FULL RESEARCH PAPER
Detection of Phytophthora nicotianae and P. palmivora in
citrus roots using PCR-RFLP in comparison with other
Kim D. Bowman Æ Æ Ute Albrecht Æ Æ James H. Graham Æ Æ
Diane B. Bright
Received: 21 June 2006/Accepted: 5 April 2007/Published online: 15 August 2007
? KNPV 2007
ra are the most important soil-borne pathogens of
citrus in Florida. These two species were detected and
identified in singly and doubly infected plants using
polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) of internal transcribed
spacer (ITS) regions of ribosomal DNA. The sensi-
tivity of the PCR-RFLP was analyzed and the
usefulness of the method evaluated as an alternative
or supplement to serological methods and recovery
on semi-selective medium. In a semi-nested PCR
with universal primers ITS4 and ITS6, the detection
limit was 1 fg of fungal DNA, which made it 1000·
more sensitive than a single-step PCR with primers
ITS4 and DC6. The sensitivity of detection for P.
nicotianae was shown to be ten-fold lower than for P.
palmivora, limiting its detection with restriction
profiles in plants infected by both fungal species.
Phytophthora nicotianae was detected with species-
specific primers in all samples inoculated with this
species despite the absence of species-specific
Phytophthora nicotianae and P. palmivo-
patterns in RFLP. In contrast, the incidence of
detection of P. palmivora in the presence of P.
nicotianae was considerably lower using plating and
morphological detection methods. Due to its high
sensitivity, PCR amplification of ribosomal ITS
regions is a valuable tool for detecting and identify-
ing Phytophthora spp. in citrus roots, provided a
thorough knowledge of reaction conditions for the
target species is established prior to the interpretation
ITS regions ? Phytophthora root rot
The genus Phytophthora causes some of the most
serious soil-borne diseases of plants worldwide
(Erwin and Ribeiro 1996). Together with the genus
Pythium and the downy mildews, Phytophthora
belongs to the class oomycetes. Although commonly
regarded as fungi, oomycetes differ from the true
fungi in various morphological, biochemical and
molecular characteristics and are more closely related
to the golden-brown and the heterokont algae (Cav-
alier-Smith 1986; Erwin and Ribeiro 1996; Govers
In Florida citrus, Phytophthora spp. cause eco-
nomic losses from damping-off of seedlings in
nurseries, foot rot of the trunk, brown rot of fruit,
and fibrous root rot in groves leading to tree decline
K. D. Bowman (&) ? U. Albrecht
US Horticultural Research Laboratory, US Department of
Agriculture, Agricultural Research Service, 2001 South
Rock Road, Fort Pierce, FL 34945, USA
J. H. Graham ? D. B. Bright
Citrus Research and Education Center, University of
Florida, Institute of Food and Agricultural Sciences, 700
Experiment Station Road, Lake Alfred, FL 33850, USA
Eur J Plant Pathol (2007) 119:143–158
and yield losses (Timmer and Menge 1988; Graham
and Menge 1999). The most important Phytophthora
spp. affecting citrus worldwide are P. nicotianae
(syn. P. parasitica), P. palmivora, and P. citrophtho-
ra. The latter species causes brown rot and gummosis
on the trunks of trees in Mediterranean climates
where winter rainfall is predominant, but P. citroph-
thora is usually not a serious problem on citrus trunks
and roots in warm subtropical areas and has not been
found in recent Florida surveys (Graham et al. 1998).
Rootstock resistance or tolerance is the principal
tactic used to manage Phytophthora diseases, but
in susceptibility or tolerance towards each Phytoph-
thora species. Many hybrids of trifoliate orange
Swingle citrumelo (Citrus paradisi · P. trifoliata),
have previously been considered tolerant towards root
rot caused by P. nicotianae (Graham 1995). However,
it is now evident that this does not hold true for P.
palmivora (Bowman et al. 2002; Graham et al. 2003;
Grosser et al. 2003; Albrecht and Bowman 2004).
Conversely, some otherrootstocksnotderived from P.
trifoliata, like Cleopatra mandarin (Citrus reticulata),
are relatively susceptible to P. nicotianae, but seem to
possess a higher tolerance to P. palmivora than
trifoliate orange (Graham et al. 2003).
Traditional methods for detection, identification
and characterization of Phytophthora spp. involve the
use of leaf and fruit baits (Grimm and Alexander
1973), plating onto semi-selective agar media (Tsao
and Guy 1977; Timmer et al. 1988), isozyme analysis
(Oudemans and Coffey 1991; Mchau and Coffey
1994; Graham et al. 1998), and serological methods
(Timmer et al. 1993; Miller et al. 1997). The most
common method of species identification is to use
morphological criteria for colonies growing on
media, which is time-consuming, requires expertise,
and can be limited by interference from fast-growing
secondary microflora (Tsao 1990; Nechwatal and
Oßwald 2001). Serological techniques facilitate
detection but are only useful at the genus level. Also,
low sensitivity and cross-reactions with other species
limit their extensive application (Mohan 1989; Miller
1996). In recent years, molecular methods have been
developed to allow species identification based on
restriction enzyme digest patterns of the internal
transcribed spacer (ITS) regions of the ribosomal
RNA genes (Cooke and Duncan 1997; Cooke et al.
2000). The internal transcribed spacer regions of
ribosomal DNA are particularly useful for species
discrimination of fungal taxa, because they evolve in
a neutral manner at a rate that approximates the rate
of speciation (White et al. 1990; Lee and Taylor
1992). Also, ribosomal RNA genes are present in
multiple copies within the genome, increasing PCR
sensitivity as compared with single-copy genes. The
development of ITS methodology has lead to exten-
sive application of PCR in various fields of research
including studies on evolution and taxonomy (Cooke
and Duncan 1997; Cooke et al. 2000), studies on
intraspecific variation (Cohen et al. 2003) and species
hybridization (Bonants et al. 2000), as well as
identification of species responsible for disease
outbreaks (Schubert et al. 1999; Nechwatal and
Oßwald 2001; Vettraiano et al. 2001). Based on
information derived from sequencing ribosomal ITS
regions, a number of species-specific primers have
also been developed, eliminating the need for gener-
ating restriction profiles (Bonants et al. 1997; Trout
et al. 1997; Bo ¨hm et al. 1999; Schubert et al. 1999;
Nechwatal et al. 2001; Grote et al. 2002).
