REVIEW ARTICLE.. PLANT DISEASE...measures depend on proper identification of diseases, as well as...

Nigerian Journal of Mycology Vol.7 (2015) 1

REVIEW ARTICLE. Niger. J. Mycol. Vol.7 , 1-13

PLANT DISEASE DIAGNOSTICS: A MOLECULAR APPROACH

Shittu, H. O.,1* Igiehon, E1, Imhangbe, O. P.2 and Odenore, V. D.3 1Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria 2Department of Science Laboratory Technology, University of Benin, Benin City, Nigeria

3Physiology Division, Nigerian Institute for Oil palm Research, Benin City, Nigeria

*Corresponding author: (E-mail: [email protected]) (Phone: 08084765588)

ABSTRACT

Accurate identification and quantification of pathogens are important aspects of plant disease

diagnostics. Traditional methods which are being used in plant disease diagnosis include plating,

microscopic, spore extraction and determination of colony forming units in bulk samples. These

techniques are slow, inconclusive, inaccurate, semi quantitative, labour intensive and often

associated with low sensitivity and specificity. Some of these limitations have prompted the

search for alternative diagnostic approaches. Here, we give a highlight of molecular diagnostic

methods and classified them into three. Nucleic acid sequence amplification based methods,

which use specific DNA sequence; antibody-based approaches, which rely on the use of specific

enzymes; and polymorphism-based methods, which rely on the differences occurring in the

pathogen DNA; all of these for the detection and quantification of pathogens. The molecular plant

diagnostic methods are faster, more accurate, sensitive, with high level of specificity than the

traditional methods, although, they are also associated with some challenges.

Keywords: Diagnostics, Traditional, Molecular, Nucleic acid, Serological, Polymorphism

INTRODUCTION

The term “Diagnostics” simply refers to the techniques used in identifying a plant

disease, either by the type of symptoms expressed or by determining the individual

species, races, strains, or isolates of the causative agent, such as viral, bacterial, fungal or

nematode pathogens (Cullen et al., 2005). The term “diagnosis” refers to the process of

identifying or trying to identify a disease. Diagnostic techniques are used to carry out

diagnosis. Analysis of soil sample for pathogenic microorganisms is considered also as

part of diagnosis (Mauchline et al., 2002). Both identification and quantification of

pathogens are necessary in most confirmatory diagnostic studies. Plant disease diagnosis

Molecular approach of plant disease diagnostics


is important for both research purposes and agricultural practices. In scientific studies, a

researcher studying a particular pathological phenomenon such as susceptibility,

resistance or tolerance may want to determine the amount of pathogen under

investigation, either in plant material or soil sample in order to correlate it with other

measurable parameters such as symptom score, disease resistance, yield gain/loss (Robb

et al., 2009;Shittu et al., 2009a). In agricultural practices, diagnosis enables farmers to

detect plant disease well on time before serious damage is caused. Early and non-

destructive diagnostics of perennial crop disease is a major concern in precision farming

and sustainable agriculture (Camille et al., 2010). Diagnosis is crucial in determining the

essential disease control methods required to stop further spread of the disease. Control

measures depend on proper identification of diseases, as well as the causative agents. The

latter is important because without its proper identification, disease control measures can

be a waste of time and money which may lead to further plant losses (Ellis et al., 2008).

In the diagnosis of plant diseases, signs and symptoms are looked out for, but

sometimes, neither symptoms nor signs provide specific or characteristic information to

describe the causative agent of an infectious plant disease (Riley et al., 2002). In this

case, it becomes necessary to carry out plant diagnostic methods in identifying the

causative pathogen(s). The lack of rapid, accurate and reliable means by which plant

pathogens can be identified and quantified have been the main limitation in plant disease

diagnostics and management (Lievens et al., 2006). Several traditional methods have

been used in plant diagnosis, but most of these are associated with several limitations. At

present, molecular tools are being employed in plant disease diagnosis and this is usually

referred to as molecular plant disease diagnostics. Richard and Peter (2005) opined that to

effectively detect and quantify pathogens, it is necessary to perform diagnosis at the

molecular level, hence molecular diagnostics which is currently being employed in plant

disease diagnosis. This review is an attempt to recount some traditional diagnostic

methods, their limitations and showcase some molecular diagnostic alternatives recently

developed.

