This presentation deals with modern (Serological, Molecular and other latest) detection techniques for seed borne fungal pathogens.

1. MODERN TECHNIQUES FOR
DETECTION OF SEED BORNE
FUNGAL PATHOGENS
Submitted to: Dr Narender Kumar Bharat
Submitted by: Paranjay Rohiwala
Admission no.: H-2019-40-D

2. INTRODUCTION
 Seed-borne pathogens possess a serious threat to seedling
establishment.
 Close association of the pathogens with seeds facilitates their
long-term survival, introduction into new areas and widespread
dissemination.
 Under such conditions, elimination is the most effective disease
management strategy accomplished by using seed detection
assay.
 Transboundary spread of pathogens is a major concern today.
 Precise detection methods are essential for seed-borne pathogens
to support seed health strategies.
 The considerable advancement in molecular biology has
facilitated rapid identification/detection of seed-borne pathogens.

3.  Methods of detection have to fulfil six main requirements:
 (i) Specificity – the ability to distinguish a particular target
organism from others occurring on tested seeds.
 (ii) Sensitivity – the ability to detect organisms at low
incidence in seed stocks.
 (iii) Speed – less time requirements, to enable prompt
action against the target pathogen(s).

4.  (iv) Simplicity – minimization of a number of
examination stages to reduce error and enable
testing by a staff not necessarily highly qualified.
 (v) Cost-effectiveness – costs should determine
acceptance to the test.
 (vi) Reliability – methods must be sufficiently
robust to provide repeatable results within and
between samples of the same stock regardless who
performs the test.

5. WHY DETECTION OF SEED-BORNE FUNGAL
PATHOGENS IS IMPORTANT?
A. Seed-borne fungal pathogens-serious threat to seedling
establishment and hence may lead to crop failure.
B. Seeds -long-term survival of pathogens and vehicle for
their introduction into newer areas and their widespread
dissemination.
C. May cause catastrophic losses to food crops and hence
directly linked to the food security.
D. Unlike infected vegetative plant tissues, infested seeds
can be asymptomatic, making visual detection
impossible.
E. Additionally, fungal pathogen’s populations on seeds
may be low, and the infested seeds may be non-
uniformly distributed within a lot.

6. MODERN DETECTION METHODS FOR
SEED-BORNE FUNGAL PATHOGENS

7. Serology-based assays
•Serological seed assays rely on antibodies (polyclonal or monoclonal)
generated against unique antigens on the surfaces of plant pathogens
(Hampton et al., 1990).
•Antibodies bind strongly and specifically to their antigens and can
subsequently be detected by the enzymatic digestion of substrates or
fluorescent tags.
•Serology based seed tests have several formats including the widely
applied enzyme linked immunosorbent assay (ELISA) (McLaughlin and
Chen, 1990) and immuno fluorescence microscopy (Franken, 1992).
Advantages:
Serological assays do not require pure isolations of the pathogen and, hence, are
applicable to biotrophic and necrotrophic seedborne pathogens.

8. ELISA has been used for detection of :
Barley seeds using a polyclonal antibody (PAb) raised against Penicillium
aurantiogriseum var. melanoconidium in indirect ELISA test.
Rice and corn seeds colonizing fungi, viz. Aspergillus parasiticus, Penicillium
citrinum and Fusarium oxysporum, were detected by employing double antibody
sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test.
Karnal bunt disease of wheat caused by Tilletia indica detected using SDS-PAGE
analysis (Kutilek et al. 2001).
Ustilago nuda causes loose smut in barley and it is an internally seed-borne
pathogen. Eibel et al. (2005b) employed a DAS-ELISA test with biotinylated
detection antibodies to detect loose smut pathogen in naturally infected barley
seeds.
Phomopsis longicolla causing Phomopsis seed decay of soybean detected using
indirect ELISA and a modified immunoblot assay, named as seed immunoblot
assay (SIBA),(Gleason et al. 1987).

