Content uploaded by Vaibhav K. Singh
Author content
All content in this area was uploaded by Vaibhav K. Singh on Aug 18, 2021
Content may be subject to copyright.
107
© Springer Nature Singapore Pte Ltd. 2020
R. Kumar, A. Gupta (eds.), Seed-Borne Diseases of Agricultural Crops:
Detection, Diagnosis & Management,
https://doi.org/10.1007/978-981-32-9046-4_5
R. Kumar (*) · A. Gupta
ICAR-Indian Agricultural Research Institute, Regional Station, Karnal, Haryana, India
S. Srivastava
School of Agriculture, Lovely Professional University, Phagwara, Punjab, India
G. Devi
ICAR-Indian Wheat and Barley Research Institute, Karnal, Haryana, India
V. K. Singh · M. S. Gurjar · R. Aggarwal
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, India
S. K. Goswami
ICAR-National Bureau of Agriculturally Important Microorganisms,
Mau Nath Bhanjan, Uttar Pradesh, India
5
Diagnosis andDetection ofSeed-Borne
Fungal Phytopathogens
RavindraKumar, AnujaGupta, SewetaSrivastava,
GeetaDevi, VaibhavKumarSingh, SanjayKumarGoswami,
MalkhanSinghGurjar, andRashmiAggarwal
Abstract
Food losses due to crop infections caused by different pathogens such as bacte-
ria, viruses and fungi are persistent issues in agriculture for centuries across the
globe. The timely detection and appropriate identication of casual agents asso-
ciated with diseases of crop plants or seeds are considered to be the most impor-
tant issue in formulating the management strategies. Seed health testing to detect
seed-borne pathogens is an important step in the management of crop diseases.
Specicity, sensitivity, speed, simplicity, cost-effectiveness and reliability are the
main requirements for the selection of seed health test methods. Examples of
frequently used seed assays include visual examination, selective media, seed-
ling grow-out and serological assays which, while appropriate for some patho-
gens, often display inadequate levels of sensitivity, specicity and accuracy.
Polymerase chain reaction (PCR) has emerged as a tool for the detection of
microorganisms from diverse environments. Thus far, it is clear that nucleic acid-
based detection protocols exhibit higher level of sensitivity than conventional
methods. Unfortunately, PCR-based seed tests require the extraction of PCR-
quality DNA from target pathogens in backgrounds of saprophytic organisms
108
and inhibitory seed-derived compounds. The inability to efciently extract PCR-
quality DNA from seeds has restricted the acceptance and application of PCR for
the detection of seed-borne pathogens. To overcome these limitations, several
modied PCR protocols have been developed including selective target colony
enrichment followed by PCR (Bio-PCR). These techniques seek to selectively
concentrate or increase target organism populations to enhance detection and
have been successfully applied for detecting fungi in seed. Ultimately, improved
protocols based upon PCR, ELISA, etc. will be available for the detection of all
seed-borne pathogens and may supersede conventional detection methods. This
chapter provides a comprehensive overview of conventional and modern tools
used for the early detection and identication of seed-borne fungal pathogens.
5.1 Introduction
Seed-borne pathogens possess a serious threat to seedling establishment. Close
association of the pathogens with seeds facilitates their long-term survival, intro-
duction into new areas and widespread dissemination. Under such conditions, elim-
ination is the most effective disease management strategy accomplished by using
seed detection assay to screen and reject infested seed lots before sowing/planting
or its distribution to the farmers or seed growers. Transboundary spread of patho-
gens is a major concern today. Precise detection methods are essential for seed-
borne pathogens to support seed health strategies. While choosing a method, it is
essential to see that it is reliable, less time-consuming, cost-effective, reproducible
and sensitive. The conventional seed health testing methods are being used in the
identication of fungus up to species level. The considerable advancement in
molecular biology has facilitated rapid identication/detection of seed-borne patho-
gens. Over 100years of seed health studies, many new methods were developed, or
older methods were modied, but all of them used for the detection and identica-
tion of seed-borne organisms have to full six main requirements (Ball and Reeves
1991):
(i) Specicity– 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).
(iv) Simplicity– minimization of a number of examination stages to reduce error
and enable testing by a staff not necessarily highly qualied.
(v) Cost-effectiveness– costs should determine acceptance to the test.
(vi) Reliability– methods must be sufciently robust to provide repeatable results
within and between samples of the same stock regardless who performs the
test.
R. Kumar et al.
109
5.2 Why Detection ofSeed-Borne Fungal Pathogens is
Important?
• Seed-borne fungal pathogens present a serious threat to seedling establishment
and hence may contribute as potential factor in crop failure.
• Seeds not only facilitate the long-term survival of these pathogens but also may
act as a vehicle for their introduction into newer areas and their widespread
dissemination.
• Seed-borne fungal pathogens are able to cause catastrophic losses to food crops
and hence directly linked to the food security.
• Unlike infected vegetative plant tissues, infested seeds can be asymptomatic,
making visual detection impossible.
• Additionally, fungal pathogen’s populations on seeds may be low, and the
infested seeds may be non-uniformly distributed within a lot.
5.3 Detection Methods forSeed-Borne Fungal Pathogens
The following methods are used to detect seed-borne fungal pathogens which
include conventional and modern methods.
5.3.1 Conventional Detection Methods
5.3.1.1 Visual Examination ofDry Seeds
The rst step of the detection of seed-borne pathogens is examination of dry seeds
with unaided eye (naked eye) or with the magnifying glasses (hand lens). In certain
cases, infected seeds exhibit different characteristic symptoms produced by various
seed-borne fungal pathogens on seed surface, viz. seed rot, seed necrosis, shrunken
seed, seed discolouration, shrivelling, etc. (Table5.1 and Fig. 5.1). Besides these
symptoms, dry seeds are examined for the presence of admixtures such as sclerotia,
fungal fructication such as pycnidia and acervuli, smut balls and smut sori, etc. In
this method stereoscopic microscope, hand lens or naked eye can be used for a
sample consisting of 400 or more seeds. By this examination someadditional sig-
nicant risks can alsobe eliminated, e.g. weed seed contaminants, insect pests and
abnormal seeds. Seed may be soaked in water or other liquids to make pathogen
structures, e.g. pycnia, and symptoms, i.e. anthracnose, on the seed coat more
visible.
Visual examination method may be coupled with automatic devices that sort
seeds based on visuals of physical characteristics (Paulsen 1990; Walcott et al.
1998) to reduce seed lot infestation. But, as a limitation, these systems usually have
low detection sensitivity, which makes these devices less useful in decision-making
system for rejection of seed lots. Additionally, seeds infested by fungi, bacteria and
viruses may display no macroscopic symptoms, making visual or physical inspec-
tion of seeds useless as a detection assay.
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
110
Table 5.1 Visual sign/symptoms of some major seed-borne fungi on various crop seed
S.
no. Crop
Visual sign or symptom on
seed Possible fungi associated References
1. Barley Scald symptoms Rhynchosporium secalis Lee etal.
(1999)
2. Carrot Seed rot Alternaria radicina Gaur (2011)
3. Celery Pycnidia embedded in the
seed coat
Septoria apii Horst (2008)
4. Cereals Normal seed is replaced by
sori of spores
Smut, bunt or ergot in
cereals
Warham etal.
(1996)
5. Chick pea Small and wrinkled seed Fusarium oxysporum f.
sp. ciceri
Khare (1996)
Ashy brown discolouration
in seeds
Ascochyta rabiei Khare (1996)
Blackish seed coat Alternaria alternata Khare (1996)
Reduction in seed size Ascochyta rabiei Gaur (2011)
6. Chilli Acervuli and microsclerotia Colletotrichum dematium Kumar etal.
(2004)
7. Coriander Hypertrophied seed Protomyces macrosporus Khare (1996)
8. Crucifers Reduction in seed size and
seed rot
Phoma lingam Gaur (2011)
Shrivelling Alternaria brassicae, A.
raphani and A. alternata
Rude etal.
(1999)
9. Dolichos
lablab
Red discolouration around
micropyle (red nose)
Stemphylium botryosum Gaur (2011)
Brown spot Colletotrichum
lindemuthianum
Gaur (2011)
10. Lentil Ashy brown discolouration
in seeds
Ascochyta fabae f. sp.
lentis
Khare (1996)
11. Maize White streaks with black
spore masses near the tips
Nigrospora sp. Agarwal and
Sinclair (1997)
Seeds exhibit white streaks Fusarium moniliforme Khare (1996)
Seed rot Fusarium graminearum Gaur (2011)
12. Onion Seed rot Alternaria porri Gaur (2011)
Shrunken seeds Peronospora destructor Gaur (2011)
13. Pea Brown spot Ascochyta pisi Gaur (2011)
Seed rot Mycosphaerella pinodes Gaur (2011)
14. Peanut Speckles Cylindrocladium
parasiticum
Randall-
Schadel etal.
(2001)
15. Rice Light pink discolouration Fusarium graminearum Sachan and
Agarwal (1995)
Ash grey discolouration Alternaria alternata Sachan and
Agarwal (1995)
Black discolouration, dark
brown spots and light to
dark brown dot-like spots
Helminthosporium oryzae
[Cochliobolus
miyabeanus]
Sachan and
Agarwal (1995)
Light brown discolouration Sarocladium oryzae Sachan and
Agarwal (1995)
(continued)
R. Kumar et al.
111
5.3.1.2 Microscopic Examinations
(a) Examination of Seed Washings
This method is used to detect seed-borne pathogens which are loosely present on
the seed surface. This method is mostly used for the detection of fungi causing
smuts, bunts, downy mildew, powdery mildew and rust with the important excep-
tion of loose smut of wheat and barley which are internally seed-borne diseases. For
seed washing test, seed samples (50 seeds) are placed in test tubes containing sterile
distilled water (10ml) and a few drops (10–20) of 95% ethyl alcohol or a detergent.
The sample tubes are agitated in a mechanical shaker for 10 min. The aqueous
Table 5.1 (continued)
S.
no. Crop
Visual sign or symptom on
seed Possible fungi associated References
16. Sesame Hyphae and sclerotia on
seed coat
Macrophomina
phaseolina
Khare (1996)
17. Sorghum Completely deformed Acremonium sp. Agarwal and
Sinclair (1997)
Shrunken seeds Sclerospora sorghiaGaur (2011)
18. Soybean Purple stain Cercospora kikuchii Murakishi
(1951) and
Khare (1996)
Fine cracks and mould,
starting near the hilum
Phomopsis longicolla Li (2011)
19. Wheat Bunted seed Tilletia tritici Warham (1986)
Shrivelled rough scabby
appearance
Fusarium sp. Warham etal.
(1996)
Black point on seed Alternaria alternata Khare (1996)
aNot seed-borne but affect seed
Fig. 5.1 Visual symptoms caused by fungal pathogens on seeds after harvesting (a–d) and in
standing crops (e–h): Karnal bunt of wheat (a), false smut of paddy (b), kernel smut/bunt of paddy
(c–d), loose smut of wheat (e), ergot of pearl millet (f), green ear disease of pearl millet (g), smut
disease of pearl millet (h)
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
112
suspension is then centrifuged at 1000rpm for 10min. The supernatant is poured
off and the pellet is re-suspended in 2ml of sterile water. Spores or fungal structure
present in the suspension can be viewed by examining a few drops of the suspension
under the light microscope.
(b) NaOH Seed Soak Method
The NaOH seed soak method was rst used by Agarwal and Srivastava (1981).
This method is applied for the detection of Karnal bunt of wheat and bunt (kernel
smut) of paddy. In this method seeds are soaked in 0.2–0.3% NaOH solution for
24h at 25–30 °C.Next day the solution is decanted and the seeds are thoroughly
washed in tap water. After washing, the seeds are spread over blotter paper so that
the excess moisture is absorbed by blotter. Now the seeds are examined visually.
The wheat seeds showing black to shiny black discolouration may contain Karnal
bunt infection of Tilletia indica. This may be conrmed by rupturing suspected seed
with a ne needle in a drop of water, the bunt spores (teliospores) will be released,
if the suspected seed is infected. Similarly, the infection in paddy seeds due to bunt
or kernel smut disease of paddy caused by Tilletia barclayana can also be detected.
Likewise, by treating the rice seeds with NaOH (0.2%), the infection by
Trichoconiella padwickii could be inferred by the change of colour of the diseased
portion of infected seeds to black (Singh and Maheshwari 2001).
(c) Whole Embryo Count Method
This method is used when seed-borne infection is deep seated in the seed tissues
such as embryo in case of loose smut of wheat and barley. The embryo count method
was rst used by Skvortzov (1937) for detecting loose smut pathogen Ustilago nuda
var. tritici. He dissected the embryos, macerated them with NaOH and then stained
them with aniline blue. This method is completed in 3–4days. This method was
modied by Agarwal etal. (1978) as follows (Fig.5.2):
5.3.1.3 Incubation Methods
(a) Testing on Agar Media: In agar tests seeds are incubated on agar media for a
particular length of time and optimum temperature under alternating light and
dark cycles. The associated fungi are detected based on their morphological and
habit characters on seed surface and colony characters on the medium. It is used
to detect Alternaria, Bipolaris, Curvularia, Fusarium, etc. in infected seeds.