Evaluating promising new rootstock candidates for
their resistance or tolerance to Phytophthora diseases
is one important objective of the citrus breeding
programmes in Florida (Bowman et al. 2001, 2002,
2003; Grosser et al. 2003; Albrecht and Bowman
2004). An early step in the evaluation process is the
screening of newly developed rootstocks in the
greenhouse under controlled environmental condi-
tions. The application of molecular detection methods
may facilitate the rapid testing of large numbers of
plants and the selection of new candidate rootstocks
for their resistance or tolerance to Phytophthora
diseases before proceeding with the more time-
consuming examination under natural field condi-
tions. A number of studies have been conducted using
molecular techniques for detection of citrus phy-
tophthoras (Zheng and Ward 1998; Cohen et al.
2003; Ippolito et al. 2002, 2004). Whereas the studies
of Zheng and Ward (1998) and Cohen et al. (2003)
focused on the taxonomic variation of Phytophthora
spp. using cultured isolates from different geographic
locations, Ippolito et al. (2002) tested detection
sensitivity of Phytophthora spp. from soil and
infected root material using species-specific primers.
However, except for Ippolito et al. (2004) who used
species-specific primers in a multiplex PCR assay,
144 Eur J Plant Pathol (2007) 119:143–158
detection procedures were limited to individual
pathogens only and did not address complications
which may arise when testing for multiple pathogenic
organisms coexisting in the tissue.
This study investigates whether molecular meth-
ods based on ITS-fingerprinting can be effectively
used for the detection and identification of Phytoph-
thora spp. of citrus under controlled conditions in the
greenhouse. The main objective was to apply existing
molecular detection methods in addition to traditional
procedures and to evaluate their value for breeding
programmes. In contrast to most studies on plant
pathogens, experimental treatments included the
simultaneous infection of citrus seedlings by P.
nicotianae and P. palmivora, since both are associ-
ated with citrus decline in Florida and are frequently
isolated from the same location in the field. The
sensitivity of molecular detection was tested and
compared with results obtained from morphological
analyses using selective plating techniques and
Materials and methods
Isolates of P. nicotianae (Pn198) and P. palmivora
(Pp99-59-1) were used in three experiments. Phy-
tophthora nicotianae isolate Pn198 was isolated in
2000 from citrus roots in one of the greenhouses at
the Citrus Research and Education Centre in Lake
Alfred (Polk County, Florida), while P. palmivora
isolate Pp99-59-1 was isolated in 1995 from citrus
fruit in a field site in the same county. In addition, an
isolate of P. citrophthora (M140), isolated in 1988
from soil under citrus trees in California, was used for
molecular studies regarding the specificity of PCR
reactions. Isolates were maintained on 20% clarified
V8 juice (Campbell, Camden, NJ) agar at 18?C with
annual transfers to fresh medium.
Seedlings of the three-rootstock genotypes: Cleopatra
mandarin, sour orange (Citrus aurantium) and Swin-
gle citrumelo were evaluated. The ages of seedlings
upon initiation of the studies were 12 weeks
14 weeks (experiment 3).
1), 17 weeks(experiment2) and
Inoculation of plants
Inoculum of Phytophthora spp. was prepared by
growing cultures on sterilized millet (Panicum
ramosum) seeds. Seeds (Brown Top Millet, Seedland
Inc., Wellborn, FL) were soaked overnight in deion-
izedwater (75 ml water/100 ml
autoclaved twice with 24 h between cycles. For
experiments 1 and 2, seeds were inoculated by adding
two agar discs of 5 mm diam from five day-old
actively growing P. nicotianae or P. palmivora
cultures per 100 ml of seeds. For dual inoculations
one disc of each species per 100 ml of seeds was
used. Millet seeds were incubated for 10–15 days at
23–25?C in the dark under sterile conditions. For the
third experiment the amount of inoculum for P.
palmivora was increased by 50% to compensate for
the apparently slower growth of this species under the
conditions used in this study. Millet seeds for control
treatments were prepared in the same manner, but
were not inoculated with Phytophthora spp.
A commercially available soil-less potting med-
vermiculite (Pro-Mix BX, Premier Horticulture Inc.,
Red Hill, PA) was filled into 63 cm · 40 cm · 22
cm drained plastic tubs to a depth of 15 cm. Millet
seeds were incorporated at a rate of 20 ml inoculum
per 1,000 ml potting medium, and tubs were filled to
a final depth of 18 cm. Citrus seedlings of all three
rootstock genotypes were planted into the tubs with
five randomized replicates for each rootstock in each
tub. One tub was prepared for each treatment in
experiments 1 and 2, resulting in a total of 60 plants
per experiment, and three tubs were prepared for each
treatment in experiment 3, resulting in a total of 180
plants. Treatments were potting medium containing
(1) non-inoculated millet seeds, (2) millet seeds
inoculated with P. nicotianae, (3) millet seeds
inoculated with P. palmivora, and (4) millet seeds
inoculated with P. nicotianae and P. palmivora.
Tubs were placed into non-draining 75 cm ·
50 cm · 15 cm plastic trays and arranged in a
completely randomized order on the greenhouse
benches. Seedlings were grown under natural light
conditions in anenclosed
Eur J Plant Pathol (2007) 119:143–158145
maximum photosynthetic photon flux (PPF) of
1,200–1,300 lmol s?1m?2. Potting medium was
kept at near field capacity by maintaining water in
the trays at a level of 2.5 cm above the bottom of the
tubs. Tubs were irrigated with a water-soluble
fertilizer mix, 20N-10P-20K (Peters Professional,
The Scotts Company, Marysville, OH) once every
week, applied at a rate of 500 mg N l?1water.