9. Types of ELISA
1) Direct ELISA
• A direct ELISA is a plate-based immunosorbent assay.
• For the detection and quantification of a specific analyte
(e.g. antigens, antibodies, proteins, hormones, peptides,
etc.) from within a complex biological sample.
• Simplest and quickest to perform.
• In a direct ELISA, antigen is immobilized directly onto the
surface of a multi-well microtiter plate and then
complexed with an enzyme-labeled primary antibody
specific for the antigen.

10. • Once the enzyme-labeled primary antibody binds to the
antigen, the conjugated primary antibody catalyzes a
reaction with its respective substrate
• Resulting in a visible colorimetric output that is
measured by a spectrophotometer or absorbance
microplate reader.

11. 2) Indirect ELISA
• Indirect ELISA is a two-step ELISA which involves two
binding process of primary antibody and labeled
secondary antibody.
• The primary antibody is incubated with the antigen
followed by the incubation with the secondary antibody.
• In the indirect ELISA test, the sample antibody is between
the antigen coated on the plate and an enzyme-labeled,
anti-species globulin conjugate.

12. •The addition of an enzyme substrate chromogenic reagent
causes color to develop.
•This color is directly proportional to the amount of bound
sample antibody.

13. 3) Sandwich ELISA
• The sandwich ELISA quantify antigens between two layers
of antibodies (i.e. capture and detection antibody).
• The antigen to be measured must contain at least two
antigenic epitope capable of binding to antibody, since at
least two antibodies act in the sandwich.
• Either monoclonal or polyclonal antibodies can be used as
the capture and detection antibodies in Sandwich ELISA
systems.
• Monoclonal antibodies recognize a single epitope that
allows fine detection and quantification of small differences
in antigen.

14. • A polyclonal is often used as the capture antibody to pull
down as much of the antigen as possible.
• The advantage of Sandwich ELISA is that the sample does
not have to be purified before analysis, and the assay can
be very sensitive (up to 2 to 5 times more sensitive than
direct or indirect ELISA)

15. Limitations:
1. The unavailability of species-specific antibodies.
2. The detection thresholds of serology-based assays vary
significantly based on the quality of the antibody and the testing
format.
3. Finally, with serology-based assays it is possible to detect
nonviable pathogens which results in erroneous (false positive)
interpretation.

16. Nucleic Acid-Based Detection Methods
Generally, nucleic acid-based techniques resulting in a
high level of sensitivity and specificity are used for
species-specific detection of seed-borne pathogens.
Through these techniques, very small quantities of
samples or tissues are sufficient for the detection of
pathogens in seeds of various crops.
In recent times nucleic acid-based detection methods
have become the preferred choice for detection,
identification and quantification of seed-borne fungal
pathogens.

17. In molecular detection several strategies, viz.
1. Polymerase chain reaction (PCR)
i. Bio-PCR,
ii. Immuno magnetic separation (IMS)
iii. Magnetic capture hybridization-PCR,
2. Loop-mediated isothermal amplification,
3. Rapid Cycle Real-time PCR
4. Multiplex PCR
5. DNA Chip (microarray) technology
6. DNA barcoding
7. Next Generation Sequencing (NGS)
are available for the detection and identification of pathogens which
involves propagation of putative pathogen propagules on a culture
medium and subsequent PCR on washes from the culture plates,
often using nested PCR primer pairs and sometimes without DNA
extraction.

18. 1. Polymerase chain reaction-based seed detection
assays
 Polymerase chain reaction (PCR) is the in-vitro, primer-
directed, enzymatic amplification of nucleic acids (Erlich et al.,
1988; Saiki et al., 1988). This technique has been used in many
diverse applications including diagnosis of plant diseases.
 For PCR, primers (small oligonucleotide probes) designed to
anneal to specific DNA sequences in the target organism’s
chromosomal DNA or RNA, hybridize with and direct
amplification of millions of copies of the target sequence.
 This amplified DNA can be visualized after electrophoresis in
ethidium bromide stained agarose gels.

