Detection of Seedborne

Pathogens
Ron R. Walcott

ADDITIONAL INDEX WORDS. PCR, MCH-PCR, IMS-PCR, BIO-PCR, Botrytis allii, bacterial fruit
blotch, Allium cepa, Acidovorax avenae subsp. citrulli, Citrullus lanatus

SUMMARY. Plant pathogens present a serious threat to seedling establishment and the potential
for plant disease epidemics under greenhouse conditions is great. Hence, pathogen exclusion
by detection and elimination of infested seedlots remains a requisite tactic for seedling produc-
tion and disease management. Unfortunately, the numbers of contaminated seed within a lot
may be low and infested seed may be asymptomatic making their detection difficult. To
address these issues seed detection assays have been developed, but many of them have short-
comings that reduce their effectiveness. Examples of frequently used seed assays include visual
examination, selective media, seedling grow-out and serological assays which, while appropri-
ate for some pathogens, often display inadequate levels of sensitivity and specificity. Recently,
the polymerase chain reaction (PCR) has emerged as a tool for detecting microorganisms in
many diverse environments. Thus far, it is clear that DNA-based detection systems exhibit
higher levels sensitivity than conventional techniques. Unfortunately, PCR-based seed tests
require the extraction of PCR-quality DNA from target organisms in backgrounds of
saprophytic organisms and inhibitory seed-derived compounds. The inability to efficiently
extract PCR-quality DNA from seeds has restricted the acceptance and application of PCR for
seed detection. To overcome these limitations several modified PCR protocols have been
developed including selective target colony enrichment followed by PCR (BIO-PCR) and
immunomagnetic separation and PCR. These techniques seek to selectively concentrate or
increase target organism populations to enhance detection and have been successfully applied
for detecting bacteria in seed. Other techniques with great potential for rapid detection of
seedborne pathogens include magnetic capture hybridization and PCR, and DNA-chip
technology. Ultimately, PCR will be available for the detection of all seedborne pathogens and
may supersede conventional detection methods.

S
eedborne pathogens present a serious threat to seedling estab-
lishment. Close association with seeds facilitates the long-term
survival, introduction into new areas and widespread dissemina-
tion of pathogens. Under greenhouse conditions, the risks of signifi-
cant economic losses due to diseases are great because factors includ-
ing high populations of susceptible plants, high relative humidity, high
temperatures and overhead irrigation, promote explosive plant disease
development. Under these conditions, the most effective disease man-
agement strategy is exclusion which is accomplished by using seed
detection assays to screen and eliminate infested seedlots before plant-
ing. The following will explore the current state of seed detection
technology and include recent advances. A summary of the features of
each assay is presented (Table 1).

Department of Plant Pathology, The University of Georgia, 4315 Miller Plant Sciences Building, Athens, GA 30602; e-mail
[email protected].

