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Plant Pathology (2016) Doi: 10.1111/ppa.12515

REVIEW

Diagnostic methods for detecting fungal pathogens on

vegetable seeds

V. Mancini, S. Murolo and G. Romanazzi*

Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, Via Brecce Bianche, 60131 Ancona, Italy

Early diagnosis of seedborne fungal pathogens is particularly important, as often, infected seeds appear symptomless;
seed diagnosis can avoid uncontrolled propagation of pathogens through long-distance exchange of such material. This
will prevent economic losses and unnecessary use of fungicides, so reducing costs and the introduction of toxic sub-
stances into the environment. Traditional techniques for detection of seedborne fungi are based on incubation and
grow-out methods. Although these are frequently used because of their simplicity of application, they are time-consum-
ing, require mycological skills, and are sometimes not sensitive enough to low levels of seed infection. Recently, new
identification techniques, based on DNA analysis, have been applied and are very efficient due to high sensitivity and
specificity. The most common technique is conventional PCR, while other recent techniques include nested PCR, to
obviate low levels of target pathogens, multiplex PCR, to detect several pathogens simultaneously, real-time PCR, to
quantify fungi on seeds, and magnetic-capture hybridization PCR. The main drawbacks of molecular methods are the
inability to distinguish between vital and non-vital inocula, and the difficulty in obtaining quality DNA template, due
to PCR inhibitors in seeds. To reduce inhibitory effects, several modified PCR protocols, such as loop-mediated isother-
mal amplification, and non-destructive testing methods have been developed. Loop-mediated isothermal amplification
and next-generation sequencing have been widely applied in nucleic acid analysis and, given the numerous advantages
provided, their application can be substantially extended in the future for detection of fungal pathogens in seeds.

Keywords: Alternaria spp., Botrytis spp., diagnosis, PCR, seedborne fungi

minimum requirements for adequate seed testing. Over

Introduction
the last few decades, the advent of serological and
The movement of plant germplasm and any other raw molecular tools has promoted efforts to set up assays
plant material can pose great risks for the spread of dis- with specific technical aspects (e.g. specificity, sensitiv-
eases and pests (Gergerich et al., 2015). In particular, ity, robustness) and economic demands (e.g. short diag-
seeds represent a particularly efficient vehicle to disperse nosis time, high-throughput, minimum taxonomic
seedborne pathogens. Unlike infected vegetative plant tis- expertise, minimum cost; Walcott, 2003; Lievens &
sues, infected seeds are in most cases symptomless; addi- Thomma, 2005).
tionally, low percentages of seed infection can result in Considering that accurate and rapid identification of
severe crop losses (Pellegrino et al., 2010), particularly if these organism(s) is essential to satisfy phytosanitary reg-
the pathogens are quarantine microorganisms. ulations, and consequently for effective disease control,
The close association of fungi with seeds facilitates the present review outlines and critically discusses the
long-term survival and widespread dissemination of such traditional and innovative diagnostic techniques that
pathogens. Many countries have formulated legislation have been applied to seedborne fungal pathogens.
that helps to limit or prevent the introduction of exotic
pathogens into new areas, and these are generally sup-
Conventional Fungal Detection Assays
ported by detection techniques. However, the low inocu-
lum levels and the often non-uniform distribution within
Visual examination
seed lots make the testing of seeds for pathogens a diffi-
cult task. Some fungal pathogens can cause symptoms on seeds
The detection approaches that are applied to fungal that are visible to the naked eye, or at least under low
seed pathogens have been developed using different magnification (Chen et al., 2007). Sometimes symptoms
technologies. However, few of these can satisfy the can be attributed to certain fungal species, such as dis-
colouration, shrivelling and cracks on soybean seeds that
are typical of Phomopsis spp. and Cercospora kikuchii
*E-mail: g.romanazzi@univpm.it (Murakishi, 2002; Li, 2011), and the speckles on peanut