19. As a result of this great potential, over the past 20 years, many PCR-
based assays have been reported for the identification of seedborne
pathogens;
e.g.
Ascochyta lentis in lentil seeds
Alternaria radicina in carrot seeds,
Alternaria brassicae in cruciferous seeds,
Leptosphaeria maculans in canola seeds, and
Phoma valerianella in lamb’s lettuce seeds
PCR-based methods also provide rapid and unequivocal identification
of F. oxysporum f. sp. basilici from basil seeds, unlike the
conventional detection methods that cannot distinguish between
pathogenic and non-pathogenic F. oxysporum isolates (Baayen, 2000;
Chiocchetti et al., 2001).

20. Advantages of PCR
 speed (completed within 2 to 3 h)
 specificity (DNA probes can be designed to amplify nucleic acids
from the desired genus, species, subspecies, race, etc.)
 sensitivity (single copies of nucleic acids can be detected after
amplification)
 easy and objective result interpretation (the presence of a DNA
fragment of specific size indicates the presence of the pathogen).

21. Lack of acceptance
• Many seed types contain compounds (e.g., tannins, phenolic
compounds, phenolic compounds) that inhibit DNA amplification.
• This lead to false-negative results when PCR is attempted directly
on seed extracts.
• To circumvent, elaborate DNA extraction and purification steps are
employed.
• It reduce assay sensitivity, efficiency and also require potentially
harmful chemicals e.g. chloroform and phenol.
• Finally, DNA from nonviable cells or tissues can yield false-positive
results in seed assays, so it is necessary to confirm positive results
by recovering the target organism.

22. •Management decisions based on these types of errors may result in
the unnecessary destruction of healthy seedlings and subsequent
financial losses for commercial seedling producers.
To overcome such problems, several modifications have been
developed.
These include
i. BIO PCR
ii. Nested PCR
iii. Immuno magnetic separation (IMS)
iv. Magnetic capture-hybridization (MCH)

23. i. BIO-PCR.
Seeds are infested with fungi at very low levels, and
consequently the DNA of the pathogen is not sufficient for the
subsequent reactions (De Boer et al., 1995).
To overcome this problem, Schaad et al. (1995) developed a
highly sensitive PCR technique, named BIO-PCR.
This consists of a pre-assay incubation step to increase the
biomass of the fungal pathogen on the seeds, which is then
followed by DNA extraction and amplification by PCR.
Target cell enrichment followed by PCR or BIO-PCR improves
the efficiency and sensitivity of PCR by allowing target
pathogen populations to increase in a pre enrichment phase,
before DNA extraction and PCR.

24.  Selective pre enrichment increases pathogen populations relative to non
target microorganisms and
 Results in higher quantities of target DNA, which ultimately results in
higher sensitivity.
 Additionally, during incubation and enrichment on artificial media,
inhibitory compounds are adsorbed or diluted during cell harvest, and do
not interfere with DNA amplification.
 For this technique: in the case of seedborne are incubated under
conditions of high relative humidity to increase target fungus mycelial
mass before DNA extraction and PCR (Pryor and Gilbertson, 2001).

25. BIO-PCR has been developed for the detection of black rot
(Alternaria radicina) of carrot (Daucus carota).
This technique has been reported to significantly improve
the sensitivity and the ease of implementation of PCR,
It has detection limits of 2 to 3 cfu/mL (Schaad et al., 1999).
In BIO-PCR the target organism must grow on the selective
medium before it can be detected by PCR.
Bio-PCR has been proven successful in the
detection/identification of Tilletia indica teliospores in wheat seed
samples (Schaad et al. 1997).