(b) Blotter Testing: Doyer (1938) and de Temp (1953) were rst to adopt blotter
paper method in seed health management. This test is used to detect infection of
seeds, and in certain cases, infection of the germinated seedlings can also be
detected by this method. Blotter method is the most widely used seed health
assay. Mainly this method is of two types:
(i) Standard Moist Blotter (SMB) Method
In the standard blotter test, seeds are sown in Petri dishes containing 1–3
layers of water or buffer-soaked absorbing (blotting) paper or cellulose pads
R. Kumar et al.
113
for a couple of days depending on the fungus and type of seed tested
(Marcinkowska 2002). In general, 10–20 non-sterilized seeds (depending
on the seed size) are placed equidistant from each other in Petri dish and
incubated at 25±2°C with alternate cycles of 12h of light and 12 h ofdark-
ness for 7–10days. In the blotter test, seeds are subjected to conditions that
enable pathogen growth and expression during the incubation period
(Fig.5.3). After the incubation period, the seeds are examined under a ste-
reomicroscope for the presence of fungal colonies, and their characteristics
are recorded for the identication of the fungal pathogens.
The seed must be surface-sterilized prior to its placement on blotter
paper in Petri dish, if the internally seed-borne fungal pathogens are to be
Fig. 5.2 General protocol for whole embryo count method for detection of loose smut of wheat
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
114
detected. Seeds may be immersed in a NaOCl solution containing 1% chlo-
rine for 10min or in 1.7% NaOCl solution for 1min followed by immersion
in 70% chlorine for 10min (ISTA 1966). The germination of seeds may be
obstructedby wetting the blotting paper with 0.1–0.2% 2,4-D.This proce-
dure has been used for the detection of Leptosphaeria maculans (anamorph
Phoma lingam) in crucifer seeds (Hewett 1977) and for routine seed health
testing of common bean and soybean (Dhingra etal. 1978).
(ii) Deep Freezing Blotter (DFB) Method
The DFB method is used to detect a wide range of fungi which are able to
grow easily from seeds in the presence of humidity. After plating seeds as
described in the SMB method, the Petri dishes are incubated at 20±2°C
for 24h and then transferred to a −20°C freezer for 24h followed by incu-
bation at 20±2°C for 5days under cool white uorescent light with alter-
nating cycles of 12h light and 12h darkness. Pure cultures are obtained
through hyphal-tip and single-spore isolation techniques and maintained on
carrot potato agar (CPA) slants for further studies. Fungi are identied using
cultural, biochemical, macromorphological and micromorphological char-
acteristics as described by Raper and Fennel (1965), Booth (1971), Ellis
(1971) and Domsch etal. (1980).
Fig. 5.3 Incidence of seed
mycoora on pea seeds
under blotter testing
method after 10days of
incubation
R. Kumar et al.
115
The percentage of seed infection in each sample and the percentage of
infection in each region are determined by the following formulae:
Mean rate of seed infectionNumber of seedsonwhich afungal
sspecies identified
Number of seeds tested
100
Mean of regional infectionFrequency of sampleonwhich afung
uus identified
Number of samples collected
100
For species-level identication, the fungi are isolated on potato dextrose
agar (PDA) and maintained at 24±1°C for 7–10days. The identication is
conducted using colony colour, colony texture pattern, arrangement of
spore on the conidiophores, spore shape and size (Watanabe 2002; Leslie
and Summerell 2006; Utobo etal. 2011).
The blotter method has been coupled with scanning electron microscopy (SEM)
for the detection of seed-borne fungi (Alves and Pozza 2009). The seeds of common
bean (Phaseolus vulgaris L.), maize (Zea mays L.) and cotton (Gossypium hirsutum
L.) were submitted to the standard blotter test. The specimens were prepared and
observed with the standard SEM methodology. It was possible to identify Fusarium
sp. on maize, C. gossypii var. cephalosporioides and Fusarium oxysporum on cot-
ton and Aspergillus avus, Penicillium sp., Rhizopus sp. and Mucor sp. on common
bean (Alves and Pozza 2009).
(c) Seedlings Symptoms Test and Grow-Out Test
Seedlings symptoms test is based on the characteristic symptoms produced by
seed-borne fungi on growing seedlings under controlled conditions, whereas in
grow-out test, plants are grown beyond the seedling stage in near-optimum condi-
tions of temperature and moisture in sterile medium, i.e. sand, and water-agar
medium, and the seedlings/plants are observed for symptoms of the fungal patho-
gens. It can facilitate the detection of a number of fungal pathogens associated with
seed rotting and other symptoms at seedling stage, e.g. fungal pathogens causing
seedling diseases as Alternaria, Bipolaris, Fusarium, Pyricularia, etc. This method
involves the planting of a certain number of seeds, preferably on sterile soil for
determining the number of infected plants and calculating the percent infected
plants out of the total number of seed sown. These test results are helpful in assess-
ing eld performance and estimating the number of infection loci/unit area, if the
seed lot under investigation is used for cultivation by farmers. Infection of soybean
seeds by Colletotrichum truncatum was detected by this method (Dhingra etal.
1978). This method is very effective in the case of non-cultivable obligate pathogens
causing downy mildew diseases. However, it requires large greenhouse space, and
also it is time-consuming, making it unsuitable for testing a large number of seed
lots. There are a number of seedling symptoms and grow-out tests as follows:
(i) Test Tube Agar Method
This method was developed by Khare, Mathur and Neergaard in 1977. It is
used for the detection of Septoria nodorum in wheat seeds and is very
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
116
useful for assaying the small quantity of high cost material. In this method
infection of root can also be examined. Fungal pathogens of cereals like
Drechslera sp., Bipolaris sp. and Septoria sp. can be easily detected. Steps
used in procedure are as follows:
1. 15 ml water agar is taken in test tube, sterilized and solidied with a
slight slant.
2. One seed is sown in each test tube and incubated at 28 ± 1 °C with
12hours alternating cycles of light and darkness.
3. Seedlings are examined after 14days for the typical symptoms of dis-
ease in the coleoptiles.
4. The symptoms can be easily studied being visible on roots as well as on
green parts.
(ii) Hiltner’s Brick Stone Method
It was developed by Hiltner in 1917. Sterile crushed brick stone with a
maximum piece size of 3–4mm is used to ll in plastic pots up to ¾th of
their capacity. The crushed brick stone in the pot is saturated with water and
seeds are placed 1cm deep. The pots are kept in darkness at room tempera-
ture, and observations for disease symptoms are recorded after 2weeks by
removing the seedlings. It is a good method for testing eld performance
giving information on seedling symptoms. It is also used for testing treated
seed.
(iii) Sand Method
This method is similar to Hiltner’s brick stone method except that in place
of sterile crushed brick stone, sterilized sand is used.
(iv) Standard Soil Method
A pre-sterilized uniform soil mixture containing four parts clay, six parts
peat and essential amount of fertilizer is lled in plastic multi-pot trays.
After sowing the seed, appropriate moisture should be maintained. The
symptoms are observed after incubation for 2–4weeks depending on the
kind of seed and temperature.
(d) Selective Media
It is a direct method of seed testing in which seed-borne pathogens are allowed
to grow on specic media. The use of selective media for the detection of pathogens
is more reliable than blotter or agar method. This can be done by directly plating
surface-sterilized seed samples or seed wash liquid onto articial media, followed
by adequate incubation under favourable conditions. Once a fungal pathogen is iso-
lated, it can be identied by its cultural, morphological or biochemical characteris-
tics. Selective articial media are developed that use antibiotics, fungicides, selected
carbon and nitrogen sources and other inhibitory compounds to retard the growth of
non-target microora while allowing the target pathogen to grow. For example,
R. Kumar et al.
117
potato dextrose agar is useful for the detection of Septoria nodorum in wheat, while
PCNB agar is a selective medium for the detection of Fusarium species in cereals.
The list of some selective and semi-selective media for different seed fungi is given
in Table5.2.
Table 5.2 General/selective media and temperature requirements that favour the development of
seed-borne fungal pathogens
S.
no.
Name of seed-borne
fungal pathogen Nutrient medium
Incubation
temp. (°C) References
1. Botrytis cinerea Selective media: Botrytis
selective medium (BSM) and
Botrytis spore trap medium
(BSTM)
25°C Edwards and
Seddon (2001)
2. Botrytis cinerea Potato dextrose agar (PDA) 20–22°C Mirzaei etal.
(2008)
3. Botryodiplodia
theobromae
PDA 28°C Fu etal. (2007)
4. Lasiodiplodia
theobromae
Selective medium 25°C Cilliers etal.
(1994)
5. F. oxysporum f. sp.
niveum
PDA/lima bean agar 25°C Zhang etal.
(2005)
6. F. oxysporum f. sp.
cucurbitae
Fusarium selective medium
(FSM)
25–37°C Mehl and
Epstein (2007,
2008)
7. F. solani f. sp.
cucurbitae
Fusarium selective medium
(FSM)
22°C Mehl and
Epstein (2007)
8. Trichoconiella
padwickii
Semi-selective media 28–30°C Muthaiyan
(2009)
9. Fusarium
graminearum
Semi-selective media (NSA,
SRA-FG)
25–28°C Segalin and
Reis (2010)
10. Fusarium species Semi-selective medium (MGA
2.5+carnation leaves)
25°C Thompson
etal. (2013)
11. Exserohilum
turcicum
Semi-selective medium
(DRR-Reis)
25±2°C De Rossi and
Reis (2014)
12. Alternaria
brassicicola
(crucifer seeds)
Semi-selective media (CW
medium)
24°C Wu and Chen
(1999)
13. Fusarium species in
cereals
Dichloran chloramphenicol
peptone agar (DCPA)
25°C Andrew and
Pitt (1986)
14. Phoma betae Hold fast method 20°C Mangan (1971)
15. Fusarium
moniliforme
Modied Czapek’s dox agar
medium (MCZA)
26–28°C Agarwal and
Singh (1974)
16. Pyricularia oryzae Guaiacol agar 25°C Kulik (1975)
17. Stagonospora
nodorum
SNAB (Stagonospora nodorum
agar for barley)
20°C Cunfer and
Manandhar
(1992)
18. Septoria nodorum
[Stagonospora
nodorum]
Selective media SNAW 20°C Manandhar and
Cunfer (1991)
(continued)
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
118
5.3.2 Serological Detection Techniques
The seed-infecting fungal communities may comprise the saprobes which can grow
rapidly over the target fungal pathogens. These fast-growing saprobes arrest their
isolation; and examination of their morphological characteristics becomes difcult
and confusing. In the case of rice seed-borne pathogens, such situation exists, where
about 30 fungal phytopathogens infecting rice have been reported to be seed-borne
(Mew etal. 1988). Additionally, the presence of very closely related strains, race or
even fungal species on the seeds makes the detection morphologically almost
impossible. Therefore, more sensitive techniques such as immunoassay and nucleic
acid-based protocols are needed to overcome this issue. Since pure culture of the
pathogens is not needed in serological detection protocols, these techniques could
be applied to detect biotrophic as well as necrotrophic seed-borne pathogens
(Mancini etal. 2016).
After the rst use of enzyme-linked immunosorbent assay (ELISA) by Clark and
Adams (1977), employed for successful detection of plant viruses, this technique
has been widely adopted and modied based on requirement of the assays (Fang
and Ramasamy 2015). This serological method is used for the identication of dis-
eases based on antibodies and colour change in the assay. Serological assays depend
on antibodies generated against specic antigens of plant pathogens. The antibodies
bind specically to its antigens and consequently are detected by the enzymatic
digestion of substrates. Polyclonal and monoclonal antibodies have been produced
against fungal antigens present in culture ltrate, cell fractions, whole cells, cell
walls and extracellular components (Narayanasamy 2005).
Species of several seed-borne fungi like Aspergillus, Penicillium and Fusarium
have been demonstrated to be potential mycotoxin producers. A monoclonal antibody
(MAb) capable of reacting with antigens of 10 eld fungi and 27 storage fungi was
generated. The presence of fungal pathogens in barley seeds was detected using a
polyclonal antibody (PAb) raised against Penicillium aurantiogriseum var.
Table 5.2 (continued)
S.
no.
Name of seed-borne
fungal pathogen Nutrient medium
Incubation
temp. (°C) References
19. F. graminearum Toxoavin-based selective
medium
25°C Jung etal.
(2013)
20. Curvularia lunata CS medium with 200ppm
carbendazim +200ppm
streptomycin and CR medium
with 200ppm carbendazim
+200ppm rifampicin
25°C Deshpande
(1993)
21. Rhynchosporium
secalis
Lima bean agar medium 15–20°C Lee etal.
(1999)
22. Fusarium species in
cereals
PCNB agar 22–25°C Pastircak
(2007) and
Alborch etal.
