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LITERATURE REVIEW
BIOL7011: MOLECULAR
DIAGNOSTIC IN PLANT
PROTECTION
NAME: ESAVA TABUA KORANIU
THE UNIVERSITY OF
QUEENSLAND
PROGRAMME: MASTER OF
AGRICULTURE SCIENCE
CAMPUS: St. LUCIA
A Comparison of Plant Pathogen Diagnostic Techniques: Traditional
versus Molecular Diagnostic Technique
ABSTRACT
The ability to identify plant pathogens responsible for crop diseases is the main objective of
plant pathology. Without this ability, we will not be able to understand the diseases and most
importantly, the best way to control it. (Mccartney et al, 2003). Bacterial leaf spot caused by
Pseudomonas sp. is a common disease of brassica crops. Diagnosis of leaf spot can be carried
out in several ways. This include the conventional way which has been used in earlier years
until the present as well as the latest method which has been increasingly used. A common
test used for the presence of Pseudomonas sp. is streaming which can be identified under the
microscope. Not all bacterial infections show streaming and it may not be visualized without
special microscope. Therefore, serological tests, which are enzyme-linked, and physiological
assays are usually available to diagnose some common and economically important bacteria.
Molecular tests such as the polymerase chain reaction (PCR), are becoming more readily
available and used. According to Schaad et al (2001), diagnostic tests are still evolving and
tend to improve over the years. The paper will evaluate, traditional and molecular diagnosis
techniques used to detect Pseudomonas bacteria in brassica crops and suggest a best method
that can be used to suit any given condition.
INTRODUCTION
Plant diseases is a major limiting factor of crop productivity. In order to identify plant
disease, there is a need to correctly identify the pathogen. Current technologies, such as
polymerase chain reaction (PCR), requires a relatively large amount of target tissue and rely
on multiple assays to accurately identify distinct plant pathogens. (Khiyani et al, 2014). The
traditional diagnostic methods has been slowly replaced by the more advanced techniques as
they are more time consuming and lack high sensitivity. On the other hand, there is a need to
develop a low-cost methods to improve the accuracy and rapidity of plant pathogens
diagnosis. Traditional approaches to disease diagnosis involves the interpretation of visual
symptoms from the plant. For example, leaf spot. This is usually followed by laboratory
identification in which selective media and microscopy can be used to confirm the diagnosis.
The major practice which distinguish traditional diagnostic technique is the isolation and
inoculation of pathogen onto healthy crop to find out the presence of the target pathogen.
This method is still the cheapest and most appropriate to use especially in developing
countries. (Stowell & Gelernter, 2001). However, the drawbacks in conventional or
traditional methods has resulted in a search for other alternative diagnostic techniques which
can be taught easily to staffs. Such an accurate technology may help to design a proper
integrated disease management system which may modify crop environments to adversely
affect crop pathogens. This review will focus on the development of two molecular
diagnostic techniques which are classified as modern techniques as well as the traditional
technique which is a common technique which has been used in many countries. The two
modern techniques will be discussed in detail i.e. Nucleic Acid Based Diagnosis and
Antibody Based technique. The review will also compare and contrast traditional and modern
diagnostic techniques.
1.0
Traditional Technique of Detecting Pseudomonas. Spp on Brasicca
Visual symptoms on the leaf of the crop is the first detection where researchers can
draw their assumption from. There is a need to have deep knowledge on plant
symptoms in order to identify the possible causal agent. (Galea, 2013). There is a high
tendency of the symptoms been displayed by the plant to have similar characteristics
with nutrient deficiency or climatic interference. Therefore, there is a need for further
test the diseased material to further prove the suspected pathogen.
The Koch’s postulate experiment is
the common practice for traditional
method which takes several days or
weeks to complete. In order to prove
the presence of Pseudomonas
bacteria on the leaf, an Ooze test is
necessary to check for any signs of
streaming from the leaf material.
This is done by addition of two
drops of distilled water to the
diseased material under sterile
conditions and placed in under the
microscope to look for any signs of
streaming. Subculturing is also
Close-up of a leaf spot caused by Pseudomonas spp. on a
conducted in order to isolate a pure
colony from the diseased material.
Once isolation has been done, inoculation of the healthy plant will be carried out in
order to find symptoms on the leaf which will resemble the initial diseased plant. If
the symptoms are found again on the healthy plants, then this will prove positive for
the presence of Pseudomonas spp. bacteria on the leaf which causes leaf spot.
Brassica leaf. (Source: Cynthia M. Ocamb, May 2014)
Figure 2 above, shows the steps of isolation of bacteria from diseased material and used to inoculate on
healthy plant. (Source: Tabua, E, 2014)
Figure 3 shows healthy plants being inoculated with isolated pathogen to prove its presence on the plant.
(Source: Tabua, E, 2014). The test is positive as the healthy leaves shows symptoms which conclude that
Pseudomonas bacteria is the cause of leaf spot.
Ward (2004) stated that although traditional method is cheap and appropriate to be
used, he also argue that they need skilled and specialised expertise which takes many
years to train them in order to acquire skills needed.