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 Conventional seed production of a bluish-green fluores- (McLaughlin and Chen, 1990) and
detection assays cent pigment on King’s B medium immunofluorescence microscopy
(King et al., 1956) in the case of fluo- (Franken, 1992). Serological assays do
Testing seeds for plant pathogens rescent Pseudomonas spp. or the pro- not require pure isolations of the patho-
can be a difficult task. Unlike infected duction of dark, muriform conidia in gen and, hence, are applicable to
vegetative plant tissues, infested seeds the case of Alternaria spp. Unfortu- biotrophic and necrotrophic seedborne
can be asymptomatic, making visual nately, seeds may be contaminated by pathogens. Currently serology is the
detection impossible. Additionally, saprophytic microorganisms (nonpatho- most widely used detection assay for
pathogen populations on seeds may be gens) that grow as well as, or better than seedborne viruses and it has proven to
low, and infested seeds may be target organisms on nutrient-rich, arti- be sensitive and robust (Barba, 1986;
nonuniformly distributed within a lot. ficial media. The excessive growth of Barba, 1986; Bossennec and Maury,
Many detection assays exist for different saprophytic organisms including 1978; Delecolle et al., 1985; Falk and
seedborne pathogens, however, few sat- Rhizopus spp., Penicillium spp., and Purcifull, 1983; Pasquini et al., 1998).
isfy the minimum requirements for ad- yeasts make it impossible to identify Serology has also been widely used for
equate seed tests. Ideally, seed assays pathogens that may be present. The the detection of bacterial and fungal
should be sensitive, specific, rapid, ro- inability to identify the unique charac- plant pathogens, but the unavailability
bust, inexpensive and simple to imple- teristics of the target pathogens in the of species-specific antibodies is a limi-
ment and interpret. Seed assays have presence of contaminating microor- tation (Franken et al., 1993; Frommel
been developed based on different tech- ganisms lead to inaccurate assessments and Pazos, 1994; Higley et al., 1993;
nologies including visual examination; of seedlot infestation. To overcome Linfield et al., 1995; McLaughlin and
selective media; seedling grow-out tests this problem, selective artificial media Chen, 1990; Rajeshwari et al., 1998; ).
and serological techniques. While these are developed that use antibiotics, fun- Additionally, the detection thresholds
tests have been used for many years, gicides, selected carbon and nitrogen of serology-based assays vary signifi-
some of them have shortcomings that sources and other inhibitory com- cantly based on the quality of the
make them less than ideal. Brief de- pounds to retard the growth of non- antibody and the testing format. Fi-
scriptions of these assays including their target microflora while allowing the nally, with serology-based assays it is
advantages and disadvantages are dis- pathogen to grow. Many selective and possible to detect nonviable patho-
cussed below. semiselective media have been devel- gens which results in erroneous (false-
VISUAL EXAMINATION. In some oped for seedborne fungi and bacteria positive) interpretation.
cases infected seeds display character- (Chang et al., 1991; Franken et al.,
istic symptoms, including discolora- 1991; Kritzman and Netzer, 1978; Seedling grow-out assay