Traditional Plant Disease Diagnostics

Traditional diagnostic methods involve the identification of a pathogen based on

its phenotype or the ability to grow on a certain media, to metabolize a given chemical

compound, etc. (Lauri and Mariani, 2009). The use of plating method for diagnosis is one

of the oldest techniques and it involves spreading macerated infected plant parts such as

stem, petiole, leaves and roots on selective media. This method relies on the isolation of

pathogen from diseased plant materials on nutrient plates for identification and semi-

quantification (Anderson and Hoyos, 1987). Morphological identification of symptoms

and reproductive fruiting bodies is another traditional diagnostic method that has been

Shittu,Igiehon, Imhangbe, & Odenore


used in the past. This technique involves morphological identification of causative

pathogens via symptoms appearance, morphology of reproductive structure of fungi

associated with disease tissues (Riley et al., 2002). Many fungi produce fruiting bodies,

but some do not. If fruiting structures are associated with disease tissue, they may be

sufficient to permit identification, which is usually the case with the smut, rust, powdery

mildew, downy mildew fungi and frequently with many other pathogens (Agrios, 2005).

In the absence of fruiting structures, isolation and growth on specialized media and in

controlled environment (optimal temperature, humidity and photoperiod) may induce

reproduction. Prior to isolating plant parts from plant materials, particularly when seeking

an organism(s) implicated in a plant disease, the tissues should be examined for fruiting

bodies, mycelia, and bacteria cells.

Another plant disease diagnostic technique is the use of microscopy. This method

involves the collection of diseased plant parts which are sectioned, stained and viewed

under the microscope. The number of colonized cells is determined in the tissue cross

sections (Tsai and Erwin, 1975). In this method, free hand sections are enough in the

identification of fungi that produce fruiting bodies. Paraffin sections or other mechanical

slide sections are essential in any detailed host-pathogen relationship study. Elderberry

pith or carrot root can be used for free hand sectioning. Elderberry pith can be kept in 70

% alcohol prior to sectioning. Small portions of the materials to be sectioned are enclosed

between split halves of the pith or carrot roots. A new razor blade is used to make

sections across the material. Thereafter, suitably thin sections are transferred directly to a

drop of mounting media on a microscope slide. If a stereoscopic binocular is available,

materials can be sectioned, while being viewed through the microscope. This technique is

particularly useful when fruiting bodies are present on firm host tissue. After materials

are sectioned and stained, they are mounted on slides and colonies are hand counted

through the light microscope. This usually includes a count of both living and dead cells

together (Tuite, 1969;Agrios, 2005).

Spore extraction is another traditional method used for diagnosis. This procedure

involves picking up the spores of the fruiting bodies of an organism that is sporulating on

the diseased material with fine needle, micro-spatula or loop with the aid of a

stereoscopic microscope. The spores are then streaked on agar directly or added to 1–2

drops of sterile water on the surface of acidified agar, and streaked from the drops.

Fruiting bodies which do not ooze out spores are usually transferred through 3 or 4

adjoining drops of sterile water on a previously flamed slide to reduce contaminants

before being crushed. Spores thus released may be streaked on agar. Frequently, fungi

such a Cercospora, Septoria and Kabatiela, which are not fast growing organisms, are

isolated from the host by direct isolation or spore extraction method. The growth of the



spores on the nutrient medium is later used for the identification of the causative

pathogen (Tuite, 1969).