(2010)
R. Kumar et al.
119
melanoconidium in indirect ELISA test. A clear linear relationship was recorded
between absorbance and fungal population increase, suggesting the utility of these
antibodies for a broad-spectrum assay to determine the fungal content in seeds (Banks
et al. 1993). 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. The
absorbance values of ELISA were in good correlation with concentration of mould
growth, and the sensitivity of this DAS-ELISA was 1μg/ml (Chang and Yu 1997).
Karnal bunt disease of wheat caused by Tilletia indica is an internationally quar-
antined fungal disease with a signicant impact on international wheat trade as well
as quality and quantity of wheat seed. SDS-PAGE analysis suggested that T. indica
has a protein of 64kDa weight with antigenic properties. Antibodies specic to this
protein specically reacted with pathogen’s teliospores in a microwell sandwich-
ELISA and dipstick immunoassay. The detection limit of both of these immunoas-
says was 1.25ng/well of puried T. indica protein or 40ng/well of crude spore
extract, which distinguished Karnal bunt from all wheat smuts and, to some degree,
the rice smut, T. barclayana (Kutilek etal. 2001). Ustilago nuda causes loose smut
in barley and it is an internally seed-borne pathogen. Eibel etal. (2005b) employed
a DAS-ELISA test with biotinylated detection antibodies to detect loose smut
pathogen in naturally infected barley seeds.
Phomopsis longicolla is a seed-borne fungal pathogen causing Phomopsis seed
decay of soybean, a major concern for quality seed production in soybean (Glycine
max L.). This pathogen was detected using indirect ELISA and a modied immunob-
lot assay, named as seed immunoblot assay (SIBA). The comparative efciency of
both detection assays was evaluated. The problems with nonspecic interference
occurred during ELISA test could be solved by employing seed immunoblot assay
(SIBA) for detection. In SIBA, infected soybean seeds are transferred to nitrocellu-
lose paper on which the mycelium of P. longicolla grows out forming a clearly visi-
ble coloured blotch on the nitrocellulose paper after the assay. Since the viable spores
can only produce the mycelium, SIBA test is capable of differentiating the living and
dead spores of the pathogen, which is a distinct advantage of this technique. In con-
trast ELISA test results do not offer such vital information (Gleason etal. 1987).
Similarly, wheat seeds with different grades of Karnal bunt (Tilletia indica) infection
could be readily detected by seed immunoblot binding assay (SIBA). After the
immuno-processing, coloured imprints were produced on nitrocellulose paper on
which infected wheat seeds were placed for vigour test, indicating the presence of
viable teliospores of Tilletia indica in the wheat seed lots tested (Kumar etal. 1998).
Two methods, viz. PCR-based assay and DAS-ELISA, were developed and
evaluated for the detection of Tilletia caries (syn. T. tritici), a seed-borne fungus
causing common bunt in wheat. Double-antibody sandwich enzyme-linked
immunosorbent assay (DAS-ELISA) was performed using biotinylated detection
antibodies. The presence of bunt pathogen could be detected by PCR in shoots as
well as in leaves of infected wheat plants. Except for the closely related T. con-
troversa, no cross-reactions with other fungi were observed with both methods.
The analysis of results obtained from DAS-ELISA of plant shoots revealed that
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
120
articial inoculation of seeds with T. caries at EC 10 was efcient in infecting all
host population, with a great variability in the inoculum of pathogen. ELISA
employed in the assay was found most suited than PCR assay, allowing precise
quantication of the amount of fungal antigen present in the plant (Eibel etal.
2005a). Agar plating method and DAS-ELISA were compared for the detection
of Macrophomina phaseolina, a seed-borne fungal pathogen and causal agent of
root rot diseases in wide host range. The presence of the pathogen was conrmed
in four out of ve lots by both the detection methods; however, DAS-ELISA
format revealed more sensitivity in the detection of the pathogen in higher per-
centage of seeds as compared to agar plating method which additionally required
much time and is inconvenient (Afouda etal. 2009).
Instead of successful detection of some seed-borne fungi, we may conclude
that serological techniques have limited values in the detection of seed-borne
fungal pathogens, since they contain many nonspecic antibodies which may
cause cross- reactions with related and unrelated species concealing the effects of
specic antibodies (Dewey 1992; Miller etal. 1992). These assays are widely
applied to detect seed-borne viruses, but insufciency of species-specic anti-
bodies is a major limitation in its wide application for the detection of seed-borne
fungal pathogens. Besides, serology-based assays can also detect non-viable fun-
gal propagules, which can lead to imprecise interpretations (Mancini etal. 2016).
5.3.3 Nucleic Acid-Based Detection Methods
Generally, nucleic acid-based techniques resulting in a high level of sensitivity and
specicity are used for species-specic detection of seed-borne pathogens. Through
these techniques, very small quantities of samples or tissues are sufcient 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, identication
and quantication of seed-borne fungal pathogens. In molecular detection several
strategies, viz. polymerase chain reaction (PCR), multiplex PCR, magnetic capture
hybridization-PCR, Bio-PCR, loop-mediated isothermal amplication, real-time
PCR and DNA barcoding, are available for the detection and identication of patho-
gens 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.
5.3.3.1 Polymerase Chain Reaction (PCR)
Molecular-based methods began after the introduction of PCR in the mid-1980s.
Over the past few decades, considerable advancement has taken place in the devel-
opment of molecular diagnostics for the detection of pathogens in seeds (Molouba
etal. 2001). Potential benets (e.g. rapid, same-day analysis, specic and sensitive
tests) this new technology offers, make it extremely attractive. PCR is a method
which enables amplication and multiplication, up to a manifold, of the target
sequence of DNA.In fungi, internal transcribed spacer (ITS) region has been widely
R. Kumar et al.
121
used to design specic primers to detect the presence of seed-borne infection of
fungi. PCR procedures/protocols have been developed for the detection of several
seed-borne fungal pathogens associated with seeds of various commercially impor-
tant crops (Mancini etal. 2016).
Rhynchosporium secalis, a causative agent of barley scald disease, overwinters
in the plant debris, and this pathogen is capable of infecting barley seeds without
producing conspicuous symptoms, or it may induce typical scald symptoms on
seeds. A PCR-based detection method was developed, and in this assay, pathogen-
specic primer pairs derived from the ITS region of rDNA of R. secalis were effec-
tive in detecting this pathogen in the symptomless seed infections. The detection
assay revealed the presence of the diagnostic band in the symptomless seeds of
susceptible cultivar (Lee etal. 2001). Further, a primer set (RS1 and RS3) derived
from the internal transcribed spacer (ITS) regions of ribosomal RNA genes of this
pathogen was used to quantify the inoculum of seed-borne infection caused by
Rhynchosporium secalis in barley using competitive PCR (Lee etal. 2002).
A complex of three species, viz. Alternaria brassicae, A. brassicicola and A.
japonica, are responsible for the black spot disease of crucifers. To restrict the trans-
boundary spread of this disease by infected seeds, it is essential to ensure the
absence of these pathogenic Alternaria species in seed shipments, which constitutes
the disease management strategies. A PCR-based diagnostic technique was devel-
oped using specic primers developed from sequence analysis of internal tran-
scribed spacer (ITS) regions of nuclear rDNA of Alternaria brassicae, A. brassicicola
and A. japonica. This protocol was able to detect these pathogens in DNA extracted
from seed macerates (Iacomi-Vasilescu etal. 2002). Another attempt was made to
detect Alternaria brassicae, an important seed-borne fungal incitant of the black
spot disease of crucifers using a polymerase chain reaction (PCR)-based assay. A.
brassicae-specic primers sets were designed on the basis of the sequences of two
clustered genes potentially involved in pathogenicity. The designed two sets of
primers were used for conventional and real-time PCR assay. By both the detection
methods, A. brassicae was specically detected using DNA extracted from seed
(Guillemette etal. 2004).
Rice blast disease, a serious threat to rice production, is caused by Magnaporthe
grisea. A PCR-based detection protocol was developed using primers designed on
the basis of nucleotide sequences of the mif 23, an infection-specic gene of M.
grisea. The primers amplied target DNA from genetically and geographically
diverse isolates of M. grisea, but not from DNA of other fungi tested, proving the
specicity of the primers. The detection limit was ~20pg of pathogen DNA.This
PCR-based seed assay was capable in detecting M. grisea in rice seed lots with
infestation rates as low as 0.2% (Chadha and Gopalakrishna 2006).
Septoria tritici (teleomorph, Mycosphaerella graminicola) is an economically
important pathogen causing leaf blotch disease in wheat. Septoria tritici, naturally con-
taminating wheat seeds, was detected employing conventional PCR assay. The species-
specic primers developed from strict alignment of ITS and α-tubulin sequences of
Septoria tritici were used in the diagnostic assay. A single DNA fragment was ampli-
ed from DNA of S. tritici, but not from DNA of wheat seeds or other fungi selected,
the detection limit being 0.5pg of pathogen DNA (Consolo etal. 2009).
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
122
Downy mildew disease caused by biotrophic obligate oomycete Peronospora
arborescens (Berk.) is one of the most economically important diseases of opium
poppy (Papaver somniferum L.) worldwide. This pathogen was detected in opium
poppy seeds using sensitive nested PCR assay. Two primers designed from the
sequences of ITS region of rDNA improved the pathogen detection sensitivity sig-
nicantly up to 1000-fold compared with single PCR employing same primers. The
frequent detection of P. arborescens in seeds suggested the likely threat posed by
this incitant for rapid spread through the seeds (Montes-Borrego etal. 2009).
The fungus Corynespora cassiicola is responsible for target spot disease in soy-
bean in Brazil. This pathogen can be transmitted by seeds and is able to cause severe
damage in this crop. Though early diagnostic of the disease by conventional seed
testing is possible, species-level detection through these methods is time- consuming
and cumbersome. A PCR-based assay was employed using specic GA4-F/GA4-R
primers for the detection of C. cassiicola in pure culture and in soybean seeds. The
pathogen could be detected in infected and inoculated seed samples at the low level
of 0.25% (Sousa etal. 2016).
Species-specic detection of Diaporthe phaseolorum and Phomopsis longicolla,
responsible for soybean seed decay, was achieved using polymerase chain reaction-
restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry (Zhang
etal. 1999). An ultrasonic processor was used to break the seed coats and cells, enabling
the extraction of fungal DNA from soybean seeds. Three TaqMan primer/probe sets
were designed, based on DNA sequences of the ITS regions of ribosomal DNA.Primer/
probe set PL-5 amplied a 96bp fragment within the ITS 1 region of P. longicolla, D.
phaseolorum var. caulivora, D. phaseolorum var. meridionalis and D. phaseolorum
var. sojae. An 86bp DNA fragment was obtained within the ITS 2 region of P. longi-
colla by the set PL-3, whereas set DPC-3 was able to produce 151bp DNA fragment
within the ITS 2 region of D. phaseolorum var. caulivora. The detection sensitivity of
TaqMan primer/probe sets was as low as 0.15fg (four copies) of plasmid DNA.When
using PCR-RFLP for Diaporthe and Phomopsis detection, the assay was able to detect
as little as 100pg of pure DNA.However, TaqMan detection provided the fastest results
of all the methods tested (Zhang etal. 1999).
Tilletia indica, the incitant of Karnal bunt disease, can be correctly identied
based on morphological features but the germination of teliospores is time-
consuming. A polymerase chain reaction (PCR) detection assay was employed
using species-specic primers designed from rDNA-ITS region and 2.3kb mtDNA
fragment of this pathogen. The primer set from ITS region could specically amplify
570bp amplicon of T. indica, whereas primer set derived from mtDNA was able to
amplify 885bp amplicon of KB pathogen only (Thirumalaisamy etal. 2011). This
assay was able to avoid delay in detection and wrong identication of closely related
species of T. indica from wheat seed lots.
Spot blotch disease of wheat caused by Bipolaris sorokiniana is one of the
important diseases of wheat. This disease development may take place through
seed-borne infections. A quick and reliable PCR-based diagnostic assay was devel-
oped to detect B. sorokiniana using a pathogen-specic marker derived from
genomic DNA. A PCR-amplied DNA amplicon (650 bp) was obtained in B.
R. Kumar et al.
123
sorokiniana isolates employing universal rice primer (URP 1F), and it was cloned
in pGEMT easy vector and sequenced. A primer pair RABSF1 and RABSR2, of six
primers designed based on sequences of PCR-amplied DNA amplicon, amplied
a DNA sequence of 600 bp in B. sorokiniana isolates. The pathogen could be
detected specically in a mixed population comprising of total 74 isolates of B.
sorokiniana, Bipolaris spp. and other pathogens infecting wheat and other hosts.
This single DNA fragment was amplied only from DNA of B. sorokiniana, but not
from DNA of other species of Bipolaris genus and other pathogenic fungi tested,
suggesting the specicity of the detection assay, the detection limit being 50pg of
genomic DNA (Aggarwal etal. 2011).