tion and shriveling. Examples of such Lorbeer and Tichelaar, 1970; The seedling grow-out assay is a
seedborne diseases include purple seed Toussaint et al., 2001). Unfortunately, direct measure of the the seedlot’s
stain (Cercospora kikuchii) (Murakishi, development of such media is time ability to transmit a disease. To con-
2002), and advanced stages of consuming and requires specific knowl- duct this assay, seedlot samples are
Phomopsis seed decay (Phomopsis edge of the nutritional requirements planted under greenhouse conditions
longicolla) of soybean (Glycine max), and chemical tolerances of the target conducive to disease development and
and Cylindrocladium black rot organism, relative to the nontarget after germination, seedlings are ob-
(Cylindrocladium parasiticum) of pea- seed microflora. Employing selective served for the development of symp-
nut (Arachis hypogeae) (Randall- media also requires 2 to 4 d for patho- toms. Seedling grow-out is one of the
Schadel et al., 2001). In these cases gen growth and the test operator must most applicable and widely used seed
seedlot infestation can be reduced by be familiar with the range of cultural detection assays (Capoor et al., 1986;
using automatic devices that sort seeds characteristics associated with the Lee et al., 1990; Yang et al., 1997) but
based on visual of physical characteris- pathogen. Finally, while selective me- for successful implementation, infected
tics (Paulsen, 2002; Walcott et al., dia can be applied for certain bacteria seedlings must display obvious and
1998). These systems usually display and fungi, it cannot be applied for characteristic symptoms. Unfortu-
low detection sensitivity. Additionally, nonculturable obligate parasites, e.g., nately, this is not always the case as
seeds infested by fungi, bacteria and viruses, nematodes and certain fungi some diseases have nondistinct symp-
viruses may display no macroscopic and bacteria. toms, e.g., wilting, chlorosis etc. An-
symptoms, making visual or physical other drawback of the seedling grow-
inspection of seeds useless as a detec- Serology-based assays out assay is that large seed samples
tion assay. Serological seed assays rely on (10,000 to 50,000 seeds in the case of
S ELECTIVE MEDIA . A direct antibodies (polyclonal or monoclonal) bacterial fruit blotch (Acidovorax
method of testing seeds is by allowing generated against unique antigens on avenae subsp. citrulli) of watermelon
pathogens to grow from them onto the surfaces of plant pathogens (Hamp- (Citrullus lanatus) must be tested to
appropriate artificial media. This can ton et al., 1990). Antibodies bind statistically ensure that one infested
be done by directly plating surface- strongly and specifically to their anti- seed can be detected. In addition to
sterilized seed samples or seed-wash gens and can subsequently be detected losses associated with the destructive
liquid onto artificial media, followed by the enzymatic digestion of sub- testing of expensive seeds, assaying
by incubation under adequate condi- strates or fluorescent tags. Serology- this quantity of seeds requires large
tions. Once a pathogen is isolated it based seed tests have several formats areas of greenhouse space and adequate
can be identified by its cultural or including the widely applied enzyme- labor for assay set up and evaluation.
biochemical characteristics e.g. the linked immunosorbent assay (ELISA) The seedling grow-out assay is also

