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101
Detection and Diagnosis of Important
Soil-Borne Pathogens 5
K. Jayalakshmi, Nazia Manzar, B. K. Namriboi, Abhijeet S. Kashyap,
Pramod K. Sahu, R. Saranya, Yalavarthi Nagaraju, N. Sharath,
Amrita Gupta, and Alok K. Singh
Abstract
The agricultural industry has serious economic concerns globally because soil-
borne plant diseases can result in catastrophic losses in crop yields, both in terms
of quantity and quality. If a suitable and precise management approach is to be
optimized, early, quick, and reliable pathogen identification is crucial. Histori-
cally, the most popular techniques for diagnosing plant diseases have relied on
labour-intensive, time-consuming colony-based morphological approaches. For
precise disease diagnosis and detection, technologies based on nucleic acids are
now often utilized. Innovative molecular tools for pathogen detection and differ-
entiation have been made possible by current developments in standard and
variable PCR methods, including nested, quantitative, magnetic capture
hybridization (MCH); multiplex, biological, post, and isothermal amplification;
development of DNA and RNA-based probes; and next-generation sequencing
(NGS). These nucleic acid-based detection techniques are used to identify symp-
tomatic and asymptomatic infections caused by culturable and non-culturable
fungal pathogens. Even though molecular diagnostic methods have made
K. Jayalakshmi · N. Sharath
ICAR-Directorate of Onion Garlic Research, Pune, Maharashtra, India
N. Manzar (✉) · A. S. Kashyap · P. K. Sahu · Y. Nagaraju · A. K. Singh
ICAR-National Bureau of Agriculturally important Microorganisms, Mau, Uttar Pradesh, India
B. K. Namriboi
Department of Plant Pathology, GBPUA & T, Pantnagar, Uttarakhand, India
R. Saranya
ICAR-Central Arid Zone Research Station, Jaisalmer, Rajasthan, India
A. Gupta
ICAR-Indian Institute of Seed Sciences, Mau, Uttar Pradesh, India
#The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2023
U. B. Singh et al. (eds.), Detection, Diagnosis and Management of Soil-borne
Phytopathogens,https://doi.org/10.1007/978-981-19-8307-8_5
significant strides recently, there is still more to be done regarding their develop-
ment and use in plant diseases. Molecular methods that are more consistent,
efficient, and user-friendly than conventional methods are needed for soil-borne
pathogen diagnosis. These approaches have high significance because of their
unique specificity in separating related species at various taxonomic levels.
Scientists are currently working on the problem of creating efficient tools for
plant disease molecular diagnostics. This chapter talks about current
developments in the creation and application of molecular methods to detect
several soil-borne plant diseases.
102 K. Jayalakshmi et al.
Keywords
Soil-borne plant pathogens · Diagnosis · Molecular identification · Polymerase
chain reaction · Next-generation sequencing
5.1 Introduction
Healthy crops are crucial for food quality and life in sustainable farming. In reality, a
problem is “detected”by objectively observing the symptoms it causes, but a
problem is “diagnosed”by identifying the particular pathological condition causing
it. The foundation for a healthy crop, aid in risk management, and ensure safety
during agricultural production are diagnosing or quickly identifying plant pests and
diseases.
A rising concern is that the biggest threat to international agriculture is soil-borne
diseases (Singh et al. 2018; Kashyap et al. 2021). Agriculture today makes up around
half of the land that is livable worldwide. In previous centuries, as the human
population grew, the area covered by crops progressively rose. Rice, wheat, and
maize were cultivated on an estimated 540 million hectares worldwide, according to
McDonald and Stukenbrock (2016), and plant diseases can significantly lower crop
yield. Similar to this, diseases and pests connected with maize, rice, wheat, potatoes,
greengram and soybean generate yearly losses of between 17 and 30 per cent
worldwide (Manzar et al. 2022a; Kashyap et al. 2022a; Manzar et al. 2021; Reznikov
et al. 2018; Savary et al. 2019).
Food security and safety are provided through systematic crop disease control in
agriculture, which is essential for the expanding world population (FAO 2018;
Sarrocco and Vannacci 2018). Most of the harm is caused by the unintentional
spread of invasive alien disease species into new locations due to international
commerce and transportation (Ghelardini et al. 2017). In the Great Irish famine
(caused to the late potato blight (Phytophthora infestans)) of Britain and Ireland
(between 1845 and 1849), nearly one million people lost their lives (Cox and Large
1960). Recently, Pyricularia graminis f. sp. tritici, a blast disease that originated in
South America, damaged more than 15,000 acres of wheat in Bangladesh (Callaway
2016). Xylella fastidiosa, a xylem-inhabiting plant pathogen, severely damaged
olive trees in Italy since 2013. According to genetic research, Italian strains were
comparable to Central American isolates (Marcelletti and Scortichini 2016;
Giampetruzzi et al. 2017). The planting material (white pine blister) exchange
transferred the pathogen Cronartium ribicola from Europe to Northern America.
At the same time, the subspecies Americana of the Dutch elm disease fungus
Ophiostoma novo-ulmi arrived with rock elm logs in Europe from North America
(Ghelardini et al. 2017).
5 Detection and Diagnosis of Important Soil-Borne Pathogens 103
Climate changeinfluences plant-pathogen interactions, viz. the spread of diseases
in agriculture can be attributed to increased temperatures, climatic extremities, and
changes in yearly precipitation patterns. Most of the cultivating soil is also planted
with monocultures or even just one genotype, creating a genetically homogeneous
environment that makes it simple for host-specific crop diseases to spread
(Schmidhuber and Tubiello 2007). Developing quick, effective, affordable tools
for early pathogen identification and control is essential. Early disease identification
is important since using chemicals or biological agents to cure a significant disease
incidence with increased severity and incidence harms the environment and natural
ecology (Padaria et al. 2016; Sharma et al. 2017). Using the resistant germplasm as
the first line of defence is the most efficient strategy to combat plant diseases
(Sharma et al. 2010). However, integrated disease management systems rely heavily
on the availability of quick, accurate, and focused disease detection technologies
without resistant strains (Tarafdar et al. 2018).
Plant pathogen detection and identification, such as commencing sampling and
reaction inhibition, provide several challenges. The development of sensitive and
targeted molecular techniques has transformed the identification of soil-borne
pathogens in recent years. All practising plant pathologists will soon be exposed to
the quick and exciting changes in diagnosis. The immunological and nucleic acid-
based tests, in particular, are currently accessible for various bacteria. While con-
ventional methods like baiting, culturing, and microscopic observations are still in
use and serve as the backbone of plant pathologists, molecular techniques are readily
accessible. The most important approaches for novel molecular methods to identify
soil-borne diseases and their utility in agriculture are covered in this chapter. In
addition to its other benefits, such as facilitating a quicker diagnosis without the need
of a diagnostic laboratory, on-site diagnosis can aid in early illness assessment in
domains depending on their relevance.
