European Journal of Plant Pathology 110: 893–908, 2004.

2004 Kluwer Academic Publishers. Printed in the Netherlands.

Real-time quantitative PCR: a new technology to detect and study phytopathogenic

and antagonistic fungi

Leonardo Schena, Franco Nigro, Antonio Ippolito and Donato Gallitelli

Department of Plant Protection and Applied Microbiology, University of Bari, Via Amendola 165/A,
70126 Bari, Italy (Phone: +39-0805443055; Fax: +39-0805442911; E-mail: [email protected])

Accepted 16 March 2004

Key words: detection, gene expression, molecular beacons, scorpion PCR, SYBR Green I, TaqMan

Abstract

Real-time PCR technologies open increasing opportunities to detect and study phytopathogenic and
antagonistic fungi. They combine the sensitivity of conventional PCR with the generation of a specific
fluorescent signal providing real-time analysis of the reaction kinetics and allowing quantification of specific
DNA targets. Four main chemistries are currently used for the application of this technique in plant
pathology. These chemistries can be grouped into amplicon sequence non-specific (SYBR Green I) and
sequence specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods. Amplicon sequence non-
specific methods are based on the use of a dye that emits fluorescent light when intercalated into double-
stranded DNA. Amplicon sequence specific methods are based on the use of oligonucleotide probes labelled
with a donor fluorophore and an acceptor dye (quencher). The fluorescent signal eliminates the requirement
for post-amplification processing steps, such as gel electrophoresis and ethidium bromide staining. This
significantly reduces time and labour required for the analysis and greatly increases the throughput of PCR
testing as an automated diagnostic system, making it suitable for large-scale analyses. Furthermore, the use
of different fluorescent dyes facilitates the detection of several target microrganisms in a single reaction
(multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high throughput quantification
of target fungal DNA in various environmental samples, including hosts tissues, soil, water and air, thus
opening new research opportunities for the study of diagnosis, inoculum threshold levels, epidemiology and
host–pathogen interactions. Moreover, the quantification of specific mRNA transcription by real-time
PCR is being increasingly applied to the study of changes in gene expression in response to phytopatho-
genic and antagonistic fungi.

Introduction the technique possesses the potential to detect a

single target molecule in a complex mixture, and it
Conventional-PCR has emerged as a major tool is rapid and versatile. Depending on the design of
for the diagnosis and study of phytopathogenic the primers, both narrow and broad selectivities
fungi and has contributed to the alleviation of are possible, thus enabling the detection of a single
some of the problems associated with the detec- pathogen at the species or strain level. However,
tion, control and containment of plant pathogens despite these advantages, the adoption of PCR for
(Henson and French, 1993; Martin et al., 2000). routine detection of plant pathogens has been slow
PCR is a sensitive technology that offers several due to technical limitations related to the post-
advantages over the traditional methods of diag- amplification procedures, which are necessary to
nosis: micro-organisms do not need to be cultured, detect amplicons. In fact, although PCR
894

techniques can considerably reduce the time nee- dimers, must be prevented by accurate primer de-
ded for diagnosis compared to conventional cul- sign and condition optimisation. At the end of the
turing methods, they still require additional work reaction, products of different length and/or se-
when Southern blot or sequencing are required to quence can be observed as distinct fluorescent
identify the PCR products. Furthermore, conven- peaks by heating the reaction from 30–40 to 95 C
tional PCR is unreliable for quantitative analysis whilst continuously monitoring the fluorescence
(Ginzinger, 2002). (melting curve analysis). In fact, plotting the first
Several reviews on the use of real-time PCR in negative derivative of fluorescence vs. temperature,
fields other than plant pathology have been pub- the point at which dsDNA melts will be observed
lished recently (Bustin, 2000, 2002; Didenko, 2001; as a drop (peak) in fluorescence (many of the
Lyon, 2001; Schmittgen, 2001; Snider et al., 2001; machines will do this calculation in the analysis
Ahmed, 2002; Broude, 2002; Ginzinger, 2002). In software). If a single peak representing the specific
plant pathology, reviews of interest have focused products is observed, SYBR Green I is a simple
on crop biosecurity (Schaad et al., 2003) and on and reliable low-cost method for monitoring PCR
the detection of seed-borne fungal pathogens amplicons and for quantifying template DNA.
(Taylor et al., 2001a) and phytopathogenic bacte- However, since different amplicons will melt at
ria (Schaad and Frederick, 2002). However, real- different temperatures, melting curve analyses also
time PCR detection systems are available for enable the use of SYBR Green I in multiplex
viruses (Schoen et al., 1996; Eun et al., 2000; Fi- detection.
netti Sialer et al., 2000a, b; Roberts et al., 2000;
Boonham et al., 2002; Korimbocus et al., 2002), Amplicon sequence specific methods
nematodes (Ciancio et al., 2000; Bates et al., 2002),
bacteria (Weller et al., 2000; Hermansson and There are several amplicon sequence specific
Lindgren, 2001) and fungi (Table 1). detection methods based on the use of oligonu-
cleotide probes labelled with a donor fluorophore
and an acceptor dye (quencher). A fluorophore is a
Real-time technologies molecule that absorbs light energy and is promoted
to an excited state. In the absence of a quencher the
Four main chemistries are utilised to detect and fluorophore falls back to the ground state and re-
study phytopathogenic and antagonistic fungi leases the excess of energy as light at a longer
(Table 1). These chemistries can be grouped into wavelength (fluorescence). Quenchers are mole-
amplicon sequence non-specific and sequence cules that can accept energy from a fluorophore
specific methods (Mackay et al., 2002). and dissipate the energy by proximal quenching or
by fluorescence resonance energy transfer (FRET)
Amplicon sequence non-specific methods (Didenko, 2001). In proximal quenching, the flu-
orophore is in close proximity with a quencher and
Included in this group are detection methods the energy is transferred from the fluorophore to
based on the use of dyes that emit fluorescent light the quencher and dissipated as heat (collisional
when intercalated into double-stranded DNA quenching). As a result, no fluorescence is ob-
(dsDNA). The most utilised intercalating dye is served. In FRET quenching, the fluorophore
SYBR Green I (Morrison et al., 1998) although transfers its energy to the quencher (which may be
other intercalating dyes, such as ethidium bromide another fluorophore), and the energy is released as
(Higuchi et al., 1992) and YO-PRO-1 (Ishiguro light of a longer wavelength (Didenko, 2001).
et al., 1995) have been used. In solution, the un- There are a number of different fluorophore/
bound dye exhibits very little fluorescence, but this quencher combinations that can be used, provid-
is enhanced upon DNA-binding. As a conse- ing that spectral overlap between the fluorescent
quence, the fluorescence is proportional to the dye and the quencher molecule is ensured. Flu-
amount of total dsDNA in the reaction. Since orophores such as FAM, TET, TAMRA, HEX,
these dyes do not discriminate between the differ- JOE, ROX, CY5 and Texas Red and quenchers
ent dsDNA molecules, in a PCR reaction, the such as TAMRA, DABCYL and Methyl Red are
formation of non-specific amplicons, as well as of the most utilised. Furthermore, a new class of
 895