ª 2016 British Society for Plant Pathology 1

2 V. Mancini et al.

seeds that are caused by Cylindrocladium parasiticum (El-Nagerabi & Elshafie, 2000; Ora et al., 2011), or 10–
(Randall-Schadel et al., 2001). 90% ethanol (Vallad et al., 2005).
In most cases, identification of a pathogen based only In blotting tests, low temperatures or chemicals can be
on such symptoms is not recommended, as these can be applied to prevent seed germination, which can interfere
common to several fungi (Andersen & Leach, 1961). with the growth of the seedborne fungi; these treatments
Examination of seeds under a stereoscopic microscope disrupt the vitality of the seed tissues (Ha et al., 2009).
allows detection of the presence of overwintering or For example, the sodium salt of the herbicide (2,4-
reproductive structures of fungi, such as the crust of oos- dichlorophenoxy) acetic acid monohydrate (2,4-D) is
pores of Peronospora manshurica on soybean seeds often used to moisten the filter paper in blotting tests
(Agarwal et al., 2006), the acervuli and microsclerotia of (Kumar et al., 2004; Alves & Pozza, 2009); the freeze
Colletotrichum dematium on chilli seeds (Kumar et al., blotter test includes an additional step where seeds are
2004), and the pycnidia of Septoria apii embedded in the left to soak for 24 h and then kept at 20 °C for 24 h
seed coat of celery seeds (Horst, 2008). However, when to kill the embryo (Du Toit et al., 2005; Elwakil et al.,
seeds that are infected by fungi do not show macroscopic 2009).
symptoms, no sort of visual inspection has a rational The agar media most commonly used for incubation
basis as a detection assay (Walcott, 2003). tests are the non-selective potato dextrose agar and malt
Seed washing techniques can be used in combination extract agar (Roy et al., 2000; Marcinkowska, 2002;
with visual examination and are useful for testing for Nasir, 2003; Ora et al., 2011). However, fungal repro-
surface-borne pathogenic fungi (Rao et al., 2006), or ductive structures are not always produced on these sub-
when seedborne fungi are present as microscopic spores strates. To stimulate fungal sporulation, a medium that
on the seed surface (Agarwal et al., 2006). These tech- is less rich in nutrients is more advisable, such as water
niques allow the removal of spores, hyphal elements, or agar or cornmeal agar (Roy et al., 2000; Afouda et al.,
other fungal structures from the seeds by washing and 2009).
stirring them in distilled water (Reeves, 1998). Following Acidified agars and selective and semiselective media
the washing, the resulting suspension can be decanted can be used to reduce bacterial contaminants (Pyndji
and examined under a stereomicroscope, or the pellet, et al., 1987) and saprophytic microorganisms, which can
obtained after a centrifugation, can be examined under a prevent the growth of the pathogens. Many selective and
compound microscope. The number of spores for a semiselective media have thus been developed for specific
known amount of seed can also be determined micro- seedborne fungi, through the addition of antibiotics, such
scopically with a haemocytometer counting chamber as streptomycin sulphate (Pellegrino et al., 2010) and
(Andersen & Leach, 1961). penicillin G (Peres et al., 2002; Nasir, 2003); fungicides,
such as benomyl (Soteros, 1979); other specific chemi-
cals, such as bromophenol blue (Peres et al., 2002); or
Incubation methods
inhibitory compounds (Marcinkowska, 2002; Walcott,
Incubation methods are the most frequently used tech- 2003). A selective medium has been developed specifi-
nique for detection of seed-transmitted pathogens, espe- cally for Fusarium oxysporum that reduces the develop-
cially for high-incidence fungi that can occur on seed ment of fungal contaminants and allows F. oxysporum
samples at a level >1%, also providing information colonies to grow rapidly (Komada, 1975; Chiocchetti
about the viability of the inoculum on the seeds (Marcin- et al., 1999; Vannacci et al., 1999; Garibaldi et al.,
kowska, 2002). Seeds can be incubated in Petri dishes 2004). Selective media are generally more practical for
that contain medium-impregnated filter paper (i.e. blot- fungal detection, which then do not require further steps
ting) or different agar media, under conditions that will for their identification.
promote fungal growth and sporulation. Such samples At the end of these incubation periods, the seeds are
are generally exposed to light–dark cycles of 12/12 h. individually examined under a stereomicroscope (Cap-
The times and temperatures for plate incubations vary pelli & Covarelli, 2005; Alves & Pozza, 2009) and the
depending on the fungal species, but generally this ranges fungal colonies that have developed on agar media can
from 2 to 10 days, with a temperature from 20 to 28 °C be identified according to macroscopic and microscopic
(Shovan et al., 2008; Elwakil et al., 2009; Naqvi et al., features (Peres et al., 2002; Pryor & Gilbertson, 2002;
2013). A standard sample for testing uses 400 seeds Ora et al., 2011).
(Marcinkowska, 2002; Rao et al., 2006), although this The blotter and agar-plate methods are simple and
will depend on the seed size. inexpensive ways to detect seedborne fungi that respond
Saprophytic microorganisms can often be present on by sporulation (Rao et al., 2006). However, the blotter
seed surfaces. To limit or eradicate them, the seeds can test does not always provide adequate conditions for the
be surface sterilized if the pathogen to be detected is development of mycelial growth, sporulation, or for the
inside the seed (El-Nagerabi & Elshafie, 2000; Du Toit symptoms of the pathogen to appear on the seeds. For
et al., 2005; Rodrigues & Menezes, 2005). Different sur- these characteristics, it is preferable to incubate the seeds
face disinfectants can be used for this, such as 0.5–1.0% on agar media, where as well as developing into charac-
sodium hypochlorite, which is the most common (Roy teristic colonies, greater numbers of fungal colonies can
et al., 2000; Peres et al., 2002), 0.1% mercuric chloride be formed (Marcinkowska, 2002; Nasir, 2003).

Plant Pathology (2016)