Martin et al (2013) further supported Ward’s argument by stating that it is time
consuming for culturing of pathogen which is not recommended when rapid diagnosis
is required. Martin further states that ‘the results may not be conclusive as different
pathogens may cause similar symptoms’. Traditional methods may not be accurate
enough as closely related organisms will be hard to identify based on their
morphological characters. These common problems have resulted in the development
of diagnostic based on antibodies as well as nucleic acid technologies. (Duncan and
Torrance, 1992)
2.0
Nucleic Acid Based Diagnostics (PCR)
This method has been increasingly used over the years to develop assays for plant
pathogens. (Schots et al, 1994). According to Schots et al, the method is sensitive and
highly specific. Most assays developed for fungi and bacteria were found to detect
pathogen DNA which are more stable than RNA and easily prepared. This method
synthesise millions of specific DNA sequence. Edel (1998), explained that, ‘DNA
strands are separated by heating to 950 Celsius then temperature is reduced to 650
Celsius. This action will allow the binding of two primers at each end of the region.
These primers give PCR their specificity. The second strand of DNA is generated at
the temperature of 720 Celsius which extend from the primer by using a thermostable
DNA polymerase and dNTPs. The number of copies of the target DNA sequence will
be doubled at the end of the first cycle of PCR reaction. The whole process will be
repeated many times so that millions of copies of sequence been produced. This will
be placed in PCR machines that are computer controlled and giving out the results.
3.0
Antibody Based Technique
Antibodies are molecules that can be used to produce mammalian immune systems
which are important to identify invading organisms. If antibodies are generated to
recognise specific antigens then they can be used as a diagnostic tool. (Nayaranasami,
2011). According to Barker, 1996, there are three ways of antibodies production. For
example, Polyclonal antibodies are made through injection extracts from pathogen
into an animal, then collect blood from it later. The blood samples will then be
allowed to clot and the serum collected from it can contain antibodies for the tested
extract.
Monoclonal antibodies can be made by fusing antibody producing cells from the
inoculated animal’s spleen with cultured myeloma cells. This results in the generation
of many hybrid line cells which will produce a different single antibody. The
individual cell lines will then be propagated and the single monoclonal antibodies are
harvested from the culture medium.
Another format is known as the plate-trapped antigen (ELISA) in which the microtitre
plate wells is directly coated with the test sample. This test involves the enzyme
mediated colour change reaction which aim to detect antibody binding. The colour
change is usually measured in a computer controlled reader which usually determine
the amount of pathogen present. This is a simple and cheap method that can be used
to process many samples.
The above two techniques can be used to determine the presence of Pseudomonas
spp. Bacteria in suspected plant materials. (Voller and Bidwell, 1985)
4.0
Comparison of Diagnostic Techniques
Immunological and nucleic acid techniques are both molecular techniques which have
comparative advantage over traditional method. Traditional method is a cheap method
and the resources can be available to be used. For molecular diagnostic technique,
there are some factors to be considered in order to use the recommended techniques.
The staffs needs to be skilful whereby proper training is needed, the costs is very high
due to the high technological facilities been used. Also, a combination of diagnostic
techniques can be the best approach. For example, immunocapture can be used for
improvement of specificity of PCR assays thus overcoming problems with inhibitors
in the sample. There has been significant advances in plant pathogen diagnostics. The
discussed method of molecular diagnostic techniques i.e. antibody and nucleic acid
based diagnosis have been increasingly used in many developed countries. The
introduction of real time PCR machines has also boost major improvement in nucleic
acid based methods of detection. The volume of samples been tested each day has
also increased with molecular diagnostic techniques.
Considering the importance of the latest development of diagnostic techniques, I
believe that this is the best way forward for all countries. Even though, these
techniques are found to be practiced mainly in developed countries due to the high
cost of equipment and facilities which may be little expensive to be used in
developing countries, I do believe that there is a need for international co-operation
through research and education in order to upgrade skills of responsible staffs in
developing countries as well as installation of machines to be used in many
laboratories in developing countries. It is believed that the cost of newer techniques
will decrease over time due to the increase usage of techniques and a wider choice
from manufacturers due to improvement in technologies.
Though, traditional method is widely practiced around the world especially in
developing countries, there is a need to consider the many advantage of molecular
diagnostic techniques which tend to save time and energy, improve accuracy and
specificity, and also allow rapid on site detection by unskilled personnel. Service,
1998 also pointed out that due the development of new technologies, it will enable to
detect several organisms simultaneously in the same sample as compared to one target
species at a time. Example, multiplex PCR which can detect thousands of gene
sample at a time.
5.0
Conclusion
In conclusion, the new molecular based diagnostic techniques which have been
developed can be more rapid, accurate and sensitive as compared to the traditional
methods. There is still problems that needs to be considered before they can be
implemented. Antibody techniques can be little difficult to generate enough specific
antibodies for the target organisms but is forecasted to be improved over time after
continuous research. The nucleic acid based technique has limited target DNA
sequence databases which usually prevent the ability to design proper diagnostic
probes or primers. More effort is needed to generate a more sophisticated DNA
database sequence. Cost of new techniques will also decrease due to an increased
usage of machines and the availability from wider choice of manufacturers. The
discussed modern diagnostic technologies will have an increasingly important role in
pathological research in the near future which can also lead to more efficient control
strategies. The increase application of diagnostic methods to inoculum detection can
also allow progress to be made to improve our understanding on the temporal and
spatial dynamics of development thus opening new opportunities for disease
forecasting and management. Leaf spot is a common disease that is caused by
Pseudomonas spp. bacteria that usually attack many brassica crops. It is believed that
the continuous usage of modern diagnostic techniques will improve farmer’s control
over the disease as they will be able to apply the proper chemical on time to the
affected crop instead of waiting for a longer period of time for results to be known
when using traditional method to detect the pathogen. This will save time for the
farmer’s thus increasing productivity of farm.
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