Table 1. Example of real time-PCR applications for the study and detection of phytopathogenic or antagonistic fungi

Pathogen/antagonist Crop Chemistry Reference

Aphanomices euteiches Alfalfa TaqMan Vandemark et al. (2002)

Aspergillus flavus Maize, pepper and paprika TaqMan Mayer et al. (2003)
Aspergillus fumigatus, Geotricum Air, water and dust TaqMan Haugland et al. (2002)
candidum, Candida albicans,
Stachybotrys chartarum
Aureobasidium pullulans Table grape and sweet Scorpion-PCR Schena et al. (2000), Finetti
cherries Sialer et al. (2000a), Schena
et al. (2002a)
Blumeria graminis f. sp tritici Wheat SYBR Green I Fraaije et al. (2002)
Cladosporium sp., Ramularia Peduncolate oak SYBR Green I Heuser and Zimmer, (2002)
sp., and Microsphaera alphitoides
Colletotrichum coccodes Potato and soil TaqMan Cullen et al. (2002)
Diaporthe phaseolorum and Soybeen TaqMan Zhang et al. (1999)
Phomopsis longicolla
Fusarium solani f.sp phaseoli Bean and soil SYBR Green I Filion et al. (2003a), Filion
and Glomus intraradices et al. (2003b)
Fusarium species Wheat SYBR Green I Schnerr et al. (2001)
Glomus mosseae, Phytophthora Various hosts TaqMan Böhm et al. (1999)
infestans, and P. citricola
Helmintosporium solani Potato and soil TaqMan Cullen et al. (2001)
Heterobasidion annosum Norway spruce TaqMan Hietala et al. (2003)
Magnaporthe grisea Rice SYBR Green I Qi and Yang (2002)
Phaeocryptopus gaeumannii Douglas-fir SYBR Green I; TaqMan Winton et al. (2002), Winton
et al. (2003)
Phakopsora pachyrhizi and Soybean TaqMan Frederick et al. (2002)
P. meibomiae
Phytophthora infestans Potato SYBR Green I Avroa et al. (2003)
Phytophthora medicaginis Alfalfa TaqMan Vandemark and Barker (2003)
Phytophthora nicotianae Citrus roots and soils Scorpion-PCR Ippolito et al. (2000), Ippolito
et al. (2004)
Pyrenophora spp. and P. graminea Barley seeds SYBR Green I Bates et al. (2001), Taylor
et al. (2001b)
Pyrenophora teres Barley Scorpion-PCR Bates and Taylor (2001)
Rizoctonia solani Potato and soil TaqMan Lees et al. (2002)
Rosellinia necatrix Various hosts and soils Scorpion-PCR Schena et al. (2002b), Schena
and Ippolito (2003)
Septoria tritici, Stagonospora nodorum, Wheat SYBR Green I Fraaije et al. (2001)
Puccinia striiformis and P. recondita
Spongospora subterranea Potato, soil and water TaqMan Van de Graaf et al. (2003)
Stachybotrys elegans SYBR Green I Morissette et al. (2003)
Suillus bovines and Paxillus involutus Soil Two fluorogenic probes Landeweert et al. (2003)
Tilletia indica and T. walkeri Wheat TaqMan Frederick et al. (2000)
Various pathogens Arabidopsis thaliana SYBR Green I Brouwer et al. (2003)
Verticillium dahliae Olive and soil Scorpion-PCR Nigro et al. (2002)

quencher that has no native fluorescence has be- entire visible spectrum and into the infrared (Bu-
come available recently as traditional quenchers stin, 2002).
can suffer from a number of drawbacks including The advantage of fluorogenic probes over DNA
intrinsic fluorescence (i.e. TAMRA) or poor binding dyes is that specific hybridisation between
spectral overlap with the fluorescent dye (i.e. probe and target DNA sequence is required to
DABCYL and Methyl Red). The new quenchers generate a fluorescent signal. Furthermore, fluor-
are defined as dark quenchers or black hole ogenic probes can be labelled with different dis-
quenchers (BHQ) and are suitable for a wide range tinguishable reporter dyes to amplify and detect
of dyes since they can quench fluorescence over the two or more distinct sequences in a single PCR
896