5.2 Major Plant Pathogens Causing Soil-Borne Diseases
Numerous soil-borne plant diseases have been identified, and high disease-
suppression soils have been found. Even in the presence of pathogen inoculum
and favourable conditions for the development of illness, disease growth is restricted
in these soils. Even while the fundamental processes at work in these soils aren’t
fully understood and are known to differ according to the pathosystem, it is assumed
that the suppressive effect is complex in nature, coming from a combination of
general and specific suppression.
104 K. Jayalakshmi et al.
Numerous crop species are adversely affected by the diversity of soil-borne
diseases, including root, vascular, and seed rot, which can be caused by fungi,
bacteria, phytoplasmas, viruses, protozoa, and nematodes. Frequent severe crop
destruction results in significant annual economic losses. It might be challenging
to see soil-borne bacteria with the naked eye. They are tiny, relying on the biotic and
abiotic components of the soil to exist, and complete their life cycle in the soil. The
principal soil-borne fungi—Phytophthora,Rhizoctonia,Fusarium,Pythium,
Verticillium, and Armillaria—infect roots, resulting in root rot, wilt, yellowing,
stunting, and dieback, which eventually cause the death of the plants. Armillaria
and Rhizoctonia induce root rot, Verticillium and Fusarium cause wilt, and
Phytophthora causes late blight (Armillaria is a honey mushroom that produces
brackets or flowers at the base of a tree). Compared to fungus, bacteria are a less
common kind of soil-borne disease. Erwinia,Rhizomonas,Ralstonia and Strepto-
myces are a few soil-borne bacterial pathogens that cause the diseases soft rot, corky
root, bacterial wilt and scab (Kashyap et al. 2022a). Viral infections seldom spread
through the soil because they need live plant tissue, although they have been seen to
move on fungus or nematodes and enter through water. Soil-dwelling creatures
called plant pathogenic nematodes mostly affect roots. They cause branching and
swelling by feeding on the roots’terminals.
5.3 Traditional Methods for Soil-Borne Pathogen Detection
Isolation and cultivating, reinoculation, microscopic techniques, and biochemical
testing in the laboratory are conventional/traditional ways of diagnosing soil-borne
pathogens. These procedures have tremendous utility since they are reasonably
priced and not technically difficult. They need a high level of competence in
interpretation and analysis, are time-consuming, and are usually slow. Additionally,
taxonomy and fungal plant pathology knowledge and skills are required. Conidia,
sclerotia, or mycelia, and symptoms that develop after infection, have historically
served as the foundation for diagnosing or identifying a fungal condition. This
disease diagnosis is usually cumbersome and impractical when quick results are
sought (Sharma et al. 2015). They are not suitable for quick diagnosis or large-scale
sample analysis, and producers must rely on specialist diagnostic facilities because
they are not easily accessible.
Furthermore, correct identification needs the assistance of trained and experi-
enced people because eye inspection is usually inadequate. Making timely disease
management decisions may be more challenging due to the chance that the pathogen
would remain dormant in plant tissue (Tarafdar et al. 2013; Tarafdar et al. 2013). It
can be difficult to differentiate between many plant diseases due to their physical
resemblance. Examples are the Macrophomina phaseolina and the Phoma species
(Somai et al. 2002). A thorough understanding of taxonomy is required for determi-
nation. Identifying various populations of the same pathogen with diverse features,
such as toxin production, fungicide resistance, or variations in virulence, can some-
times be getting difficult. For a high number of samples, this approach proved
inadequate. Additionally, quarantining pathogens to lower the danger of illness and
the spread of the inoculum necessitates using exact, quick detection techniques.
5 Detection and Diagnosis of Important Soil-Borne Pathogens 105
5.4 Immunological/Serological Detection of Soil-Borne
Pathogens
Immunological methods’underlying notion of antigen-antibody interaction has
many drawbacks, including low test sensitivity and affinity and the possibility of
contamination. Due to advancements over the past 10 years, it is now possible to
detect and quantify several hazardous species using immunological approaches,
including nematodes and mycoplasmas. For more than 20 years, immunological
techniques have been researched. Furthermore, fungus’s high inconsistency and
phenotypic serological flexibility have rendered plant disease detection ineffective
(Luchi et al. 2020; Meng and Doyle 2002). Applying and developing cutting-edge
and efficient diagnostic procedures to prevent fungal plant disease is essential. As a
result, molecular approaches that make it easier to identify and quantify pathogens
are being used to diagnose soil-borne infections. The drawbacks of traditional and
serological diagnostic techniques can be overcome by molecular testing.
Beginning in the 1970s, the use of antibodies in serological detection systems for
the rapid and precise diagnosis of diseases accelerated with the advent of monoclo-
nal antibody technology. Soil-borne bacteria can be discovered if bacterial antigens
are used to generate antibodies. These methods were used as laborious analytical
instruments. This requires using specific antibodies to find the matching antigens in
test samples. Each antibody has a distinct antigen-specific binding site. Monoclonal
antibodies, which may be produced indefinitely and are highly specific when utilized
in immunological testing, allow for identification at the genus, species, and isolate
levels (Hardham et al. 1994).
Serological diagnostic methods provide several advantages. Antibodies may take
weeks to produce, but if properly kept, they are frequently stable for a long period
and produce effects quickly. They have not yet been fully utilized in diagnosing
plant diseases other than viruses and bacteria, although they offer a wide range of
applications for the general and accurate detection of unique epitopes of numerous
soil-borne microorganisms. Tests for antibodies have significantly improved. They
can now distinguish between strains within a species, are nanogram sensitive, and
take less time to conduct in lab and field settings. Second, diagnosis depends only on
a structural element of the organism, such as the coat protein, which offers very little
information about the virus.
Thirdly, serology is only useful when an antigen that can be used to create an
antiserum is accessible or when the antiserum is ready. Finally, serology is worthless
for identifying as-yet-unidentified soil-borne diseases. The capacity to recognize
IgM or rising antibody titres determines how a serological diagnostic is organized.
Serological methods are used to diagnose the majority of prevalent bacterial illnesses
that are transmitted through soil. The antibody-antigen combination may be used in
various ways due to its endurance. The enzyme-linked immune sorbent assay
(ELISA), which comes in various formats and offers numerous endpoint detection
choices, is the most significant. The ELISA can measure a pathogen’s presence and
offer proof of it.