26. BIO-PCR has several advantages over traditional PCR:
1. Increased sensitivity,
2. Elimination of PCR inhibitors
3. Detection of viable cells only, thus avoiding false positives due to
detection of DNA from dead cells.
4. No need to identify the pathogen based on colony morphology since
specific PCR primers are used.
Disavantages
1. More expensive than conventional PCR, as selective media are used
2. The disadvantages of BIO-PCR include the need for a semi selective
medium for each pathogen. As mentioned earlier, semi selective media
require specific knowledge about the nutritional requirements and
chemical tolerances of the target organism and usually take time to
develop.
3. it usually requires from 5 to 7 days for the fungal growth, which
significantly increases the time required for completion of the assays.

27. ii. Nested PCR
 Another way to obviate the low levels of the target fungus on seeds
is the use of nested PCR.
 This procedure can improve the sensitivity and specificity of the
assay, thus allowing detection of a target DNA at several-fold lower
levels than for conventional PCR (Chiocchetti et al., 2001).
 Indeed, nested PCR has been used to detect DNA levels of 10 fg for
Colletotrichum lindemuthianum in bean seeds (Chen et al., 2007),
and of 1 fg for F. oxysporum f. sp. lactucae in lettuce seeds
(Mbofung & Pryor, 2010).
 However, this molecular assay is more labour intensive, more
costly, and more prone to contamination than conventional PCR
(McCartney et al., 2003; Atkins & Clark, 2004; Tomlinson et al.,
2005).

28.  This PCR increases the specificity of DNA amplification,
by reducing background due to non-specific amplification
of DNA.
 Two sets of primers are used in two successive PCRs.
 In the first reaction, on pair of primers “outer pair” is used
to generate DNA products, which besides the intended
target, may still consist of non-specifically amplified DNA
fragments.
 The product(s) are then used in second PCR after the
reaction is diluted with a set of second set “nested or
internal” primers whose binding sites are completely or
partially different from and located 3’ of each of the
primers used in the first reaction.

30.  Nested PCR means that two pairs of PCR primers were used
for a single locus.
 The first pair amplified the locus as seen in any PCR
experiment.
 The second pair of primers (nested primers) bind within the first
PCR product and produce a second PCR product that will be
shorter than the first one.
 The logic behind this strategy is that if the wrong locus were
amplified by mistake, the probability is very low that it would
also be amplified a second time by a second pair of primers.
 This method can be used for detection of Colletotrichum
lindemuthianum in bean and Fusarium oxysporum f. sp.
lactucae in the seed of lettuce.

31. iii. IMMUNOMAGNETIC SEPARATION AND PCR
(IMS-PCR)
Immunomagnetic separation refers to the use of microscopic magnetic
beads (IMBs) coated with antibodies produced against a specific
microorganism, to selectively sequester target cells from suspensions
containing heterogenous cell mixtures (Olsvik et al., 1994; Safarik and
Safarikova, 1999).
Captured cells can be recovered on selective media or DNA can be
extracted from them and used for PCR.
This technique has been employed widely for the detection of
microorganisms from a variety of backgrounds.

32. IMMUNOMAGNETIC SEPARATION AND PCR

33. iv. MAGNETIC CAPTURE HYBRIDIZATION PCR
(MCH-PCR).
Similar in format to IMS-PCR.
But MCH-PCR uses single stranded DNA probes to capture and
concentrate specific DNA fragments that can then be used as
templates for PCR.
MCH-PCR is a relatively new technique, first described in 1995 for
the detection of Pseudomonas fluorescens in non sterile soil
(Jacobsen, 1995).
This technique has subsequently been developed for the detection of
fungi, viruses and bacteria in water, wood, soil and food, all of
which contain PCR-inhibitory compounds (Chen et al., 1998).
 Recently, a research has been done to develop a MCH-PCR assay
for the detection of Botrytis aclada (causal agent of Botrytis neck
rot) on onion seed.