reaction tube, without melting curve analysis F = Fluorophore

A FRET
(multiplex PCR). The high specificity of these Ps = Probe sequence
methods enables the discrimination of single base Q = Quencher
Ps
F Q Pf = Primer forward
pair mismatches. Specific methods are thus ideal Pr = Primer reverse
for genotyping single nucleotide polymorphisms
(SNPs) and mutation analyses (Thelwell et al., B FRET
2000; Lyon, 2001). 5' Pf 3' Taq Ps
F Q
Amplicon sequence specific methods include
TaqMan (Livak et al., 1995), Molecular beacons T
(Tyagi and Kramer, 1996), and Scorpion PCR Taq
3' Pr 5'
(Whitcombe et al., 1999). The probes used to
monitor DNA amplification in a PCR are cleaved F
in the reaction (TaqMan) or undergo a confor- C
mation change in the presence of a complementary 5' Pf 3' Taq Q
DNA target (Molecular beacons and Scorpion- 3' 5'
T
PCR) that separate fluorophore and quencher. In
TaqMan, the fluorophore is quenched by FRET, Taq
3' Pr 5'
whereas in Molecular beacons and Scorpion-PCR,
fluorescence quenching is proximal, due to the Figure 1. TaqMan. This system consists of a single stranded
close contact of fluorophore and quencher. sequence (probe sequence) with a fluorophore and a quencher
attached respectively to the 5¢ and the 3¢ end (A). The probe
sequence binds to the amplicon during each annealing step of
TaqMan the PCR (B) and generates a signal through the 5¢–3¢ exonu-
In the TaqMan system (Holland et al., 1991; Livak clease activity of the Taq DNA polymerase that degrades the
et al., 1995), the probe is a sequence of 25–30 probe sequence and releases both fluorophore and quencher
nucleotides in length labelled with a reported dye into the solution (C). Once cleaved from the rest of the probe
at the 5¢ end, and a quencher at the 3¢ end (Fig- sequence, the 5¢ dye is freed from the quenching effect (via
FRET).
ure 1). During amplification, the probe binds to its
target sequence and is cleaved by the 5¢ nuclease
activity of Taq DNA polymerase when the enzyme forming a hairpin-like structure. Due to the stem
extends from an upstream primer into the region structure, when the probe is not hybridised, flu-
of the probe. The cleavage separates the quencher orophore and quencher are kept in close proximity
from the reporter dye and restores fluorescence. and the energy is dissipated as heat (Didenko,
This dependence on polymerisation ensures that 2001). When a Molecular beacon encounters the
cleavage of the probe occurs only if the target se- target DNA a probe-hybrid longer and more stable
quence is being amplified. While the probe is in- than the stem-hybrid is formed. Consequently, a
tact, the proximity of the quencher greatly reduces conformational change occurs; arm sequences are
the fluorescence emitted by the reporter dye by forced apart and the quenching effect drops down
FRET through space. Additional reporter dye resulting in detectable fluorescence. Because the
molecules are cleaved from their respective probes hairpin shape is very thermostable molecular bea-
in each cycle thus causing an increase in fluores- cons must have a high specificity to hybridise to a
cence intensity proportional to the amount of target. This makes the chemistry appropriate for
amplicons produced. the detection of single nucleotide differences in
mutation and SNP analyses. Molecular beacons
Molecular beacons have been utilised frequently to detect plant viruses
Molecular beacons (Tyagi and Kramer, 1996) are in combination with Nucleic Acid Sequence-Based
stem-and-loop shaped hybridisation probes with a Amplification (NASBA) (Gonçalves et al., 2002).
fluorescent dye covalently attached on one end and
a quencher covalently attached on the opposite end Scorpion-PCR
(Figure 2). The loop fragment of the probe is Two different formats of Scorpion primer are
complementary to a sequence of the template and available: (i) the ‘stem-loop format’ (Whitcombe
the two ends are complementary to each other et al., 1999; Thelwell et al., 2000) and (ii) the
 897