106 K. Jayalakshmi et al.
5.5 Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is a different method that uses the studied antibody colour change to
determine the presence of soil-borne pathogens. The target epitopes (antigens)
from viruses, bacteria, and fungi are accurately bound using this method by
antibodies bound to an enzyme. The interaction between the substrate and the
immobilized enzyme causes colour changes, which may be used to identify
substances. Specific monoclonal and recombinant antibodies easily available on
the market can greatly improve ELISA performance. Specific monoclonal antibodies
have been used in ELISA to achieve lower detection limits in the region of
105–106 CFU/mL. For the on-site detection of plant diseases, tissue print-ELISA
and lateral flow devices have been developed. Although it cannot be used to
diagnose infections early on before symptoms appear because the sensitivity for
bacteria is so low (105–106 CFU/mL), it may be used to confirm plant illnesses once
visible signs appear. The ELISA tests can be classified as a direct, indirect, sand-
wich, or competitive ELISAs depending on the antigen-antibody combination.
5.5.1 Direct ELISA
A target protein (or a target antibody) placed on the surface of microplate wells is
treated with an enzyme-labelled target antibody (or a specific antigen to the target
antibody). The activity of the microplate well-bound enzyme is evaluated after
washing.
5.5.2 Indirect ELISA
The primary antibody is treated with a target protein immobilized on the surface of
microplate wells before being incubated with a secondary antibody against it. After
washing, the activity of the microplate well-bound enzyme is measured. Even
though indirect ELISA requires more steps than direct ELISA, the primary antibody
does not need to be labelled because labelled secondary antibodies are commercially
available.
5.5.3 Sandwich ELISA
A second antibody that is also specific to the target protein but has been enzyme-
labelled is used to treat a target protein-specific antibody placed on the surface of
microplate wells. The activity of the microplate well-bound enzyme is evaluated
after washing.
5 Detection and Diagnosis of Important Soil-Borne Pathogens 107
The enzyme-labelled antibody (green) and the immobilized antibody must iden-
tify various target protein epitopes (orange). Sandwich ELISA is more selective than
direct ELISA because it combines antibodies to two different epitopes on the target
protein. Sandwich ELISA is beneficial when extreme accuracy is needed.
5.5.4 Competitive ELISA
An antibody that is specific for the target protein and has been immobilized on the
surface of microplate wells is used to treat samples that contain the protein and a
known amount of the target protein. The activity of the microplate well-bound
enzyme is measured after the procedure. The sample will seem lighter when there
are less antibody-bound enzyme-labelled antigens present. When it is low, on the
other hand, more enzyme-labelled antigen is bound to antibodies, which results in a
deeper colour. When the target antigen in a sandwich ELISA test is a small molecule
like dioxin, histamine, or a pesticide, two antibodies cannot attach to it simulta-
neously. Competitive ELISA may be used to measure low molecular weight targets.
5.5.5 Phage Display
Phage display-based antibody engineering has the potential to revolutionize the
production of antibodies by making the process faster and more affordable than
current monoclonal antibody techniques (Mitchell et al. 1997; Wilson and Finlay
1998; Aujame et al. 1997). To produce foreign proteins (antibodies) as fusions to
phage coat proteins, cloning sites that have been introduced to filamentous phage
vectors are used in this technique. Before being chosen for certain proteins with
particular binding capabilities, Escherichia coli cells are transformed with phage
libraries and cultured in culture. The technique has been used for diagnosing plant
diseases and general plant biology. For example, Ralstonia solanacearum Race
3 and Black Currant Reversion Associated Virus have been detected using phage
display to create particular antibody fragments that can be used in ELISA (Griep
et al. 1998). Due to the ability to manufacture specific antibodies in large quantities
without the need of expensive hybridoma technology or test animals, antibodies will
soon be available at a greatly reduced cost.
5.6 Lateral Flow Devices
The lateral flow device is one of the most extensively used diagnostic tools available
to farmers today (LFD). These devices are simple to use and swiftly generate
results—typically in less than 10 min. The LFDs that can be purchased commercially
to identify viral infections in plants are the most beneficial. As little as 3 ng mL
-1
of
antigen may be detected by an LFD-based test for Rhizoctonia solani, which is
equivalent to the sensitivity of conventional ELISA methods (Thornton 2008). In
contrast to the plant viruses and bacterial pathogens that are typically the objectives
of commercial LFD-based tests and for which specific antibodies are frequently
available, this work focused on a soil-borne plant pathogenic fungus. The develop-
ment of species-specific antibodies against fungi has proven to be more difficult;
however as was already said, some targets have achieved success.
108 K. Jayalakshmi et al.
5.7 Biochemical Methods for Soil-Borne Pathogen Detection
Biochemical traits specific to each creature can be used to identify it. On one end of
the scale, certain qualities are shared by large populations while, on the other, some
are exclusive to individual populations within the species. In order to determine the
taxonomic rank at which an organism is categorized, it is essential to characterize the
pathogen. Embracing gel electrophoresis for soluble protein analysis are bacteria and
fungi. It is crucial to standardize these procedures since gene expression is a
characteristic of all of them and may be affected by environmental factors.
Similar to this, Pernezny et al. (1995) used substrate to pinpoint Xanthomonas
campestris as the bacterial species in charge of a serious outbreak of bacterial spots
in Florida lettuce crops; the pathovar presence was determined to be vitians by its
fatty acid composition. In some situations, the creation of unusual metabolites by an
organism can be utilized to identify it.
For instance, identifying Aspergillus flavus strains capable of making aflatoxin
was aided by synthesizing volatile C15H24 compounds, including alpha-gurjunene,
trans-caryophyllene, and cadinene. Non-toxic strains did not create these chemicals
(Zeringue et al. 1993). When identifying bacterial plant pathogens using fatty acid
profiles (FAME Analysis), the bacterium is often grown in pure cultures first. Wet
cells are methylated and saponified to around 40 mg. By using an ether-hexane
combination to extract the fatty acid methyl esters (FAME), gas chromatography is
used to examine the results.
Because the fatty acid profiles of the field-collected strains most closely mirrored
that of this pathovar, Xanthomonas campestris pv. vitians was discovered to be the
pathogen that produced an outbreak of a bacterial spot on lettuce (Pernezny et al.
1995). The four species of the Erwinia herbicola group and the five species of the
Erwinia amylovora group could be distinguished in more detailed research by Wells
et al. (1994). When electrophoresizing, soluble proteins from plant diseases usually
produce intricate patterns that can be used for identification. Proper staining methods
may be able to disclose a particular protein dye, which, for example, may include
enzyme activity, rather than utilizing a broad protein stain like Coomassie Blue.