Determination of the number of colony forming units (cfu) in bulk samples is

another traditional diagnostic technique less commonly used. Colony forming unit are

individual colonies of microorganisms on a nutrient medium or diseased material. A

colony of bacterium or yeast refers to individual cells of same organisms growing

together. For moulds, a colony is a group of hyphae (filaments) of the same mould

growing together. Colony forming unit are used as a measure of the number of

microorganisms present in or on the surface of a diseased sample, which may be reported

as cfu per unit weight, cfu per unit area, cfu per unit volume, depending on the type of

sample tested (Agrios, 2005). Unlike direct microscopic counts, where all cells, dead and

living are counted, determination of cfu measure viable cells, and the technique may also

be used to measure the degree of contamination in samples such as fruits, soil,

vegetables, etc.

Limitations of Traditional Plant Disease Diagnostic Methods

The traditional techniques of identifying plant pathogens are fundamentally

valuable; however, they are time consuming, difficult, inaccurate, unreliable, unspecific,

semi quantitative, and may lead to inconsistent results. For example plating technique is

difficult and required special handling of sample (Martin et al., 2004). The isolation of

pathogens followed by biochemical and pathogenicity test is time consuming which often

takes days or weeks to complete. This is not acceptable when rapid high throughput

diagnosis is required in the sense that before disease is correctly diagnosed vast amount

of crops are lost (Yachum et al., 2013).

Symptoms based identification and characterization is inaccurate and unreliable due to

incipient infection (Kamle et al., 2013). Most times neither signs nor symptoms provide

specific or characteristic information to describe the causative agent of an infectious plant

(Riley et al., 2002). Similar symptoms can be caused by different pathogens or

physiological conditions (Ward et al., 2004). Morphological identification of

reproductive structures of pathogens is not only time consuming, but also difficult and

require expensive knowledge and experience in recognizing detailed disease features

(Thermis et al., 2005). Not all pathogenic fungi produce reproductive structures (Agrios,

2005). Traditional methods may not be sensitive enough especially where the detection of

pre-symptomatic infection is needed (Ward et al., 2004). Sensitivity and specificity are

numeric measures of effectiveness of a detection system in plant diagnostics (Malorny et

al., 2003). Diagnostic specificity is defined as a measure of the degree a particular

diagnostic method is able to detect the target pathogen in a sample, which may result in

false negative responses (Malorny et al., 2003). Thus, the ability to detect true and

precisely the target microorganism without any interference from other non-target



organisms is known as a high degree of diagnostic accuracy. Traditional methods

generally require skilled and specialized microbiological expertise, which often take

many years to acquire (Ward et al., 2004). More so, traditional techniques for diagnosis

are usually labour intensive and may not be effective in diagnosing certain crops. For

example, many Oil palm plantations suffer from ganoderma, which is a fungal infection.

An efficient tool to manage this plague without great loss in production or large use of

chemicals is limiting in the traditional diagnostic methods (Bridge et al., 2000).

Besides all these limitations, in order to truly certify isolated/associated organisms

as the causal agents of disease, plant pathologists commonly employ or apply the Koch’s

Postulates to confirm pathogenicity (Agrios, 2005). The postulates are based on the

principles of isolating pathogens from diseased host, establishing the organism in pure

culture and reintroducing the isolate into a disease free host with the expectation of

classical symptom observed at the first. Surely, all the above are also cumbersome, time

consuming and resource consuming procedures. Other limitations of traditional

diagnostic methods may include their semi quantitative nature to determine the amount of

pathogen in infected samples of which results obtained are usually inaccurate and may

vary from one trial to the other. These limitations have thus prompted the search for

alternative plant disease diagnostic techniques (Ward et al., 2004).

Molecular Plant Disease Diagnostics

Molecular plant disease diagnostics employ techniques of molecular biology in

the identification and quantification of pathogens (Shittu, 2012). These techniques are

more recent, sensitive and accurate. The high degree of sensitivity of molecular methods

made pre-symptomatic detection and quantification of pathogens possible (Degefu,

2008). In this review, molecular disease diagnostic techniques have been classified into

three: nucleic acid sequence amplification-based (NASAB), antibody–based and

polymorphism–based detection and quantification methods.