reaction is effectively instantaneous and occurs

Ps Ps = Probe sequence prior to any competing or side reactions such as
A
S = Stem target amplicon re-annealing or inappropriate
F = Fluorophore target folding. This leads to stronger signals, more
Q = Quencher
S S Pf = Primer forward
reliable probe design, shorter reaction times and
Pr = Primer reverse better discrimination (Thelwell et al., 2000).
5' F Q 3'
T = Template DNA Comparison among Scorpion primers and alter-
Pf native chemistries showed that TaqMan probes
have high backgrounds and moderate signal
T strength, molecular beacons have low background
Pr
B
fluorescence and low signal strength, Scorpion
primers have low backgrounds and high signal
F Q
strength (Thelwell et al., 2000).
3' A further improvement of the ‘stem-loop’ for-
5' S Ps S
mat has been achieved with the ‘duplex format’. In
3'
T Pr
5' this format the probe element has a fluorophore
attached at its 5¢-end and is annealed to a com-
Figure 2. Molecular beacon. This system consists of a hairpin plementary oligonucleotide bearing a quencher at
loop structure (probe sequence), a 6-base-long stem that hold the 3¢-end (Figure 4). The mechanism of action is
the probe sequence in the hairpin configuration and a fluoro- quite similar to the stem-loop format, since the
phore and a quencher linked, respectively, to the 5¢ and the 3¢
end of the stem (A). Molecular beacons generate signals when
intramolecular probe-target interaction, which is
hybridise to the amplified template DNA and distance of the 5¢ the most important feature of the Scorpion system,
dye molecule from the quenching molecule is great enough to is maintained in both formats. However, in stan-
have a specific increase of fluorescence (B). This hybridisation is dard Scorpions the quencher and fluorophore re-
favoured over the intramolecular binding of the complementary main within the same strand of DNA and some
target strand, however, if there is no template DNA the hairpin
configuration is restored and the fluorescence quenched.
quenching can occur even in the open form. In
duplex format, the quencher is on a different oli-
gonucleotide and separation between the quencher
‘duplex format’ (Solinas et al., 2001). In both and fluorophore is greatly increased, thus
formats the probe element is covalently linked to decreasing the quenching when the probe is bound
one of the two primers and quenching is due to the to the target (Solinas et al., 2001).
close proximity between fluorophore and quencher
as in molecular beacons. Instrumentation and primer design
The stem loop format is composed of a PCR
primer and a Molecular beacons tail, linked to the In last few years several real-time thermal cyclers
5¢ end via a ‘PCR stopper’ that prevents poly- with multiplexing and high throughput applica-
merase read-through (Figure 3). The probe ele- tions have been proposed on the market. Some of
ment (loop) is complementary to a sequence newly them are suitable for small batches of samples but
synthesised by the DNA polymerase as a contin- are fast, thus allowing the user to run different
uation of the linked primer. During amplification, parameters each time. Other instruments are suit-
Scorpion primer is incorporated into the PCR able for large batches but are less flexible (Bustin,
product and in the annealing phase the probe se- 2002). All instruments are true ‘real-time system’
quence in the Scorpion tail curls back to hybridise in that they collect data (monitoring fluorescence)
to the target sequence in the PCR product. This during each PCR cycle. Recently, a portable
hybridisation event opens the hairpin loop, elimi- thermocycler has been developed (Schaad and
nates the quenching of the donor fluorophore and Frederich, 2002).
an increase in signal is observed. Thus, the Scor- Detailed information on how to design Taq-
pion-primer approach uses a unimolecular mech- Man, Molecular beacons, and Scorpion primers
anism in which the hybridisation reaction occurs can be found on the Eurogentec documentation
within the same strand. The benefits of a unimo- catalogue (http://www.eurogentec.com/code/en/
lecular rearrangement seem significant, since the page_07.asp?Page=409). Briefly, the simplest way
898

Ps = Probe sequence A
S = Stem Ps
F = Fluorophore
Q = Quencher
B = Blocker S S
Pf = Primer forward 5' F Q B Pf 3'
Pr = Primer reverse
B T = Template DNA
A = Amplified DNA T
F S
5' Pr
C
Ps Ps
3'
Pf
B S S
S
Q 5' B Pf A 3'
F Q
T 3' 5'

F D
5'
S

Ps
E B F
Q S S 5'
Ps
B Pf A 3' Pf
SQ 3'
A

Figure 3. Scorpion PCR (stem loop format). This system is similar to Molecular beacons (Figure 2), however the hairpin loop is linked
to the 5¢ end of a primer via a PCR blocker (A). Step B: Initial denaturation of target and scorpion sequence. Step C: Annealing of the
primer element of the Scorpion to the target DNA and extension of a new DNA fragment to which Scorpion remains attached. Step D:
Denaturation of DNA. Step E: During annealing extension the Scorpion probe binds to its complement within the same strand of
DNA. This hybridisation event opens the hairpin loop so that fluorescence is no longer quenched and a specific increase of fluorescence
is achieved.

to design TaqMan probes is to use the Primer the amplicon and make the reaction fast and
ExpressTM software, which contains all the efficient.
necessary parameters (http://www.appliedbiosys-
tems.com/support/tutorials/#). A commercial
program to assist with the design of Molecular Identification of fungal specific target sequences
Beacon is also available (http://www.premier-
biosoft.com/molecularbeacons.html). No specific In real-time PCR, as well as in conventional PCR,
software is available at present to design Scorpion two main strategies can be utilised to identify the
primers. However, since for Scorpion the folding specific target sequences needed for the detection
of the primer and probe is the crucial feature, the and identification of fungal species and/or strains:
use of a folding program available on the website (i) amplifying and sequencing conserved genes,
(http://www.bioinfo.rpi.edu/applications/mfold/old/ common to all fungi, with universal primers, (ii)
rna/form1.cgi) can greatly help in the design. amplifying unknown genomic regions with non-
To design primers and/or probes, it is useful to specific random primers (McCartney et al., 2003).
choose regions that are unlikely to cause second- The nuclear-encoded ribosomal RNA genes
ary structures and that have a GC content (rDNA) provide attractive targets since they are
comprised between 20% and 80%. Primers should highly stable, possess conserved as well as variable
have a similar Tm, a length comprised between 17 sequences, and can be amplified and sequenced
and 27 bases and not able to form dimers. It is with universal primers (White et al., 1990; Henson
important to keep the amplicons short (70–250 and French, 1993). They also occur in multiple
bases), to allow the probe sequences to success- copies (up to 200 copies per haploid genome) ar-
fully compete with the complementary strand of ranged in tandem repeats with each repeat
 899

Q = Quencher A Q F
F = Fluorophore 3' 5'
Ps = Probe sequence
C Ps
C = Oligonucleotide bearing a quencher
B = Blocker
Pf = Primer forward 5' B Pf 3'
Pr = Primer reverse
T = Template DNA
A = Amplified DNA T
Pr
Q
B F C 3' Q F 5'
5' 3' C
Ps C Ps
Pf
B B Pf A 3'
5'
T T

F D
5'

Ps E
B
B Pf Ps 5' F
Pf A 3' 3'
A

Figure 4. Scorpion PCR (duplex format). The unimolecular probing mechanism is basically the same as in the stem loop-format
(Figure 3), however the quencher is on a different oligonucleotide chain (A). Step B: Initial denaturation. Step C: Annealing of the
primer element of the Scorpion to the target DNA and extension of a new DNA fragment to which Scorpion remains attached. Step D:
Denaturation of DNA. Step E: During annealing extension the Scorpion probe binds to its complement within the same strand of
DNA. This hybridisation event prevents annealing of the probe sequence with the complementary oligonucleotide bearing the
quencher so that fluorescence is no longer quenched and a specific increase of fluorescence is achieved.