Before each fertilizer application, water was allowed
to drain from the tubs. Four weeks (experiment 1),
6 weeks (experiment 2) and 7 weeks (experiment 3)
after initiating the experiment, plants were extracted
from the medium. Roots were washed thoroughly
with tap water to remove adhering particles and
blotted dry. For the morphological detection of
Phytophthora spp., seven root segments of 1–2 cm
length were excised from each plant and plated onto
medium (Timmer et al. 1988). Species were identi-
fied based on sporangium morphology and colony
characteristics (Erwin and Ribeiro 1996). Fresh
weight of shoots and roots was measured and roots
were kept at ?80?C until used for DNA extraction
and enzyme-linked immunosorbent assays (ELISA).
Samples of potting medium were taken from each tub
and the propagules per cm3of medium determined by
dilution-plating as described by Graham (1995).
Citrus roots were ground in liquid nitrogen with a
mortar and pestle. One hundred milligrams of ground
tissue was used for DNA extraction. DNA was
extracted using the Plant DNeasy Mini Kit (Qiagen,
Valencia, CA) according to the manufacturer’s
instructions. DNA was quantified at 260 nm using a
Beckman Coulter DU 640 spectrophotometer.
Fungal cultures were grown at room temperature in
the dark in 100 ml 20% clarified V8 juice. After 7–
10 days, mycelium was harvested by filtration,
washed with sterile deionized water (sdw), freeze-
dried and stored at ?20?C. Fungal tissue was ground
in liquid N with a mortar and pestle in preparation for
DNA extraction. Twenty milligrams of tissue was
used for extraction and quantification of DNA as
PCR amplification of ribosomal ITS regions as
described by Cooke et al. (2000) was performed
with the primers ITS4 (50-TCCTCCGCTTATTGA-
TATGC-30) and DC6 (50-GAGGGACTTTTGGGT
AATCA-30), which specifically amplify ribosomal
DNA from the major pathogenic oomycete groups
Pythium, Phytophthora and the downy mildews
(Bonants et al. 1997). PCR products were then
amplified in a second, semi-nested round, using
AGGTGAAGTCGTAACAAGG-30). Second round
PCR amplifications of the ITS regions specific for
P. nicotianae were performed with primer pair
PNIC1 (50-CAATAGTTGGGGGTCTTATT-30) and
developed by Grote et al. (2002).
All primers were purchased from MWG-Biotech
(High Point, NC).
PCR reactions were performed in a total volume of
25 ll using the Platinum SuperMix (Invitrogen,
Carlsbad, CA) composed of 22 mM Tris–HCl (pH
8.4), 55 mM KCl, 1.65 mM MgCl2, 220 lM dGTP,
220 lM dATP, 220 lM dTTP, 220 lM dCTP, and
22 U/ml recombinant Taq DNA polymerase provid-
ing an automatic ‘hot start’. Each reaction included
1 ll of DNA template, equivalent to 20–30 ng of
total DNA, and 1 ll of each primer at a final
concentration of 0.5 lM each. Amplification was
carried out in a PTC-100 or PTC-200 Thermal Cycler
(MJ Research, Reno, NV) using 0.2 ml tubes. An
initial denaturation step at 94?C for 3 min was
followed by 35 cycles of annealing at 60?C for
30 s, extension at 72?C for 1 min and denaturation at
94?C for 30 s before a final extension step at 72?C for
10 min. In nested PCR, 1 ll of the first round
amplification product was used for the second PCR
round. Five ll of PCR products were separated by
146Eur J Plant Pathol (2007) 119:143–158
electrophoresis in 2% agarose gels (Amresco) for
120 min at 5–6 V cm?1, stained with ethidium
bromide and visualized under UV light (Fluor S
Imaging System, Biorad, Hercules, CA). All PCR
reactions included multiple negative controls using
sdw in place of DNA template.
Ten ll of the amplification products generated with
primer pair ITS4 and ITS6 were digested with
restriction enzymes AluI, MspI and TaqI (New
England BioLabs, Beverly, MA) in a total volume
of 20 ll according to the manufacturer’s instructions.
Reaction products were analyzed by electrophoresis
in 2.5% agarose gels (Amresco) for 2–3 h at 5–
6 V cm?1. Restriction fragment patterns were com-
pared with data provided by Cooke et al. at
Sensitivity of detection
To determine the sensitivity of PCR detection for
each Phytophthora sp., ten-fold serial dilutions
ranging from 1 ng to 0.1 fg of fungal genomic
DNA were prepared in sdw or DNA extracts from
healthy citrus roots at a concentration of 10 ng ll?1.
Nested PCR was performed as described above. To
determine the sensitivity of simultaneous detection of
two Phytophthora spp. with RFLP, samples were
prepared using different ratios of DNA of all three
Phytophthora spp. Ratios tested were 99:1, 49:1, 9:1,
1:1; 1:9, 1:49, and 1:99 for all combinations of
species. DNA stocks of each species were adjusted
previously to 1 ng ll?1of DNA. Nested PCR and
digests were performed as described above.