 Diagnostic methods for seedborne fungi 3

To overcome this problem, the cetyl trimethylammo-

Serological Fungal Detection Assays
nium bromide (CTAB) method can be applied with the
Immunological methods for the detection of seed- addition of particular chemical and enzymatic treat-
transmitted pathogens are based on the use of polyclonal ments (Terry et al., 2002). To remove polysaccharides
or monoclonal antibodies that specifically bind to a tar- and proteins, treatments with enzymes such as pecti-
get antigen, allowing the pathogen to be subsequently nase, cellulase, hemicellulose, a-amylase, proteinase K
detected by enzymatic conversion of substrates or using and glycoside hydrolases can be used (Rether et al.,
fluorescent tags. The immunological method most com- 1993; Demeke & Jenkins, 2010). Alternatively, the high
monly used is the double antibody sandwich–enzyme- levels of polysaccharides in soybean and chickpea seeds
linked immunosorbent assay (DAS-ELISA; Ward et al., during DNA extraction can be precipitated using high
2004). Serological assays do not require pure isolation of concentrations of NaCl and Sarcosyl (Sharma et al.,
the pathogen, and hence they are applicable to bio- 2002). There are several other methods for reducing the
trophic as well as necrotrophic seedborne pathogens. effect of PCR inhibitors. A silica matrix purification can
These assays are widely applied to detect seedborne be used instead of ethanol precipitation to recover the
viruses, but less so for fungal plant pathogens, due to the DNA. The DNA recovered from lettuce seeds using a
lack of species-specific antibodies. Furthermore, serology- silica matrix does not contain the brown pigments that
based assays can also detect non-viable pathogens, which are responsible for inhibition of DNA amplification,
can result in erroneous interpretations (Walcott, 2003; and the DNA of the target fungus, F. oxysporum f. sp.
Afouda et al., 2009). lactucae, can be consistently amplified without the
requirement for further dilution (Demeke & Jenkins,
2010; Mbofung & Pryor, 2010). Commercial DNA
Molecular Fungal Detection Assays
extraction kits can also efficiently remove most PCR
inhibitors (Ma & Michailides, 2007); this has allowed
Conventional PCR
the detection of several fungal pathogens from seeds,
PCR has numerous positive characteristics, including such as Ascochyta rabiei from chickpea seeds (Phan
rapidity, specificity, sensitivity and easy interpretation, et al., 2002), L. maculans from canola seeds (Chen
which make it suitable for detection of seedborne patho- et al., 2010), and Botrytis spp. from onion seeds (Chil-
gens (Walcott, 2003; Ward et al., 2004). vers et al., 2007). However, their cost is higher than
As a result of this great potential, over the past that of CTAB methods.
20 years, many PCR-based assays have been reported for Reagents can be added to PCR mixture buffers, to
the identification of seedborne pathogens; e.g. Ascochyta allow consistent amplification of the target DNA frag-
lentis from lentil seeds, Alternaria radicina from carrot ment from undiluted DNA extracts from seeds. To detect
seeds, Alternaria brassicae in cruciferous seeds, Lep- A. radicina on carrot seeds efficiently, Pryor & Gilbert-
tosphaeria maculans from canola seeds, and Phoma vale- son (2001) reported that the addition of 0.2% skimmed
rianella from lamb’s lettuce seeds (Hussain et al., 2000; milk in the PCR mixture allowed consistent amplification
Pryor & Gilbertson, 2001; Guillemette et al., 2004; of the target fungal DNA from the undiluted DNA
Landa et al., 2007; Chen et al., 2010; Pellegrino et al., obtained from infected seeds. Bovine serum albumin
2010). PCR-based methods also provide rapid and improves the sensitivity for the detection of F. oxyspo-
unequivocal identification of F. oxysporum f. sp. basilici rum f. sp. basilici from basil seeds (Pasquali et al., 2006)
from basil seeds, unlike the conventional detection and Alternaria alternata and A. radicina from infected
methods that cannot distinguish between pathogenic and carrot seeds (Konstantinova et al., 2002). The use of 8%
non-pathogenic F. oxysporum isolates (Baayen, 2000; glycerol has been reported to improve the amplification
Chiocchetti et al., 2001). efficiency and specificity of the detection of Diaporthe
One of the most important advantages that molecular- phaseolorum and Phomopsis longicolla in soybean seeds
based detection techniques have over conventional diag- (Zhang et al., 1999). The addition of bovine lacto
nostic methods is the ability, in principle, to distinguish transfer technique optimizer (BLOTTO) in PCR reaction
between closely related organisms. However, the pres- buffers, which comprises skimmed milk powder, phos-
ence of compounds within the seeds that can inhibit phate-buffered saline and sodium azide, can also attenu-
DNA amplification, resulting in false negatives, can be a ate inhibitory effects of polyphenolic and other
problem. To overcome these limitations, several modifi- compounds derived from plant tissues (De Boer et al.,
cations have been developed, starting from DNA extrac- 1995), although to date this has not been tested in
tion, to the amplification of the DNA. amplification reactions with DNA extracted from seeds.
The DNA recovered from seeds after phenol-chloro- However, while these reagents have proven to be effec-
form extraction and ethanol precipitation often contains tive for the elimination of the effects of PCR inhibitors,
PCR inhibitors that cannot be removed by repeated their usefulness and their optimal concentrations have to
washing with 70% ethanol or by repeated DNA precip- be tested in each case, as some of these reagents might
itation. Dilution of DNA extracts has been shown to themselves inhibit PCR amplifications when they are
eliminate the effects of PCR inhibitors, but it also used at concentrations that are too high (Wilson, 1997;
reduces the PCR sensitivity (Demeke & Jenkins, 2010). Koonjul et al., 1999).

Plant Pathology (2016)