consisting of the 18S small subunit (SSU), the in rDNA exists within the IGS regions. Compared
5.8S, and the 28S large subunit (LSU) genes sep- to ITS regions, IGS pose more difficulties for
arated by internal transcribed spacer (ITS) regions amplification and sequencing, however, they can
(ITS1 and ITS2) (Bruns et al., 1991; Liew et al., be useful when there are not enough differences
1998). Another part of rDNA is the spacer be- available across the ITS. As an example, ITS re-
tween the LSU and SSU genes, known as inter- gions are very similar in V. dahliae and V. albo-
genic spacer (IGS) or the non-transcribed spacer atrum, therefore it is difficult to design primers to
(NTS). In some species of Phytophthora and differentiate these two species (Nazar et al., 1991).
Pythium the IGS contains the 5S gene (Howlett et More differences are available across the IGS re-
al., 1992) which is not present in fungi such as gions (Pramateftaki et al., 2000) enabling the
Verticillium dahliae and V. albo-atrum (Morton et development of conventional and Scorpion prim-
al., 1995). The rDNA-conserved regions, allow ers to identify and detect V. dahliae and V. albo-
probes or primers from one species to be used to atrum (Nigro et al., 2002).
detect the equivalent ribosomal genes from other Among conserved genes, the b-tubulin has been
species, genera, families, or even kingdoms (White used frequently to develop diagnostics for fungi
et al., 1990). However, the equivalent DNA frag- (McKay et al., 1999; Fraaije et al., 2001). This gene
ments detected in different organisms have many can be amplified quite easily with universal primers
sequence differences that can be exploited in the (Glass and Donaldson, 1995) and have sequences
identification. Among the variable regions, ITS are that can be useful when ITS sequence variation
the most widely sequenced in fungi (Henson and is not suitable for production of a taxon-specific
French 1993; Cullen et al., 2001, 2002; Ippolito diagnostic. Other genes used as diagnostic targets
et al., 2002; Lees et al., 2002; Schena and Ippolito, include ascomycete mating-type genes (Dyer et al.,
2003). The greatest amount of sequence variation 2001; Foster et al., 2002), the elictin gene (Lacourt
900

and Duncan, 1997) and the laccase gene (Hietala bacterial diseases by using a portable real-time
et al., 2003). PCR apparatus suggest the problem is being
Specific target sequences can also be identified overcome (Schaad and Frederich, 2002).
by amplifying random regions of the fungal gen- The potential of real-time PCR as large-scale
ome with PCR-based techniques such as random detection method for phytopathogenic and
amplified polymorphic DNA (RAPD) and arbi- antagonistic fungi can be further improved by the
trarily primed PCR (AP-PCR) (Welsh and McC- use of multiple primers to amplify and detect
lelland, 1990; Williams et al., 1990). PCR amplified multiple templates within a single reaction (mul-
fragments are separated by gel electrophoresis to tiplex real-time PCR). In conventional multiplex
identify unique PCR bands that can be purified, PCR, discrimination relies on obtaining products
cloned, sequenced and used as probes and/or as a of different sizes that can be distinguished by gel
target to design specific sequence characterised electrophoresis. However, different size products
amplified region (SCAR) primers. This approach amplify with different efficiencies. In contrast, with
requires more time and experience compared to real-time specific detection methods, differentia-
the amplification of conserved genes, since the tion can be achieved using different fluorescent
analysis of a large number of isolates belonging to dyes, and therefore amplicons of the same length
both the species of interest and the related species can be used. A limit to the use of multiplex real-
is necessary. As no information is available on the time PCR is the limited number of fluorophores
localisation of the amplified fragments in the available, however the discovery and application
genome, extensive analyses are required to assess of non-fluorescent quencher (dark quencher) has
reproducibility and specificity over time. However, made available wavelength emission that were
SCAR primers with different levels of specificity previously occupied by the emission of the
have been used for a large number of fungi and quenchers themselves (Mackay et al., 2002). This
shown to be particularly useful when closely re- advance has permitted the inclusion of a greater
lated species or specific strains need to be identi- number of spectrally discernible oligoprobes per
fied. Specific SCAR primers were used to monitor reaction, and highlighted the need for a single
biocontrol fungal strains released in the field (Pa- dark quencher, which can quench a broad range
avanen-Huhtala et al., 2000; Schena et al., 2000, of emission wavelengths. From a practical point of
2002a), the ectomycorrhizal fungus Tuber borchii view, although several fluorophores have been
(Bertini et al., 1998), and phytopathogenic fungi used simultaneously in human medicine (Ugozzoli
(Taylor et al., 2001b; Vandemark and Barker, et al., 2002), only two fluorophores have been re-
2003). ported in plant pathology for the detection of
fungi with specific real-time detection methods
(TaqMan, Molecular beacons and Scorpion-
Qualitative detection of fungi PCR). This is partially due to reduced sensitivity
of multiplex PCR compared to separate amplifi-
Compared to conventional PCR, real-time PCR cations. Furthermore, multiplex PCR is less reli-
has significant advantages. This technique elimi- able for quantitative analyses. A very high level of
nates the requirement for post-amplification pro- sensitivity is essential to detect soilborne and
cessing steps and significantly reduces the time and wood colonising fungi that can be present at very
labour, greatly increasing the throughput of PCR low levels. TaqMan probes labelled with different
testing as an automated diagnostic system suitable fluorophores enabled the simultaneous detection
for large-scale analyses (Schena et al., 2002b; of host (Douglas-fir) and pathogen (Phaseocryp-
Schena and Ippolito, 2003). Furthermore, without topus gaeumanii) DNA whereas two different
ethidium bromide, health risks for operators and Scorpion primers enabled the identification of
environmental contamination are reduced. To Phytophthora nicotianae and P. citrophthora in a
date, the main constraint to the diffusion of real- single tube (Winton et al., 2002; Ippolito et al.,
time PCR is the requirement for dedicated, 2004).
sophisticated spectrofluorometric thermal cyclers, Real-time PCR seems to be more sensitive than
which are expensive to purchase and to maintain. conventional PCR. Amplification of Rhizoctonia
However, the recent results on the diagnosis of solani target DNA extracted from soil was
 901

