Plant Pathology (2005) 54, 650– 656 Doi: 10.1111/j.1365-3059.2005.01211.

Pathogenicity of branched broomrape (Orobanche ramosa)

Blackwell Publishing, Ltd.

populations on tobacco cultivars

H. Buschmann*†, G. Gonsior and J. Sauerborn

University of Hohenheim, Institute for Plant Production and Agroecology in the Tropics and Subtropics (380), Garbenstrasse 13, 70599
Stuttgart, Germany

Parasitic weed species of the genus Orobanche are a serious threat for the production of several crops in Europe, Africa
and Asia. In contrast to other broomrape species of agronomic importance, O. ramosa (branched broomrape) has a
broad host range and in Europe particularly affects hemp, tobacco, tomato and, in recent times, oilseed rape. Two sep-
arate sets of experiments investigated the effect of two populations of O. ramosa on nine tobacco cultivars grown in
Europe and belonging to the three major tobacco types: Virgin (flue-cured), Burley (light air-cured) and dark air-cured
under standardized glasshouse conditions. The two broomrape populations were discriminated by means of polymor-
phic DNA fragments obtained by PCR of the intersimple sequence repeat regions (ISSRs). The Orobanche populations
exhibited different levels of pathogenicity but all various tobacco cultivars were susceptible. Dark air-cured tobacco
cultivars were the least susceptible to both broomrape populations. Virgin and Burley tobacco cultivars were more
susceptible to one population of O. ramosa.

Keywords: ISSR-PCR, microsatellites, Nicotiana tabacum, parasitic angiosperm, resistance, tobacco cultivars

carota), aubergine (Solanum melongena), hemp (Cannabis

Introduction
Within the plant kingdom, various families independently
developed parasitic lifestyles (Kuijt, 1969). The members
of the genus Orobanche lack chlorophyll and a well-
developed root system and rely completely on the uptake
of water, mineral nutrients and assimilate from the host
plant. The transport of these nutrients occurs via a contact
organ, the haustorium, directly from the host roots.
Depending on the level of soil infestation and subsequent
infection, the consequences for parasitized host plants are
wilting, reduction of biomass and, in seriously affected crops
or with additional abiotic stresses, death. Of approxi-
mately 170 different species of Orobanche, six severely
parasitize crop plants [O. aegyptiaca, O. cernua, O. crenata,
O. cumana, O. minor and O. ramosa (Sauerborn, 1991;
Parker, 1994)] and the latter, branched broomrape, can
have a major impact on affected crops. The distribution of
this species covers central and southern Europe, northern
Africa and Asia. Additionally, this species was introduced
by humans to southwestern Australia, South and North
America and South Africa. Orobanche ramosa has a
broad host range, including, for example, carrot (Daucus
*To whom correspondence should be addressed.
†E-mail: hbuschma@uni-hohenheim.de
Accepted 14 March 2005
sativa), lentil (Lens culinaris), potato (Solanum tuberosum),
tomato (Lycopersicon esculentum) and tobacco (Nico-
tiana tabacum) (Musselman & Parker, 1982; Labrada &
Perez, 1988a; Parker & Riches, 1993). In central Europe
the major hosts of branched broomrape are tomato and
tobacco. Recently, it has also increasingly parasitized
oilseed rape (Brassica napus ssp. napus) and is widespread
in western France (Gibot-Leclerc et al., 2003).
As a result of the direct connection to the host, a pre-
dominantly subterranean life and durable, numerous and
small seeds, control of Orobanche spp. is very difficult by
means of agronomic practices and the application of her-
bicides (Dhanapal et al., 1996). A more efficient way of
limiting the effects of these parasitic angiosperms is the
prevention of infection of plants and subsequent infesta-
tion of sori by developing resistant crops (Rubiales, 2003).
To date, apparently only Palakarcheva et al. (1987) have
screened tobacco for sources of resistance to O. ramosa.
In field experiments these authors evaluated 29 Bulgarian
cultivars and interspecific hybrids over 2 years for their
susceptibility to branched broomrape, but did not identify
any resistance. Although Joel & Portnoy (1998) investi-
gated expression, no defence mechanisms have been iden-
tified. Resistance breeding in tobacco has not focused on
the Orobanche problem, even though there are several
different types and cultivars of commercial tobacco and
many wild relatives available.