4 V. Mancini et al.

PCR has numerous advantages, but the presence of increases the time required for completion of the assays
low levels of the target fungus on the seeds can be a (Walcott, 2003).
problem and may result in a false negative. The major
disadvantage of this technique is that it does not provide
Nested PCR
accurate information concerning the percentage of con-
taminated seeds, which is an important parameter for Another way to obviate the low levels of the target fun-
seed growers and seed trading companies. gus on seeds is the use of nested PCR. This procedure
can improve the sensitivity and specificity of the assay,
thus allowing detection of a target DNA at several-fold
BIO-PCR
lower levels than for conventional PCR (Chiocchetti
Seeds are often infested with fungi at very low levels, et al., 2001). Indeed, nested PCR has been used to detect
and consequently the DNA of the pathogen is not suffi- DNA levels of 10 fg for Colletotrichum lindemuthianum
cient for the subsequent reactions (De Boer et al., 1995). in bean seeds (Chen et al., 2007), and of 1 fg for F.
To overcome this problem, Schaad et al. (1995) devel- oxysporum f. sp. lactucae in lettuce seeds (Mbofung &
oped a highly sensitive PCR technique, named BIO-PCR. Pryor, 2010). However, this molecular assay is more
This consists of a pre-assay incubation step to increase labour intensive, more costly, and more prone to con-
the biomass of the fungal pathogen on the seeds, which tamination than conventional PCR (McCartney et al.,
is then followed by DNA extraction and amplification by 2003; Atkins & Clark, 2004; Tomlinson et al., 2005).
PCR. Initially, this technique was applied mainly for
phytopathogenic bacteria, as these are easily and rapidly
Real-time PCR
cultured over 2–3 days in a growth medium (Weller
et al., 2000); later, this was also shown to be effective Although conventional PCR is relatively sensitive and
for fungi (Munkvold, 2009). specific as well as being rapid, the main disadvantage is
Several incubation methods have been tested to facili- that it only gives qualitative data. Real-time PCR is able
tate rapid and consistent fungal growth, one of which to generate a specific fluorescent signal detected by an
includes the incubation of seeds in liquid fungal growth integrated fluorometer to provide real-time analysis of
medium (Phan et al., 2002). However, the fungal growth reaction kinetics and so allows quantification of specific
may be limited or suppressed by the presence of bacteria DNA targets (Atkins & Clark, 2004; Schena et al.,
and the addition of antibiotics to the substrate might not 2004; Ward et al., 2004).
always suppress bacterial growth, which thus decreases With limited sample manipulation, real-time PCR sig-
the reliability of the PCR assay (Pryor & Gilbertson, nificantly reduces the risk of false positives due to cross-
2001; Mbofung & Pryor, 2010). The use of specific contamination of the reaction mixtures (Cullen et al.,
media can avoid this problem, increasing the biomass of 2001; Gachon et al., 2004; Tomlinson et al., 2005). This
fungal pathogens, as seen for L. maculans and A. rabiei technique is less time-consuming than other assays (Guil-
from rapeseeds (Taylor, 1993) and chickpea seeds (Phan lemette et al., 2004; Chilvers et al., 2007), and is charac-
et al., 2002), respectively. terized by a high sensitivity in the order of magnitude of
Other pre-assay methods include an incubation on a few femtograms of DNA (Chen et al., 2013).
moistened filter paper, or just on the surface of a plastic When applied to infected seeds, quantitative real-time
Petri dish under conditions of high humidity, such as PCR assays, with the use of SYBR Green dye and Taq-
used to increase the low levels of target F. oxysporum f. Man-labelled probes, have shown high sensitivity for the
sp. lactucae on lettuce seeds (Mbofung & Pryor, 2010) detection and quantification of seedborne pathogens. For
and A. radicina on carrot seeds (Pryor & Gilbertson, example, quantitative real-time PCR has been used to
2001). The absence of nutrients allows the development detect and quantify Verticillium dahliae on spinach seeds
of fungal growth on the seed surface, with little or no (Duressa et al., 2012), A. brassicae on cruciferous
bacterial growth (Pryor & Gilbertson, 2001). Also, it is seeds (Guillemette et al., 2004), Botrytis spp. on onion
important to determine the incubation period precisely seeds (Chilvers et al., 2007), and C. lindemuthianum on
(Schaad & Frederick, 2002), in order to identify a com- dry bean seeds (Chen et al., 2013). Multiplex real-time
promise between growth of the target microorganism PCR uses multiple primers (together with probes in the
and limitation of other saprophytic fungi (Phan et al., TaqMan assay) in the same reaction, to reduce costs and
2002). labour. To ensure adequate specificity and sensitivity,
BIO-PCR has several advantages over traditional and comparable amplification efficiency of different
PCR: increased sensitivity, elimination of PCR inhibi- pathogens in real-time PCR assays, it is critical to choose
tors, and detection of viable cells only, thus avoiding the appropriate target DNA fragments for the design of
false positives due to detection of DNA from dead cells the primers and probes.
(Marcinkowska, 2002). The negative aspects of BIO-
PCR are that it is more expensive than conventional
Magnetic-capture hybridization PCR
PCR, especially if selective media are used (Schena
et al., 2004), and that it usually requires from 5 to Magnetic-capture hybridization (MCH)-PCR combines
7 days for the fungal growth, which significantly initial DNA extraction with a purification step that

Plant Pathology (2016)