achieved with real-time PCR, but not using con- generation of a fluorescent signal in the early
ventional PCR (Lees et al., 2002). Similarly, the stages of PCR (Mumford et al., 2000).
same level of sensitivity for specific detection of A major limitation to the use of conventional as
Helminthosporium solani in soil and in tubers was well as real-time PCR to detect phytopathogenic
obtained with a TaqMan-based PCR (Cullen and antagonistic fungi by purely molecular meth-
et al., 2001). Combining two sequential amplifi- ods is the lack of discrimination between living and
cations with conventional (first amplification) and dead material. As a result, molecular profiling of
labelled primers (second amplification) a further natural samples can be expected to give different
increase of sensitivity can be achieved without results compared with those obtained by tradi-
loosing the advantages of real-time PCR. Such tional isolation techniques (Bridge and Spooner,
approach (nested Scorpion-PCR) enabled the 2001). Nucleases are widely diffused in the envi-
detection of Rosellinia necatrix (Schena et al., ronment and can degrade DNA after the death of
2002b; Schena and Ippolito, 2003), V. dahliae microrganisms. However, the degradation rate
(Nigro et al., 2002), P. nicotianae, and P. citroph- strongly depends on environmental conditions.
thora (Ippolito et al., 2000) in naturally infected Schena and Ippolito (2003) found that DNA of
substrates (soils, roots, bark, and/or woody tis- R. necatrix is degraded rapidly in soil and minimize
sues). Nested Scorpion-PCR provided higher lev- the risks of false positives due to the presence of
els of sensitivity than conventional detection dead cells. However, further research is necessary
methods and required much less time. to assess the persistence of the DNA under differ-
One of the main disadvantages of fungal diag- ent environmental conditions and in relation to the
nosis based on conventional PCR is the risk of structures produced by the pathogens. Indeed, al-
false positive due to cross contamination of reac- though several reports indicate that nucleic acids
tion mixtures. Although false positives can result are quickly digested by DNases in the soil (England
from sample-to-sample contamination, a more et al., 1998), other studies have shown that DNA
serious source of false positive is the carry-over of can persist in soil for long period of time by
DNA from a previous amplification of the same forming complexes with soil components (England
target (Kwok, 1990). In real-time PCR, the re- et al., 1997). A possible approach to avoid false
duced level of sample manipulation reduces the positive due to the detection of DNA in dead cells
potential of cross contamination. When a double is the combination of real-time PCR with baiting
amplification is required (nested PCR) cross con- (BIO-PCR). The biological amplification of the
tamination can be a problem due to increased target microrganisms increase sensitivity of the
handling. However, considering that the quantity detection and reduce problems with DNA extrac-
of amplified product after the first amplification is tion from complex environmental samples, such as
usually very low and that tubes remain closed after soil, since nucleic acids do not need to be concen-
the second amplification, the risks of cross con- trated before PCR (Schaad and Frederich, 2002).
taminations in real-time nested PCR remain much BIO-PCR seems to be particular suitable for phy-
lower compared to conventional PCR. topathogenic bacteria since they are easily and
A variety of naturally occurring compounds, quickly cultured in liquid and/or on solid nutritive
such as humic acids, tannins, and lignin associated media (Weller et al., 2000). This technique has also
compounds, can interfere with PCR reactions and been utilised to detect phytopathogenic fungi, such
inhibit the amplification (Cullen and Hirsch 1998; as R. solani (Lees et al., 2002) and P. nicotianae
Bridge and Spooner, 2001; Ippolito et al., 2002), (Ippolito et al., 2000) in naturally infested soils.
although such problems have been partially over- Compared to real-time PCR alone, BIO-PCR re-
come by development and optimization of the quires more time for analysis (microrganisms needs
initial DNA extraction methods. Real-time PCR to be grown on nutritive media for 1–4 days) and is
seems to be less affected by inhibitors than con- more expensive (especially if selective media are
ventional PCR, because they mainly affect the late utilised). An alternative strategy could rely on the
cycles of PCR, which are critical for product use of RNA rather DNA as target molecule for
accumulation. In contrast, product accumulation diagnosis. RNA is degraded quickly in dead cells
is not required to give positive results in real-time and does not interfere with the analysis. In order to
assays, because detection is achieved through the be used as target molecule by PCR, RNA must first
902