4250 Australian isolates of Rhizoctonia solani were divided into 10 groups, termed
zymograms, by MacNish et al. (1994), who stained for pectic enzymes.
5 Detection and Diagnosis of Important Soil-Borne Pathogens 109
5.8 Molecular Methods for Soil-Borne Pathogen Detection
Many experts agree that nucleic acid (NA)-based methods are among the best for
finding soil-borne plant infections. More contemporary methods, including immu-
nological methods, DNA/RNA probe technologies, and polymerase chain reaction
(PCR) amplification of nucleic acid sequences, are increasingly being used to
identify plant diseases (Manzar et al. 2022a). These techniques have a number of
benefits over traditional diagnostic techniques, including the fact that they are more
accurate, faster, and easier to use without specialized taxonomic expertise. More
significantly, these techniques make it possible to identify bacteria that cannot be
grown. Furthermore, molecular identification techniques aid in the discovery of new
diseases with unidentified aetiologies. These instruments might be employed to
accurately gauge the biomass of infections and confirm their presence (Biswas
et al. 2012a, 2012b; Sharma et al. 2012b).
5.9 Nucleic Acid-Based Detection Techniques for Soil-Borne
Pathogens
Most NA-based detection techniques, particularly those that employ PCR, are rapid,
specialized, and sensitive. This provides a more robust diagnosis. While molecular
testing verifies the diagnosis for other diseases or determines whether litigation is
feasible, traditional procedures are helpful for different conditions. It is challenging
to separate pathogens taxonomically because many plant pathologists cannot swiftly
differentiate important disease taxa like Pythium or Phytophthora by visual inspec-
tion. To help create a genome database, various bacteria, even nonsporulating ones,
can be awarded species I.D.s as sequencing expertise increases.
Diagnosticians and other applied plant pathologists are mainly situated to
increase the genetic library for plant diseases due to their exposure to various
conditions on diverse hosts. Sequencing the ITS or mitochondrial genes may be
helpful since it provides a DNA fingerprint for many plant illnesses. Many of these
diseases must be cultivated before being detected. The study of this area may easily
recognize these sequences. Massive sequencing technology advancements have
profoundly influenced genomic research and considerably increased the throughput
of cost-effective sequences. The pyrosequencing method of DNA sequencing is built
on the sequencing-by-synthesis methodology. The management of fungal plant
diseases currently does not make extensive use of pyrosequencing technologies.
5.10 Polymerase Chain Reaction (PCR)
For developing monoclonal antibodies and using the polymerase chain reaction to
amplify nucleic acid sequences, J.F. Kohler, C. Milstein, and K. Mullis were
awarded two Nobel Prizes in 1984 and 1993 (PCR). A thermostable DNA polymer-
ase catalyses an exponential amplification of a target DNA strand in the polymerase
chain reaction (PCR), the mainstay of NA-based disease detection. This valuable and
inexpensive molecular method can duplicate or amplify tiny fragments of DNA or
RNA. By connecting two synthetic oligonucleotides, or “primers”, to the target
genomic sequence and extending them using a Taq polymerase, this in vitro ampli-
fication technique amplifies a single copy of the nucleic acid target (a thermostable
DNA polymerase). Because of the DNA hybridization and replication fidelity, PCR
was initially used to detect illnesses caused by bacteria and viruses. These days, both
plant illnesses and diseases transmitted through the soil are frequently identified
using it. Due to its exceptional sensitivity, advanced PCR methods, such as reverse-
transcription PCR (RT-PCR), have also been used in addition to traditional PCR
technology for the identification of plant pathogens. The many PCR types used in
pathogen detection are described in the section below.
110 K. Jayalakshmi et al.
5.11 Random Amplified Polymorphic DNA (RAPD)
The Random Amplified Polymorphic DNA (RAPD) technique is a simple, rapid,
and inexpensive way to amplify a tiny amount of total genomic DNA at low
annealing temperatures. It uses short synthesized oligonucleotides of random
sequences as primers. A somewhat unique profile pattern is visible when the ensuing
PCR product is resolved. As a result, RAPD markers have established themselves as
useful tools for studying the genetics of fungal populations (Nasir and Hoppe 1991).
This marker makes it possible to detect even the smallest DNA changes in the
organism. For molecular taxonomy, genomic mapping, and evolutionary studies,
several fungal species have been identified using RAPD (Nasir and Hoppe 1991). By
examining DNA products created by RAPD, it has been possible to learn about the
variation and segregation of genetic traits among strains.
5.12 Restriction Fragment Length Polymorphism (RFLP)
The phylogenetic separation, description, and categorization of soil-borne illnesses
is made possible by nuclear ribosomal DNA (rDNA) amplified using restriction
fragment length polymorphism (RFLP) (RFLP). Restriction fragment length
polymorphisms in DNA encoding specific genes can be used to identify the species
of a pathogen. This method of identifying a species depends on having a good
database on the variability in fragment length polymorphisms that may be found
among isolates of individual species because conspecific isolates may differ in the
presence or absence of specific restriction sites, changing the RFLP banding.
As an illustration, Camele et al. (2005) employed thorough RFLP of
PCR-amplified rDNA to identify and separate 10 Phytophthora species infecting
different crops, enabling selective identification of these Phytophthora spp. The
restriction patterns of 27 other Phytophthora species were identified and used to
amplify and further digest the amplicons generated by PCR using Phytophthora-
specific primers (Drenth et al. 2006,2006). Following analysis of the ITS region
using PCR-RFLP, several anastomosis groups were discovered in Rhizoctonia
solani isolates (Pannecoucque and Hofte 2009). The ability to discriminate between
pathogenic and non-pathogenic Pythium myriotolum strains was also made feasible
(Gómez-Alpízar et al. 2011). Sharma et al. have identified the genetic diversity in
populations of M. phaseolina, a PCR-amplified rDNA-targeting microbe isolated
from chickpea (2012a).
5 Detection and Diagnosis of Important Soil-Borne Pathogens 111
5.13 Amplified Fragment Length Polymorphism (AFLP)
A PCR-based tool and variation of the RFLP, the amplified fragment length poly-
morphism (AFLP) is used in genetic research, DNA fingerprinting, and the practice
of genetic engineering. It has been used to distinguish between different species,
although it is most usually employed to examine genotypic diversity in a population
(Gargouri et al. 2006). Infections connected to recent disease outbreaks, such as
sorghum ergot, can be traced back to their geographic source using the latter trait.