 Diagnostic methods for seedborne fungi 5

includes hybridization with a single-stranded DNA probe analysis. Therefore, given the numerous advantages pro-
on magnetic beads, with the subsequent PCR amplifica- vided by LAMP, its application can be extended to a lar-
tion of the relevant DNA region. This technology can ger number of types of plant tissues, including seeds.
capture specific target DNA, thus facilitating detection of
specific DNA sequences by PCR (Jacobsen, 1995; Reeves,
Next-generation sequencing (NGS)
1998). MCH-PCR has been successfully used to detect
pathogenic microorganisms in materials that contain After a first application in basic biological research, NGS
PCR inhibitory compounds, e.g. Botrytis aclada in onion technologies have been extended to other fields of appli-
seeds (Walcott et al., 2004). Also, a MCH multiplex cation, which have included plant disease diagnosis.
real-time PCR assay has been developed to detect two Methods based on NGS technologies have been reported
different pathogens, Didymella bryoniae and Acidovorax for the identification and characterization, in particular,
avenae subsp. citrulli, from watermelon and melon seeds of viruses that affect plants, and less so for pathogenic
(Ha et al., 2009). However, the adoption of MCH-PCR bacteria and fungi, where a powerful and generic front-
is still limited, although, in addition to the above-men- line screen has been described (Adams et al., 2009,
tioned advantages of this application, its improved detec- 2013; Kreuze et al., 2009; Rwahnih et al., 2009; Cantu
tion of pathogens will facilitate future research. et al., 2011). The application of Life Sciences 454
sequencing allowed analysis of RNAs from a grapevine
showing Syrah decline symptoms, which revealed a mul-
Loop-mediated isothermal amplification
tiple virus infection, including a novel virus (Rwahnih
Loop-mediated isothermal amplification (LAMP) of et al., 2009). As NGS has been a valuable technique for
DNA is a recent technology that was developed by rapid identification of disease-causing agents from
Notomi et al. (2000) as a simple, cost-effective and rapid infected plants, it can also be applied to the detection of
method for specific detection of genomic DNA. LAMP fungal pathogens in seeds. This technique has been
uses a set of four or six primers and a thermophilic applied to study the mycobiome of wheat seed, using
DNA polymerase from Geobacillus stearothermophilus 454 pyrosequencing, allowing the identification of sev-
that has strand displacement activity to amplify DNA eral fungal genera (Nicolaisen et al., 2014). In view of
with high specificity and in less than 1 h (Mumford this technology’s great potential, the major sequencing
et al., 2006). This technology has several positive fea- platforms used for genome and other sequencing applica-
tures: all of the reactions can be carried out under tions, 454 sequencing, AB SOLiD technology and Illu-
isothermal conditions; it does not require expensive mina/Solexa sequencing, are described below.
equipment; and there are fewer preparation steps com- The first NGS technology that was proposed by Roche
pared to conventional PCR and real-time PCR assays. for the market was 454 sequencing, which bypasses clon-
Furthermore, as well as being highly specific, the amplifi- ing steps by taking advantage of PCR emulsion, a highly
cation efficiency of LAMP is extremely high, which pro- efficient in vitro DNA amplification method. It is based
vides improved sensitivity, and robustness to the on colony sequencing and pyrosequencing. The pyrose-
inhibitors that usually adversely affect PCR methods (Fu quencing approach is a sequencing-by-synthesis technique
et al., 2011). LAMP products can be visualized by gel that measures the release of pyrophosphate by producing
electrophoresis, by the use of magnesium pyrophosphate, light, due to the cleavage of oxyluciferin by luciferase.
which promotes precipitation of amplified DNA (Fukuta Currently, the 454 platform can produce 80–120 Mb of
et al., 2003; Nie, 2005), with a real-time turbidity reader sequence in 200- to 300-bp reads in a 4 h run (Moro-
(Fukuta et al., 2004; Mori et al., 2004; Thai et al., zova & Marra, 2008; Barba et al., 2014).
2004), or with the addition of an intercalating dye, such AB/SOLiD technology is sequencing by oligonucleotide
as SYBR Green I, which produces a colour change if the ligation and detection (SOLiD). It depends on ligation-
LAMP reaction is positive (Iwamoto et al., 2003; Mum- based chemistry with di-base labelled probes and uses
ford et al., 2006). minimal starting material. Sequences are obtained by
To date, the LAMP method has been applied in vari- measuring serial ligation of an oligonucleotide to the
ous assays for the diagnosis of bacterial and viral infec- sequencing primer by a DNA ligase enzyme. Each SOLiD
tions of humans and animals and the detection of plant run requires 5 days and generates 3–4 Gb of sequence
pathogenic bacteria and viruses in host tissues and in data with an average read length of 25–35 bp (Mardis,
insect vectors (Fukuta et al., 2003, 2004; Nie, 2005; 2008; Morozova & Marra, 2008).
Okuda et al., 2005). The first applications of LAMP for Illumina/Solexa sequencing is similar to the Sanger-
fungi were reported only recently. LAMP was very effec- based methods, because it uses terminator nucleotides
tive for the detection of Fusarium graminearum in total incorporated by a DNA polymerase. However, Solexa
genomic DNA isolated from ground wheat grains (Nies- terminators are reversible, allowing continuation of poly-
sen & Vogel, 2010) and from contaminated and germi- merization after fluorophore detection and deactivation.
nated wheat seeds (Abd-Elsalam et al., 2011), thus Sheared DNA fragments are immobilized on a solid sur-
demonstrating its use for early detection of toxigenic face (flow-cell channel), and solid-phase amplification is
Fusarium species in cereals. Over the last 10 years, the performed. At the end of the sequencing run (4 days),
LAMP technique has been widely applied in nucleic acid the sequence of each cluster is computed and subjected

Plant Pathology (2016)