34. To conduct MCH-PCR, infested onion seeds were crushed and DNA was
extracted using a Mini-Beadbeater (Biospec Products Inc., Bartlesville,
Okla.).
Double-stranded target DNA molecules were denatured by boiling and
magnetic beads coated with the capture probe were incubated with the
DNA suspensions for 2 h at 62 ºC (143.6 °F).
During this time, the capture probe hybridized with singles tranded target
DNA fragments.
After incubation, the beads were washed and the captured DNA was used
for PCR.
Development of the MCH-PCRbased seed detection assay is still in
progress, however, detection thresholds of 10 conidia/ mL in the presence
of PCR-inhibiting onion seed wash can be observed.

36. MCH-PCR has been successfully used to detect pathogenic
microorganisms in materials that contain PCR inhibitory compounds, e.g.
Botrytis aclada in onion seeds (Walcott et al., 2004).
Also, a MCH multiplex real-time PCR assay has been developed to detect
two different pathogens, Didymella bryoniae and Acidovorax avenae sub
sp. citrulli, from watermelon and melon seeds (Ha et al., 2009).

37. 2. Loop-mediated isothermal amplification
 Loop-mediated isothermal amplification (LAMP) of DNA is a recent
technology that was developed by Notomi et al. (2000) as a simple, cost-
effective and rapid method for specific detection of genomic DNA.
 LAMP uses a set of four or six primers and a thermophilic DNA
polymerase from Geobacillus stearothermophilus that has strand
displacement activity to amplify DNA with high specificity and in less
than 1 h (Mumford et al., 2006).
 This technology has several positive features: all of the reactions can be
carried out under isothermal conditions; it does not require expensive
equipment; and there are fewer preparation steps compared to
conventional PCR and real-time PCR assays.
 Furthermore, as well as being highly specific, the amplification efficiency
of LAMP is extremely high, which provides improved sensitivity, and
robustness to the inhibitors that usually adversely affect PCR methods (Fu
et al., 2011).

39.  LAMP products can be visualized by gel electrophoresis, by the use of
magnesium pyrophosphate, which promotes precipitation of amplified
DNA, with a real-time turbidity reader or with the addition of an
intercalating dye, such as SYBR Green I, which produces a colour change
if the LAMP reaction is positive.
LAMP was very effective for the detection of Fusarium graminearum in
total genomic DNA isolated from ground wheat grains (Niessen & Vogel,
2010) and from contaminated and germinated wheat seeds (Abd-Elsalam
et al., 2011), thus demonstrating its use for early detection of toxigenic
Fusarium species in cereals.

40. some other fungal pathogens which could be detected by
employing LAMP assay
1. Fusarium oxysporum f. sp. ciceris, the incitant of
Fusarium wilt,
2. Tilletia indica causing Karnal bunt,
3. Phomopsis longicolla responsible for the
deterioration in the seed quality of soybean and
4. Colletotrichum truncatum causal organism of
anthracnose in soybean.

41. 3. RAPID-CYCLE REAL-TIME PCR
 Recent advances in PCR, in the form of rapid-cycle real-time PCR promise
to eliminate many of these barriers and make PCR more accessible for seed
detection.
 With real-time PCR, DNA amplification is coupled with the production of a
fluorescent signal that increases proportionally with the numbers of
amplicons produced (Kurian et al., 1999; Cockerill and Smith, 2002).
 The fluorescent signal is monitored on a computer in real-time and
provides an indirect visual representation of DNA amplification.
 Detection of amplified DNA can be accomplished by staining with SYBR
Green I (Molecular Probes Inc., Eugene Ore.) that binds double-stranded
DNA indiscriminately or with the use of specific reporter probes like
TaqMan (Taylor et al., 2002).