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 Table 1. General features of seed detection assays including the time required for completion, sensitivity, ease of application,
specificity, and applicability for the detection of fungi, bacteria and viruses.

Ease
Assay Time of
specificity required Sensitivity application Specificity
Visual examination 5–10 min Low Simple and inexpensive (requires experience) Low
Semiselective media 2–14 d Moderate Simple and inexpensive Low–moderate
Seedling grow-out assay 2–3 weeks Low Simple, inexpensive and robust low
Serology-based detection 2–4 h Moderate–high Simple, moderately expensive and robust Moderate–high
Conventional DNA extraction and
polymerase chain reaction (PCR) 5–6 h High Complicated; easy to interpret expensive Very high
BIO-PCR (selective target colony
enrichment followed by PCR) 3–4 d Very high Complicated, expensive Very high
IMS-PCR (immunomagnetic
separation and PCR) 2–5 h Very high Complicated, expensive very high
MCH-PCR (magnetic capture
hybridization and PCR) 2–5 h Very high Complicated expensive Very high
Real-time PCR 40–60 min Very high Complicated, expensive Very high
DNA microarrays 6h Very high Complicated; very expensive Very high

time consuming, requiring up to 3 and direct amplification of millions of sitivity and efficiency, but also require
weeks for seedling germination and copies of the target sequence. This the use of potentially harmful chemicals
symptom development. Finally, seed amplified DNA can be visualized after e.g. chloroform and phenol. Finally,
test evaluators must be familiar with electrophoresis in ethidium bromide- DNA from nonviable cells or tissues can
the symptoms associated with each stained agarose gels. PCR has many yield false-positive results in seed assays,
disease. This can be difficult since each beneficial characteristics that make it so it is necessary to confirm positive
disease has a range of possible symp- highly applicable for detecting results by recovering the target organ-
toms that are influenced by environ- seedborne pathogens. These include ism. Management decisions based on
mental conditions. Hence, for the seed- speed (completed within 2 to 3 h); these types of errors may result in the
ling grow-out assay, greenhouse con- specificity (DNA probes can be de- unnecessary destruction of healthy seed-
ditions must be strictly regulated to signed to amplify nucleic acids from lings and subsequent financial losses for
ensure consistent results. In large the desired genus, species, subspecies, commercial seedling producers.
greenhouses this can be a challenge race, etc.); sensitivity (single copies of To unlock the invaluable poten-
and it can lead to erroneous test re- nucleic acids can be detected after tial of PCR for detecting seedborne
sults. Also, because of the variations in amplification) and easy and objective pathogens, several modifications have
seedling symptom expression it is of- result interpretation (the presence of a been developed to specifically over-
ten necessary to isolate the pathogen DNA fragment of specific size indi- come the above-mentioned limitations.
from suspected seedlings for confir- cates the presence of the pathogen). These include enrichment or BIO-
mation. These extra steps further pro- Because of this great potential, over PCR, immunomagnetic separation
long the time required to complete the the past 10 years many PCR-based (IMS) and magnetic capture-hybrid-
seedling grow-out assay. Residual con- assays have been reported for seedborne ization (MCH). The following discus-
tamination and cross-contamination pathogens (Audy et al., 1996; Frederick sion will describe these techniques.
between spatially separated seedlots et al., 2002; Hadas et al., 2001; Hussain BIO-PCR. Target cell enrichment
are also issues of concern under green- et al., 2000; Pasquini et al., 1998; followed by PCR or BIO-PCR im-
house conditions. Prosen et al., 1993; Zhang et al., 1999). proves the efficiency and sensitivity of
Despite the potential improvements PCR by allowing target pathogen
Polymerase chain over conventional assays, PCR-based populations to increase in a
reaction-based seed seed assays have not been widely preenrichment phase, before DNA ex-
adopted by commercial and govern- traction and PCR. Selective preenrich-
detection assays
ment testing agencies in the U.S. One ment increases pathogen populations
Polymerase chain reaction (PCR) reason for this lack of acceptance is relative to nontarget microorganisms
is the in-vitro, primer-directed, enzy- that many seed types contain com- and results in higher quantities of tar-
matic amplification of nucleic acids pounds (e.g., tannins, phenolic com- get DNA, which ultimately results in
(Erlich et al., 1988; Saiki et al., 1988). pounds, phenolic compounds) that higher sensitivity. Additionally, dur-
This technique has been used in many inhibit DNA amplification resulting in ing incubation and enrichment on ar-
diverse applications including diagno- false-negative results when PCR is at- tificial media, inhibitory compounds
sis of plant diseases. For PCR, primers tempted directly on seed extracts. To are adsorbed or diluted during cell
(small oligonucleotide probes) de- circumvent the inhibitory effects of harvest, and do not interfere with DNA
signed to anneal to specific DNA se- seed compounds, elaborate DNA ex- amplification. For this technique, seed
quences in the target organism’s chro- traction and purification steps are em- samples are washed or crushed in an
mosomal DNA or RNA, hybridize with ployed, that not only reduce assay sen- appropriate buffer to extract seedborne

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 Applicability for
Comments Fungi Bacteria Viruses
Relies on presence of symptoms on seeds that may not always be present, low reliability X X X
Many good selective media exist but they may be difficult to develop X X
Requires large areas of greenhouse space and ability to recognize symptoms X X X
Very reliable and widely used for viruses; however, sensitivity and specificity is of concern for bacteria X X X

Affected by inhibitors in seeds; lengthy DNA extraction procedures are required X X X

Cannot be applied to obligate parasites X X

Highly sensitive but requires polyclonal antibodies X X X

Applicable to all pathogens but subject to false positives due to detection of DNA from nonviable pathogens X X X
While actual PCR time is short, DNA extraction is still required X X X
Will allow simultaneous testing for multiple pathogens; many of the same limitations as PCR X X X