(a) Nucleic Acid Sequence Amplification-Based Techniques

These techniques rely on the recognition of specific DNA sequences such as the

internal transcribed spacer regions (ITS1 and ITS2), intergenic spacer region (ISR) of the

rRNA genes (Shittu et al., 2009b), cytochrome oxidase genes (COX I and II genes) of the

mitochondria (Martins and Tooley 2003), pathogenicity genes like hrPL gene and Tox

gene, genes encoding secondary metabolites that function as virulence factor (Schaad et

al., 2003). These gene sequences are very specific to different pathogenic organisms.

Among the NASAB techniques, polymerase chain reaction (PCR) is commonly used and

is the most potent molecular diagnostic tool.

Polymerase Chain Reaction (PCR) is used to amplify a segment of DNA strand

from a template (target) to generate several thousand to million copies of the starting



amount (products) (Shittu, 2012). PCR is now the basic technique for the development of

most molecular diagnostic methods due to its simplicity, cost-effectiveness and

adaptability to large scale screening (Mauer, 2006). According to Lauri and Mariani

(2009), in diagnostic PCR, specific primers directed against the DNA of the organism to

be detected are used. For example (Fig. 1), primers specific for the ITS regions (P1 and

P2) or ISR (P3 and P4) of the target pathogen are made. The homology between primers

and target DNA confers specificity to the amplification. The presence of the

amplification product at a given reaction conditions, reveals the organism in the test

sample. The technique is also quantitative, as it is often used to quantify the amount of

pathogen in infected plant or soil samples. In diagnostic PCR, the amount of pathogen

DNA is determined and used as an estimate of the amount of pathogen biomass in

infected plant samples. A homologous or heterologous internal control DNA of known

concentration is usually included in each PCR reaction to allow accurate quantification of

the pathogen DNA and detection of inhibiting compounds as demonstrated in Figure 2

(Shittu et al., 2009b). Other NASAB methods often used in molecular diagnostics include

ligation-mediated amplification method (Wu and Wallace, 1989) and transcription-based

amplification method (Kwoh et al., 1989).

Figure 1: Structure of rDNA showing tandem repeat arrangement of genes. ITS1: Internal transcribed spacer region 1; ITS2: Internal transcribed spacer region 2; ISR: Intergenic spacer region; P1 and P3: forward primers; P2 and P4: reverse primers (Source: Shittu et al., 2009b).

Figure 2: PCR diagnostic assay for quantifying amount of Verticillium dahliae race 1 in infected tomato stems. For

fungal DNA measurements, plant extracts were used as templates (lanes b and c) to amplify the 18-28S intragenic

region (ITS1 and ITS2 regions) (Figure 1; primers P1 and P2) in the presence of a truncated internal control template.



Products were fractionated on 2 % agarose gels, to differentiate the 544 bp fungal DNA from the 391 bp internal

control as indicated on the right. Purified fungal DNA and internal control are included in lanes (a) and (d),

respectively; a reaction without template (lane e) and fragment length markers (M) also are included. The DNA bands

were quantified using Molecular Analyst Software as a measure of the amount of pathogen in plant stems (Adapted

from Shittu et al., 2009b).



b)Antibody-Based (Serological) Diag-nostic Techniques

These techniques are immunodiagnostic assays that rely on detection of protein

component of pathogens with the help of specific antibodies that are produced by

immune system in response to pathogenic attack. Antibodies refer to group of molecules,

which are produced by mammalian immune systems that are used to identify invading

organisms or substances. If antibodies that recognize specific antigens associated with a

given plant pathogen can be generated, such antibodies can be used as the basis for a

diagnostic tool (Ward et al., 2004). Among the serological techniques, enzyme linked

immunosorbent assay (ELISA) has found wide spread application in the detection and