Seed-borne fungal pathogen, Alternaria radicina, causes black rot disease of
carrot. A PCR-based seed assay was developed for the detection of A. radicina from
infested carrot seed. PCR primers used in assay were designed based on a cloned
random amplied polymorphic DNA (RAPD) fragment of this pathogen. This seed
assay was coupled with 5-day incubation under high humidity conditions to increase
the fungal biomass. PCR amplication of the target A. radicina DNA sequence was
improved by the addition of skim milk to the PCR reaction mixture. This PCR-
based assay was able to detect the pathogen (Alternaria radicina), from seed lots
with infestation rates as low as 0.1% (Pryor and Gilbertson 2001).
Pyrenophora graminea, a fungal incitant of leaf stripe disease of barley, is trans-
mitted entirely by seed, and it cannot infect the leaf directly. This pathogen could be
detected employing RAPD primers designed using a sequence-characterized ampli-
ed region (SCAR) approach. Out of 60 RAPD primers, a set of P. graminea-
specic primers (PG2 F/R) was obtained that amplied a single DNA fragment
(435bp) from 37 isolates of P. graminea tested, but not from other Pyrenophora
spp. or saprophytes isolated from barley seed. The diagnostic assay was completed
within 25min (including melting point analysis) using a LightCycler, capable of
measuring emission of uorescence from the binding of SYBR Green I dye to the
PCR products. The rapidity was coupled with the closed ‘in-tube’ detection of PCR
products which reduces the chances for contamination (Taylor etal. 2001b).
Anthracnose is mainly a seed-borne disease caused by Colletotrichum lindemu-
thianum in bean (Phaseolus vulgaris). The pathogen could be detected employing a
rapid, specic and sensitive PCR-based detection method. Based on data analysis of
sequences of rDNA region consisting of the 5.8S gene and internal transcribed spac-
ers (ITS) 1 and 2 of 4 C. lindemuthianum races and 17 Colletotrichum spp. down-
loaded from GenBank, 5 forward primers were designed. One forward primer
showing specicity of the detection was selected for use in combination with ITS 4
to specically detect C. lindemuthianum. A 461bp specic DNA band was obtained
from the genomic DNA template of 16 isolates of C. lindemuthianum, but not from
other Colletotrichum species or 10 bean pathogens. A nested PCR protocol was
applied to enhance the sensitivity of detection, which enabled the detection of as
little as 10fg of C. lindemuthianum genomic DNA and 1% infected seed powder.
This detection assay could be accomplished within 24h against a 2-week period
required for culturing the pathogen, and this protocol required no specialized taxo-
nomic expertise (Fig.5.4) (Chen etal. 2007).
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
124
Fusarium wilt of lettuce (Lactuca sativa) is a serious threat for the lettuce pro-
duction around the world. The fungal incitant of the disease Fusarium oxysporum
f. sp. lactucae has the potential for seed-borne spread. Fusarium oxysporum f. sp.
lactucae was detected in the seed of lettuce using a nested polymerase chain reac-
tion (nPCR)-based assay. Sequences of intergenic spacer region of the rDNA were
used to design three primers for PCR amplications. A PCR product (2270bp)
was generated using primer pair GYCF1 and GYCR4C in the rst amplication.
A 936bp DNA fragment was amplied employing the forward primer GYCF1
and the nested primer R943in the second amplication. The nPCR protocol suc-
cessfully detected the target sequence in genomic DNA of Fusarium oxysporum
f. sp. lactucae at 1fg/μl. The nPCR seed assay was coupled with a 4-day incuba-
tion under high humidity conditions to increase fungal biomass for DNA extrac-
tion. In seed lots known amounts of F. oxysporum f. sp. lactucae-infested seed
were mixed with non- infested seed, and this assay detected the pathogen from
seed lots with infestation rates as low as 0.1% (Mbofung and Pryor 2010).
5.3.3.2 Multiplex PCR
Multiplex polymerase chain reaction (multiplex PCR) is a valuable molecular tool
which offers simultaneous amplication of several DNA amplicons of different
sizes, within single PCR reaction (Sint etal. 2012). The multiplex PCR technique
was rst described by Chamberlain etal. (1988). This technique has various appli-
cations and is being commonly used for the identication and detection of patho-
gens, gene deletion analysis, high-throughput SNP genotyping, linkage and
mutation analyses, etc. Since multiplex PCR consists of multiple primer sets within
a single PCR mixture to produce amplicons of different sizes that are specic to
different DNA sequences, it is capable of detecting several seed-borne pathogenic
fungi simultaneously with high sensitivity. Preferably, potential seed health tests
can be designed as multiplex assays for particular crops, with their ability to detect
all seed-borne pathogens required for phytosanitary purposes.
Multiplex PCR was employed to differentiate members of two groups belonging
to Aspergillus avus. The rst Aspergillus avus group encloses A. avus and A.
parasiticus as aatoxin producers, and the second group includes A. oryzae and A.
Fig. 5.4 Detection of Colletotrichum lindemuthianum-specic DNA fragment in bean seed pow-
der using nested PCR assay. Lanes: M, 100bp DNA ladder (Invitrogen); 1, 100%; 2, 80%; 3, 60%;
4, 40%; 5, 20%; 6, 10%; 7, 8%; 8, 6%; 9, 4%; 10, 2%; 11, 1%; 12, 0%; 13, anthracnose-resistant
bean genotype G2333; 14, negative control (water replaced genomic DNA as the template); 15,
positive control (C. lindemuthianum DNA). (Courtesy of Chen etal. 2007)
R. Kumar et al.
125
sojae which are best known for their capability to ferment soybean to prepare vari-
ous food products. Aatoxigenic strains could be detected, and their differentiation
was possible employing the multiplex PCR protocol that minimized the risk of a
genotype being a phenotypic producer of aatoxin. This assay was based on four
genes involved in aatoxin biosynthesis, viz. norsolorinic acid reductase (nor-1),
versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-
1) and a regulatory protein (apa-2) (Chen etal. 2002). Multiplex PCR could suc-
cessfully detect Fusarium species within Fusarium head scab complex (Waalwijk
etal. 2003) and Rhynchosporium secalis, a seed-borne fungal incitant causing eco-
nomically important leaf blotch disease of barley (Fountaine etal. 2007). There may
be chances that a seed may harbour very closely related species of fungi. The occur-
rence of very closely related fungal species on the seed may lead to overlapping
while simultaneous detection. In multiplex PCR, possible problem of overlapping
of amplicons with similar sizes could be overcome using primers, already dyed with
different colour uorescent dyes (Sint etal. 2012).
Comparison of two PCR-based protocols was done for the detection of Fusarium
verticillioides and Fusarium subglutinans, important fungal pathogens of maize and
other cereals worldwide. PCR-based protocols were used for the identication of
these pathogens targeting the gaoB gene, which codes for galactose oxidase. The
designed primers recognized isolates of F. verticillioides and F. subglutinans obtained
from maize seeds from several regions of Brazil but did not recognize other Fusarium
spp. or other fungal genera. A multiplex PCR diagnostic protocol was capable to
simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans
growing in articially or naturally infected maize seeds (Faria etal. 2012).
Fusarium culmorum causes disease complex, viz. seed rot, seedling blight and
ear rot in maize. The multiplex PCR assay was standardized targeting trichothecene
metabolic pathway genes, viz. Tri6, Tri7 and Tri13, for the detection of trichothe-
cene (DON/NIV) chemotypes and rDNA gene for the specic detection of Fusarium
culmorum species in freshly harvested maize seeds. The analysis of primers
employed in multiplex PCR assay revealed that 94 isolates were able to produce
deoxynivalenol/nivalenol DON/NIV.The practical usefulness of mPCR assay was
validated by comparing these results with high-performance thin-layer chromatog-
raphy (HPTLC) and found that mPCR results equivocally matched with the HPTLC
chemical analysis for eld samples (Venkataramana etal. 2013).
5.3.3.3 Magnetic Capture Hybridization (MCH)-PCR
Interference caused by inhibitory compounds of seed extracts in conventional PCR
is a major limitation affecting both assay sensitivity and reliability. The magnetic
capture hybridization-PCR (MCH-PCR) has the advantage that the MCH process
puries and concentrates the DNA of interest while removing non-target DNA and
other substances that can inhibit the in vitro enzymatic manipulation of nucleic
acids that are normally found in complex sharing biological material. In MCH-
PCR, magnetic beads coated with single-stranded DNA probes are used to capture
DNA fragments which are further used for PCR amplication. This technique has
been successfully used to detect fungi, viruses and bacteria in materials containing
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
126
PCR inhibitory compounds (Jacobsen 1995). With the use of MCH-PCR, the detec-
tion sensitivity can be enhanced up to 10- to 100-fold as compared to conventional
PCR protocol.
Complex of three species of Botrytis, viz. B. aclada, B. allii and B. byssoidea, are
responsible for neck rot disease in onion. The pathogens of this disease are transmit-
ted by onion seeds. An MCH-PCR diagnostic protocol was employed for the rapid
and sensitive detection of B. aclada in onion seed samples. The DNA of B. aclada
could be detected using MCH-PCR diagnostic protocol, in aqueous solutions pre-
pared from seed extract with detection limit as 100fg of fungal DNA/ml. MCH-
PCR protocol was more sensitive and efcient than normal PCR, in detecting the
fungus B. aclada in seed lots with 4.8% and 9.9% infection in naturally infested
seeds. MCH-PCR detection assay could be completed within 24 hours against a
10–14-days period required in conventional methods to test onion seeds (Walcott
etal. 2004). Two important seed-borne pathogens of cucurbits could be detected by
employing a magnetic capture hybridization (MCH) multiplex real-time PCR assay.
This assay offered the improved simultaneous detection of two different pathogens
in cucurbit seed lots, viz. Acidovorax avenae subsp. citrulli, a causal agent of bacte-
rial fruit blotch, and Didymella bryoniae, a fungal incitant of gummy stem blight
disease (Ha etal. 2009).
5.3.3.4 Bio-PCR
Bio-PCR enables the enhancement of fungal biomass since seed-borne fungi gener-
ally infect the host seeds at very low concentration of inoculum, and therefore the
DNA of the fungal pathogen is not enough for the subsequent reactions, limiting the
use of conventional PCR-based detection. This technique was developed by Schaad
etal. (1995) primarily to detect a seed-borne bacterium from bean seed extracts.
Later, this technique was also proved to be efcient for detection of fungi (Munkvold
2009). Bio-PCR is mainly applied for fungi and bacteria. In this detection method,
a pre-assay incubation step is coupled with PCR process. Bio-PCR consists of the
preventive growth of non-target pathogens on selective medium and selective
increase in the biomass of target microorganisms, followed by DNA extraction and
amplication by PCR (Schaad etal. 1995).
Bio-PCR has been proven successful in the detection/identication of Tilletia
indica teliospores in wheat seed samples (Schaad etal. 1997). In Bio-PCR, standard
deep-freeze blotter method was utilized as pre-assay incubation to increase the fun-
gal biomass of Alternaria dauci and A. radicina from infected seeds of carrot and
was detected using of specic primers of A. dauci and A. radicina during the PCR
assay (Konstantinova etal. 2002).
Though Bio-PCR offers several advantages over conventional PCR assay, like it
is highly sensitive, eliminates PCR inhibitors and avoids false positives due to dead
cells since it detects live cells only (Marcinkowska 2002), there are some limitations
of Bio-PCR also, viz. if selective media are used, the method becomes more expen-
sive and pre-assay requires 5–7-days period to increase fungal growth, which sub-
stantially increases the time required for the completion of the assays (Mancini
etal. 2016).
R. Kumar et al.
127
5.3.3.5 Loop-Mediated Isothermal Amplification (LAMP)
Loop-mediated isothermal amplication (LAMP) is one of the novel nucleic acid
amplication technologies that enables the synthesis of large amounts of DNA in a
short period of time with high specicity. This technique was developed by Notomi
etal. (2000) as a simple, cost-effective and rapid method for the specic detection
of genomic DNA (Mancini etal. 2016). In the future it may be a potential alternative
to PCR, since LAMP protocol does not require thermocycler apparatus. LAMP uses
a pair of four or six oligonucleotide primers with eight binding sites hybridizing
specically to diverse areas of a target gene and a thermophilic DNA polymerase
from Geobacillus stearothermophilus for DNA amplication. Additionally, being a
highly specic diagnostic protocol, the amplication efciency of LAMP is
extremely high, which provides improved sensitivity, and can overcome the prob-
lem of inhibitors that usually adversely affect PCR methods (Fu etal. 2011). LAMP
products can be visualized by gel electrophoresis, using magnesium pyrophosphate,
which enhances precipitation of amplied DNA (Fukuta etal. 2003; Nie 2005),
with a real-time turbidity reader (Fukuta etal. 2004; Mori etal. 2004), or with the
addition of an intercalating dye, such as SYBR Green I, which produces a colour
change in the presence of target phytopathogen (Iwamoto etal. 2003; Mumford
etal. 2006).
