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1943 7714 Article p40
1943 7714 Article p40
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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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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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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