6 V. Mancini et al.

to quality filtering to eliminate low quality reads. A typi- whereby up to 100 pathogens can be analysed simultane-
cal run yields about 40–50 Mb (typical read length of ously in a single run, and its rapid analysis, it is an
50–300 bp; Varshney et al., 2009; El-Metwally et al., attractive technology for laboratory-based testing of sam-
2014). ples (De Boer & L
opez, 2012).
The availability of these NGS assays means that they
should now be used to examine the presence of patho-
Diagnosis of Important Pathogens Transmitted
gens on or in seeds, especially 454 sequencing that has
by Seeds of Vegetable Crops
already been proven to identify fungi on seeds; they may
prove useful in future for routine seed diagnosis. In the following subsections, the conventional and
molecular diagnostic techniques will be examined for
each of the pathosystems given in Table 1.
Other newly developed diagnostic techniques
A recently applied tool that can reveal the presence of
Daucus carota/Alternaria dauci, Alternaria radicina
pathogenic fungi on seeds is known as the ‘biospeckle’
and Alternaria carotiincultae
laser technique. This technique is based on the optical
phenomenon of interference that is generated by a laser Alternaria leaf blight and alternaria black rot are the
light that interacts with the seed coat. Examination of most destructive of the diseases of carrot, and they are
seeds under laser light allows the identification of areas caused by Alternaria dauci and A. radicina, respectively.
with different activities (Braga et al., 2005; Rabelo et al., These diseases have spread to all carrot production areas
2011). As fungi present on the seeds have biological in the world, and they commonly occur when carrots are
activity, this method can detect their presence on seeds. cultivated under conditions of moderate temperatures,
Another tool that can distinguish infected seeds from where the leaves are exposed to prolonged periods of
healthy seeds is a multispectral vision system, e.g. wetness due to rainfall, dew or sprinkler irrigation
VideometerLab instrument, useful to determine the col- (Pryor, 2002; Rogers & Stevenson, 2010). Alternaria
our, texture and chemical composition of seed surfaces. carotiincultae is another species that was identified more
The combinations of the features from images captured recently on wild carrot; it is similar in many respects to
by visible light wavelengths and near-infrared wave- A. radicina, which causes similar disease symptoms (Sim-
lengths were valuable in the separation of healthy spi- mons, 1995).
nach seeds from seeds infected by Stemphylium The efficiency of methods for the detection of
botryosum, Cladosporium spp., Fusarium spp., Verticil- Alternaria spp. on carrot seeds is influenced by the loca-
lium spp. or A. alternata (Olesen et al., 2011). A similar tion of the pathogens on the seed, and by the presence of
approach has already been used in a study on malting saprophytic microorganisms. To suppress or eliminate
barley, to detect Fusarium spp. (S. Bodevin, Carlsberg non-target microorganisms, surface sterilization allows
Research Centre, Copenhagen, Denmark, personal com- more accurate assessment of the incidence of these
munication). pathogens. Rinsing carrot seeds in 0.1% sodium
Luminex has developed novel technology that, since hypochlorite has been reported to increase the recovery
2008, has been applied in plant pathogen diagnosis (Ishii of A. dauci in a 2,4-D blotter assay, and to reduce seed
et al., 2008). The Luminex PCR system is based on the saprophytes (Strandberg, 2002). However, external
hybridization between a biotin-labelled PCR product and decontamination can partially suppress the target fungi,
an oligonucleotide probe coupled to distinct beads that thus reducing its incidence. To overcome these draw-
specifically recognize differences in nucleotide sequences. backs, the application of heat treatment methods at
As laborious and time-consuming procedures are not 100 °C for 1 h allows the detection of high levels of seed
required, the potential of this technique is high and its infection without compromising the presence of patho-
application may be useful in the context of seed diagno- gens, while at the same time reducing the masking effects
sis. The Luminex xMAP system represents an alternative of the seed microflora (Soteros, 1979).
to ELISA; this technology is based on the use of anti- To allow fungal sporulation, and consequently the
body-coated paramagnetic microspheres (immunobeads) identification of some species, the standard deep-freeze
that are stained internally with fluorochromes. These blotter method for assaying carrot seeds for Alternaria
beads act as microscopic ELISA wells. Initially, these spp. infection is effective (Konstantinova et al., 2002;
techniques were applied in the clinical setting (De Boer Bulajic et al., 2009). The identification is based on the
& L
opez, 2012), and they are now used for plant pathol- different morphological features of the reproductive
ogy applications; e.g. for multiplex detection of seed- structures of these species: A. radicina forms non-catenu-
borne viruses and bacteria (Peters et al., 2007; late, multicellular, beakless conidia, while A. dauci forms
Bergervoet et al., 2008). This Luminex xMAP technology solitary, multicellular conidia with long beaks. The coni-
has been applied for seed detection of several viral dial dimensions of A. radicina are less than those of A.
pathogens, including Lettuce mosaic virus and Pepino dauci (Konstantinova et al., 2002; Pryor & Gilbertson,
mosaic virus (Van der Burg, 2009). To date, this technol- 2002; Farrar et al., 2004). The morphological features of
ogy has not been tested on fungal pathogens in seeds, A. carotiincultae differ from those of A. radicina: greater
but in view of its enormous multiplexing capacity, mean conidium length, fewer obovoid and subspherical

Plant Pathology (2016)

 Diagnostic methods for seedborne fungi 7

Table 1 Features of the different seed-detection assays and their effectiveness in detecting seedborne fungal pathogens of vegetable seeds

Diagnostic method Time required Sensitivity Specificity Ease of implementation Examples of pathogens detecteda

Visual examination Very high Low 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 Very high Low Low Mycological skills required Peronospora manshurica/soybean seed
technique
Freeze blotter Low Low/moderate Moderate Mycological skills required Alternaria dauci, Alternaria radicina/carrot
incubation seed; Leptosphaeria maculans/
Brassicaceae seed
Agar medium Low Low/moderate Moderate Mycological skills required Alternaria dauci, Alternaria radicina,
incubation Alternaria carotiincultae/carrot seed;
Verticillium dahliae, Fusarium spp./
Cucurbitaceae seed; Botrytis spp./onion
seed
Serology-based assay High Moderate/high Moderate/high Ease of interpretation Macrophomina phaseolina/cowpea seed
Conventional PCR Moderate/high High High Molecular biology skills Alternaria brassicae, Leptosphaeria
required, ease of maculans/Brassicaceae seed;
interpretation Ascochyta lentis/lentil seed; Alternaria
radicina/carrot seed; Phoma
valerianella/lamb’s lettuce seed;
Fusarium oxysporum f. sp. basilici/basil
seed
BIO-PCRb Moderate Very high High Molecular biology skills Alternaria dauci, Alternaria radicina/carrot
required, ease of seed; Alternaria brassicae,
interpretation Leptosphaeria maculans/Brassicaceae
seed; Ascochyta rabiei/chickpea seed;
Fusarium oxysporum f. sp. lactucae/
lettuce seed
Nested PCR Moderate Very high High Molecular biology skills Colletotrichum lindemuthianum/bean
required, ease of seeds; Fusarium oxysporum f. sp.
interpretation lactucae/lettuce seeds
Real-time PCR High Very high High Molecular biology skills Alternaria brassicae, Plasmodiophora
required 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
MCH-PCRc High Very high High Molecular biology skills Didymella bryoniae/Cucurbitaceae seed;
required Botrytis spp./onion seed
Laser biospeckle High High High Technological skills Fusarium oxysporum, Aspergillus flavus,
technique required Sclerotinia spp./bean seed
Videometer High High High Technological skills Stemphylium botryosum, Cladosporium
required spp., Fusarium spp., Verticillium spp.,
Alternaria alternata/spinach seed

a
All of these pathogens are referenced in the text.
b
BIO-PCR, with pre-PCR incubation step to increase biomass of the fungal pathogen.
c
MCH-PCR, magnetic-capture hybridization PCR.