Fusarium graminearum is the major causative agent among the species complex
of Fusarium head blight of small cereals and is potential producer of the mycotox-
ins, viz. deoxynivalenol, nivalenol and zearalenone. The pathogen could be detected
by employing LAMP assay, based on the gaoA gene (galactose oxidase) of Fusarium
graminearum. Amplication of DNA during the reaction was indirectly detected in
situ by using calcein uorescence as a marker, circumventing the use of time-
requiring electrophoretic analysis. The LAMP protocol was able to detect the pres-
ence ~2pg of puried target DNA per reaction within 30min, specically from
DNA of F. graminearum (Niessen and Vogel 2010).
Fusarium oxysporum f. sp. ciceris (Foc), the incitant of Fusarium wilt, is both a
soil-borne and seed-borne fungus. It is one of the most devastating pathogens of
chickpea, causing major economic losses ranging from 10% to 40% worldwide. It is
estimated to cause a 10–15% yield loss annually in India (Haware and Nene 1982).
A loop-mediated isothermal amplication (LAMP) assay was developed targeting
the elongation factor 1 alpha gene sequence for visual detection of Foc. The LAMP
reaction was optimal at 63°C for 60min. In the presence of hydroxynaphthol blue
(HNB) added before amplication, DNA of Foc developed a characteristic sky blue
colour, whereas this colour was absent in the DNA of six other plant pathogenic
fungi. Later, gel electrophoresis analysis conrmed the results obtained with LAMP
and HNB.The detection limit of this LAMP assay for Foc was 10fg of genomic
DNA per reaction, against 100pg of conventional PCR (Ghosh etal. 2015).
Karnal bunt in wheat caused by a fungus Tilletia indica is a quarantine disease,
and therefore timely and specic detection of the pathogen is very essential. The
pathogen could be detected specically with rapidity employing the loop-mediated
isothermal amplication (LAMP) at 62°C.Four major unique regions were identi-
ed in T. indica through analysis of alignment of the mitochondrial DNA of T.indica
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
128
and T. walkeri. Six LAMP primers designed from one of these major unique regions
in T. indica could amplify T. indica DNA.Among 17 isolates of T. indica, T. walkeri,
T. horrida, T. ehrhartae and T. caries, this protocol offered highly specic detection
of T. indica. Endpoint detection with the naked eye could be possible using the uo-
rescent chemical calcein. The diagnostic assay could be completed in 30min, offer-
ing similar sensitivity as with conventional PCR.The specicity issues that occurred
during PCR-based detection protocols due to the high DNA homology of T. indica
with other Tilletia species, especially T. walkeri, could be solved by employing this
technique for detection (Gao etal. 2016).
Phomopsis longicolla is an important seed-borne fungal pathogen responsible
for the deterioration in the seed quality of soybean. The pathogen could be detected
employing loop-mediated isothermal amplication (LAMP) diagnostic assay based
on transcription elongation factor 1-α (TEF1-α), identied as a suitable target for
the detection of P. longicolla. This LAMP diagnostic assay, with great specicity,
was capable to detect all 54 isolates of P. longicolla from the rest of the 41 isolates
of other fungi tested. Before the amplication of LAMP products, hydroxynaphthol
blue (HNB) was added, and a sky blue colour was only developed in the presence of
P. longicolla, while other fungal isolates failed to show colour change. The detec-
tion limit of the assay was 100pg/μL fungal DNA, and additionally the assay also
detected P. longicolla from diseased soybean tissues and residues from different
origins (Dai etal. 2016).
Anthracnose is a worldwide occurring fungal disease of soybean. This disease is
primarily caused by Colletotrichum truncatum. Rapid and direct detection of the
pathogen in diseased soybean tissues could be possible employing a loop-mediated
isothermal amplication (LAMP) assay. A pair of species-specic primers was
designed using the target gene Rpb1 (that codes for the large subunit of RNA poly-
merase II). During the screening, species-specic primers amplied the genomic
DNA of Colletotrichum truncatum at 62°C over 70min. The presence of C. trun-
catum could be conrmed by a yellow-green colour (visible to the unaided eye),
developed in LAMP reaction products after addition of SYBR Green I dye. This
Rpb1-Ct-LAMP assay could successfully diagnose soybean anthracnose in eld
samples collected from various locations of China and was able to detect C. trunca-
tum in soybean seeds from farmers’ markets, the detection limit being 100pg (per
μL genomic DNA of pathogen) (Tian etal. 2017).
5.3.3.6 Real-Time PCR
It is a laboratory technique of molecular biology based on PCR, which consists of
amplication and simultaneous detection or quantication of targeted DNA mole-
cule. Real-time PCR consists of coupling DNA amplication with uorescent sub-
stances which can be easily measured, giving an indirect measurement of DNA
amplication. This is the case of TaqMan (Heid etal. 1996; Taylor etal. 2001a)
where uorescence is directly linked to the excision of reporter dye molecules,
which is directly related to DNA amplication. In contrast to other detection tech-
niques, a much quicker and more sensitive, quantitative assay could be provided by
real-time PCR assays.
R. Kumar et al.
129
Detection through real-time PCR was reported for Didymella bryoniae, an incit-
ant of gummy stem blight of cucurbits (Ling etal. 2010). A real-time uorescent
polymerase chain reaction (PCR) assay was developed using SYBR Green chemis-
try to quantify three species of Botrytis, viz. B. aclada, B. allii and B. byssoidea,
associated with onion (Allium cepa) seed that are also able to induce neck rot of
onion bulbs (Chilvers etal. 2007). The nuclear ribosomal intergenic spacer (IGS)
regions of target and non-target Botrytis spp. were sequenced and aligned and used
to design a primer pair specic to B. aclada, B. allii and B. byssoidea. The primers
reliably detected 10fg of genomic DNA per PCR reaction extracted from pure cul-
tures of B. aclada and B. allii (Chilvers etal. 2007).
Simultaneous detection of Pantoea ananatis and Botrytis allii was performed in
onion seeds using magnetic capture hybridization and real-time PCR (Ha and
Walcott 2008). Montes-Borrego etal. (2011) have achieved real-time PCR quanti-
cation of Peronospora arborescens, the opium poppy downy mildew pathogen, in
seed stocks and symptomless infected plants. Ioos etal. (2012) have used duplex
real-time PCR tool for sensitive detection of the quarantine oomycete Plasmopara
halstedii in sunower seeds. A real-time PCR assay utilizing SYBR Green was
developed to detect V. dahliae associated with spinach seed (Duressa et al. 2012).
More recently, a multiplex TaqMan real-time PCR assay was developed for the
detection of spinach seed-borne pathogens, viz. Peronospora farinosa f. sp. spina-
ciae, Stemphylium botryosum, Verticillium dahliae and Cladosporium variabile,
that cause economically important diseases on spinach (Feng et al. 2014). They
tested TaqMan assays on DNA extracted from numerous isolates of the four target
pathogens, as well as a wide range of non-target, related fungi or oomycetes and
numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR
assays were evaluated by detecting two or three target pathogens simultaneously.
Singular and multiplex real-time PCR assays were also applied to DNA extracted
from bulked seed and single spinach seed (Feng etal. 2014). Fusarium oxysporum
f. sp. phaseoli is a devastating pathogen that can cause signicant economic losses
and can be introduced into elds through infested common bean (Phaseolus vul-
garis) seeds. Robust seed health testing methods can be helpful in preventing long-
distance dissemination of this pathogen by contaminated seeds. A rapid real-time
PCR assay (qPCR) protocol was developed for the detection and quantication of
Fusarium oxysporum f. sp. phaseoli in common bean seeds. SYBR Green and
TaqMan qPCR methods were compared directly using primers based on the Fop
virulence factor ftf1. Both qPCR assays detected infection in seed at low levels
(0.25%); however, the TaqMan assay was found more reliable at quantication than
the SYBR Green assay (Sousa etal. 2015). To ensure adequate specicity and sen-
sitivity and comparable amplication efciency of different pathogens in real-time
PCR assays, it is critical to choose the appropriate target DNA fragments todesign
the primers and probes (Mancini et al. 2016). Recently, a real-time PCR-based
marker was developed for the detection of teliospores of Tilletia indica in soil
(Gurjar etal. 2017).
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
130
5.3.3.7 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 etal.
2003). The nuclear ribosomal internal transcribed spacer (ITS) region is a recently
proposed DNA barcode marker for fungi (Schoch etal. 2012). Identication of
universal barcoding regions is important to detect seed-borne fungi. Internal tran-
scribed spacer (ITS) has been used as the primary barcode marker for fungi based
on its ability to successfully identify inter- and intraspecic variation among a
wide range of fungi. An ideal barcoding gene should be sufciently conserved to
be amplied with wide range of primers, however divergent enough to identify
closely related species. Other applications include, for example, identifying plant
leaves even when owers or fruits are not available and identifying insect larvae
(which may have fewer diagnostic characters than adults and are frequently not
well-known) (Kress etal. 2005). Barcoding regions of some important seed-borne
fungi are given in Table5.3.
5.3.4 Next-Generation Sequencing (NGS)
After its rst application in basic biological research, NGS technologies have been
extended to other elds of application, which have included plant disease diagnosis. As
NGS has been a valuable technique for the rapid identication of disease- causing
agents from infected plants, it can also be applied to the detection of fungal pathogens
in seeds. This technique has been applied to study the mycobiome of wheat seed, using
454 pyrosequencing, allowing the identication of several fungal genera (Nicolaisen
etal. 2014). In view of this technology’s great potential, the major sequencing plat-
forms used for genome and other sequencing applications, 454 sequencing, AB/SOLiD
technology and Illumina/Solexa sequencing are described below.
The rst NGS technology that was proposed by Roche for the market was 454 sequenc-
ing, which bypasses cloning steps by taking advantage of PCR emulsion, a highly efcient
in vitro DNA amplication method. It is based on colony sequencing and pyrosequencing.
The pyrosequencing approach is a sequencing-by- synthesis technique that measures the
release of pyrophosphate by producing light, due to the cleavage of oxyluciferin by lucif-
erase. Currently, the 454 platform can produce 80–120Mb of sequence in 200 to 300bp
reads in a 4h run (Morozova and Marra 2008; Barba etal. 2014).
AB/SOLiD technology is sequencing by oligonucleotide ligation and detection
(SOLiD). It depends on ligation-based chemistry with di-base labelled probes and
uses minimal starting material. Sequences are obtained by measuring serial ligation
of an oligonucleotide to the sequencing primer by a DNA ligase enzyme. Each
SOLiD run requires 5days and generates 3–4Gb of sequence data with an average
read length of 25–35bp (Mardis 2008; Morozova and Marra 2008).
Illumina/Solexa sequencing is similar to the Sanger-based methods, because it
uses terminator nucleotides incorporated by a DNA polymerase. However, Solexa
R. Kumar et al.
131
terminators are reversible, allowing continuation of polymerization after uorophore
detection and deactivation. Sheared DNA fragments are immobilized on a solid sur-
face (ow-cell channel), and solid-phase amplication is performed. At the end of
the sequencing run (4days), the sequence of each cluster is computed and subjected
to quality ltering to eliminate low-quality reads. A typical run yields about 40–50Mb
(typical read length of 50–300bp; Varshney etal. 2009; El-Metwally etal. 2014).
The availability of these NGS assays means that they should now be used to
examine the presence of pathogens on or in seeds, especially 454 sequencing that
has already been proven to identify fungi on seeds; they may be proved useful in the
future for routine seed diagnosis.
Table 5.3 Barcoding regions of some seed-borne fungi
S.
no. Barcoding regions Seed-borne fungi References
1. Translation elongation factor-1
alpha (TEF-1 alpha)
Fusarium spp. Amatulli etal.
(2010)
2. Intergenic spacer sequence
(IGS)
Fusarium verticillioides González-Jaen
etal. (2004)
3. Internal transcribed spacers
(ITS) region
Alternaria alternata, A. infectoria
and A. triticina
Links etal. (2014)
Peronospora arborescens Landa etal.
(2007)
Albugo candida Robideau etal.
(2011)
Pseudoperonospora cubensis Robideau etal.
(2011)
4. Lpv gene Phytophthora cinnamoni Kong etal. (2003)
5. Cytochrome C oxidase 1 Aspergillus spp. Geiser etal.
(2007)
Albugo candida Robideau etal.
(2011)
Penicillium spp. Seifert etal.
(2007)
Pseudoperonospora cubensis Robideau etal.
(2011)
6. Pot2 transposon Magnaporthe oryzae Kachroo etal.
(1994)
7. LSU regions Albugo candida Robideau etal.
(2011)
Pseudoperonospora cubensis Robideau etal.
(2011)
8. α-Tubulin sequences Septoria tritici and
Rhynchosporium secalis
Rohel etal. (1998)
9. NADH dehydrogenase Fusarium sp.Kamil etal.
(2015)
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
132
5.3.5 Other Newly Developed Diagnostic Techniques
5.3.5.1 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 identication of areas
with different activities (Braga etal. 2005; Rabelo etal. 2011). As fungi present on
the seeds have biological activity, this method can detect their presence on seeds.