Despite being an effective diagnostic tool, AFLP analysis takes a lot of time, requires
complex technical skills, and is not suited for everyday use in diagnostic clinics.
5.13.1 Simple Sequence Repeats (SSR)
Simple sequence repeats (SSRs), often referred to as microsatellites or short tandem
repeats (STRs), are repeating patterns made up of one to six nucleotides that are
found in every eukaryotic genome. They are known for producing the best and most
precise markers, which are frequently applied in soil-borne diseases to identify
genetic changes between even among closely related species (Prospero et al.
2004). The distribution of these nucleotide units across the genome is essentially
random, and their recurrence patterns may differ from person to person. To produce
PCR products of various lengths, one can employ primers that surround such varied
locations.
Microsatellites are a common genetic marker used for DNA fingerprinting due to
their extraordinary versatility. The abundance of thousands of potentially polymor-
phic markers and a high degree of polymorphism in SSRs are advantages. SSR
markers are a reliable solution for a broad range of applications, such as genome
analysis and genetic mapping (Szabo and Kolmer 2007). Microsatellite markers
exclusive to the Phytophthora ramorum pathogen were used in the additional study
to discriminate between the A1 and A2 mating types of isolates from this disease that
originated in two distinct countries.
5.13.2 Multiplex PCR
Using a single reaction mixture and many primer pairs, the multiplex PCR test
enables the simultaneous amplification of numerous pathogens (Sint et al. 2012).
The generated amplicons can then be separated and shown using electrophoresis.
The multiplex test requires the creation of primers, and specific sets of primers
should have equivalent annealing temperatures for effective amplification. It makes
it possible to accurately and simultaneously detect several DNA or RNA targets
using a single procedure. It is advantageous in plant pathology because sensitive
detection is necessary to produce pathogen-free plant material, and different soil-
borne pathogens frequently infect a single host. Wheat (Sun et al. 2018),strawberries
(Li et al. 2011), and turfgrass are a few examples of hosts where several infections
can be found at the same time in a single multiplex PCR test (Asano et al. 2010).
112 K. Jayalakshmi et al.
5.13.3 Real-time PCR
Real-time PCR, which is based on the nucleic acids of bacteria, fungi, and viruses, is
used to rapidly identify plant illnesses. The important component in managing plant
diseases is detection and pathogen quantification (Le Floch et al. 2007; Lees et al.
2002). Real-time PCR has significantly improved pathogen identification and quan-
tification, while quantification based on culture techniques is frequently considered
inaccurate and unreliable (Tarafdar et al. 2018). Real-time PCR differs from end-
point PCR in that each PCR cycle includes a measurement of the amplified PCR
product. Since the exponential phase of the reaction is being monitored as it
progresses, real-time PCR allows for accurate template quantification. Real-time
PCR is gaining popularity for identifying and quantifying a variety of pathogenic
fungus, oomycetes, bacteria, nematodes, viruses, and biocontrol agents that affect
plants. A specific increase in fluorescence during PCR amplification can be used to
identify pathogenic fungi.
5.13.4 Colony PCR
This efficient method for crude mycelium-based amplification utilizes the
ITS1–5.8S-ITS2 section of the fungal ribosomal DNA cluster. PCR generally has
a high success rate. This method ought to be widely applied to streamline molecular
taxonomic studies and enable more in-depth, sequence-based analyses of fungal
isolates. The data were directly obtained from fungal hyphae without any prior DNA
extraction or other processing. It is possible to successfully amplify DNA from the
fungus Cladosporium,Geomyces,Fusarium, and Mortierella. Yeasts discovered in
the soil may always have their DNA enhanced. Mutualistic Basidiomycota and
Ascomycota were also successfully amplified without the need for DNA extraction
from cleaned mycorrhized root tips, and Tuber melanosporum fruiting bodies could
be swiftly recognized using a direct PCR using species-specific primers (Walch et al.
2016;Bonito 2009).
5 Detection and Diagnosis of Important Soil-Borne Pathogens 113
5.13.5 Nested PCR
Nested PCR is an endpoint PCR variation that uses two sets of primer pairs for two
rounds of PCR amplification to boost specificity and sensitivity. Nesting makes it
easier to employ non-specific PCR primers for amplifying different pathogens in the
first round of PCR, followed by the use of pathogen-specific primers in the second
round. The main goals of the PCR modification were to improve sensitivity and
specificity. Two primer sets are used to carry out two successive PCR reactions,
treating the results of the first round of amplification with the same treatment in the
second round (Ni et al. 2011; Grote et al. 2002; Kamolvarin et al. 1993).
5.13.6 Bio PCR
The bio-PCR test amplifies the endpoint PCR technique, which involves a pre-assay
incubation step in a sick sample to increase the biomass of the causal agent. This
method focuses solely on the target pathogens by cultivating the target pathogen in a
growing medium that prevents the growth of non-target microorganisms to maxi-
mize detection.
5.14 DNA or RNA Probe-Based Assays
5.14.1 In Situ Hybridization
Using the in situ hybridization (ISH) technique, the mRNAs present in the fixed
sample may be identified. The main goal of this test is to design an antisense small-
scale RNA probe that will bind the target mRNA (interesting sequence). But it’s also
feasible to use cDNA probes and artificial oligonucleotide probes. Because they are
detectable and straightforward to measure for, the radioactive isotopes 35S, 125I,
and 32P are widely employed to label probes. Tyramide, bromodeoxyuridine, biotin,
digoxigenin, alkaline phosphatase, and digoxigenin can all be used to label
nonisotopic probes. Photographic, X-ray film autoradiography, liquid emulsion,
and microscopic techniques are a few examples of signal detecting techniques.
5.14.2 Fluorescent In Situ Hybridization
Fluorescent in situ hybridization (FISH) is a cutting-edge approach for the diagnosis
of plant diseases that are still relatively new. The specificity of DNA sequences is
combined with the sensitivity of fluorochrome-based detection methods (Hijri 2009;
Cui et al. 2016). Using DNA or RNA probes that are fluorescently coloured either
directly or indirectly, FISH assays may locate specific DNA or RNA sequences in
cells or tissues (Shakoori 2017). Using wide-field epifluorescence or confocal laser
scanning microscopy, stained cells from the standard FISH methods are seen when
fluorescently mono-labelled oligonucleotide probes hybridize the ribosomal RNA
(rRNA) of microbial cells (Lukumbuzya et al. 2019). The rRNA sequences of plants
that have been infected with a pathogen are specific to that pathogen. FISH can
recognize this specific information provided by RNA (Fang and Ramasamy 2015).