conidia, and greater frequency of conidia that are pro- growth on selective media is subject to fewer variations
duced in chains of two, or less commonly, three (Pryor than methods based on the production and identification
& Gilbertson, 2002). of the conidia, such as the freeze blotter method (Pryor
The production of conidia by many Alternaria spp. is et al., 1994; Lopes & Martins, 2008). For the detection
subject to the influence of a number of environmental of A. dauci on infested carrot seeds, a modified agar
conditions, such as temperature and quantity and quality allows high levels of seed infection to be obtained, thus
of light. Thus, this can affect the sensitivity and repro- providing a more sensitive assay than the blotter method.
ducibility of seed assays performed on blotter paper. The Alternaria radicina can be identified through the use of
identification of Alternaria spp. based on its vegetative selective media created specifically for this species (Pryor

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8 V. Mancini et al.

et al., 1994) or the addition of selective fungicides and primer pairs that were designed on the basis of the
antibiotics to the medium (Strandberg, 2002). These sup- sequences of two clustered genes that are potentially
press the growth of seed saprophytes but not of the tar- involved in pathogenicity. Before the DNA extraction
get pathogen, thus eliminating the additional step of from seeds, an incubation step of a few days is advisable
pretreating the seeds before the assay. to increase the fungal biomass, using 2% malt extract,
Selective media based upon carrot leaf extracts com- 2% dextrose and 0.1% peptone liquid culture medium.
bined with fungicides and/or bactericides have been The mycelia and conidia can then be separated from the
designed for both A. radicina and A. dauci, although seeds and used for DNA extraction (Guillemette et al.,
these are particularly sensitive for the detection of A. 2004).
dauci on infested carrot seeds, through the promotion of To detect L. maculans in seed samples, the filter
profuse sporulation (Strandberg, 2002). Alternaria dauci paper/freeze method can be used, although PCR with a
colonies are brown or dark brown with olive-grey aerial species-specific primer pair is equally effective and less
mycelia, and in comparison to the other Alternaria spp., time-consuming (Mahuku et al., 1996; Chen et al.,
A. dauci colonies can be recognized by the production of 2010). BIO-PCR with a specific medium for L. maculans
a brown diffusible pigment in the medium (Lopes & can help to increase the biomass of this fungus on Brassi-
Martins, 2008). Alternaria radicina isolates grow slowly caceae seeds (Taylor, 1993).
with irregular colony margins and produce dendritic A real-time PCR-based protocol that uses specific pri-
crystals and a diffusible yellow pigment (Pryor & Gil- mers has been developed for the quantification of P.
bertson, 2002). brassicae resting spores that occur as external contami-
Although A. radicina and A. carotiincultae are very nants on canola seeds. Quantitative PCR analysis can be
similar species, they can be differentiated on the basis of performed on undiluted DNA extracted from the seeds,
their culture characteristics on acidified potato dextrose or after dilution to avoid problems due to PCR inhibitors
agar. Unlike colonies of A. radicina, those of A. carotiin- (Rennie et al., 2011).
cultae grow more rapidly and do not produce crystals or
pigment. These different characteristics might be asso-
Cucurbitaceae/Didymella bryoniae, Verticillium
ciated with the production of radicinin (which is sig-
dahliae and Fusarium spp.
nificantly higher in A. radicina isolates than A.
carotiincultae isolates), thus providing a useful diagnostic Gummy stem blight (black rot) is a serious foliar disease
characteristic (Park et al., 2008). that affects Cucurbitaceae crops. The disease is caused
In BIO-PCR, an incubation step of the seeds can by the fungus D. bryoniae, which produces lesions on
increase the fungal biomass. Then, the use of specific pri- the stems and leaves. Didymella bryoniae can also be
mers for A. dauci and A. radicina during the PCR assays is spread by seeds because it is located both externally on
highly sensitive, with detection of even low infection and internally in the seeds (Sudisha et al., 2006). Verti-
levels, and differentiation between these Alternaria spp. cillium dahliae and Fusarium spp. are destructive soil-
(Konstantinova et al., 2002). During the DNA extraction, borne and seedborne pathogens that can infect many
the use of a silica matrix allows higher yields of DNA to economically important agricultural crops worldwide,
be obtained. The target A. radicina DNA sequence is con- including Cucurbitaceae (Trionfetti Nisini et al., 2002;
sistently amplified if the DNA extracts from infested seeds Rampersad, 2008).
are diluted with Tris-EDTA (TE) buffer, or with the addi- PCR-ELISA has been used successfully to detect D. bry-
tion of skimmed milk to the PCR mixture (Pryor & Gil- oniae on crude extracts of fungal samples isolated from
bertson, 2001). Data obtained using the blotter method infected cucurbit plants. Although less sensitive than gel
and incubations on selective media are similar to those electrophoresis, PCR-ELISA is a highly specific, simple
obtained after the application of molecular methods, and rapid assay, and it can also be used to identify D. bry-
although the molecular methods are less time-consuming. oniae in cucurbit seed lots (Somai et al., 2002). To obtain
greater sensitivity, specific real-time PCR can be used
(Ling et al., 2010), and if other pathogens are present in
Brassica spp./Alternaria brassicae, Leptosphaeria
addition to D. bryoniae, MCH-multiplex real-time PCR
maculans and Plasmodiophora brassicae
can be applied to identify all of the microorganisms in
Alternaria brassicae and L. maculans are the causal agents these seeds. This last technique is 10-fold more sensitive
of black spot disease and blackleg disease, respectively. than direct real-time PCR (Ha et al., 2009).
These are important seedborne pathogens that affect Bras- Verticillium dahliae infections on seeds affect the seed
sica spp., and can cause serious reductions in crop yields coat, cotyledons and radicle (Maruthachalam et al.,
(Howlett et al., 2001; Lancaster, 2006). Another economi- 2013). For its isolation from pumpkin seeds, a semiselec-
cally important disease of the Brassicaceae family is club- tive medium can be used (Termorshuizen et al., 1998),
root, which is caused by the obligate parasite P. brassicae, and then the colonies obtained can be subcultured onto
a soilborne, and also seed-transmissible, pathogen (Dixon, potato dextrose agar medium for species identification
2009; Hwang et al., 2012). (Rampersad, 2010). Direct incubation of dissected water-
For the identification of A. brassicae, PCR and real- melon seeds (i.e. without the testa) on Komada’s selec-
time PCR assays have been applied, using two specific tive medium for Fusarium spp., and incubation of entire