Enzyme-linked immunosorbent assay (ELISA)
For the quantification of Phytophthora spp., a mul-
tiwell-test system (Agdia Incorporated, Elkhart, IN)
using a polyclonal Phytophthora antibody and a
monoclonal alkaline phosphatase-conjugated second-
ary antibody was used. For each reaction, 1 ml of
extraction buffer GEB2 was added to 100 mg of root
tissue, previously ground with liquid N using a
mortar and pestle. Samples were mixed by vortexing
for 10 s, incubated at room temperature for 15 min
and centrifuged at 20,800 g for 5 min. One-hundred
microliters of supernatant was used for each well; all
samples were tested in duplicate. Linearity of the test
system and detection threshold were determined
using ten-fold serial dilutions of extracts derived
from freeze-dried mycelia of P. palmivora and P.
nicotianae ranging from 1 pg to 10 mg of fungal
tissue. All assays were performed according to the
manufacturer’s instructions. Absorbance was mea-
sured at 405 nm using a Spectra Max Pro 190
Microplate Spectrophotometer (Molecular Devices
Corp., Sunnyvale, CA) in combination with the
software Soft Max Pro, Version 2.6.
Growth data and immunological data were tested by
analysis of variance using Statistica version 6.0
(SNK) test was used for mean comparison when the
F-test was significant at P < 0.05.
Identification of Phytophthora species
First round amplification of DNA from P. nicotianae,
P. palmivora, or P. citrophthora with primer pair
ITS4 and DC6 typically generated a PCR product of
about 1,300 bp (Fig. 1a). After second round ampli-
fication with the universal primers ITS4 and ITS6, a
PCR product of about 900 bp was obtained, which is
in the range of band sizes typically observed for
Phytophthora (Fig. 1b).
Nested PCR of DNA from P. nicotianae with primer
pair PNIC1 and PNIC2 produced an amplification
product of about 750 bp (Fig. 1c). A few non-specific
bands were observed when template concentrations
were high. No visible product specific for P.
Eur J Plant Pathol (2007) 119:143–158 147
nicotianae was obtained for DNA from P. palmivora,
and P. citrophthora.
Phytophthora nicotianae was identified by the pres-
ence of restriction fragments of 745 bp, 117 bp and
52 bp after digest with AluI and fragments of 404 bp,
390 bp and 120 bp after digest with MspI (Fig. 2).
The two bands of 404 bp and 390 bp after MspI
digest generally appeared as one broad band under
the electrophoresis conditions used in this study.
Phytophthora palmivora was identified by the pres-
ence of restriction fragments of 501 bp, 160 bp, and
157 bp after digest with AluI, with the latter two
bands appearing as one broad band, and fragments at
508 bp and 389 bp after digest with MspI. Restriction
patterns obtained after digest with TaqI revealed
profiles typical for both pathogens but did not aid in
species discrimination and were therefore not used
Sensitivity of PCR detection
First round amplification of decreasing concentra-
tions of fungal DNA prepared in sdw resulted in a
PCR product still visible at 1 pg of fungal DNA
derived from P. nicotianae (Fig. 3a). PCR products
obtained from DNA of P. palmivora and P. citroph-
thora were still detectable at 0.1 pg of fungal DNA.
Lower amounts of template generally yielded ampli-
fication products of lesser intensity. After nested PCR
with primer pair ITS4 and ITS6, P. nicotianae was
detectable at 10 fg of DNA thus increasing sensitivity
of detection by 100-fold (Fig. 3b). Phytophthora
palmivora was detectable at 1 fg of DNA, 1/10th of
the threshold for P. nicotianae. The detection limit
for P. citrophthora was the same as that for P.
palmivora (data not shown). Detection of P. nicoti-
anae with species-specific primers PNIC1 and PNIC2
after nested PCR was possible down to 1 fg of fungal
DNA (Fig. 4). Results obtained from serial dilutions
Fig. 1 (a) Amplification product of DNA from P. palmivora
using primers ITS4 and DC6 (b). (b) Amplification product of
DNA from P. palmivora after nested PCR with primers ITS4
and ITS6 (b). (c) Amplification product of DNA from P.
nicotianae (Pn) after nested PCR with primers PNIC1 and
PNIC2 (b); no product was obtained with DNA from P.
citrophthora (Pc) and P. palmivora (Pp). M, 100 bp ladder;
molecular weights in bp are indicated on the left
Fig. 2 Restriction profiles of Phytophthora species obtained
after digestion of ITS4/ITS6-amplification products with AluI,
MspI, and TaqI. Pn, P. nicotianae; Pp, P. palmivora; Pc, P.
citrophthora. M, 50 bp ladder; molecular weights in bp are
indicated on the left
148 Eur J Plant Pathol (2007) 119:143–158
of fungal DNA prepared in citrus DNA extracts were
identical to those obtained from dilutions prepared in
sdw (data not shown).
When using restriction patterns to simultaneously
identify two Phytophthora spp. in DNA preparations
containing varying proportions of template, the
following was observed. Due to the lower sensitivity
of ITS primers, P. nicotianae was only detectable in
mixed preparations when DNA was present in ratios
equivalent to or above 9:1 (Fig. 5). In samples
containing equal amounts of P. nicotianae and P.
palmivora or P. citrophthora DNA, bands specific for
P. nicotianae were never detected. Samples contain-
ing DNA of P. palmivora and P. citrophthora in
equal proportions produced restriction patterns spe-
cific for both species. At ratios above 9:1, only bands
specific for the species in excess were clearly
Linearity of the assay was determined for absorbance
values ranging from 0.1 to 1.8 corresponding to 1 ng
to 1 mg of freeze-dried fungal tissue, with a detection
threshold of approximately 0.1 ng of fungal tissue.
No differences in sensitivity of the system were
observed for the three Phytophthora spp. tested.