5.3.5.2 Videometer Lab Instrument
One more recently developed tool called videometer lab instrument that can distin-
guish infected seeds from healthy seeds is a multispectral vision system also useful
to determine the colour, texture and chemical composition of seed surfaces (Boelt
etal. 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 etal. 2011). 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 etal. 2015).
5.4 Summary
Seed health has become an important quarantine issue, mainly in the international
movement of seeds and germplasm exchange. Thus, it is essential to make sure that
no potentially damaging pathogens are established on seeds. Conventional seed
detection methods including visual examination, selective media, seedling grow-out
assay and the serological assays have been used extensively, but all have limitations
like inefciency and sensitivity. The molecular methods have shown great potential
for improving pathogen detection in seeds as it embodies many of the key charac-
teristics including specicity, sensitivity, rapidity, ease of implementation, interpre-
tation and applicability. PCR and its modications including Bio-PCR and
MCH-PCR may offer opportunities to evade inhibitory compounds while improv-
ing detection of seed-borne pathogens. Further, reduced cost and more efciency
will ultimately allow DNA-based detection methods to replace the vast range of
seed detection assays presently engaged and will provide advanced detection abili-
ties essential for healthy seedling establishment. A comparative analysis of various
seed detection methods based on the time required for completion, sensitivity, ease
of application and specicity along with the examples of fungi detected on seeds
using the particular technique has been summarized below in Table5.4.
R. Kumar et al.
133
Table 5.4 General features of seed detection assays including the time required for completion, sensitivity, ease of application, specicity and applicability
for the detection of fungi on seed (Walcott 2003; Mancini etal. 2016)
Type of assay
Time
required Sensitivity
Ease of
application Specicity
Ease of
implementation Examples
Visual examination 5–10min Low Simple and
inexpensive
(requires
experience)
Low Mycological skills
required
Phomopsis spp., Cercospora kikuchii,
Peronospora manshurica/soybean seed;
Cylindrocladium parasiticum/peanut seed;
Colletotrichum dematium/chilli seed; Septoria
apii/celery seed
Seed washing
technique
10–30min Low Simple and
inexpensive
Low Mycological skills
required
Peronospora manshurica/soybean seed
Semi-selective media 2–14days Moderate Simple and
inexpensive
Low–
moderate
Mycological skills
required
Alternaria brassicicola (crucifer seeds)
Seedling grow-out
assay
2–3weeks Low Simple,
inexpensive
and robust
Low Mycological skills
required
Fungal seedling diseases caused by Alternaria,
Bipolaris, Fusarium, Pyricularia, etc.
Freeze blotter
incubation
1week Low/
moderate
Simple and
inexpensive
Moderate Mycological skills
required
Alternaria dauci, Alternaria radicina/carrot
seed; Leptosphaeria maculans/Brassicaceae seed
Agar medium
incubation
5–7days Low/
moderate
Simple and
inexpensive
Moderate Mycological skills
required
Alternaria dauci, Alternaria radicina, Alternaria
carotiincultae/carrot seed; Verticillium dahliae,
Fusarium spp./Cucurbitaceae seed; Botrytis spp./
onion seed
Serology-based assay 2–4h Moderate–
high
Simple,
moderately
expensive and
robust
Moderate–
high
Ease of
interpretation
Macrophomina phaseolina/cowpea seed
(continued)
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
134
Type of assay
Time
required Sensitivity
Ease of
application Specicity
Ease of
implementation Examples
Conventional DNA
extraction and PCR
5–6h High Complicated;
easy to
interpret,
expensive
Very high Molecular biology
skills required, ease
of interpretation
Alternaria brassicae, Leptosphaeria maculans/
Brassicaceae seed; Ascochyta lentis/lentil seed;
Alternaria radicina/carrot seed; Phoma
valerianella/lamb’s lettuce seed; Fusarium
oxysporum f. sp. basilici/basil seed
Bio-PCR (selective
target colony
enrichment followed
by PCR)
5–7days Very high Complicated,
expensive
Very high Molecular biology
skills required, ease
of interpretation
Alternaria dauci, Alternaria radicina/carrot
seed; Alternaria brassicae, Leptosphaeria
maculans/Brassicaceae seed; Ascochyta rabiei/
chickpea seed; Fusarium oxysporum f. sp.
lactucae/ lettuce seed
MCH-PCR (magnetic
capture hybridization
and PCR)
2–5h Very high Complicated,
expensive
Very high Molecular biology
skills required
Didymella bryoniae/Cucurbitaceae seed; Botrytis
spp./onion seed
Nested PCR 5–6h Very high Complicated,
expensive
High Molecular biology
skills required, ease
of interpretation
Colletotrichum lindemuthianum/bean seeds;
Fusarium oxysporum f. sp. lactucae/lettuce seeds
Real-time PCR 40–60min Very high Complicated,
expensive
Very high Molecular biology
skills required
Alternaria brassicae, Plasmodiophora brassicae/
Brassicaceae seed; Didymella bryoniae/
Cucurbitaceae seed; Botrytis spp./onion seed;
Verticillium dahliae/spinach seed; Colletotrichum
lindemuthianum/bean seed; Fusarium oxysporum
f. sp. basilici/basil seed
DNA microarrays 6hours Very high Complicated,
expensive
Very high Molecular biology
skills required
Botrytis cinerea, B. squamosa and Didymella
bryoniae
Laser biospeckle
technique
High High Complicated,
expensive
High Technological
skills required
Fusarium oxysporum, Aspergillus avus,
Sclerotinia spp./bean seed
Videometer High High Complicated,
expensive
High Technological
skills required
Stemphylium botryosum, Cladosporium spp.,
Fusarium spp., Verticillium spp., Alternaria
alternata/spinach seed
Table 5.4 (continued)
R. Kumar et al.
135
5.5 Challenges andFuture Directions
Besides the unique advantages offered by the various seed/plant disease detection
methods, each method has its own limitations. Before adopting these assays, it is
critical to rigorously evaluate their applicability, precision and accuracy in real-
world, high-throughput testing of naturally infested seeds. To ensure that these
assays work, they must be validated in stringent multilaboratory tests which evalu-
ate their reproducibility and repeatability. Only assays evaluated in this manner
should be considered for testing of commercial seeds.
5.6 Conclusion
In this chapter, we reviewed the currently existing methods for detection of seed-
borne fungal phytopathogens. Although the conventional methods are widely used
for the detection of seed-borne fungal phytopathogen presently, they are relatively
difcult to operate, require expert technicians and are time-consuming for data anal-
ysis. Ultimately, improved protocols based upon PCR, ELISA, etc. will be available
for the detection of all seed-borne pathogens and may supersede conventional
detection methods.
References
Afouda L, Wolf G, Wydra K (2009) Development of a sensitive serological method for specic
detection of latent infection of Macrophomina phaseolina in cowpea. J Phytopathol 157(1):15–
23. https://doi.org/10.1111/j.1439-0434.2008.01453.x
Agarwal VK, Sinclair JB (1997) Principles of seed pathology, 2nd edn. CRC Press, Boca Raton,
p539
Agarwal VK, Singh OV (1974) Routine testing of crop seeds for Fusarium moniliforme with a
selective medium. Seed Res 2:19–22
Agarwal VK, Srivastava AK (1981) A simpler technique for routine examination of rice seed lots
for rice bunt. Seed Technol News 11(3):1–2
Agarwal VK, Verma HS, Singh SB (1978) Techniques for detection of loose smut infection in
wheat seeds. Seed Technol News 8(3):1
Aggarwal R, Gupta S, Banerjee S etal (2011) Development of a SCAR marker for detection of
Bipolaris sorokiniana causing spot blotch of wheat. Can J Microbiol 57(11):934–942. https://
doi.org/10.1139/w11-089
Alborch L, Bragulat MR, Cabanes FJ (2010) Comparison of two selective culture media for
the detection of Fusarium infection in conventional and transgenic maize kernels. Lett Appl
Microbiol 50(3):270–275. https://doi.org/10.1111/j.1472-765X.2009.02787.x
Alves MDC, Pozza EA (2009) Scanning electron microscopy applied to seed-borne fungi exami-
nation. Microsc Res Tech 72(7):482–488. https://doi.org/10.1002/jemt.20695
Amatulli MT, Spadaro D, Gullino ML etal (2010) Molecular identication of Fusarium spp. asso-
ciated with bakanae disease of rice in Italy and assessment of their pathogenicity. Plant Pathol
59(5):839–844. https://doi.org/10.1111/j.1365-3059.2010.02319.x
Andrew S, Pitt JI (1986) Selective medium for isolation of Fusarium species and dematiaceous
hyphomycetes from cereals. Appl Environ Microbiol 51(6):1235–1238
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
136
Ball SFL, Reeves JC (1991) The application of new techniques in the rapid testing for seed-borne
pathogens. Plant Var Seeds 4:169–176
Banks JN, Cox SJ, Northway BJ (1993) Polyclonal and monoclonal antibodies to eld
and storage fungi. Int Biodeterior Biodegradation 32(1–3):137–144. https://doi.
org/10.1016/0964-8305(93)90046-5
Barba M, Czosnek H, Hadidi A (2014) Historical perspective, development and applications of
next-generation sequencing in plant virology. Viruses 6(1):106–136. https://doi.org/10.3390/
v6010106
Boelt B, Shrestha S, Salimi Z et al (2018) Multispectral imaging – a new tool in seed quality
assessment? Seed Sci Res 28(3):222–228. https://doi.org/10.1017/S0960258518000235
Booth C (1971) The genus Fusarium. Commonwealth Mycological Institute, Kew, p237
Braga RA Jr, Rabelo GF, Granato LR etal (2005) Detection of fungi in beans by the laser biospeckle
technique. Biosyst Eng 91(4):465–469. https://doi.org/10.1016/j.biosystemseng.2005.05.006
Chadha S, Gopalakrishna T (2006) Detection of Magnaporthe grisea in infested rice
seeds using polymerase chain reaction. J Appl Microbiol 100:1147–1153. https://doi.
org/10.1111/j.1365-2672.2006.02920.x
Chamberlain JS, Gibbs RA, Ranier JE etal (1988) Deletion screening of the Duchenne muscular
dystrophy locus via multiplex DNA amplication. Nucleic Acids Res 16(23):11141–11156.
https://doi.org/10.1093/nar/16.23.11141
Chang GH, Yu RC (1997) Rapid immunoassay of fungal mycelia in rice and corn. J Chin Agric
Chem Soc 35:533–539
Chen RS, Tsay JG, Huang YF etal (2002) Polymerase chain reaction-mediated characterization
of molds belonging to the Aspergillus avus group and detection of Aspergillus parasiticus in
peanut kernels by multiplex polymerase chain reaction. J Food Prot 65(5):840–844
Chen YY, Conner RL, Gillard CL etal (2007) A specic and sensitive method for the detection of
Colletotrichum lindemuthianum in dry bean tissue. Plant Dis 91(10):1271–1276. https://doi.
org/10.1094/PDIS-91-10-1271
Chilvers MI, du Toit LJ, Akamatsu H etal (2007) A real-time, quantitative PCR seed assay for
Botrytis spp. that cause neck rot of onion. Plant Dis 91(5):599–608. https://doi.org/10.1094/
PDIS-91-5-0599
Cilliers AJ, Swart WJ, Wingeld MJ (1994) Selective media for isolating Lasiodiplodia theobro-
mae. Plant Dis 78:1052–1055
Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immuno-
sorbent assay for the detection of plant viruses. J Gen Virol 34(3):475–483
Consolo VF, Albani CM, Berón CM etal (2009) A conventional PCR technique to detect Septoria
tritici in wheat seeds. Australas Plant Pathol 38(3):222–227. https://doi.org/10.1071/AP08099
Cunfer BM, Manandhar JB (1992) Use of a selective medium for isolation of Stagonospora nodo-
rum from barley seed. Phytopathology 82:788–791. https://doi.org/10.1094/Phyto-82-788
Dai T, Shen H, Zheng XB (2016) Establishment and evaluation of a TEF1-α based loop-mediated
isothermal amplication assay for detection of Phomopsis longicolla. Australas Plant Pathol
45(3):335–337. https://doi.org/10.1007/s13313-016-0415-6
De Rossi RL, Reis EM (2014) Semi-selective culture medium for Exserohilum turcicum isolation
from corn seeds. Summa Phytopathol 40(2):163–167. https://doi.org/10.1590/0100-5405/1925
de Temp J (1953) The blotter method of seed health testing programme. ISTA 28:133–151
Deshpande GD (1993) Development of medium for selective expression of Curvularia lunata in
sorghum seed health testing. J Maharashtra Agric Univ 18:142–143
Dewey FM (1992) Detection of plant-invading fungi by monoclonal antibodies. In: Duncan JM,
Torrance L (eds) Techniques for the rapid detection of plant pathogens. Blackwell Scientic
Publications, Oxford, pp47–63
Dhingra OD, Sediyama C, Carraro IM etal (1978) Behavior of four soybean cultivars to seed
infecting fungi in delayed harvest. Fitopatol Bras 3:277–382
Domsch KH, Gams W, Anderson TH (1980) Compendium of soil fungi, vol 1. Academic Press,
London, p860
R. Kumar et al.
137
Doyer LC (1938) Manual for the determination of seed-borne diseases. International Seed Testing
Association, Wageningen, p59
Duressa D, Rauscher G, Koike ST etal (2012) A real-time PCR assay for detection and quanti-
cation of Verticillium dahliae in spinach seed. Phytopathology 102(4):443–451. https://doi.
org/10.1094/PHYTO-10-11-0280
Edwards SG, Seddon B (2001) Selective media for the specic isolation and enumeration of
Botrytis cinerea conidia. Lett Appl Microbiol 32(2):63–66
Eibel P, Wolf GA, Koch E (2005a) Detection of Tilletia caries, causal agent of com-
mon bunt of wheat, by ELISA and PCR. J Phytopathol 153(5):297–306. https://doi.
org/10.1111/j.1439-0434.2005.00973.x
Eibel P, Wolf GA, Koch E (2005b) Development and evaluation of an enzyme-linked immunosor-
bent assay (ELISA) for detection of loose smut of barley (Ustilago nuda). Eur J Plant Pathol
111:113–124. https://doi.org/10.1007/s10658-004-1421-z
El-Metwally S, Osama OM, Mohamed H (2014) Next generation sequencing technologies and chal-
lenges in sequence assembly. Springer, NewYork. https://doi.org/10.1007/978-1-4939-0715-1
Ellis MB (1971) Dematiaceous Hyphomycetes, 1st edn. Commonwealth Mycological Institute,
Kew, p608. isbn-13: 978-0851986180
Fang Y, Ramasamy RP (2015) Current and prospective methods for plant disease detection.