bacteria. Aliquots of the seed wash are medium for each pathogen. As men- watermelon seeds (Walcott and Gitaitis,
spread onto semiselective media and tioned earlier, semiselective media re- 2000). Bacterial fruit blotch poses a
incubated for 2 to 3 d. Colonies are quire specific knowledge about the nu- perennial threat to the US watermelon
then harvested and after DNA extrac- tritional requirements and chemical seed and transplant industry (Latin
tion, PCR is conducted with specific tolerances of the target organism and and Hopkins, 1995). To use IMS,
primers. In the case of seedborne fungi, usually take time to develop. Fortu- seeds are washed or crushed in buffer
seeds are incubated under conditions nately, in the case of BIO-PCR, there and seed debris is removed. Bacteria in
of high relative humidity to increase is no need to identify the pathogen the seed wash are pelleted by centrifu-
target fungus mycelial mass before based on colony morphology since gation and resuspended in buffer. IMBs
DNA extraction and PCR (Pryor and specific PCR primers are used. As such coated with specific antibodies are in-
Gilbertson, 2001). it is only necessary for the selective cubated with the seedwash, during
BIO-PCR has been developed for media to retard the growth of nontar- which time the antibodies bind target
the detection of bacterial fruit blotch get organisms. BIO-PCR also requires bacteria. After immunocapture, the
of watermelon, halo blight (Pseudomo- two to three days for bacteria and 5 to IMBs are immobilized with a magnet
nas syringae subsp. phaseolicola) of 7 d for fungi to grow, significantly and washed thoroughly to eliminate
beans (Phaseolus vulgaris), bacterial increasing the time required for assay inhibitors and nontarget bacteria. IMBs
ring rot (Clavibacter michiganensis completion. Another critical drawback can then be spread onto selective agar
subsp. sepidonicum) of potato of BIO-PCR is that it cannot be used medium and incubated until bacterial
(Solanum tuberosum) and black rot (Al- for obligate parasites (e.g., viruses). As colonies can be observed. Alternatively
ternaria radicina) of carrot (Daucus such, it is limited primarily to readily captured bacteria can be lysed by boil-
carota) (Pryor and Gilbertson, 2001; culturable bacteria and fungi. ing to release DNA that can be used for
Schaad et al., 1995, 1999). This tech- IMMUNOMAGNETIC SEPARATION PCR.
nique has been reported to significantly AND PCR (IMS-PCR). Immunomagnetic IMS-PCR significantly improves
improve the sensitivity and the ease of separation refers to the use of micro- the detection efficiency and sensitivity
implementation of PCR, displaying de- scopic magnetic beads (IMBs) coated over conventional PCR (Walcott and
tection limits of 2 to 3 cfu/mL (Schaad with antibodies produced against a Gitaitis, 2000). IMS consistently re-
et al., 1999). The advantages of BIO- specific microorganism, to selectively covered viable target colonies from
PCR have also been demonstrated in a sequester target cells from suspensions suspensions containing 10 target cfu/
comparison between direct PCR, double containing heterogenous cell mixtures mL. Additionally, at least 10-fold more
antibody sandwich ELISA and (Olsvik et al., 1994; Safarik and CFUs were recovered by IMS than by
semiselective media (Wang et al., 1999) Safarikova, 1999). Captured cells can direct spread-plating. The frequency
for the detection of Xanthomonas be recovered on selective media or at which IMS-PCR could detect sus-
albilineans, the causal agent of leaf scald DNA can be extracted from them and pensions with 10 target cfu/mL was
of sugar cane (Saccharum spp.). Finally, used for PCR. This technique has been 43%, however this improved to 83%
with BIO-PCR the target organism must employed widely for the detection of for suspensions with 10,000 cfu/mL.
grow on the selective medium before it microorganisms from a variety of back- Finally, IMS-PCR proved to be more
can be detected by PCR. Hence, only grounds including food, and feces sensitive and reliable than
viable colonies are detected as opposed (Vernozy-Rozand et al., 1997; hexacetyldimethylethyl ammonium
to DNA from nonviable cells. Widjojoatmodjo et al., 1992). Recently bromide (CTAB)-DNA extraction
The disadvantages of BIO-PCR IMS-PCR was developed for the de- (Ausubel et al., 1987) followed by
include the need for a semiselective tection of A. avenae subsp. citrulli in direct PCR and ELISA for the detec-