be reverse transcribed into DNA (RT-PCR) (see et al., 2003), Fusarium solani f. sp. phaseoli and the
below). arbuscolar mycorrhizal fungus Glomus intraradices
(Filion et al., 2003a). Unlike baiting and cultural
methods, real-time PCR is not affected by external
Quantitative detection of fungi factors such as other fungal species that could
conceal the presence of the pathogen on agar-
An accurate, reliable, and high throughput quan- based media. Therefore real-time PCR has the
tification of target DNA requires a real-time PCR potential for determining the soil inoculum
approach (Lie and Petropoulos, 1998; Schmittgen, threshold levels necessary for the disease develop-
2001). Several amplification methods have been ment in a number of host–pathogen combinations.
developed with conventional PCR for quantitative Similar research should allow the development of
analysis of phytopathogenic fungi (competitive predictive diagnostic test to identify high-risk fields
PCR) (Hadidi et al., 1995; Mahuku and Platt, where pathogens inoculum is above threshold
2002). These methods are laborious and not en- values (Cullen et al., 2001, 2002). Quantitative
ough accurate because amplification efficiency de- analyses are of basic importance if there is a
creases during later PCR cycles (Ginzinger, 2002). threshold level of pathogen propagules that trigger
As previously described, in real-time PCR application of chemical or other control means.
the amplicons are measured at an early stage of the For example in citrus, fungicide application is
reaction when the efficiency is still constant. The thought to be economically justified if the Phy-
number of PCR cycles necessary to generate a tophthora population is higher than 15–20 propa-
fluorescent signal significantly above the noise le- gules per gram of soil (ppg) for susceptible
vel (Cycle threshold, Ct) is inversely related to the rootstocks (Lutz and Menge, 1986; Magnano di
log of the initial amount of target molecules. San Lio et al., 1988) and 30 ppg for the resistant
Quantification is automatically determined by ones (Ippolito et al., 1991).
associate software interpolating Ct values of un-
known samples with standard curves prepared Quantification of biocontrol agents
from known quantities of target DNA.
Biocontrol agents need to be monitored to evalu-
Quantification of soil-inhabiting fungi ate their ability to colonise the environment, to
tolerate climate changes and to assess environ-
A research area of particular interest is the quan- mental risks (Gullino et al., 1995). A strain of
tification of individual fungal species in the soil. A Aureobasidium pullulans (L47), effective against
high and significant correlation was found between postharvest rot of fruits and vegetables has been
inoculum density of P. nicotianae assessed with a monitored and quantified on the carpoplane of
selective medium (propagules per gram of soil), table grapes and sweet cherries by Scorpion-PCR
and the corresponding Ct values (Ippolito et al., (Schena et al., 2002a). The results from molecular
2004). Similarly, using a R. necatrix infested soil analysis were correlated significantly with the col-
serially mixed with different amounts of uninfested ony forming units, assessed by culturing the
soil a high correlation was found between the antagonist on a semiselective medium. Similarly,
dilution factor (% of infested soil) and the cycle compared to dilutions on selective media, real-time
threshold (Schena and Ippolito, 2003). The bio- PCR provided a more sensitive method to detect
mass of two ectomycorrhizal fungal species (Suil- and quantify in soil a potential biocontrol agent
lus bovinus and Paxillus involutus) was detected in (Plectosphaerella cucumerina) of potato cyst nem-
samples containing a complex microbial commu- atodes (Atkins et al., 2003).
nity and showed increasing amounts of S. bovinus
DNA and decreasing amounts of P. involutus Fungal DNA quantification in host tissues
DNA over the time (Landeweert et al., 2003).
Other methods used to quantify pathogen DNA in The quantification and the assessment of the col-
soil have been developed for H. solani (Cullen onization rate of plant pathogens in host tissues is
et al., 2001), Colletotrichum coccodes (Cullen et al., one of the most attractive applications of real-time
2002), Spongospora subterranea (van de Graaf PCR. Quantitative detection methods are available
 903

to quantify H. solani and C. coccodes in potato authors demonstrated that quantitative PCR
tubers (Cullen et al., 2001, 2002), G. mosseae, could be used to study mechanisms of resistance to
Phytophthora infestans and P. citrophthora in dif- the pathogen. Low levels of pathogen DNA in
ferent hosts (Böhm et al., 1999), Diaporte phaseo- resistant plants would characterise a mechanism
lorum and Phomopsis longicolla in soybean seeds that result in the inhibition of pathogen multipli-
(Zhang et al., 1999), R. necatrix in roots and bark cation, whereas the presence of relatively high
of different hosts (Schena and Ippolito, 2003), amounts of pathogen DNA should indicate a
P. nicotianae and P. citrophthora in citrus roots mechanism based on tolerance rather than on true
(Ippolito et al., 2004), Pyrenophora teres on in- resistance.
fected barley seed (Bates et al., 2001; Bates and
Taylor, 2001), and Aspergillus flavus on maize,
pepper, and paprika (Mayer et al., 2003). Using Quantification of mRNA transcription
two probes labelled with different fluorophores it
was possible to quantify simultaneously host Among the different real-time PCR applications,
(Douglas-fir) and pathogen (Phaeocryptopus the quantification of messenger RNA (mRNA) to
gaeumannii) DNA (Winton et al., 2002). In this evaluate gene expression is one of the most
case, detection of host DNA provided an endog- promising. If mRNA is being measured the
enous reference that served as an internal positive experiment must contain a reverse transcription
control to adjust variations introduced by sample- step in which RNA is converted to cDNA (RT-
to-sample differences in DNA extraction and PCR PCR). Once the cDNA has been synthesised a
efficiency. Compared to three different alternative standard quantitative real-time PCR reaction can
methods (fruiting body abundance, ergosterol be run and quantification carried out. Primers
content and dot blot analysis), TaqMan real-time should bind to separate exons to avoid false po-
PCR provided a more accurate calculation of the sitive results, arising from amplification of con-
relative growth of the fungus within Douglas-fir taminating genomic DNA. If the intron/exon
needles and was the only method able to quantify boundaries are unknown, or when targeting an
the pathogen early in the disease cycle (Winton intron-less gene, it is necessary to treat the RNA
et al., 2003). sample with RNAse-free DNAse. Generally, two
Due to its high sensitivity and reproducibility, quantification strategies can be performed: abso-
real-time PCR is ideal to detect minor changes of lute and relative quantification (Livak and Sch-
host resistance and susceptibility. Blast resistance mittgen 2001; Pfaffl and Hageleit, 2001; Pfaffl
levels of rice cultivars were more accurately eval- et al., 2002). In the absolute quantification the
uated with real-time PCR, since by the time in mRNA copy number is determined by comparison
which lesions on leaves just became visible, the with appropriate external calibration curves. The
growth of Magnaporthe grisea was 80 times higher relative quantification is based on the expression
in susceptible than in resistant cultivar (Qi and ratio of a target gene versus a reference gene.
Yang, 2002). In Norway Spruce (Picea abies) col- Several housekeeping non-regulated reference gene
onised with Heterobasidion annosum, pathogen have been proposed for mammalian systems (Bu-
DNA was more restricted and localised in the le- stin, 2002), whereas much less information is
sion of a clone with high resistance, whereas in a available for filamentous fungi and plant hosts.
clone with low resistance, the fungus was detected The b-tubulin and the actin genes seem to be
until the visible end of the lesion (Hietala et al., appropriate to normalise results from gene
2003). Similarly, in alfalfa plants infected with expression in both filamentous fungi (Semighini
Aphanomyces euteiches (Vandemark et al., 2002) et al., 2002) and plant hosts (Bézier et al., 2002).
and with Phytophthora medicaginis (Vandemark The quantification of mRNA to assess the effect
and Barker, 2003), significant correlations were of different treatments on its transcription level is
found between the amount of pathogens DNA and widely and increasingly used in medicine (Bustin,
disease severity, suggesting that real-time PCR can 2000, 2002; Snider et al., 2001; Ginzinger, 2002)
be utilised for the selection of resistant plants also and it is likely that it would be adapted to plant
when samples are indistinguishable based on the pathology. In fact, although microarrays are the
visual assessment of disease severity. The same most powerful methods for determining gene
904