Southern tomato blight is brought on by the disease Sclerotium rolfsii, which can be
found in soil. The FISH technique that used an oligonucleotide probe dyed with Cy3
and Cy5 successfully identify soil smears in DNA isolation with 0.06 pg L
-1
of
S. rolfsii (Milner et al. 2019). FISH’s most vital points are repeatability, sensitivity,
specificity, precision, and speed (Bozorg-Ghalati et al. 2019). In mixed-species
specimens, it could also pinpoint the primary pathogens and offer details on resolu-
tion and morphology (Frickmann et al. 2017). A common pitfall that reduces test
specificity is false-positive results using autofluorescence materials.
114 K. Jayalakshmi et al.
5.15 Isothermal Amplification Techniques
5.15.1 Loop-Mediated Isothermal Amplification (LAMP)
Due to its outstanding efficacy, specificity, ease of use, and speed, LAMP requires
four primers, two long outside and two short inside, each recognizing six different
sequences in the target DNA. DNA synthesis will begin when the target sequence
hybridizes with the first inner primer, which comprises a sense and antisense DNA
sequences. The single-stranded DNA produced by the outer primer serves as a
template for the creation of a DNA molecule with a loop structure by the second
inner and outer primers. The term “strand-displacement DNA synthesis”refers to
this procedure. The constant cycle reaction causes products with repeated target
DNA sequences of varying lengths to accumulate.
The reaction tube is incubated at 63–65 °C in a standard water bath or heat block
in a laboratory setting to maintain a constant temperature. Unaided eyes can perceive
the amplified product as a white precipitate or a yellow-green-coloured solution after
adding SYBR green to the reaction tube. The primary benefit of LAMP is that it may
be completed rapidly and at a constant temperature. Since it uses an expedient
isothermal technique, it is ideal for plant pathogen identification at the point of
care in the field.
It also has a high amplification efficiency and sensitivity since it can generate
many PCR products from a small quantity of DNA input. Due to the assay requiring
only a few pieces of essential equipment, this process is also affordable. The
sensitivity of hybridization assays, such as LAMP-ELISA hybridization and
LAMP paired with colorimetric gold nanoparticle hybridization probes, may be
improved by using amplicons containing many inverted repeats produced by
LAMP, according to specific reports. The electrochemical sensor, in conjunction
with LAMP offered a reliable platform for pathogen detection due to its outstanding
sensitivity, which allowed it to recognize as little as ten copies of pathogen genomic
DNA. LAMP-biosensor technology has a significant potential for in-field testing,
detection, and identification of plant diseases (Tsugunori et al. 2000; Fukuta et al.
2003; Ghosh et al. 2016; Ghosh et al. 2017).
5 Detection and Diagnosis of Important Soil-Borne Pathogens 115
5.15.2 Rolling Circle Amplification
Rolling circle amplification is a widely used isothermal enzymatic assay that utilizes
DNA or RNA to diagnose plant diseases. In addition to RCA, several techniques,
like direct sequencing and RFLP, have effectively discovered and classified plant
diseases with much less time and price than conventional methods. The main
components required for this experiment are deoxynucleotide triphosphates, a circu-
lar template, a short DNA/RNA primer, and a homologous buffer. For 40 Fusarium
strains, naked eye viewing of the RCA result has been made possible by adding
fluorescent dye to the reactions (Davari et al. 2012). Ligating padlock probes with
RCA has also been shown to detect fungal infections (Najafzadeh et al. 2011). The
RCA test offers the advantages of simplicity, efficacy, and lack of temperature
cycling apparatus (Dong et al. 2013; Goo and Kim 2016). Using this method, it is
also possible to analyse gene expression, single nucleotide polymorphism, mRNA
splicing, and post-translational modifications of protein molecules (Gao et al. 2019).
5.16 DNA-Based Point-of-Care Diagnostic Methods
Diagnostic tests that can be performed at the point of care (POC) and without costly
equipment are desperately needed. Despite having several advantages over other
technologies, PCR-based methods are much less effective for POC applications
because they require energy to carry out the temperature modifications necessary
for DNA amplification. The best way to overcome this constraint uses isothermal
DNA amplification. For instance, POC detection of pathogen DNA utilizing
isothermal amplification combined with lateral flow strips and portable fluorometers
has been accomplished.
•POC—DNA Extraction methods: To successfully extract DNA from plant
tissues, it is necessary to be able to properly remove a variety of contaminants
that may otherwise interfere with the DNA amplification process. A rapid and
efficient DNA extraction method using a lateral flow device (LFD) has been
devised for POC testing and plant pathogen identification.
•In an extraction buffer, the sample is agitated with metal ball bearings before the
lysate is transferred to the release pad of an LFD nitrocellulose membrane. The
membrane is then added to the DNA amplification process using PCR or another
isothermal amplification technique after being partly removed. It is possible to do
the extraction outside since the isolated DNA is very stable on the membrane at
ambient temperature.
•Another method uses a simple dipstick composed of cellulose, which can analyse
plant samples in as little as 30 s. Plant tissues are macerated by giving them a
116 K. Jayalakshmi et al.
vigorous 8–10 s shake in a tube with extraction buffer and one or two ball
bearings. Before entering the tube containing the amplification mix, the sample
is first put in a cellulose dipstick tube and three times rinsed with wash buffer in a
separate tube. The technique works on various domesticated species, including
mature tree leaves and notoriously tricky tissues such as rice, tomato, and
sorghum (mandarin, lime, and lemon). It is compatible with a variety of amplifi-
cation methods, such as PCR, LAMP, and RPA, and it may be used to detect
pathogen DNA and RNA in tissues that have been infected.
5.17 Recent Advances in Soil-Borne Pathogen Detection
5.17.1 Ancillary Ways of Pathogen Detection
Thermography, fluorescence imaging, hyperspectral imaging, and gas chromatogra-
phy are a few techniques for indirectly identifying infections.
Thermography is a promising method for evaluating the heterogeneity in the
infection of soil-borne diseases and can record changes in the surface temperature of
plant leaves and canopies. Thermography uses thermographic cameras to record and
analyse colour variations in emitted infrared light. Plant diseases affect how much
water a plant losses when its stomata open and close (Hillnhütter et al. 2011).
Thermographic imaging shows the disease that results may be observed, and without
the effect of outside temperatures, the amount of water lost can be determined (Oerke
et al. 2006).