Growth responses of citrus rootstocks
The average fresh weights of roots and shoots from
all healthy plants ranged from 2.7 g to 10.1 g and
from 8.7 g to 24.5 g, respectively (Table 1). Results
Fig. 4 Sensitivity of nested PCR with primers PNIC1 and
PNIC2 for the detection of P. nicotianae using ten-fold serial
dilutions of fungal DNA. M, 100 bp ladder; molecular weights
in bp are indicated on the left. DNA dilutions contained citrus
DNA at a concentration of 10 ng ll?1. With increasing template
concentration non-specific amplification products appear
Fig. 5 Restriction profiles obtained after digestion of nested
ITS4/ITS6-amplification products from DNA preparations
containing varying proportions of DNA from different
Phytophthora species. (a) Restriction profiles after digest with
AluI. (b) Restriction profiles after digest with MspI. DNA
proportions were 99:1 (lane 1), 49:1 (lane 2), 9:1 (lane 3), 1:1
(lane 4), 1:9 (lane 5), 1:49 (lane 6), and 1:99 (lane 7). Pnic, P.
nicotianae; Ppal, P. palmivora; Pcitr, P. citrophthora. M,
50 bp ladder; molecular weights in bp are indicated on the left
Fig. 3 Sensitivity of simple and nested PCR for the detection
of P. nicotianae and P. palmivora using tenfold serial dilutions
of fungal DNA. (a) Simple PCR with primers ITS4 and DC6.
(b) Nested PCR with primers ITS4 and ITS6. M, 100 bp
ladder; molecular weights in bp are indicated on the left. DNA
dilutions were prepared in sterile water
Eur J Plant Pathol (2007) 119:143–158149
of MANOVA for reductions of root mass, shoot mass
and ELISA with rootstock, fungal species and
experiment as main effects were highly significant
(P < 0.001) as were interactions between root-
stock · fungal species, rootstock · experiment, and
fungal species · experiment (Table 2). MANOVA
performed for each experiment produced highly
significant (P < 0.001) results for rootstock and
fungal species (Table 3). A significant (P < 0.05)
interaction between rootstock and fungal species
were observed only in experiment 3.
Univariate results showed significant (P < 0.05)
differences of root mass reductions for rootstock and
fungal species effect in all three experiments, but no
significant interactions. Shoot mass reductions were
significantfor both effects
(P < 0.05), for fungal species in experiment 2
(P < 0.001) and for rootstock in experiment 3
(P < 0.0001). No significant interaction between
rootstock and fungal species was observed.
Infected plants from all three experiments exhib-
ited growth reductions from 16% to over 70%
(Table 4). Rootmass reductions after infection with
P. nicotianae were generally smallest (25–51%) for
seedlings of Swingle citrumelo as were shoot mass
reductions which ranged from 30% to 45%. Growth
reductions for Cleopatra mandarin and sour orange
were larger with up to 73% in sour orange after
infection with this pathogen. Similarly, lowest root
mass reductions from 28% to 38% were observed for
Swingle citrumelo after dual infection of roots with
P. nicotianae and P. palmivora. Root mass reductions
for Cleopatra and sour orange were considerably
larger and ranged from 30% to 64%. Results for shoot
mass reductions after dual infection varied little
between Swingle and sour orange (21–49%) and were
largest for Cleopatra (43–58%) with the exception of
experiment 2. Infection with P. palmivora caused
only small root mass reductions (17–21%) for
Swingle citrumelo. Shoot mass reductions after
infection with this species were between 16% and
37% for Swingle and sour orange and were generally
larger for Cleopatra mandarin, reaching up to 50%.
Univariate results for ELISA showed significant
differences for fungal species effect and rootstock ·
fungal species interaction in experiment 1 (P < 0.05).
Significant results were observed for fungal species in
experiment 2 (P < 0.0001) and for both effects and
their interaction in experiment 3 (P < 0.01).
The amount of pathogen detected in roots using
ELISA was considerably larger in all citrus seedlings
inoculated with P. palmivora, particularly in exper-
iments 1 and 2 where absorption values ranged from
0.6 to 1.0 compared with 0.3 to 0.5 for roots infected
Table 1 Average fresh weights of roots and shoots from non-
infected seedlings of three citrus rootstocks
Rootstock Fresh weight (g)
Cleopatra mandarin2.74 0.218.690.51
Sour orange4.310.499.32 0.66
Swingle citrumelo4.74 0.3510.88 1.34
Cleopatra mandarin4.30 0.36 15.87 0.81
Sour orange 10.001.46 24.46 2.44
Swingle citrumelo 9.06 1.2722.032.71
Cleopatra mandarin 4.490.27 15.750.75
Sour orange 7.11 0.5421.01 1.18
Swingle citrumelo10.05 0.5522.031.09
Table 2 Results of MANOVA for root mass reductions, shoot mass reductions and ELISA across all three experiments
Effect dfError df
Rootstock 0.66814.596 392.000.00000
Fungal Species 0.33248.046 392.000.00000
Rootstock · Fungal Species
Rootstock · Exp
Fungal Species · Exp
Rootstock · Fungal Species · Exp
0.37042.146 392.00 0.00000
0.907 0.8124 569.06 0.72711
150Eur J Plant Pathol (2007) 119:143–158
with P. nicotianae or with both species simulta-
neously (Table 4). Absorption values obtained for
experiment 3 were generally lower but indicated the
same trend. However, no clear relationship between
the growth reductions observed for the different
rootstock genotypes and the amount of pathogen
detected in the roots was observed.
Detection of P. nicotianae and P. palmivora in
In all plants from inoculated treatments, an amplifi-
cation product typical for the genus Phytophthora was
obtained after nested PCR with primers ITS4 and
ITS6 (Fig. 6). No phytophthora-specific amplification
product was detected in non-inoculated plants, though
DNA fragments of different size were observed
occasionally. Sequence analysis confirmed that these
fragments did not derive from Phytophthora or other
plant pathogenic fungi. The presence or absence of
reaction products after first round amplification with
primer pair ITS4/DC6 was inconsistent after repeated
analysis and appeared to be affected by the thermal
cycler model used for the assay.
Restriction profiles obtained with AluI and MspI
were specific for P. nicotianae in all root samples
inoculated with P. nicotianae (Fig. 7 and Table 5).