Biosensors 5(3):537–561. https://doi.org/10.3390/bios5030537
Faria CB, Abe CA, da Silva CN etal (2012) New PCR assays for the identication of Fusarium
verticillioides, Fusarium subglutinans, and other species of the Gibberella fujikuroi complex.
Int J Mol Sci 13(1):115–132. https://doi.org/10.3390/ijms13010115
Feng C, Mansouri S, Bluhm BH et al (2014) Multiplex real-time PCR assays for detection of
four seed borne spinach pathogens. J Appl Microbiol 117(2):472–484. https://doi.org/10.1111/
jam.12541
Fountaine JM, Shaw MW, Napier B etal (2007) Application of real-time and multiplex polymerase
chain reaction assays to study leaf blotch epidemics in barley. Phytopathology 97:297–303
Fu G, Huang SL, Wei JG etal (2007) First record of Jatropha podagrica gummosis caused by
Botryodiplodia theobromae in China. Aust Plant Dis Notes 2(1):75–76. https://doi.org/10.1071/
DN07030
Fu S, Qu G, Guo S etal (2011) Applications of loop-mediated isothermal DNA amplication. Appl
Biochem Biotechnol 163(7):845–850. https://doi.org/10.1007/s12010-010-9088-8
Fukuta S, Iida T, Mizukami Y et al (2003) Detection of Japanese yam mosaic virus by
RT-LAMP.Arch Virol 148(9):1713–1720. https://doi.org/10.1007/s00705-003-0134-5
Fukuta S, Ohishi K, Yoshida K etal (2004) Development of immunocapture reverse transcription
loop-mediated isothermal amplication for the detection of Tomato spotted wilt virus from chry-
santhemum. J Virol Methods 121(1):49–55. https://doi.org/10.1016/j.jviromet.2004.05.016
Gao Y, Tan MK, Zhu YG (2016) Rapid and specic detection of Tilletia indica using loop-mediated
isothermal DNA amplication. Australas Plant Pathol 45(4):361–367. https://doi.org/10.1007/
s13313-016-0422-7
Gaur A (2011) An introduction to seed pathology. Galgotia Publications Pvt Ltd, New Delhi, p351
Geiser DM, Klich MA, Frisvad JC etal (2007) The current status of species recognition and iden-
tication in Aspergillus. Stud Mycol 59:1–10. https://doi.org/10.3114/sim.2007.59.01
Ghosh R, Nagavardhini A, Sengupta A etal (2015) Development of loop-mediated isothermal
amplication (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris– wilt
pathogen of chickpea. BMC Res Notes 8:40. https://doi.org/10.1186/s13104-015-0997-z
Gleason ML, Ghabrial SA, Ferriss RS (1987) Serological detection of Phomopsis longicolla in
soybean seeds. Phytopathology 77:371–375. https://doi.org/10.1094/Phyto-77-371
González-Jaen MT, Mirete S, Patino B etal (2004) Genetic markers for the analysis of variabil-
ity and for production of specic diagnostic sequences in Fumonisin-producing strains of
Fusarium verticillioides. Eur J Plant Pathol 110(5–6):525–532. https://doi.org/10.1023/B:E
JPP.0000032392.20106.81
Guillemette T, Iacomi-Vasilescu B, Simoneau P (2004) Conventional and real-time PCR based
assay for detecting pathogenic Alternaria brassicae in cruciferous seed. Plant Dis 88:490–496
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
138
Gurjar MS, Aggarwal R, Jogawat A etal (2017) Development of real time PCR assay for the detec-
tion and quantication of teliospores of Tilletia indica in soil. Indian J Exp Biol 55(6):549–554
Ha Y, Walcott RR (2008) Simultaneous detection of Pantoea ananatis and Botrytis allii in onion
seeds using magnetic capture hybridization and real-time PCR.Phytopathology 98:S64–S65
Ha Y, Fessehaie A, Ling KS etal (2009) Simultaneous detection of Acidovorax avenae subsp.
citrulli and Didymella bryoniae in cucurbit seedlots using magnetic capture hybridization and
real-time polymerase chain reaction. Phytopathology 99(6):666–678. https://doi.org/10.1094/
PHYTO-99-6-0666
Haware MP, Nene YL (1982) Races of Fusarium oxysporum f. sp ciceri. Plant Dis 66:809–810
Hebert PDN, Cywinska A, Ball SL etal (2003) Biological identications through DNA barcodes.
Proc R Soc Lond [Biol] 270(1512):313–321. https://doi.org/10.1098/rspb.2002.2218
Heid CA, Stevens J, Livak KJ etal (1996) Real time quantitative PCR.Genome Res 6(10):986–994
Hewett PD (1977) Pretreatment in seed health testing: hypochlorite in the 2,4-D-blotter for
Leptosphaeria maculans (Phoma lingam). Seed Sci Technol 5:599
Hiltner L (1917) Technische vorschriften fur die Prufung von saatgut, gultig vom I.Juli 1916 an B
Besonderer Teil I.Getreide, Landwirtsch, Verso SIn 89:379–383
Horst RK (2008) Westcott’s plant disease handbook, 7th edn. Springer, Dordrecht
Iacomi-Vasilescu B, Blancard D, Guenard M etal (2002) Development of a PCR-based diagnos-
tic assay for detecting pathogenic Alternaria species in cruciferous seeds. Seed Sci Technol
30:87–95
Ioos R, Fourrier C, Wilson V etal (2012) An optimized duplex real-time PCR tool for sensitive
detection of the quarantine oomycete Plasmopara halstedii in sunower seeds. Phytopathology
102(9):908–917. https://doi.org/10.1094/PHYTO-04-12-0068-R
ISTA (1966) International rules for seed testing. Proc Int Seed Test Assoc 31:1–152
Iwamoto T, Sonobe T, Hayashi K (2003) Loop-mediated isothermal amplication for direct detec-
tion of Mycobacterium tuberculosis complex, M. avium and M. intracellulare in sputum sam-
ples. J Clin Microbiol 41(6):2616–2622
Jacobsen CS (1995) Microscale detection of specic bacterial DNA in soil with a magnetic capture
hybridization and PCR amplication assay. Appl Environ Microbiol 61(9):3347–3352
Jung B, Lee S, Ha J et al (2013) Development of a selective medium for the fungal pathogen
Fusarium graminearum using toxoavin produced by the bacterial pathogen Burkholderia glu-
mae. Plant Pathol J 29(4):446–450. https://doi.org/10.5423/PPJ.NT.07.2013.0068
Kachroo P, Leong SA, Chattoo BB (1994) Pot2, an inverted repeat transposon from the rice
blast fungus Magnaporthe grisea. Mol Gen Genet 245(3):339–348. https://doi.org/10.1007/
BF00290114
Kamil D, Prameela Devi T, Prabhakaran N etal (2015) NADH dehydrogenase subunit 6: a suitable
secondary barcode for speciation of genus Fusarium. J Pure Appl Microbiol 9:545–551
Khare MN (1996) Methods to test seeds for associated fungi. Indian Phytopathol 49(4):319–328
Khare MN, Mathur SB, Neergaard P (1977) A seedling symptom test for detection of Septoria
nodorum in wheat seed. Seed Sci Technol 5:613–617
Kong P, Hong CX, Richardson PA (2003) Rapid detection of Phytophthora cinnamomi using PCR
with primers derived from the Lpv putative storage protein genes. Plant Pathol 52(6):681–693.
https://doi.org/10.1111/j.1365-3059.2003.00935.x
Konstantinova P, Bonants PJM, van Gent-Pelzer MPE etal (2002) Development of specic prim-
ers for detection and identication of Alternaria spp. in carrot material by PCR and com-
parison with blotter and plating assays. Mycol Res 106(1):23–33. https://doi.org/10.1017/
S0953756201005160
Kress WJ, Wurdack KJ, Zimmer EA etal (2005) Use of DNA barcodes to identify owering plants.
Proc Natl Acad Sci U S A 102(23):8369–8374. https://doi.org/10.1073/pnas.0503123102
Kulik MM (1975) Comparison of blotters and guaiacol agar for detection of Helminthosporium
oryzae and Trichoconis padwickii in rice seeds. Phytopathology 65(11):1325–1326
Kumar A, Singh A, Garg GK (1998) Development of seed immunoblot binding assay for the detec-
tion of Karnal bunt (Tilletia indica) of wheat. J Plant Biochem Biotechnol 7(2):119–120
R. Kumar et al.
139
Kumar K, Singh J, Khare A (2004) Detection, location, transmission and management of seed-
borne Colletotrichum dematium causing die-back and anthracnose in chilli. Farm Sci J
13(2):152–153
Kutilek V, Lee R, Kitto GB (2001) Development of immunochemical techniques for
detecting Karnal bunt in wheat. Food Agric Immunol 13(2):103–114. https://doi.
org/10.1080/09540100120055675
Landa BB, Montes-Borrego M, Munoz-Ledesma FJ etal (2007) Phylogenetic analysis of downy
mildew pathogens of opium poppy and PCR based in planta and seed detection of Peronospora
arborescens. Phytopathology 97(11):1380–1390. https://doi.org/10.1094/PHYTO-97-11-1380
Lee HK, Tewari JP, Turkington TK (1999) Histopathology and isolation of Rhynchosporium seca-
lis from infected barley seed. Seed Sci Technol 27(2):477–482
Lee HK, Tewari JP, Turkington TK (2001) A PCR-based assay to detect Rhynchosporium secalis in
barley seed. Plant Dis 85:220–225. https://doi.org/10.1094/PDIS.2001.85.2.220
Lee HK, Tewari JP, Turkington TK (2002) Quantication of seedborne infection by
Rhynchosporium secalis in barley using competitive PCR.Plant Pathol 51(2):217–224. https://
doi.org/10.1046/j.1365-3059.2002.00685.x
Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell, Ames, p388
Li S (2011) Phomopsis seed decay of soybean. In: Sudaric A (ed) Soybean– molecular aspects of
breeding. In Tech, Rijeka, pp277–292
Ling KS, Wechter WP, Somai BM etal (2010) An improved real-time PCR system for broad-
spectrum detection of Didymella bryoniae, the causal agent of gummy stem blight of cucurbits.
