capital and various planting time in one particular groundnut area.
Integrated crop management to obtain optimum yield and to be environmentally
sound and undertaken in a group at larger planting area, be likely more effective to
control the mottle virus disease on groundnut (Saleh, 2003).
 Bean Common Mosaic Virus (BCMV) is one of the most widespread and
economically important seed and aphid-transmitted viruses of beans (Phaseolus
vulgaris L.). Valuable local varieties are being lost due to the high percentage of
virus-infected seeds. Bean common mosaic disease can be effectively controlled by
planting certified seeds and or by creation and use of resistant cultivars. There are
two main types of symptoms associated with this disease: common mosaic and
common mosaic necrosis. The latter symptom is caused by Bean Common Mosaic
Necrosis Virus (BCMNV), which overcomes BCMV resistance governed by the I
gene. If a cultivar has the dominant I gene, it is resistant to strains of BCMV, but
imperfectly hypersensitive to strains of BCMNV (Pasev et al., 2013).
Under natural conditions, various viruses infect gladiolus that include: Arabis
Mosaic Virus (AMV), Bean Yellow Mosaic Virus (BYMV), Broad Bean Wilt Virus
(BBWV), Cucumber Mosaic Virus (CMV), Impatiens Necrotic Spot Virus (INSV),
Ornithogalum Mosaic Virus (OrMV), Strawberry Latent Ring Spot Virus (SLRSV),
Tobacco Mosaic Virus (TMV), Tobacco Rattle Virus (TRV), Tobacco Ring Spot
Virus (TRSV), Tomato Aspermy Virus (TAV) and Tomato Spotted Wilt Virus
(TSWV). Among them, BYMV is the most prevalent in gladiolus which causes
mosaic symptoms on leaves, color-breaking in flowers and reduces plant vigor (Kaur
et al., 2015).
Koch’s postulates are a method used to determine whether or not a virus
infects a plant. Koch’s postulate developed in the 19th century as a general guide to
identify pathogens that can be isolated by certain techniques. Time in Koch time, it is
known that some infective causes are indeed responsible for a disease and do not
meet all its postulates. Efforts to run a Koch’s postulate were stronger when
diagnosed with viral illness at the end of the 19th century. At that time the virus
could not yet be seen or isolated in culture. This impedes the early development of
virology (Rivers, 1989).
Definitive for linking pathogens by observing visible symptoms, showing
Koch postulates, isolation from disease-induced individuals from pathogenic
pathogens that are then used to induce symptoms in previously healthy hosts. The
requirement, first described in the year, is intended to establish a standard
methodology as a case of cause & effect. Virus is a obligate pathogen, it is
impossible to obtain pure culture, because some viruses are difficult to transmit a
new virus-specific transmission mechanism that may be unknown. Inducers caused
by single virus species in Koch’s postulates can be met in broad interpretation, ie
pathogens isolated from symptomatic plants into experimental homes and then re-
inoculated into native host species to try and mimic early symptoms. However,
where the virus complex may affect the host or where there may be an environmental
or oral influence on the development of symptoms (temperature, humidity, time of
exposure, time in the soil or plant growth stage, etc.) that attempt to correlate
pathogen detection with symptoms using conventional cause & effect. Therefore
statistical approaches have been used to show the possible effects of one or more
pathogens on symptom expression in the sample population (Adams et al., 2014).
Koch posts can be used and applied to various fields with the applicability
according to their individual needs. Various fields that apply Koch Postulates include
the field of microbiology, agriculture, animal husbandry, and the field of food. For
example in agriculture that Koch’s postulates can be used when there is attack by
pathogens in agroforestry cultivation, then in the field of microbiology Koch’s
postulate can be used to determine the role of microbes as the cause of illness
explained clearly with certain postulates (limits) (Saleh, 2003).
Plant viruses are responsible for some diseases that may cause agricultural
production losses such as tomato plants (black spots on tomatoes), corn and
sugarcane (tumor) and potatoes (potato yolk). Viruses can cause discoloration of
plants, inhibit growth, and cause decay in plants. Some plants may act as temporary
hosts and only show symptoms without further disintegrating. Plant cells and cells
are protected from disease by impermeable cell walls. Viruses enter the plant through
injuries that other plant parasites acquire such as nematodes, fungi and most insects
that suck plant fluids. Once the plant is infected by the virus it can spread the
infection to other plants through pollen or seed (Yayan, 2012).
The cycle of plant virus infection begins with the virus entering the