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 tion of A. avenae subsp. citrulli in interest in the epidemiological signifi- tection assay. The benefits of this assay
watermelon seedlots. IMS-PCR also cance of seed borne inoculum. This include applicability to all pathogens;
facilitated the detection of A. avenae has prompted the search for more ac- rapidity, since it can be completed
subsp. citrulli in seedlots with 0%, 1%, curate and sensitive seed assays. Cur- within a day; and the ability to over-
5%, and 10% (Walcott and Gitaitis, rently, the standard assay for B. aclada come the inhibitory effects of seed
2000). Using samples of the same involves plating seeds on semiselective compounds. In contrast to IMS-PCR,
seedlots, CTAB-DNA extraction fol- media, followed by a 7 to 10 d incuba- DNA capture probes can be readily
lowed by PCR and ELISA failed to tion and subsequent observation of synthesized and may be more acces-
detect the bacterium. To date, in addi- the morphological characteristics of sible than polyclonal antibodies.
tion to A. avenae subsp. citrulli, an isolated fungi (Lorbeer and Tichelaar, Hence, this technique may be more
IMS-PCR-based seed assay has been 1970; Kritzman and Netzer, 1978). widely implemented. Unfortunately,
reported for center rot of onion (Al- While polyclonal antibodies have been MCH-PCR captures and detects DNA
lium cepa) caused by Pantoea ananatis developed for B. aclada (Linfield et as opposed to viable pathogen
(Walcott et al., 2002). al., 1995), there have been no reports propagules and it would be impossible
Despite its many advantages, how- of attempts to use them to test seeds. to determine whether amplified DNA
ever, IMS-PCR is limited by the fact For MCH-PCR, specific oligo- originated from viable or nonviable
that it relies on polyclonal antibodies nucleotide primers, Ba1r/Ba2f, de- organisms. However, this potential
for specific capture of organisms. signed based on random amplified problem could be solved by targeting
Polyclonal antibodies are not readily polymorphic DNA data (Nielsen et al., unique mRNAs for detection. Subse-
available and must be produced for 2002) were used. These primers di- quent reverse transcription PCR could
each pathogen. Additionally, since rected amplification of a 413 base pair be used for amplification and detec-
IMS-PCR relies on microscopic beads (bp) amplicon, one strand of which tion of target DNA. Since mRNA is
for pathogen capture, it is unlikely that was used to design a complementary produced only in viable cells, and de-
they would be effective at recovering 117 bp, single-stranded DNA capture grades rapidly, this would confirm the
filamentous mycelia of plant patho- probe. The capture probe was synthe- detection of only viable organisms and
genic fungi. sized with biotin attached to its 5'- cells. However, this approach would
MAGNETIC CAPTURE HYBRIDIZA- prime end (The University of Georgia, be more technically challenging and
TION AND PCR (MCH-PCR). Magnetic Molecular Genetics and Instrumenta- more costly.
capture hybridization and polymerase tion Facility, Athens) and attached to RAPID-CYCLE REAL-TIME PCR. As
chain reaction is similar in format to microscopic, streptavidin-coated mag- previously mentioned, commercial and
IMS-PCR. The techniques differ how- netic beads (Dynal Oslo, Norway). To government seed testing agencies have