Nested PCR with species-specific primers PNIC1 and
PNIC2 revealed a P. nicotianae-specific reaction
product in all samples (Fig. 6). Morphological
analyses after plating on semi-selective agar identi-
fied P. nicotianae in all plants from this treatment; no
P. palmivora was detected in any of the root
segments (Table 5). Propagule levels in potting
medium inoculated with P. nicotianae were high
with an average of 4580 cm?3of medium for all
treatments; no P. palmivora propagules were detected
All root samples inoculated with P. palmivora
restriction digest (Fig. 7 and Table 5). No amplifica-
tion products were obtained after nested PCR with
species-specific primers PNIC1 and PNIC2 (Fig. 6).
Morphological analyses revealed the presence of P.
palmivora in all root samples from experiments 2 and
3; no P. nicotianae was detected in any of the root
segments (Table 5). However, plate tests of samples
from experiment 1 indicated a strong concurrent
infection of plants with P. nicotianae, as well as P.
palmivora. Mean propagule levels of potting medium
inoculated with P. palmivora were 4020 cm?3. No P.
nicotianae was detected with the exceptionofmedium
from experiment 1, which appeared to be moderately
contaminated with this species (Table 6).
Restriction patterns obtained for all plants from
medium inoculated with both Phytophthora spp. were
specific for P. palmivora (Fig. 7). Phytophthora
detected as faint bands in a few root samples from
experiment 1. However, nested PCR with primers
PNIC1 and PNIC 2 resulted in P. nicotianae-specific
Table 3 Results of MANOVA for root mass reductions, shoot mass reductions and ELISA comparing experiments 1–3
Effect Wilks Lambda
Effect dfError df
Rootstock 0.269 10.526 68.000.00000
Fungal Species 0.22812.396 68.000.00000
Rootstock x Fungal Species0.6811.18 12 90.250.31146
Rootstock 0.468 5.236 68.000.00018
Rootstock · Fungal Species
0.707 1.05 1290.25 0.41019
Rootstock 0.703 7.966 248.00 0.00000
Rootstock · Fungal Species
0.552 14.306 248.000.00000
0.843 1.8312328.36 0.04309
Eur J Plant Pathol (2007) 119:143–158151
amplification products in all samples from this
treatment (Fig. 6; Table 5). Morphological analyses
indicated the presence of P. nicotianae in all samples
inoculated with both fungal species (Table 5). Only
one root segment of all samples from experiment 1
exhibited the presence of P. palmivora during the
plate tests. In experiment 2, P. palmivora was
detected in only very few root samples from this
Table 4 Comparison of growth response of seedlings of three citrus rootstocks and amount of pathogen detected in roots after
inoculation with Phytophthora spp
Rootstock Fungal species
Growth reductions (%)Amount of pathogen
RM Red (%) SM Red (%)ELISAa
Cleopatra mandarin Pnic5 43.50 a-c 49.81 ab0.361 c
Sour orangePnic5 52.99 ab45.45 ab 0.415 cd
Swingle citrumelo Pnic5 25.23 bc 44.56 ab 0.352 c
Cleopatra mandarin Pnic + Ppal5 64.30 a 58.20 a0.501 cd
Sour orangePnic + Ppal5 61.16 a 44.36 ab0.510 cd
Swingle citrumeloPnic + Ppal5 27.98 bc 48.77 ab0.496 cd
Cleopatra mandarin Ppal5 52.85 ab50.03 ab 1.005 a
Sour orangePpal5 37.12 a–c27.34 b 0.654 bc
Swingle citrumeloPpal5 16.88 b37.30 ab 0.839 ab
Total 45 42.45 45.090.570
Cleopatra mandarin Pnic5 53.49 ab49.31 ab0.303 b
Sour orange Pnic5 72.62 a54.37 a 0.325 b
Swingle citrumeloPnic5 51.30 a–c40.07 ab 0.395 b
Cleopatra mandarinPnic + Ppal5 63.26 ab 43.14 ab 0.398 b
Sour orange Pnic + Ppal5 62.42 ab 48.85 ab 0.293 b
Swingle citrumelo Pnic + Ppal5 37.53 bc45.07 ab 0.397 b
Cleopatra mandarinPpal5 45.39 a–c 34.14 ab 0.697 a
Sour orange Ppal5 47.34 a–c 30.16 ab0.611 a
Swingle citrumelo Ppal5 21.28 c 24.95 b0.637 a
Cleopatra mandarinPnic 1547.42 a47.86 a0.253 c
Sour orangePnic 15 44.21 a30.79 c 0.246 c
Swingle citrumeloPnic 15 43.51 a29.68 bc 0.220 c
Cleopatra mandarinPnic + Ppal15 45.23 a43.74 ab0.283 c
Sour orangePnic + Ppal 15 29.35 ab21.12 c 0.261 c
Swingle citrumeloPnic + Ppal1533.78 ab 23.94 c 0.282 bc
Cleopatra mandarin Ppal 1545.17 a 46.65 a0.459 a
Sour orangePpal1520.77 b20.28 c0.291 c
Swingle citrumeloPpal1519.40 b16.18 c 0.364 b
Comparison was by ANOVA. Different letters within columns indicate significant differences between means according to the
Student-Newman-Keuls test for P < 0.05
Mean absorbance values (405 nm) of assays performed in duplicate are presented. Pnic, P. nicotianae; Ppal, P. palmivora; RM
Red, root mass reduction; SM Red, shoot mass reduction
152 Eur J Plant Pathol (2007) 119:143–158
treatment. Each of the two Phytophthora sp. was
detected in many samples from experiment 3, though
P. nicotianae appeared to be the dominant species.
The average propagule numbers in potting medium
inoculated with both Phytophthora spp. was more
than twice as high for P. nicotianae as compared with
P. palmivora in experiment 1 and 2. Propagule levels
in experiment 3 were similar for both spp. (Table 6).
No Phytophthora spp. was detected from roots of
non-inoculated plants using immunological assays,
plating of root segments or PCR techniques (Fig. 6).