identification of plant pathogens and are commonly used (Clark and Adams, 1977). This

is due to its high sensitivity, simplicity, reproducibility, versatility in screening large

number of sample. For diagnostic purposes, antisera (polyclonal, monoclonal or phage

display) are first produced against purified proteins from the target pathogen. These

specific antibodies are then conjugated to an enzyme (Fig. 3) which can effect colour

change (direct detection). In carrying out diagnosis, diseased plant samples are prepared

to make the antigen extracts, which are then reacted with the prepared antibodies. This

process is usually done in a microtitre plate. The principal aim of this procedure is to

detect or quantify the binding of the diagnostic antibody with the target antigen, which

could be achieved by coupling the antibody to an enzyme that can be used to generate a

colour change when a substrate is added. The presence of the target pathogen in the plant

sample indicates positive and it is usually associated with a colour change which can be

measured in a computer controlled plate reader. The extent of the colour change can be

used to determine the amount of pathogen present in the plant sample.

(ELISA) has found wide applications in plant pathology. In several studies, the

technique has been used for the detection and quantification of fungal pathogens such

Vertcillium species (Fitzell et al., 1980), Phytophthora species (Amouzou-Alladaye et al.,

1988) and even endomycorrhizal fungi (Aldwell et al., 1983). It has also proven to be

very sensitive and reliable for the detection of viral pathogens such as Cucumber mosaic

virus (CMV) in different tissues (Abdulahi et al., 2001). Akinjogunla et al. (2008), in

their review on immunological and molecular diagnostic methods for detection of viruses



infecting cowpea (Vigna unguiculata) pointed out that many factors could influence the

sensitivity and reliability of ELISA method. Some of these include quality of antibodies,

preparation and storage of reagents, incubation time, temperature, selection of

appropriate parts of sample and the use of suitable extraction buffer. Other serological

techniques are Dot immune-binding assay, Imunofluoresence microscope and Lateral

flow assay.

Figure.3a: Mechanism of serological diagnostic techique. (Source: Albert et al., 1994)

c)Polymorphism-Based Diagnostic Methods

Polymorphism-based methods are techniques that exploit the variations in homologous

DNA sequence to differentiate genotypes among a pathogen population. The variation

exists as a result of differing locations of restriction enzyme sites in the DNA molecule.

Methods in this category are less often used in diagnostic purpose. An example of a

polymorphism-based technique is the restriction fragment length polymorphism assay.

This procedure involves detection of DNA polymorphism by hybridizing a chemically

labelled DNA probe to a Southern blot of DNA digested by restriction endonucleases,

resulting in differential DNA fragment profile (Agarwal et al., 2008). A variant of this

method is the amplified fragment length polymorphism (AFLP) assay, in which the

region of variability such as the ITS regions or ISR in target pathogens is first amplified

and then subjected to restriction digestion. The resulting differential restriction fragments

are used to differentiate pathogens of interest.

An application of this diagnostic assay is the use of both specific primers and

restriction enzyme analysis which is mostly applied as a standard protocol for detecting

pathogenic Ganoderma in Oil palm (Utomo et al., 2005). The result from previous studies

indicated that Ganoderma strains associated with basal stem rot disease in Oil palm



belong to a single species. Shittu et al, (2009b) used the AFLP assay for a diagnostic

approach to differentiate and quantify the virulent fungal wilt fungus, Verticillium

dahliae race 1 (Vd1) from an eggplant isolate of the same species, V. dahliae Dvd-E6

(Dvd-E6). In the studies, a 745 bp portion of the 28-18S ISR from both Vd1 and Dvd-E6

were amplified using the polymerase chain reaction (Figure 1; primers P3 and P4). The

amplified experimental DNAs were digested with SpeI restriction enzyme that cleaves at

nucleotide 873 only in the Vd1 DNA, resulting in a shorter 640 nucleotide fragment

when fractionated on 2 % agarose gels. This serves as the basis for differentiating Vd1

from Dvd-E6 as outlined in Figure 3.