ever, in that MCH-PCR uses single- conduct MCH-PCR, infested onion been slow to adopt PCR-base seed
stranded DNA probes to capture and seeds were crushed and DNA was ex- detection assays. This has been due in
concentrate specific DNA fragments tracted using a Mini-Beadbeater part to the cost of the equipment and
that can then be used as templates for (Biospec Products Inc., Bartlesville, consumables, and level of technical
PCR. MCH-PCR is a relatively new Okla.). Double-stranded target DNA expertise required to conduct the as-
technique, first described in 1995 for molecules were denatured by boiling say. Additionally, the risks of cross-
the detection of Pseudomonas and magnetic beads coated with the contamination and the need for post-
fluorescens in nonsterile soil (Jacobsen, capture probe were incubated with the PCR steps such as gel electrophoresis,
1995). This technique has subse- DNA suspensions for 2 h at 62 ºC have made the technique unattractive.
quently been developed for the detec- (143.6 °F). During this time, the cap- Recent advances in PCR, in the form
tion of fungi, viruses and bacteria in ture probe hybridized with single- of rapid-cycle real-time PCR promise
water, wood, soil and food, all of which stranded target DNA fragments. After to eliminate many of these barriers and
contain PCR-inhibitory compounds incubation, the beads were washed make PCR more accessible for seed
(Chen and Griffiths, 2001; Chen et al., and the captured DNA was used for detection. With real-time PCR, DNA
1998; de Moraes et al., 1999; Langrell PCR. amplification is coupled with the pro-
and Barbara, 2001). Despite its great Development of the MCH-PCR- duction of a fluorescent signal that
potential as a detection assay, MCH- based seed detection assay is still in increases proportionally with the num-
PCR has not been applied for the progress, however, we have observed bers of amplicons produced (Kurian et
detection of seedborne pathogens. detection thresholds of 10 conidia/ al., 1999; Cockerill and Smith, 2002).
Recently, we have initiated research to mL in the presence of PCR-inhibiting The fluorescent signal is monitored on
develop a MCH-PCR assay for the onion seed wash. Additionally, MCH- a computer in real-time and provides
detection of Botrytis aclada (causal PCR displayed a detection threshold an indirect visual representation of
agent of Botrytis neck rot) on onion of 10–13 g·mL–1 of B. aclada DNA DNA amplification. Detection of am-
seed. This economically important dis- (Walcott, unpublished data). Finally, plified DNA can be accomplished by
ease of onion causes significant the applicability of MCH-PCR for de- staining with SYBR Green I (Molecu-
postharvest economic losses and there tecting B. aclada in naturally infested lar Probes Inc., Eugene Ore.) that
is evidence of a strong relationship seelots is currently being determined binds double-stranded DNA indis-
between seed borne inoculum but thus far, positive results have been criminately or with the use of specific
postharvest losses (Maude, 1983). obtained for seedlots with 1% infesta- reporter probes like TaqMan (Applied
Speculation about the role of B. aclada- tion (determined by plating on selec- Biosystems, Foster City, Calif.) (Tay-
infested seedlots in recent neck rot tive agar media). It is clear that MCH- lor et al., 2002). TaqMan probes are
outbreaks in New York has renewed PCR has great potential as a seed de- synthesized with reporter and quencher