650 © 2005 BSPP

 Pathogenicity of Orobanche ramosa on tobacco
About 328 600 t of tobacco are produced in Europe
from an area of 122 600 hectares. The three major types
are flue-cured tobacco (Virgin, 40·4% of production),
light air-cured tobacco (Burley, 24·8% of production) and
dark air-cured tobacco (9·9% of production) (Anonymous,
2003). Since cultivation of hemp and tobacco began in
Europe, O. ramosa has spread and caused sporadic prob-
lems (Sauerborn, 1991; Demuth, 1992; Holm et al.,
1997). However, during the last decade, the incidence and
severity of O. ramosa have increased and the parasitic
weed has become a persistent problem. Without efficient
control, every O. ramosa plant can produce 100 000–
500 000 durable and very small seeds (diameter 0·3 mm),
leading to a dramatic increase of the Orobanche seed
bank in the soil (Sauerborn, 1991). Humans, machinery,
water or wind can easily disperse these seeds.
Another obstacle to effective control is the genetic vari-
ability of O. ramosa, which threatens different hosts
(Musselman & Parker, 1982) and control strategies (Dha-
napal et al., 1996). In the past, some effort was made to
describe O. ramosa populations by means of morpholog-
ical features (Gilli, 1966). Based on these characters it is
possible to distinguish distinct populations. However,
because of its parasitic lifestyle, various features commonly
used in plant taxonomy are highly reduced in Orobanche,
so that population descriptions can have limited reliability
(Roman et al., 2001). These difficulties can be overcome
by the use of molecular techniques. For molecular phylog-
enies, some researchers have used sequence data of the
internal transcribed spacers (ITSs) of nuclear ribosomal
Figure 1 Tobacco crop in the Rhine valley between Karlsruhe and Freiburg (southwest Germany) affected by Orobanche ramosa (arrowed).
© 2005 BSPP Plant Pathology (2005) 54, 650– 656
651
DNA amplified by PCR (Schneeweiss et al., 2004). Genetic
variability of Orobanche has so far been investigated by
fingerprinting techniques such as randomly amplified poly-
morphic DNAs (RAPDs; Paran et al., 1997; Gagne et al.,
1998; Roman et al., 2001). Another recent approach is
the use of intersimple sequence repeats (ISSRs). With
ISSRs, it is possible to identify polymorphisms between
microsatellite regions. These regions are hypervariable,
short stretches of DNA and characterized by mono-, di- or
trinucleotide repeats that are the targets for the ISSR-
primers (Nagaoka & Ogihara, 1997; Wolfe & Randle,
2001). Such DNA polymorphisms elucidated by ISSR-
PCR enabled Benharrat et al. (2002) to characterize
several Orobanche species and even populations.
The aim of this study was to characterize two popula-
tions of O. ramosa and evaluate their pathogenicity to dif-
ferent tobacco cultivars by ISSR-PCR.
Materials and methods
Plant material
Seeds of O. ramosa were collected in August and Septem-
ber 2002 from two broomrape-affected tobacco fields (all
Virgin cultivars) in the Rhine valley, southwest Germany
(Fig. 1). The sampling sites were Forchheim (near Karl-
sruhe) and 30 km north of Neupotz (near Speyer). For
molecular discrimination of these two branched broom-
rape populations, namely Karlsruhe and Speyer, O. ramosa
originating from Fadasi, Gezira State, Sudan (collected in
652 H. Buschmann et al.
March 2002), was used as an outgroup. In all cases, six
plant samples per location were studied.
Nine different tobacco cultivars were used as host plants:
Virgin (flue-cured) type, cvs Helena, HyV8 and Golta;
Burley (light air-cured) type, cvs HyB2, B971 and BO11;
and dark air-cured type, cvs Z992, Adonis and Badischer
Geudertheimer.
Pot and root-chamber experiments
Tobacco plants were grown in a sand /compost mixture
(1:1, v/v) in 5 kg plastic pots. The soil of each pot was