Figure 3b: Amplified fragment length polymorphic assay for differentiating Verticillium dahliae race 1 and V. dahliae

Dvd E6 (Adapted from Shittu et al., 2009b) Left panel: Undigested (lanes a and c) and digested (lanes b and d) PCR

amplified control DNAs for Dvd-E6 and Vd1, respectively, together with a reaction in the absence of template (lane

e).Right panel: Two typical mixed experimental samples (lanes b and c). An undigested control (lane a) and fragment

length markers (M) also are included. The positions of the Dvd-E6 derived fragment (745 bp) and the Vd1 derived

fragment (640) are included on the right.

The aforementioned categories of molecular techniques are more accurate, highly

sensitive, specific, quantitative and very fast compare to the traditional diagnostic

methods. The choice of a molecular technique required for a diagnostic procedure

depends on several factors. Some of the features to be considered include the available

laboratory equipment, response time, number of species that need to be detected

simultaneously, available fund and the resolution required (genera, species, strains,

pathotype).

Drawbacks on Molecular Diagnostic Methods

Molecular diagnostics offer unparalleled advantages over the traditional

techniques; nevertheless, they are also faced with some drawbacks. Molecular diagnostic

procedures involve the use of facilities ranging from simple (such as thermal cycler for



polymerase chain reaction or gel electrophoresis apparatus for gel electrophoresis) to

complex and more sophisticated equipment (such as microarray analyzer, DNA

sequencer and oligonucleotide synthesizer). Obtaining these facilities and the cost of

chemical reagents to run analysis make the molecular diagnostic methods more

expensive, although may be cost effective on the long run. Another major challenge is the

abundance of false positives and false negatives results which may arise from the

presence of DNA contaminants from the environment, in the laboratory and even in the

instruments used to prepare the reaction mix.

This may lead to false detection of a pathogen. Thus, the handling and

interpretation of diagnostic results may require special skills of well-trained laboratory

technicians. In using a NASAB technique for pathogen identification requires a priori

knowledge of the target sequence and the occasional mutation in the genomic DNA of the

pathogen can compromise the detection (Lauri and Mariani, 2009). More so, PCR

inhibition, organic and inorganic compound present in the host tissues, components of

microbial cell can inhibit PCR diagnostic methods.

Conclusion

Molecular diagnostic techniques have provided more reliable, robust, rapid, accurate,

extremely sensitive and specific means of identification and quantification of pathogens.

They should complement the traditional techniques in order to mitigate the risk of

asymptomatic infections, monitor the spread of pathogens and improve disease

management strategies.

Acknowledgment

Our great appreciations go to Dr. (Mrs.) A. P. O. Dede and Dr. B. Ikhajiagbe of the

Department of Plant Biology and Biotechnology, University of Benin, Nigeria, for their

painstaking efforts in proofreading the manuscript and offer of valuable suggestions. We

thank Prof. A. T. Adewale of the Department of Crop Science, University of Benin,

Nigeria. We also appreciate the valuable contributions of Dr. J. Robb and Dr. R. N.

Nazar of the Department of Molecular and Cellular Biology, University of Guelph,

Canada.

REFERENCE

Abdullahi, I., Ikotun T., Winter, S., Thottappilly, G. and Atiri, A. (2001).

Investigation on soil transmission of cucumber mosaic virus in cowpea.

African Crop Science Journal, 9(4): 677–684.

Agarwal, M., Shrivastava, N. and Padh, H. (2008).Advances in molecular marker

techniques and their applications in plant science. Plant Cell Report, 27(4):

617-31.



Agrios, G. N. (2005). Plant Pathology. 5th ed. Elsevier Academic Press,Burlington,

USA. 922.

Akinjogunla, O. J., Taiwo, M. A. and Kareem, K. T. (2008). Immunological and

molecular diagnostic methods for detection of viruses infecting cowpea

(Vigna unquiculata). African Journal of Biotechnology, 7(13): 2099–2103.