expression in response to microbes, false positive of precision that was impossible a few years ago.
results are relative common and require confir- The sensitivity, speed, and versatility of this tech-
mation with independent methods such as nique, together with the possibility of achieving
Northern blot analysis (Kagnoff and Eckmann, quantitative analyses, are primary factors which
2001). However, real-time quantitative PCR is will encourage its rapid and wide acceptance in
sensitive enough to detect and quantify gene plant pathology. It is likely that real-time PCR will
expression and to reveal small changes in mRNA become a standard method suitable for large-scale
level (Bustin, 2002). diagnosis of phytopathogenic and antagonistic
Plants defence themselves against fungal fungi in extension services.
pathogens as well as other biotic and abiotic stress Quantitative results provide new important
factors activating specific genes that can be re- information not achievable with conventional
vealed by quantifying mRNA (McMaugh and PCR. In particular, although still in its embryonic
Lyon, 2003). Recently, real-time quantitative RT- phase, this technology has great potential for the
PCR was used to evaluate the effect of elevated quantification of mRNA to study unknown
growth temperatures and heat stress on the mechanisms involved in the host–pathogen inter-
expression of ascorbate peroxidase genes in Ara- action as well as in the pathogen’s response to
bidopsis (Panchuk et al., 2002). The same approach environmental changes.
enabled to study the role of defense-related genes Real-time PCR technologies are also ideal for
(phenylalanine ammonia-lyase, stilbene synthase, the detection of single nucleotide polymorphisms
polygalacturonase inhibitor protein, an acidic (SNPs). These markers are increasingly used in
chitinase) in grapevine leaves and berries infected clinical diagnosis and often are associated with
by Botrytis cinerea (Bézier et al., 2002). The anal- specific diseases. Similar applications are also
ysis of Polymyxa betae glutathione-S-transferase possible in plant pathology (Bates and Taylor,
(GST) expression in infected sugar beet showed 2001) and will prove a useful instrument for the
continuing upward trend of transcript levels in study of gene flow and population genetics of
susceptible plants and extremely low levels in phytopathogenic and antagonistic fungi.
resistant ones (Kingsnorth et al., 2003). To date research has just begun to develop
Another possible application of real-time specific applications. Although the number of
quantitative RT-PCR is the quantification of the primers and fluorescent probes available for real-
expression of specific genes from fungal pathogens time PCR is still limited compared to conventional
and antagonists. The analysis of the relative tran- PCR, many more will be developed in the near
script levels of ABC transporters genes by real- future. As knowledge of the fungal genome ex-
time RT-PCR might provide useful information pands, the opportunities to use real-time quanti-
about multidrug resistance in filamentous fungi as tative PCR in a wide variety of settings will
reported for Aspergillus nidulans (Semighini et al., expand. Classical PCR protocols already exist for
2002). In P. infestans the analysis of the expression many phytopathogenic and antagonistic fungi.
of 18 genes showed that all transcripts were up Adapting these to a real-time PCR assays should
regulated in germinating cysts and 12 were found be a relatively easy task.
to be up regulated in potato leaf during the early
stages of infection (Avrova et al., 2003). Recent
studies have demonstrated that the expression of
an endochitinase gene of the mycoparasite Acknowledgements
Stachybotrys elegans increase after 2 days of con-
tact with the pathogen Rhizoctonia solani (Moris- This work was partially supported by CEGBA
sette et al., 2003). (Centro di Eccellenza di Genomica in campo
Biomedico e Agrario) research line n.7 and by
Ministero dell’Istruzione, dell’Università e della
Concluding remarks Ricerca of Italy. PRIN 2003 – Grant No.
2003074533: ‘Diseases caused by soil-borne species
Real-time PCR has an enormous potential to ad- of Phytophthora: aetiology, molecular diagnosis,
dress central questions in plant pathology at a level epidemiology, and new control means’.
 905

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