Seed Sci Technol 38(3):692–703. https://doi.org/10.15258/sst.2010.38.3.17
Links MG, Demeke T, Grafenhan T etal (2014) Simultaneous proling of seed-associated bac-
teria and fungi reveals antagonistic interactions between microorganisms within a shared epi-
phytic microbiome on Triticum and Brassica seeds. New Phytol 202(2):542–553. https://doi.
org/10.1111/nph.12693
Manandhar JB, Cunfer BM (1991) An improved selective medium for the assay of Septoria nodo-
rum from wheat seed. Phytopathology 81:771–773
Mancini V, Murolo S, Romanazzi G (2016) Diagnostic methods for detecting fungal pathogens on
vegetable seeds. Plant Pathol 65(5):691–703. https://doi.org/10.1111/ppa.12515
Mangan A (1971) A new method for the detection of Pleospora bjoerlingii infection of sugarbeet
seed. Trans Br Mycol Soc 57(1):169–172. https://doi.org/10.1016/S0007-1536(71)80096-7
Marcinkowska JZ (2002) Methods of nding and identication of pathogens in seeds. Plant Breed
Seed Sci 46(1):31–48
Mardis ER (2008) The impact of next-generation sequencing technology on genetics. Trends
Genet 24(3):133–141. https://doi.org/10.1016/j.tig.2007.12.007
Mbofung GCY, Pryor BM (2010) A PCR-based assay for detection of Fusarium oxysporum f. sp.
lactucae in lettuce seed. Plant Dis 94:860–866
Mehl HL, Epstein L (2007) Identication of Fusarium solani f. sp. cucurbitae race 1 and race 2
with PCR and production of disease-free pumpkin seeds. Plant Dis 91(10):1288–1292. https://
doi.org/10.1094/PDIS-91-10-1288
Mehl HL, Epstein L (2008) Sewage and community shower drains are environmental reservoirs
of Fusarium solani species complex group 1, a human and plant pathogen. Environ Microbiol
10(1):219–227. https://doi.org/10.1111/j.1462-2920.2007.01446.x
Mew T, Bride J, Hibino H etal (1988) Rice pathogens of quarantine importance. In: Proceedings
of international workshop on rice seed health. International Rice Research Institute, Los Banos,
pp101–115
Miller SA, Rittenburg JH, Peterson FP etal (1992) From research bench to the market place:
development of commercial diagnostic kits. In: Duncan JM, Torrance L (eds) Techniques for
the rapid detection of plant pathogens. Blackwell Scientic Publications, Oxford, pp208–221
Mirzaei S, Goltapeh EM, Shams-Bakhsh M etal (2008) Identication of Botrytis spp. on plants
grown in Iran. J Phytopathol 156(1):21–28. https://doi.org/10.1111/j.1439-0434.2007.01317.x
Molouba F, Guimier C, Berthier C (2001) Detection of bean seed-borne pathogens by PCR.Acta
Hortic 546:603–607. https://doi.org/10.17660/ActaHortic.2001.546.84
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
140
Montes-Borrego M, Munoz-Ledesma FJ, Jimenez-Díaz RM etal (2009) A nested-polymerase chain
reaction protocol for detection and population biology studies of Peronospora arborescens, the
downy mildew pathogen of opium poppy, using herbarium specimens and asymptomatic, fresh
plant tissues. Phytopathology 99(1):73–81. https://doi.org/10.1094/PHYTO-99-1-0073
Montes-Borrego M, Munoz-Ledesma FJ, Jimenez-Diaz RM etal (2011) Real-time PCR quan-
tication of Peronospora arborescens, the opium poppy downy mildew pathogen, in seed
stocks and symptomless infected plants. Plant Dis 95(2):143–152. https://doi.org/10.1094/
PDIS-07-10-0499
Mori Y, Kitao M, Tomita N et al (2004) Real-time turbidimetry of LAMP reaction for quanti-
fying template DNA.J Biochem Biophys Methods 59(2):145–157. https://doi.org/10.1016/j.
jbbm.2003.12.005
Morozova O, Marra MA (2008) Applications of next-generation sequencing technologies in func-
tional genomics. Genomics 92:255–264
Mumford R, Boonham N, Tomlinson J et al (2006) Advances in molecular phytodiagnostics–
new solutions for old problems. Eur J Plant Pathol 116(1):1–19. https://doi.org/10.1007/
s10658-006-9037-0
Munkvold GP (2009) Seed pathology progress in academia and industry. Annu Rev Phytopathol
47:285–311. https://doi.org/10.1146/annurev-phyto-080508-081916
Murakishi HH (1951) Purple seed stain of soybean. Phytopathology 41:305–318
Muthaiyan MC (2009) Principles and practices of plant quarantine. Allied Publishers Pvt. Ltd,
New Delhi, p577
Narayanasamy P (2005) Immunology in plant health and its impact on food safety. The Haworth
Press, NewYork
Nicolaisen M, Justesen AF, Knorr K etal (2014) Fungal communities in wheat grain show signi-
cant co-existence patterns among species. Fungal Ecol 11:145–153
Nie X (2005) Reverse transcription loop-mediated isothermal amplication of DNA for detection
of Potato virus Y. Plant Dis 89:605–610. https://doi.org/10.1094/PD-89-0605
Niessen L, Vogel RF (2010) Detection of Fusarium graminearum DNA using a loop-mediated
isothermal amplication (LAMP) assay. Int J Food Microbiol 140(2–3):183–191. https://doi.
org/10.1016/j.ijfoodmicro.2010.03.036
Notomi T, Okayama H, Masubuchi H et al (2000) Loop-mediated isothermal amplication of
DNA.Nucleic Acids Res 28(12):e63
Olesen MH, Carstensen JM, Boelt B (2011) Multispectral imaging as a potential tool for seed
health testing of spinach (Spinacia oleracea L.). Seed Sci Technol 39:140–150
Olesen MH, Nikneshan P, Shrestha S etal (2015) Viability prediction of Ricinus communis L. seeds
using multispectral imaging. Sensors 15(2):4592–4604. https://doi.org/10.3390/s150204592
Pastircak M (2007) Fungal diversity of winter wheat ears and seeds in Slovakia. In: Proceeding
of the cost Susvar Fusarium workshop: Fusarium diseases in cereals–potential impact from
sustainable cropping systems. Velence, Hungary, June 1–2, pp45–48
Paulsen MR (1990) Using machine vision to inspect oilseeds. Inform 1(1):50–55
Pryor BM, Gilbertson RL (2001) A PCR-based assay for detection of Alternaria radicina on carrot
seed. Plant Dis 85:18–23. https://doi.org/10.1094/PDIS.2001.85.1.18
Rabelo GF, Enes AM, Braga RA Jr etal (2011) Frequency response of biospeckle laser images
of bean seeds contaminated by fungi. Biosyst Eng 110(3):297–301. https://doi.org/10.1016/j.
biosystemseng.2011.09.002
Randall-Schadel BL, Bailey JE, Beute MK (2001) Seed transmission of Cylindrocladium parasiti-
cum in peanut. Plant Dis 85(4):362–370. https://doi.org/10.1094/PDIS.2001.85.4.362
Raper KB, Fennel DI (1965) The genus Aspergillus. Williams and Wilkins, Baltimore, p686
Robideau GP, De Cock AW, Coffey MD etal (2011) DNA barcoding of oomycetes with cyto-
chrome c oxidase subunit I and internal transcribed spacer. Mol Ecol Resour 11(6):1002–1011.
https://doi.org/10.1111/j.1755-0998.2011.03041.x
Rohel EA, Payne AC, Hall L et al (1998) Isolation and characterization of α-tubulin genes
from Septoria tritici and Rhynchosporium secalis, and comparative analysis of fungal
R. Kumar et al.
141
α-tubulin sequences. Cell Motil Cytoskeleton 41(3):247–253. https://doi.org/10.1002/
(SICI)1097-0169(1998)41:3
Rude SV, Duczek LJ, Seidle E (1999) The effect of Alternaria brassicae, Alternaria raphani
and Alternaria alternata on seed germination of Brassica rapa canola. Seed Sci Technol
27(2):795–798
Sachan IP, Agarwal VK (1995) Seed discolouration of rice: location of inoculum and inuence on
nutritional value. Indian Phytopathol 48(1):14–20
Schaad NW, Cheong SS, Tamaki S etal (1995) A combined biological and enzymatic amplica-
tion (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean extracts.
Phytopathology 85:243–248
Schaad NW, Bonde MR, Hatziloukas E (1997) Bio-PCR: a highly sensitive technique for detect-
ing seed-borne fungi and bacteria. In: Hutchins JP, Reeves JC (eds) Seed health testing. CAB
International, Wallingford, pp159–164
Schoch CL, Seifert KA, Huhndorf S etal (2012) Nuclear ribosomal internal transcribed spacer
(ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci USA
109(16):6241–6246. https://doi.org/10.1073/pnas.1117018109
Segalin M, Reis EM (2010) Semi-selective medium for Fusarium graminearum detec-
tion in seed samples. Summa Phytopathol 36(4):338–341. https://doi.org/10.1590/
S0100-54052010000400010
Seifert KA, Samson RA, de Waard JR etal (2007) Prospects for fungus identication using CO1
DNA barcodes, with Penicillium as a test case. Proc Natl Acad Sci USA 104(10):3901–3906.
https://doi.org/10.1073/pnas.0611691104
Singh D, Maheshwari VK (2001) Inuence of stack burn disease of paddy on seed health status.
Seed Res 29(2):205–209
Sint D, Raso L, Traugott M (2012) Advances in multiplex PCR: balancing primer efcien-
cies and improving detection success. Methods Ecol Evol 3(5):898–905. https://doi.
org/10.1111/j.2041-210X.2012.00215.x
Skvortzov S (1937) A simple method for detecting hyphae of loose smut in wheat grains. Zashch
Rast (Leningrad) 15:90–91
Sousa MV, Machado JC, Simmons HE etal (2015) Real-time quantitative PCR assays for the
rapid detection and quantication of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris
(common bean) seeds. Plant Pathol 64(2):478–488. https://doi.org/10.1111/ppa.12257
Sousa MV, Siqueira CS, Machado JC (2016) Conventional PCR for detection of
Corynespora cassiicola in soybean seeds. J Seed Sci 38(2):085–091. https://doi.
org/10.1590/2317-1545v38n2152049
Taylor E, Bates J, Kenyon D etal (2001a) Modern molecular methods for characterization and
diagnosis of seed-borne fungal pathogens. J Plant Pathol 83:75–81
Taylor EJA, Stevens EA, Bates JA et al (2001b) Rapid-cycle PCR detection of
Pyrenophora graminea from barley seed. Plant Pathol 50(3):347–355. https://doi.
org/10.1046/j.1365-3059.2001.00563.x
Thirumalaisamy PP, Singh DV, Aggrawal R etal (2011) Development of species-specic primers
for detection of Karnal bunt pathogen of wheat. Indian Phytopathol 64:164
Thompson RS, Aveling TAS, Blanco Prieto R (2013) A new semi-selective medium for Fusarium
graminearum, F. proliferatum, F. subglutinans and F. verticillioides in maize seed. S Afr J Bot
84:94–101
Tian Q, Lu C, Wang S etal (2017) Rapid diagnosis of soybean anthracnose caused by Colletotrichum
truncatum using a loop-mediated isothermal amplication (LAMP) assay. Eur J Plant Pathol
148(4):785–793. https://doi.org/10.1007/s10658-016-1132-2
Utobo EB, Ogbodo EN, Nwogbaga AC (2011) Seed borne mycoora associated with rice and their
inuence on growth at Abakaliki, Southeast Agro-Ecology, Nigeria. Lib Agric Res Cent J Int
2(2):79–84
Varshney RK, Nayak SN, May GD et al (2009) Next-generation sequencing technologies and
their implications for crop genetics and breeding. Trends Biotechnol 27(9):522–530. https://
doi.org/10.1016/j.tibtech.2009.05.006
5 Diagnosis andDetection ofSeed-Borne Fungal Phytopathogens
142
Venkataramana M, Shilpa P, Balakrishna K et al (2013) Incidence and multiplex PCR based
detection of trichothecene chemotypes of Fusarium culmorum isolates collected from freshly
harvested maize kernels in southern India. Braz J Microbiol 44(2):401–406. https://doi.
org/10.1590/S1517-83822013000200009
Waalwijk C, Kastelein P, de Vries I etal (2003) Major changes in Fusarium spp. in wheat in the
Netherlands. Eur J Plant Pathol 109:743–754. https://doi.org/10.1023/A:1026086510156
Walcott RR (2003) Detection of seed borne pathogens. HortTechnology 13(1):40–47
Walcott RR, McGee DC, Misra MK (1998) Detection of asymptomatic fungal infections of soy-
bean seeds by ultrasound analysis. Plant Dis 82:584–589
Walcott RR, Gitaitis RD, Langston DB (2004) Detection of Botrytis aclada in onion seed
using magnetic capture hybridization and the polymerase chain reaction. Seed Sci Technol
32(2):425–438. https://doi.org/10.15258/sst.2004.32.2.14
Warham EJ (1986) Karnal bunt disease of wheat: a literature review. Trop Pest Manage 32:229–242
Warham EJ, Butler LD, Sutton BC (1996) Seed testing of maize and wheat: a laboratory guide.
CIMMYT, Mexico, p84
Watanabe T (2002) Pictorial atlas of soil and seed fungi: morphologies of cultured fungi and key
to species, 2nd edn. CRC Press, Boca Raton, p506
Wu WS, Chen TW (1999) Development of a new selective medium for detecting Alternaria bras-
sicicola in cruciferous seeds. Seed Sci Technol 27:397–409
Zhang AW, Hartman GL, Curio-Penny B etal (1999) Molecular detection of Diaporthe phaseolo-
rum and Phomopsis longicolla from soybean seeds. Phytopathology 89(9):796–804. https://
doi.org/10.1094/PHYTO.1999.89.9.796
Zhang Z, Zhang J, Wang Y et al (2005) Molecular detection of Fusarium oxysporum f. sp.
niveum and Mycosphaerella melonis in infected plant tissues and soil. FEMS Microbiol Lett
249(1):39–47. https://doi.org/10.1016/j.femsle.2005.05.057
R. Kumar et al.