Albert, B., Johnson, A., Lewis, J., Raff, M., Robert, K. and Walter, P.

(1994)Molecular Biology of the Cell. 4th edition. Garland Science,

pp 235-245.ll, F.

Aldwell, F. E. B., Hall, I. R. and Smith, J. M. B. (1983). Enzyme-linked

immunosorbent assay (ELISA) to identify endomycorrhizal fungi. Soil

Biology and Biochemistry, 15: 377-378.

Amouzou-Alladaye, E., Dunez, J. and Clerjeau, M. (1988).Immuno-enzymatic

detection of Phytophthora fragariae ininfected strawberry plants.

Phytopathology, 78: 1022-1026.

Anderson, N. and Hoyos, G. (1987). Varietal response of potatoes to Verticillium wilt.

Valey Potato Grower, 53:10-14.

Bridge, P. D., O’Grady, E. B., Pilotti, C.A., Sander, F.R. (2000). Development of

Molecular Diagnostic for the Detection of Ganoderma Isolates Pathogenic to

Oil palm. In: Ganoderma: Disease of Perennial Crops. Flood, J. Bridge, P. D.

Holderness, M. (Editors). CABI Publishing, United Kingdom, 225p.

Camille, C. D. L., Jean-Michel, R., Simon, B., Fabrice, D., Mathieu, L., Nurul, A.S.,

Doni A.R.,andJean-Pierre, C. (2010). Evaluation of oil-palm fungal disease

infestation with canopy hyper spectral reflectance data. Sensor, 10: 734-747.

Clark, M. F. and Adams, A. N. (1977).Characteristics of the microplate method of

enz-yme-linked immunosorbent assay for the detection of plant viruses.

Journal of Genetics and Virology, 34:475-483.

Cullen, D. W., Toth, I. K., Pitkin, Y.,Boonham, N., Walsh, K., Barker, I. and Lees,

A. K. (2005). Use of quantitative molecular diagnostic assays to investigate

Fusarium dry rot in potato stocks and soil. Phytopathology, 95: 1461-1471.

Degefu, Y. (2008). Molecular diagnostic techniques and end user applications:

Potentials and challenges. Mobile Training Team’s Annual Report. 216.

Ellis, S. D., Boehm, M. J., Chatfield, J., Boggs, J. and Draper, E. (2008). Diagnosing

sick plants: fact sheet. Agricultural and Natural Resources, 10(2): 401-402.

Fitzell, R., Fahy, P. C. and Evans, G. (1980). Serological studies on some Australian

isolates of Verticillium spp. Austral Journal of Biological Sciences, 33: 115-

124.



Kamle, A., Pandey, B. K. and Muthu, K. M. (2013). A Species-specific PCR Base

Assay for rapid detection of mango Anthrose Pathogens Colletotrichum

gloeosporiode Penzan Sacc. Journal of Plant Pathology and

Microbiology, 4(6):1–5.

Kwoh, D. Y., Davis, G. .R., Whitfield, K. M., Chappelle, H. L., DiMichele,L.J.and

Gingeras, T. R. (1989). Transcription-based amplification system and

detection of amplified human immunodeficiency virus type 1 with a bead-

based sandwich hybridization format. Proceedings of the National Academy

of Sciences, U.S.A. 86:1173–1177.

Lauri, A., and Mariani, P. O. (2009). Potentials and limitations of molecular diagnostic

methods in food safety. Genes and Nutrition, 4(1): 1–12.

Lievens, B., Brouwer, M., Vanachter, A. C. R., Commue, B. P. A. and Thomma, B.

P. J. H. (2006). Real-time PCR for detection and quantification of fungi and

Oomycetes pathogens in plant and soil sample. Plant Science, 121:155 -165.

Malorny, B., Tassios, P. T., Radstrom K., Cook, N., Wagner, M. and Hoorfar, J.