42. Advantages that potentially make it more acceptable for use in
routine seed testing:
1) Rapid cycling which reduces DNA amplification time significantly.
2) Closed system which reduces the risk of cross-contamination.
3) No need for time consuming post-PCR electrophoresis to determine PCR
results
4) The use of different dyes and probes can allow for mutliplex PCR, by
which multiple pathogens can be detected in the same reaction (Wittwer
et al., 2001)
5) Real-time PCR can allow quantification of template DNA which may be
of use in determining levels of seed infestation.
6) Real-time PCR is able to generate a specific fluorescent signal detected
by an integrated fluorometer to provide real-time analysis of reaction
kinetics and so allows quantification of specific DNA targets

43. On the other hand, there are some key factors that may prevent the
immediate adoption of this technology for seed detection
(Disadvantages)
1. Real-time PCR requires thermal cyclers that are equipped to detect
fluorescence.
2. These thermal cyclers are significantly more expensive than
conventional thermal cycler.
3. Taq Man probes used here are costly.
4. Real-time PCR is subject to many of the problems that hamper
conventional PCR , including inhibition by seed-derived compounds.

44. For example,
Quantitative real-time PCR has been used to detect and quantify
i. Verticillium dahliae on spinach seeds (Duressa et al., 2012),
ii. A. brassicae on cruciferous seeds (Guillemette et al., 2004),
iii. Botrytis spp. on onion seeds (Chilvers et al., 2007), and
iv. C. lindemuthianum on dry bean seeds (Chen et al., 2013).
v. Didymella bryoniae, an incitant of gummy stem blight of cucurbits
(Ling et al. 2010).
More recently, a multiplex TaqMan real-time PCR assay was developed
for the detection of spinach seed-borne pathogens, viz.
1. Peronospora farinosa f. sp. spinaciae,
2. Stemphylium botryosum,
3. Verticillium dahliae and
4. Cladosporium variabile,
that cause economically important diseases on spinach (Feng et al. 2014).

45. 4. Multiplex real-time PCR
Multiplex real-time PCR uses multiple primers (together with
probes in the TaqMan assay) in the same reaction, to reduce costs
and labour.
To ensure adequate specificity and sensitivity, and comparable
amplification efficiency of different pathogens in real-time PCR
assays, it is critical to choose the appropriate target DNA
fragments for the design of the primers and probes.
Since multiplex PCR consists of multiple primer sets within a
single PCR mixture to produce amplicons of different sizes that are
specific to different DNA sequences, it is capable of detecting
several seed-borne pathogenic fungi simultaneously with high
sensitivity.

47. Multiplex PCR was employed to differentiate members of two
groups belonging to Aspergillus flavus.
The first Aspergillus flavus group encloses A. flavus and A.
parasiticus as aflatoxin producers, and the second group
includes A. oryzae and A. sojae which are best known for
their capability to ferment soybean to prepare various food
products.
Multiplex PCR could successfully detect Fusarium species
within Fusarium head scab complex (Waalwijk et al. 2003)
and Rhynchosporium secalis, a seed-borne fungal incitant
causing economically important leaf blotch disease of barley
(Fountaine et al. 2007).

48. 5. DNA Chip (microarray) technology
DNA chips or microarrays represent another DNA-based detection assay
that may be applied to test seeds for pathogens.
This relatively new technology relies on the unique ability of nucleic acid
molecules to hybridize specifically with molecules with complementary
sequences (Lemieux et al., 1998; Vernet, 2002).
With DNA chip technology, oligonucleotide probes are attached to small
(approximately 1 cm sq.) glass or silica-based surfaces (chips).
The power of this technique lies in the fact that hundreds to thousand of
oligonucleotides can be attached to specific, locations on each chip.
These oligonucleotides can be complementary to DNA sequences that are
unique to certain microorganisms and hence, can be used detect
pathogens in seed samples.

49. Fungal cell

50. To apply DNA chip technology, DNA or RNA must be extracted from the
sample being tested and amplified.
The amplified DNA is digested into smaller fragments that are then
labelled with fluorescent markers and hybridized with oligonucleotides
fixed to the DNA chip.
After hybridization, the chip is washed thoroughly and fluorescence,
which is directly proportional to the amount of nucleotide retained, is
measured.
If the DNA from the pathogen of interest in present in the seed sample,
then the oligonucleotide probe at the position on the chip that corresponds
to that pathogen will display fluorescence.