Another cutting-edge technique is hyperspectral imaging, which may be used to
indirectly detect plant illnesses and gather crucial information on the health of plants
over a wide spectrum of wavelengths between 350 and 2500 nm. For the diagnosis
of agricultural diseases and plant phenotyping, it is increasingly frequently utilized
in large-scale agriculture. This method allows for quick processing of imaging data
and is exceptionally accurate. Because they monitor variations in reflectance brought
on by the biophysical and metabolic impacts of infection, hyperspectral methods are
used to detect plant infections. Hyperspectral imaging methods have been used to
identify and report infections caused by Magnaporthe grisea in rice, Phytophthora
infestans in tomatoes, and Venturia inaequalis in apple trees (Delalieux et al. 2007;
Zhang et al. 2003).
5.17.2 Gas Chromatography
Identifying the volatile chemical signature of diseased plants is another non-optical
indirect way of plant disease identification. Plant pathogen infections may cause the
emission of certain volatile organic compounds (VOCs) that are highly diagnostic of
the sort of stress the plants are experiencing. When strawberries are infected with
Phytophthora cactorum, a fungus that causes crown rot, p-ethyl guaiacol and p-ethyl
phenol are released as identifiable VOCs from the damaged section of the plant/fruit.
The volatile signature of plants may be examined using gas chromatography
(GC) technology to check for a particular VOC that may indicate the presence of a
specific disease. Gas chromatography and mass spectrometry (GC-MS) are widely
employed to detect unidentified molecules in volatile samples and improve com-
pound separation and analysis effectiveness. Due to its high specificity, GC/GC-MS
can offer more accurate details on plant disease than the optical imaging-based
detection techniques listed above. The quantitative information collected from the
VOC sample shows that illnesses can be identified at various stages (Kashyap
et al. 2022a).
5 Detection and Diagnosis of Important Soil-Borne Pathogens 117
5.18 On-Site Direct Diagnosis of Plant Diseases
There are now several on-site direct diagnostic methods available. They are straight-
forward to understand and useful for farmers in making prompt decisions and early
adoption of this technology for precise disease management strategies that might
lessen the effect of plant illnesses. On-site testing can provide immediate response
without shipping the sample to an off-site laboratory if it is done “field-side”in the
farmer’s presence. Utilizing a fluorogenic probe-based test, for instance, which
entails magnetic bead-based nucleic acid extraction followed by qPCR using porta-
ble real-time PCR, Spongospora subterranean, a soil-borne disease of potatoes, may
be quickly and easily diagnosed on-site. Compared to the laboratory-based method,
the portable real-time PCR methodology can identify the pathogen with as little as
100 copies of Spongospora subterranea DNA, even when the pathogen colonization
in the host is very low. The revolutionary portable real-time PCR may be used in
place of laboratory-based methods to detect infections.
X-ray crystallography is now one of the most sophisticated techniques for
diagnosing certain diseases using a particular protein released by the pathogen or
host during contact. Using X-ray crystallography equipment at Diamond Light
Source, researchers at the Iwate Biotechnology Research Centre (Japan) found the
deadly rice blast disease Magnaporthe oryzae. The gene-for-gene paradigm was
used for the first time to identify a pathogen at the molecular level using a
crystallographic-based technique.
5.18.1 Immunofluorescence (IF)
Afluorescence microscopy-based optical method is applied to detect pathogen
infections in root tissues. Plant samples are cut into tiny tissue slices and adhered
to microscope slides for this operation. The specific antibody is detected by
conjugating a fluorescent dye to observe the distribution of the target molecule
across the sample. They are using IF, and the onion crop infection caused by Botrytis
cinerea was found. Crown rot, a novel disease in Europe, may be found using IF and
FISH together (Wullings et al. 1998). Similar to FISH, a flaw in other fluorescence-
based methods such as photobleaching results in erroneously negative
consequences. The reduction in sensitivity brought on by photobleaching may be
controlled, though, by reducing the amount of light exposure time and intensity,
increasing the concentration of fluorophores, and choosing fluorophores that are
more resistant to photobleaching.
118 K. Jayalakshmi et al.
5.18.2 Flow Cytometry (FCM)
It is a widely utilized laser-based optical technique for cell sorting, biomarker
detection, and protein modification. FCM is a unique tool for detecting plant
illnesses even though it has been used to count bacteria, distinguish between live
and non-viable bacteria, describe bacterial DNA, and examine fungal spores. It has
also been used to research antibiotic susceptibility and cell cycle dynamics.
5.18.3 Next-Generation Sequencing
Next-generation sequencing (NGS), high-throughput sequencing (HTS), and
pyrosequencing are cutting-edge diagnostic techniques that revolutionize the detec-
tion of pathogens in various plant samples. As opposed to conventional molecular
technologies, which require prior knowledge of the pathogens’sequence informa-
tion, the NGS approach is unlimited, making it possible to identify any known and
undiscovered pathogens in a single experiment. At its genomic core, phytopathogens
are a collection of soil-dwelling bacteria, and the development of NGS technology
has spawned novel methods for the detection and taxonomic identification of
phytopathogens. The organism need not be cultivated or have its past sequencing
data to apply this procedure, which takes some time but is essential for finding novel
bacteria, viruses, and viroids (only around 10% of bacteria are culturable). NGS can
quickly identify both known and unknown plant diseases. The primary steps in
DNA-based NGS include DNA isolation and fragmentation, library preparation,
massively parallel sequencing, bioinformatics analysis, variant/mutation annotation,
and interpretation. Massive parallel signature sequencing, pyrosequencing, colony
sequencing, and sequencing by oligonucleotide ligation detection (SOLID) are some
of the most frequently employed advanced sequencing methods in HTS (Rajesh and
Jaya 2017). Using RNA-sequencing, it may be possible to comprehend and study the
dynamic nature of the transcriptome (RNA-Seq). The most popular NGS platform
for RNA-Seq is the Illumina HiSeq platform, which has taken the NGS market by
storm. The most recent release for the platform was a desktop sequencer named
MiSeq (Kukurba and Montgomery 2015; Hariharan and Prasannath 2021).
When identifying early-stage infections in plants brought on by various fungal/
oomycete diseases, symptoms in the host plant are typically necessary. Several of the
abovementioned molecular and serological methods are often utilized to find these
infections. But since it may target several different pathogen loci in a plant
metagenome that is affected, next-generation sequencing (NGS) has the most poten-
tial as a diagnostic tool (Sharma et al. 2016). Finding significant eukaryotic plant
diseases with NGS has several possibilities. It may raise the fraction of NGS
readings for targets with low abundance by concentrating specific nucleic acids in
heterogeneous samples using targeted genome capture (TGC) oligonucleotide
probes. Metagenomes and the Electronic Probe Diagnostic Nucleic Acid Analysis
(EDNA) have the potential to simplify the detection of oomycete and fungal plant
diseases significantly. EDNA is more reliable than electronic probes, which simply
rely on matches between queries and metagenome data in diagnosing oomycete and
fungal plant diseases.