organoplasm through the aid of vectors or mechanical treatment. Viruses release the
viral genome (nucleic acid DNA or RNA) from the virion (uncoating) after the
cytoplasm of the host cell. Viral nucleic acids merge with the host a biolysis tract for
viral protein translation. Viral gene expression is required for viral genome
replication and viral pathogenesis. Viral genomic replication is intended for the
synthesis of new viruses (DNA or RNA) (Bos, 1983).
Plant viruses do not contain any enzymes, toxins or other substances that
other pathogens can engage in pathogenicity and cause various symptoms in their
host plants. Viral nucleic acids (RNAs) are the only & only determinants of disease,
but the presence of RNA or virions in plants, although in large quantities, is not
sufficient as the cause of disease symptoms. This is because some plants that contain
higher viral concentrations exhibit less severe symptoms than other plants with fewer
viral loads, or sometimes and sometimes they are just as virus-carrying plants that do
not indicate a magnitude (Suseno, 1990).
Common symptoms of plant viruses are external symptoms and internal
symptoms. External symptoms are symptoms of visible disease, can be seen directly
without the help of a microscope. The external symptoms are caused by primary
infections of cells inoculated by secondary infection due to the spread of the virus
from the primary infection site to other parts of the host plant. Symptoms of primary
infection in inoculated leaves are called local symptoms. These symptoms can be
distinguished by the surrounding tissue in the form of spotting. Internal symptoms of
histologic changes in plant parts infected with the virus, especially leaves, leaflets,
and branches of plants, can be divided into three groups, namely necrolysis of cell
death, Hyperplasia or excessive cell growth, as well as hypoplasia or decreased cell
growth (Akin, 2006).
The general mechanism of the spread of plant virus there are two namely
horizontal transmission and vertical transmission. Horizontal transmission is a plant
virus transmitted as a result of an external source. Viruses penetrating the protective
outer layer of the plant weather-damaged, trimming, or vector like grasses such as
bacteria, fungi and insects are usually more susceptible to viruses. Horizontal rhythm
also occurs with certain artificial methods of vegetative reproduction usually
employed by horticulturists and farmers. Plant cutting and grafting is the common
mode used plant virus can be transmitted. The vertical transmission of the virus is
inherited from the parent. The type of transmission occurs in asexual and sexual
reproduction. Asexual reproduction methods such as vegetative propagation, their
offspring evolve from and are genetically identical to a single plant. When new
plants evolve from stems, roots, tubers and others from the parent plant, the virus is
preached to the growing plants. In sexual reproduction, viral transmission occurs as a
result of seed infection (Saleh, 2003).
Beans and legumes (leguminosae) are often used for Koch’s Postulates. This
is due to the relatively rapid growth of plant so it is easy to observe the symptoms
caused when there are diseases caused by various infectious agents. The disease that
affects peanut farming in Indonesia, is generally bacterial disease, early leaf spot,
slow leaf spot, and rust each caused by Ralstonia solanacearum, Cercospora
arachidicola, Cercosporidium personatum, and Puccinia arachidis. Leaf rust disease
Puccinia arachidis is a fairly dangerous disease in peanut planting.
Pucciniaarachidis itself is an obligate parasite fungus that can not be saturated as a
saprophyte. Viruses that attack bean and legumes such as PStV and PmoV that can
cause symptoms of warts (blotch) in peanuts (Semangun, 1996).
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

1. To know the transmission of the virus we used SAP of infected plant and then
give to the health plant.
2. Based on the observation, the health plant was infected by the SAP of iinfected
plant.
B. Suggestion

The practicans must carefully when damaging the leave of health plant for
infected using the SAP of infected plant.
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 Portofolio of Koch’s Postulate Laboratory Activity

There are two kind of external symptoms on infectes plant by virus:

1. Local symptoms.
Local symptoms is the symptoms that occur only in the part which injected by the
virs. One characteristic of local symptoms is necrosis wherease the leave are turning
into brown caused by the dead cells. Chlorosis also the characteristic of local
symptoms, the damae of chlorophyll is happen.
2. Systemic symptoms.
Systemis symptoms are those involving the reaction of greater part of all the plant
such as wilting, yellowing, and dwarfing. The systemic symptoms spread around the
whole plants through vessel from injected organ to the others.