51. Advantages of this technology include simultaneous detection of a wide
range of pathogens and rapid completion time (6 h).
However, since DNA-chip technology relies in part, on DNA
amplification, it has similar limitations as those described for
conventional PCR.

52. 6. DNA Barcoding
DNA barcoding is a taxonomic method that uses a short
genetic marker in an organism’s DNA to identify it as
belonging to a particular species (Hebert et al. 2003).
The nuclear ribosomal internal transcribed spacer (ITS)
region is a recently proposed DNA barcode marker for
fungi (Schoch et al. 2012).
Identification of universal barcoding regions is important to
detect seed-borne fungi.
Internal transcribed spacer (ITS) has been used as the
primary barcode marker for fungi based on its ability to
successfully identify inter- and intraspecific variation
among a wide range of fungi.

54. An ideal barcoding gene should be sufficiently conserved to be amplified
with wide range of primers, however divergent enough to identify closely
related species.

55. 7. Next-Generation Sequencing (NGS)
Next-generation sequencing (NGS), also known as high-
throughput sequencing, is the catch-all term used to describe
a number of different modern sequencing technologies.
These technologies allow for sequencing of DNA and RNA
much more quickly and cheaply than the previously used
Sanger sequencing, and as such revolutionised the study of
genomics and molecular biology.
Such technologies include:

56. Illumina (Solexa) sequencing
Illumina sequencing works by simultaneously identifying
DNA bases, as each base emits a unique fluorescent signal,
and adding them to a nucleic acid chain.
Roche 454 sequencing
This method is based on pyrosequencing, a technique which
detects pyrophosphate release, again using fluorescence,
after nucleotides are incorporated by polymerase to a new
strand of DNA.
Ion Torrent: Proton / PGM sequencing
Ion Torrent sequencing measures the direct release of H+
(protons) from the incorporation of individual bases by DNA
polymerase and therefore differs from the previous two
methods as it does not measure light.

57. • After its first application in basic biological research, NGS
technologies have been extended to other fields of
application, which have included plant disease diagnosis.
• Valuable technique for the rapid identification of disease-
causing agents from infected plants,
• Can also be applied to the detection of fungal pathogens in
seeds.
• Has been applied to study the mycobiome of wheat seed,
using 454 pyrosequencing, allowing the identification of
several fungal genera (Nicolaisen et al. 2014).

58. Other Newly Developed Diagnostic Techniques
a. Biospeckle Laser Technique
A recently applied tool that can reveal the presence of
pathogenic fungi on seeds is known as the ‘biospeckle’
laser technique.
This technique is based on the optical phenomenon of
interference that is generated by a laser light that interacts
with the seed coat.
Examination of seeds under laser light allows the
identification of areas with different activities (Braga et al.
2005; Rabelo et al. 2011).
As fungi present on the seeds have biological activity, this
method can detect their presence on seeds.

60. b. Videometer Lab Instrument
One more recently developed tool called videometer lab
instrument that can distinguish infected seeds from healthy
seeds is a multispectral vision system also useful to
determine the colour, texture and chemical composition of
seed surfaces (Boelt et al. 2018).
The combinations of the features from images captured by
visible light wavelengths and near-infrared wavelengths were
worthwhile in the separation of healthy spinach seeds from
seeds infected by Stemphylium botryosum, Cladosporium
spp., Fusarium spp., Verticillium spp. or A. alternata (Olesen
et al. 2011).

61. • Seed quality of castor (Ricinus communis L.) based on
seed coat colour was predicted employing multispectral
imaging technology using VideometerLab instrument.
• This technology was able to distinguish viable seeds from
dead seeds with 92% accuracy, suggesting its utility for
seed deterioration caused by fungal pathogens (Olesen et
al. 2015).

62. Features of seed detection assays including time required for completion, sensitivity,
ease of application, specificity and applicability for the detection of fungi on seed