With the exception of experiment 2, no Phytophthora
spp. was detected in potting medium containing non-
inoculated millet seeds (Table 6).
Infection of citrus roots by P. nicotianae and P.
palmivora substantially reduced root and shoot mass
of seedlings of the three rootstocks, depending on
which Phytophthora spp. was present. Growth of
Fig. 6 Amplification of DNA from non-infected and infected
citrus roots. (a) Simple PCR with primers ITS4 and DC6. (b)
Nested PCR with primers ITS4 and ITS6; non-specific
amplification products are seen in some of the control plants.
(c) Nested PCR with primers PNIC1 and PNIC2. Ctrl, non-
inoculated control plants; Pnic, plants inoculated with P.
nicotianae; Ppal, plants inoculated with P. palmivora; Pnic +
Ppal, plants inoculated with P. nicotianae and P. palmivora.
Pn, control DNA from P. nicotianae; Pp, control DNA from P.
palmivora. M, 100 bp ladder
Fig. 7 Restriction profiles obtained after digestion of ITS4/
ITS6-amplification products from infected citrus roots with (a)
AluI and (b) MspI. Pnic, plants inoculated with P. nicotianae;
Ppal, plants inoculated with P. palmivora; Pnic + Ppal, plants
inoculated with P. nicotianae and P. palmivora. Pn, control
DNA from P. nicotianae; Pp, control DNA from P. palmivora.
M, 50 bp ladder
Eur J Plant Pathol (2007) 119:143–158 153
Cleopatra mandarin and sour orange was reduced
severely, whereas that of Swingle citrumelo seedlings
was much less affected. These results are in marked
contrast to previous field trials and greenhouse assays
performed in this laboratory, which demonstrated
better performanceof Cleopatra mandarinin
Table 5 Molecular analysis and recovery of Phytophthora spp. from seedlings of three citrus rootstocks
Rootstock Fungal species
PnicPpal Pnic + Ppal
Cleopatra mandarinPnic5 Pnic+ 3200
Sour orangePnic5 Pnic+ 3200
Swingle citrumelo Pnic5 Pnic+ 3400
Cleopatra mandarinPnic + Ppal5 Ppal+ 3110
Sour orangePnic + Ppal5 Ppal+ 3100
Swingle citrumeloPnic + Ppal5 Ppal+ 3000
Cleopatra mandarin Ppal5 Ppal
Sour orange Ppal5 Ppal13 170
Swingle citrumeloPpal5 Ppal13 120
Total 45237 351
Cleopatra mandarinPnic5 Pnic+ 3500
Sour orange Pnic5 Pnic+ 3000
Swingle citrumeloPnic5 Pnic+ 3300
Cleopatra mandarinPnic + Ppal5 Ppal+ 2921
Sour orangePnic + Ppal5 Ppal+ 2605
Swingle citrumeloPnic + Ppal5 Ppal+ 2642
Cleopatra mandarin Ppal5 Ppal
Sour orange Ppal5 Ppal0 340
Swingle citrumeloPpal5 Ppal0 270
Total 45179 998
Cleopatra mandarinPnic15 Pnic+ 9400
Sour orange Pnic 15Pnic+ 8900
Swingle citrumeloPnic 15Pnic+ 9500
Cleopatra mandarinPnic + Ppal 15Ppal+ 6120 13
Sour orangePnic + Ppal15Ppal+ 48 284
Swingle citrumelo Pnic + Ppal15 Ppal+ 5021 11
Cleopatra mandarinPpal 15 Ppal
Sour orangePpal 15Ppal0 770
Swingle citrumeloPpal 15Ppal0880
Results are representative for all five replicated samples per rootstock and treatment
Species-specific restriction fragment patterns obtained after digest of nested ITS4/ITS6 products with AluI and MspI
Nested PCR with ITS4/DC6 amplification products
Data denote the number of root segments of a total of 35 per rootstock in which Phytophthora was detected. Pnic, P. nicotianae;
Ppal, P. palmivora. ‘+’, PCR product present; ‘?’, PCR product absent
154Eur J Plant Pathol (2007) 119:143–158
comparison with Swingle citrumelo after inoculation
with P. palmivora (Bowman et al. 2002 and 2003;
Albrecht and Bowman 2004). This contrasting result
may be related to the source of inoculum. In the
earlier field study and greenhouse tests, plants were
inoculated with infected field roots, whereas this
study utilized cultured pure isolates of the two
Phytophthora spp. grown on millet seeds. Experi-
ments, currently in progress at our laboratory, suggest
that differences in pathogenicity of field and cultured
Phytophthora spp. may be at least partially respon-
sible for the observed differences (data not shown).
Also, other microorganisms included in the field root
inoculum may have been involved in the pathogenic
Despite the high Phytophthora damage levels,
using Phytophthora reared on millet seeds may prove
unsatisfactory as inoculum at the rate described here,
since its incorporation into the potting medium was
followed by the rapid development of unidentified
secondary organisms. Nevertheless, Phytophthora
was detected successfully in root samples from all
inoculated treatments in all three experiments using
PCR. Non-inoculated plants yielded negative results,
and no cross-reaction with other organisms present in
the medium was detected. In a single step PCR,
detection limit was determined to be at 0.1 pg to 1 pg
of purified fungal DNA. In nested PCR the pathogen
was still detectable at concentrations down to 1 fg of
DNA, hence increasing sensitivity by 100- to 1000-
fold. Tooley and Therrien (1987) estimated the
average genome size of diploid isolates of P.