Plant Pathology (2016)

 Diagnostic methods for seedborne fungi 9

seeds on the same medium or on 2% agar medium, (Mancini & Romanazzi, 2014). Seeds are highly vulnera-
allows both external and internal Fusarium spp. to be ble to infection and/or contamination, and seed diagnosis
detected. The incubation of seeds without the testa can be particularly difficult, because in most cases, seeds
allows the development of the endogenous Fusarium spp. do not show visible symptoms, unlike other plant tissues
in the seeds (Boughalleb & El Mahjoub, 2006). (Schaad et al., 2003).
Recently Cohen et al. (2014) found another pathogen Conventional seed detection assays that include visual
on cucurbits, Pseudoperonospora cubensis, which might examination and incubation methods have been exten-
be seedborne and seed-transmitted. Species-specific PCR sively used, especially in the past, before the development
assays have shown that P. cubensis occurs in the ovaries, of molecular diagnostic techniques. In recent years, these
fruit seed cavity, and seed embryos of cucurbits. molecular techniques have increasingly replaced the tra-
ditional methods, which have the drawback of being
time-consuming, laborious, and not always reliable.
Allium cepa/Botrytis spp.
Despite this, traditional methods can still be used to
Seven Botrytis spp. have been associated with diseases of ensure detection of particular fungal pathogens, with the
Allium crops: B. aclada, B. allii, B. byssoidea, B. cinerea, advantage that they are cheaper than the molecular alter-
B. porri, B. squamosal and B. tulipae (Mohan & natives. For example, to detect pathogenic species of
Schwartz, 2005). Botrytis aclada, B. allii, B. byssoidea Alternaria on infected carrot seeds, traditional methods
and B. porri are considered the primary causal agents of are still widely applied because these species show dis-
neck rot of onion, a disease that can develop during stor- tinctive morphological features that allow them to be
age and that can cause severe loss of onion bulbs. distinguished from each other. However, in many cases,
Although B. squamosa and B. tulipae have also been accurate identification of pathogenic organisms using
associated with neck rot, these species are not typically these traditional diagnostic assays is difficult, and these
the primary causes (Chilvers et al., 2004; Chilvers & Du methods normally only work well when the seed samples
Toit, 2006). contain high levels of the target pathogens. The applica-
For the determination of the health of onion seeds, tion of nucleic acid-based detection methods allows these
various procedures can be used to isolate Botrytis spp. shortcomings to be overcome, as these are more specific
The seeds can be incubated on agar medium and, to pre- and sensitive, and they provide data that are easy to
vent the onset of fast-growing fungi that can outgrow interpret in a short time. However, PCR inhibitors can
Botrytis spp. on or in the seeds, they can be plated on a limit the applicability of these molecular technologies,
selective medium such as Kritzman’s agar. This provides although the integration of conventional or real-time
several advantages over other media, such as freedom PCR with BIO-PCR and MCH-PCR allows this problem
from secondary contaminants and ease of identification to be avoided, and improves the detection of seedborne
of Botrytis spp. (Kritzman & Netzer, 1978). To detect pathogens.
propagules on seed surfaces, seeds can be rinsed under Seed health testing is a central issue in the context of
running deionized water for 1 h to remove the spores of the phytosanitary regulations set up by national govern-
fast-growing contaminating fungi. For an internal seed ments and by groups of countries, such as the European
assay, surface sterilization using a disinfectant such as Community, as a wide range of pests can be carried in
NaOCl is necessary before plating (Du Toit et al., 2004; seed lots. Sampling in seed health testing is a key factor
Chilvers & Du Toit, 2006). Colonies of Botrytis spp. (Morrison, 1999) and international organizations, such
obtained after seed incubations can be transferred to as the European and Mediterranean Plant Protection
acidified potato dextrose agar for species determination Organization (EPPO), the International Seed Health Ini-
(Du Toit et al., 2002). tiative (ISHI), the International Seed Testing Association
For rapid and sensitive detection of B. aclada in onion (ISTA) and the International Society for Plant Pathology
seeds, a MCH-PCR assay can be applied (Walcott et al., (ISPP), have the task of developing and approving stan-
2004). Real-time PCR using SYBR Green chemistry and dard methods for seed health testing and appropriate
primer pairs specific for B. aclada, B. allii and B. byssoi- seed sampling procedures, so that each test sample is as
dea can be used to quantify Botrytis spp. on onion seeds, homogeneous as possible and representative of each lot.
with the detection of as little as 10 fg genomic DNA To improve seed quality control, novel and efficient
extracted from pure cultures of B. aclada and B. allii methodologies, such as NGS and non-destructive testing
(Chilvers et al., 2007). methods, are increasingly emerging. Although these
methods are not yet widely used for seed diagnosis, due
to their great potential, they will probably be increas-
Concluding Remarks
ingly applied for the detection of pathogens on seeds.
The present review has covered the diagnostic methods
that can be used to detect and identify fungal pathogens
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