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 dye molecules at the 5' and 3' ends produce PCR-quality template DNA/ technology relies in part, on DNA
respectively. In this configuration (re- RNA. Significant benefits can be real- amplification, it has similar limitations
porter proximal to the quencher dye), ized by combining real-time PCR with as those described for conventional
there is no fluorescence but when they the other PCR modifications men- PCR. Additionally, significant tech-
are separated the reporter dye fluo- tioned above (MCH-PCR, IMS-PCR, nological expertise and expensive
resces. With the TaqMan system, the BIO-PCR). To date, real-time PCR equipment are required. Currently, few
first step in PCR is the annealing of a seed detection assays have been re- DNA-chip seed detection assays have
complementary probe to the template ported for A. avenae subsp. citrulli in been developed (J. van der Wolf, per-
DNA. Taq DNA polymerase has 5' watermelon seeds (P. Randhawa, per- sonal communication; Fessehaie et al.,
exonuclease activity (cleaves off nucle- sonal communication) and Micro- 2001). However, it is envisioned that
otides from the 5' end of nontemplate dochium nivale in wheat (Triticum this technology will be more widely
complement DNA) and during the spp.) seeds (Taylor et al., 2002). It is employed for routine seed testing in
extension step of PCR, the TaqMan likely that more real-time PCR seed the future.
probe is excised, separating the re- assays will be developed as the technol-
porter dye from the quencher mol- ogy becomes more affordable. Conclusions
ecule. This results in fluorescence that The environmental conditions in
is detected by photosensors. The in-
DNA Chip (microarray) seedling establishment systems are usu-
tensity of fluorescence is directly re- technology ally highly favorable for disease devel-
lated to the excision of reporter dye DNA chips or microarrays repre- opment. Therefore, it is critical to en-
molecules, which is directly related to sent another DNA-based detection sure that no potentially damaging
DNA amplification. Other detection assay that may be applied to test seeds pathogens are introduced on seeds.
systems including fluorescent reso- for pathogens. This relatively new tech- This can most effectively be accom-
nance energy transfer (FRET) and nology relies on the unique ability of plished by exclusion, using seed detec-
molecular beacon probes are also em- nucleic acid molecules to hybridize tion assays to identify contaminated
ployed for real-time PCR (Cockerill specifically with molecules with seedlots that can then be discarded or
and Smith, 2002). complementary sequences (Lemieux treated. Conventional seed detection
As compared to conventional et al., 1998; Vernet, 2002). With DNA assays including visual examination,
PCR, real-time PCR has several key chip technology, oligonucleotide selective media, serological assays and
advantages that potentially make it probes are attached to small (approxi- the seedling grow-out assay have been
more acceptable for use in routine seed mately 1 cm2) glass or silica-based sur- used extensively, but all have short-
testing. These include 1) rapid cycling faces (chips). The power of this tech- comings ranging from inefficiency to
which reduces DNA amplification time nique lies in the fact that hundreds to lack of specificity and sensitivity. PCR
significantly, 2) completion of PCR in a thousand of oligonucleotides can be holds great potential for improving
closed system which reduces the risk of attached to specific, locations on each pathogen detection in seeds, as it em-
cross-contamination i.e. DNA amplifi- chip. These oligonucleotides can be bodies many of the key characteristics
cation and subsequent DNA detection complementary to DNA sequences that including specificity, sensitivity rapid-
is accomplished in the same tube; 3) are unique to certain microorganisms ity, ease of implementation and inter-
there is no need for time consuming and hence, can be used detect patho- pretation and applicability. While in-
post-PCR electrophoresis to determine gens in seed samples. To apply DNA- hibitory seed compounds can limit the
PCR results; 4) the use of different dyes chip technology, DNA or RNA must applicability of conventional PCR,
and probes can allow for mutliplex PCR, be extracted from the sample being modifications including BIO-PCR,
by which multiple pathogens can be tested and amplified. The amplified IMS-PCR and MCH-PCR may pro-
detected in the same reaction (Wittwer DNA is digested into smaller frag- vide opportunities to circumvent in-
et al., 2001) and 5) real-time PCR can ments that are then labeled with fluo- hibitory compounds while improving
allow quantification of template DNA rescent markers and hybridized with detection of seedborne pathogens.
which may be of use in determining oligonucleotides fixed to the DNA IMS-PCR and MCH-PCR are par-
levels of seed infestation. On the other chip. After hybridization, the chip is ticularly attractive because they pro-
hand, there are some key factors that washed thoroughly and fluorescence, vide simple and universally applicable
may prevent the immediate adoption which is directly proportional to the formats for testing seeds for different
of this technology for seed detection. amount of nucleotide retained, is mea- culturable and nonculturable patho-
These include the facts that real-time sured. If the DNA from the pathogen gens. Further improvements in the
PCR requires thermal cyclers that are of interest in present in the seed sample, cost and efficiency will eventually al-
equipped to detect fluorescence. These then the oligonucleotide probe at the low DNA-based detection systems to
thermal cyclers are significantly more position on the chip that corresponds replace the vast array of seed detection
expensive than conventional thermal to that pathogen will display fluores- assays currently employed and provide
cyclers and TaqMan probes are costly. cence. superior detection capabilities neces-
Finally, despite the advantages listed DNA-chips are being used in many sary for healthy seedling establishment.
above, real-time PCR is subject to different fields for diagnosis (Anthony Like other fields in which patho-
many of the problems that hamper et al., 2000; Lemieux et al., 1998). gen detection is critical, seed detection
conventional PCR , including inhibi- Advantages of this technology include assays must be based on new technolo-
tion by seed-derived compounds. simultaneous detection of a wide range gies. However, before adopting these
Hence, it is still necessary to imple- of pathogens and rapid completion assays, it is critical to rigorously evalu-
ment strategies upstream of PCR that time (6 h). However, since DNA-chip ate their applicability, precision, and

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 accuracy in real-world, high through- Cockerill, F.R. and T.F. Smith. 2002. Manulis. 2001. Detection, quantification
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