infestans to be 0.52 pg of DNA per nucleus. Assum-
ing the genome size of Phytophthora spp. in this
study is similar, only 0.002 nuclei were necessary to
detect the pathogen, making nested PCR based on
ribosomal ITS regions a very powerful detection
method. These results are in agreement with other
authors who reported sensitivity thresholds of 1 to
10 pg of DNA after simple PCR (Tooley et al. 1997;
Ippolito et al. 2002) and of 0.1 fg (Judelson and
Tooley, 2000), 1 fg (Ippolito et al. 2002), 60 fg
(Grote et al. 2002) and as few as five zoospores
(Nechwatal et al. 2001) after nested PCR with
species-specific primers. Amplification of fungal
DNA was not inhibited by citrus root DNA extracts
in contrast to other studies (Schubert et al. 1999;
Grote et al. 2002), which is probably due to the
complete removal of possible inhibitory compounds
of citrus roots by silica-gel-based DNA extraction
and the use of a soil-less potting medium in the
Serological analyses using ELISA yielded positive
results for all inoculated seedlings. No Phytophthora
was detected in non-inoculated plants, which is in
agreement with results obtained from molecular
analyses. Interestingly, ELISA values were highest
for all plants inoculated with P. palmivora, indepen-
dent of the rootstock genotype. Similar observations
were made by Widmer et al. (1998), who found a
significantly higher colonization of root cells of both
susceptible and tolerant citrus hosts with P. palmi-
vora in comparison to P. nicotianae. Apparently this
fungal species causes high infection rates, which are
not always associated with plant decline.
Restriction digests of nested PCR products yielded
profiles specific for P. nicotianae only in seedlings
inoculated exclusively with this species. Roots inoc-
ulated with P. palmivora or with a combination of
both pathogens generally exhibited P. palmivora-
specific banding patterns due to the ten-fold lower
sensitivity observed for DNA from P. nicotianae
after amplification with ITS primers as compared to
Table 6 Propagule density of Phytophthora spp. in potting
medium from experiments 1–3
P. nicotianaeP. palmivora
Inoculation with P. nicotianae
Inoculation with P. palmivora
Inoculation with both species44601880
Inoculation with P. nicotianae
Inoculation with P. palmivora
Inoculation with both species
Inoculation with P. nicotianae
Inoculation with P. palmivora
Inoculation with both species29802570
Data presented are averages from three replicated treatments
Eur J Plant Pathol (2007) 119:143–158155
P. palmivora. These results were confirmed by
analyzing restriction patterns obtained from DNA
samples containing varying proportions of purified
fungal DNA from different species. Despite the
reduced sensitivity of amplification and the negative
results obtained with PCR-RFLP, P. nicotianae was
detected in all samples inoculated with P. nicotianae
individually, or in combination with P. palmivora,
after nested PCR with species-specific primers
PNIC1 and PNIC2. Species-specific DNA primers
present an ideal alternative to PCR-RFLP technology,
especially since the time-consuming steps of diges-
tion and electrophoresis to separate fragments can be
eliminated. However, the simple presence of an
amplification product of specific molecular weight
does not prove its identity, as opposed to RFLP
patterns based on sequence variations. Also, design of
species-specific primers derived from ribosomal ITS-
regions can be problematic and cross-amplification
with other closely-related species is often observed
(Tooley et al. 1997; Schubert et al. 1999; Grote et al.
2002). Our attempts to design P. palmivora-specific
primers based on ITS sequences have so far been
unsuccessful due to cross-reactions with DNA from
P. nicotianae (data not shown). Species-specific
primers derived from random amplification of poly-
morphic DNA sequences (RAPDs) or from other
DNA sequences are useful but do not always achieve
the required sensitivity necessary for disease diagno-
sis prior to a noticeable expression of disease
symptoms (Bonants et al. 1997; Lacourt and Duncan
1997; Schubert et al. 1999). Real-Time PCR, a
recently developed highly sensitive methodology,
may eliminate these problems and also permit the
quantification of individual pathogens present in the
infected tissue (Bo ¨hm et al. 1999; Schaad and
Frederick 2002; Vandemark and Barker 2003; Ippol-
ito et al. 2004).
Results from analyses of potting medium from
all treatmentswith plating
methods were generally in agreement with those
obtained using PCR-RFLP, though slight contami-
nation was detected in some of the soil samples.
Selective agar-plating of infected root segments
indicated the presence of P. nicotianae in several
samples from experiment 1 which were inoculated
with P. palmivora only and a predominance of P.
nicotianae in all samples inoculated with both
species in experiments 1 and 2. Since no P.
nicotianae was detected in roots of seedlings
exclusively inoculated with P. palmivora by PCR
analysis with species-specific primers, contamina-
tion most likely occurred at some stage during the
preparation or handling of the plates. The predom-
inance of P. nicotianae in plating assays may be
the result of much faster growth of this species in
comparison to P. palmivora on PARPH medium, a
trend that has also been observed with other strains
of these species in continuing studies in this
laboratory. Thus, the identification of Phytophthora
spp. using semi-selective agar-plating may lead to
inaccurate results in mixed infections.
The results described in this study confirm that
molecular techniques based on ITS-fingerprinting
provide powerful tools which can be applied to the
detection and identification of Phytophthora species
in screening programmes for disease resistance under
controlled conditions in the greenhouse.
The high sensitivity of molecular methods allows
the identification of less than a single cell of
pathogen. Plants can thus be diagnosed at an early
stage of infection when no visible symptoms are
observed above-ground or are clouded by similar
symptoms produced for other reasons. In addition,
the convenience of being able to freeze tissue
samples and to analyze them at a later stage is of
great importance for our breeding programmes
involving hundreds of plants, as opposed to most
traditional methods where immediate processing of
the samples is required. Despite its more limited
sensitivity, serological assays using ELISA were
shown to be very useful in detecting fungal patho-
gens at the genus-level and provide an important
tool for estimating the amount of pathogen present
in the plant tissue. The rearing of Phytophthora
species on millet seed to be used for inoculation of
potted plants appears efficient and reliable. In our
continuing experiments with this method, we are
using lower rates of millet seed inoculum in the pots
to minimize the growth of other unwanted micro-
organisms while obtaining the desired Phytophthora
the Florida Citrus Production Research Advisory Council,
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