(2003). Standardization of diagnostic PCR for the detection of foodborne

pathogens. International Journal of Food Microbiology, 83:39–48.

Martin, F. N. and Tooley, P. W. (2003).Phylogenetic relationship among Phytophthora

species inferred from sequence analysis of mitochondrially encoded

cytochrome oxidase 1 and 11 genes. Mycologia, 95:269-283.

Martin, F. N., Tooley, P. W. and Blomquist, C. (2004). Molecular diagnostics of

Phytophthora ramorum, causal agent of sudden oak death in California and

two additional species commonly recovered from diseased plant material.

Phytopathology, 94(6): 621-631.

Mauchline, T. H., Kerry, B. R. and Hirsch, P. R. (2002). Quantification in soil and the

rhizosphere of the nematophagous fungus Verticillium chlamydosporium by

compete-tive PCR and comparison with selective plating. Applied and

EnvironmentalMicro-biology, 68(4):1846–1853.

Maurer, J. (2006). PCR Methods in Foods (Food Microbiology and Food Safety).

Springer, Berlin. 258.

Richard, N. S. and Peter, R. S. (2005). A threat to global food security. Annual

Review of Phytopathology, 43:83-116.

Riley, M. B., Williamson, M. R. and Maloy, O. (2002). Plant disease diagnosis. The

Plant Health Instructor. DOI: 10.1094/PHI-I- 2002-1021-01.

Robb, J., Castroverde, D. M. C., Shittu, H. O. and Nazar, R. N. (2009). Patterns of

defence gene expression in the tomato-Verticillium interaction. Botany,

87: 993-1006.

Schaad, N.W., Frederick, R.D., Shaw, J., Schneider, W. L., Hickson, R., Petrillo, M.

and Luster, M. (2003). Advances in molecular-based diagnostics in meeting



crop biosecurity and phytosanitary issues. Annual Review of Phytopathology,

41: 305-324.

Shittu, H. O. (2012). Basic Molecular Techniques and Applications. In:Biological

Techniques and Applications. Okhuoya, J. A., Okungbowa, F. I. and Shittu,

H. O. (Editors). University of Benin Press, Nigeria.180-210.

Shittu, H. O., Shakir, A. S., Nazar, R.N. and Robb, J. (2009a).Endophyte-induced

Verticillium protection in tomato is range-restricted. Plant Signaling and

Behavior, 4(2): 160-161.

Shittu, H. O., Castroverde, D. M. C., Nazar, R. N. and Robb, J. (2009b).Plant-

endophyte interplay protects tomato against a virulent Verticillium. Planta,

229: 415-426.

Thermis, J. M., David, P. M., Zhonghus, M., Yong, L., Daniel, F. and Mark, A. D.

(2005). Conventional and molecular assays and diagnosis of crop disease

and fungicide resistance. California Agriculture 59:115-123.

Tsai, S. D. and Erwin, D. C. (1975). A method of quantifying numbers of microscelrotia

of Verticillium albo-atrum in cotton plant tissue and in pure culture.

Phytopathology, 65:1027-1028.

Tuite, J. (1969). Plant Pathological Methods. Burgess Publishing Company, 239p.

Utomo, C., Werner, S., Niepold, F., Deising, H. B. (2005). Identification of Ganoderma

the causal agent of basal stem rot disease in oil palm using a molecular

method.Mycopathologia, 159:159-170.

Ward, E., Foster, S. J., Fraaije, B. A. and McCartney, H. A. (2004). Plant pathogen

diagnostics: Immunological and nucleic acid based approaches. Annals of

Applied Biology, 145: 1–16.

Wu, D. Y. and Wallace, R. B. (1989). The ligation amplification reaction (LAR)-

amplification for specific DNA sequences using sequential rounds of

template-dependent ligation. Genomics, 4: 560–569.

REVIEW ARTICLE.. PLANT DISEASE...measures depend on proper identification of diseases, as well as...

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