5 Detection and Diagnosis of Important Soil-Borne Pathogens 119
By amplifying certain DNA regions, the PCR method may identify diseases like
bacteria, viruses, and fungus. The drawback of the approach is that the search is quite
selective since one base their study on which pathogen is most likely to be present
based on certain symptoms. NGS eliminates the need for a prior decision because it
can directly identify all possible pathogens. This rapidity is a significant asset in a
sector where time is money. The same principle applies to cultivation: the longer
something is developed, the longer it takes to battle disease. All parties in the supply
chain benefit from rapid diagnostics since they may help producers, importers, and
exporters save much money. The disadvantage of this strategy is the time and effort
required to generate and assess a large number of sequences.
5.18.4 Disease Diagnostics Kits
Biotechnology has made it feasible to develop diagnostic tools which assist farmers
worldwide in managing various diseases that affect their crops. Thanks to improved
diagnostic techniques that take up less processing time, infections may be identified
with greater precision. The fast identification of DNA or proteins particular to each
disease, ailment, or condition is how these diagnostics function. A qualified person
must use the tools and procedures. Diagnostic kits offer a large selection of ELISA
kits for plant pathogen detections with good test performance characteristics for the
precise, quick, simple, and high-throughput identification of the organisms that
cause plant disease. Compared to conventional diagnostic procedures and
PCR-based approaches, immunological techniques based on ELISA kits provide
several benefits. A range of ELISA-based rapid test strips with obvious colour
change indicators is now readily available due to the usage of lateral flow devices
(LFD), which are designed for on-site, accurate, and quick diagnosis of plant
diseases by untrained workers.
5.19 ELISA (Enzyme-Linked Immunosorbent Assay) Kits
The ability of an antibody to recognize a particular protein fragment or antigen
linked to a plant pathogen is the basis for ELISA kits. The kits are simple to use and
take around 5 min to measure sickness in the field. Additionally, they don’t require
specialized knowledge or pricey laboratory equipment. Several ELISA test kits are
available; infections are already caused by pathogens such Erwinia amylovora,
Ralstonia solanacearum,Phytophthora sp., etc., in grains, root crops, ornamentals,
fruits, and vegetables.
120 K. Jayalakshmi et al.
5.19.1 Direct Tissue Blotting
Additionally, this approach searches for plant pathogens using specific antibodies.
Before introducing antibodies, samples of the diseased tissue are pressed onto
specialized paper to be tested for protein content. The antibody-pathogen combina-
tion is then exposed to a dye-inducing reagent for reaction. The colour reaction
shows a positive result and the presence of the pathogen in the affected tissue.
5.19.2 DNA/RNA Probes
An additional set of tools that may be used to identify plant diseases are nucleic acid
(DNA/RNA) probes. These probes are nucleic acid fragments arranged like the
DNA or RNA of the pathogen. Since the sequences complement one another, the
probes may be utilized to identify specific diseases (Goodwin et al. 1989).
5.19.3 Squash Blot Method
The squash blot method uses a specific type of paper called a membrane to “squash”
plant tissue from a plant that is suspected of having a disease. A probe that can
interact with the DNA or RNA of the plant pathogen alleged to be present in the
tissue is then applied to this membrane. The binding will occur when there exist
complementary sequences. A colour reaction demonstrates the existence of the
disease after adding several more chemicals to the membrane, which indicates that
the probe and the pathogen DNA/RNA have formed a bond. Lack of a colour
reaction means a bad result or the absence of sickness.
5.19.4 Use of Pocket Diagnostic Rapid Test Strips for Plant Diseases
Different lateral flow rapid test strips identify various plant pathogens. After break-
ing it up into small bits, place the sample in the container with the buffer and ball
bearings. Shake the sample in the liquid for about a minute to break it up. While
drawing liquid into the pipette, watch out for sample debris and air bubbles. To
acquire reliable findings in less than 10 min, add 2 drops to the sample well of the
testing device while maintaining levelness.
5 Detection and Diagnosis of Important Soil-Borne Pathogens 121
5.19.5 PCRD-Nucleic Acid Detection
The traditional method for confirming the presence of nucleic acid following DNA
amplification in PCR is DNA agarose gel electrophoresis. PCRD offers a rapid and
simple alternative to gel electrophoresis that may be finished in minutes without
requiring expensive equipment, exposure to intercalating dye, or UV radiation.
PCRD is a nucleic acid lateral flow immunoassay (NALFIA), which may be used
in conjunction with PCR, loop-mediated isothermal amplification (LAMP),
recombinase polymerase amplification (RPA), or helicase-dependent amplification
(HDA). The PCRD format may be used by large throughput laboratories and small
field-based laboratories.
5.19.6 Diagnostic Kits’Advantages
Quick tests that may be performed in the field in a matter of minutes allow for
making judgements on the spot, which is favourable for yield since it enables the
implementation of management measures earlier than if a sample were sent to the
lab. A rapid test can lower the cost per sample since fewer samples must be sent to
the lab, reducing the cost per sample.
5.20 Conclusions
It is now feasible to quickly and precisely identify the major genera and species of
disease-causing organisms by combining contemporary, sophisticated immunologi-
cal, and nucleic acid-based methods. Due to their high sensitivity and accuracy,
monoclonal antibodies and PCR-based techniques can potentially displace current
technologies. Thanks to NA-based methodologies, often regarded as fast pathogen
detection tests, an increasing range of strategies are now accessible for addressing
disease challenges that are of relevance in applied plant pathology programmes.
Molecular processes may be put to use right now to advance our lab’s technical
capabilities and get ready for any threats. Given that these techniques are a bit
challenging and time-consuming for data analysis, they must be carried out by
qualified specialists.
Additionally, since the majority of these approaches do not give real-time detec-
tion, early warning systems and in-field testing are less suitable for them. Any
pathogen detection methodology’s limitations must be understood for optimal
implementation, and NA-based procedures are no exception. Utilizing the right
parameters is crucial when using NA-based tools to assure accuracy. Understanding
the reliability of customary laboratory techniques and the need to accumulate several
lines of evidence is also necessary for critically using such technologies. Modern,
cutting-edge techniques have reproducible sensitivity and are frequently noticeably
quicker than traditional techniques. Prompt assessment of fungal resistance levels
may also help in the creation of successful resistance management techniques.
However, there is still a glaring knowledge deficit in this field of research since no
single technique can satisfy the growing need for speedier, more efficient, reproduc-
ible, and sensitive results.
122 K. Jayalakshmi et al.
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