mixed with 20 mg of O. ramosa seeds (about 1000 seeds
per kg of soil).
Root chambers were filled with autoclaved sand and
glass fibre filter paper and prepared as described elsewhere
(Linke & Vogt, 1987). The O. ramosa seeds were evenly
spread on the glass filter paper.
Pot and root-chamber experiments were conducted
under glasshouse conditions with 25/15°C day/night tem-
perature and a 12-h light regime with a light density of
100 µE m−2 s−1. Pots and root chambers were connected to
an automatic drip irrigation system. Plants were fertilized
every second week with 40 mL of a 2% liquid fertilizer
(Wuxal®, Bayer).
Three months after the experimental start date the
branched broomrape plants emerged and the mean sever-
ity of infection of tobacco (six plants of each cultivar) was
evaluated by washing out the host root system and count-
ing O. ramosa shoots. Harvested host and O. ramosa
material was dried at 80°C for 72 h for determination of
dry weight.
Seed germination and development of O. ramosa were
observed in root-chamber experiments according to
methods of Linke et al. (2001) for all Orobanche–tobacco
associations. Germination rates of Orobanche popula-
tions were determined using the synthetic germination
stimulant GR24 (provided by Professor B. Zwanenburg,
Nijmegen, Netherlands) (Linke et al., 2001).
DNA isolation and ISSR-PCR
For molecular analysis, a bulk sample of O. ramosa plants
of the same population were analysed. Closed O. ramosa
flowers (0·2 g) were ground in liquid nitrogen. DNA was
isolated from the plant cells using the Invisorb Spin Plant
Mini Kit (Invitek, Berlin, Germany) according to the man-
ufacturer’s instructions. Genomic DNA was stored in
TE buffer at −20°C. The isolated genomic DNA was
quantified spectroscopically with a Specord 50 UV-Vis-
spectrophotometer (Analytic Jena). PCR used 25 µ L aliquots
containing 20 ng template DNA, 4 µm primer, ddH2O
and puReTaq™ Ready-To-Go™ PCR beads (Amersham
Bioscience). DNA variations in O. ramosa populations
were determined using nine different ISSR primers: non-
anchored primers (CAG)5 (CAA)5 (GACA)4 and (GATA)4;
and anchored primers (CA)6RG (CTC)4RC (CT)8TC
(CA)6AC and (AG)8YA (Qiagen). The ISSR-PCR was
conducted in a Mastercycler Personal thermal cycler
(Eppendorf ) with the following programme: an initial
denaturing step 94°C (1 min); 35 cycles of denaturing (94°C,
1 min), annealing [64°C for (CAG)5, 57°C for (CAA)5, 58°C
for (GACA)4, 42°C for (GATA)4, 52°C for (CA)6RG,
52°C for (CTC)4RC, 57°C for (CT)8TC, 49°C for
(CA)6AC, 56°C for (AG)8YA, each for 1 min] and exten-
sion (72°C, 3 min); and a final extension step (72°C, 5 min).
After adding 5 µL Roti-Load-DNA loading buffer (Carl
Roth GmbH, Karlsruhe, Germany) to the vial, an aliquot
of the PCR products and a DNA-marker (pBR328 mix I,
Carl Roth GmbH) were separated on a 3% agarose gel
(Agarose Resophor, Eurobio) in 1× Tris-borate-EDTA
(TBE) buffer at 50 V for 3 h. After electrophoresis, DNA
was stained with ethidium bromide and visualized by a
UV-transilluminator. The sizes of the ISSR-PCR products
were calculated with the help of the SigmaGel software
package ( Jandel Scientific). The amplification was repeated
five times to evaluate the reproducibility of the ISSR-PCR.
Data analysis
The experimental design involved completely randomized
treatments with six replicates per Orobanche population/
tobacco– cultivar association and repeated twice. Using
Minitab (1998) for statistical analysis, all data were sub-
jected to analysis of variance. Significant differences in the
mean values were determined by Tukey’s honest signifi-
cant differences (HSD) test at a significance level of 0·05.
Results
Orobanche ramosa material collected from affected
Virgin-tobacco fields at two locations from neighbouring
regions in Germany exhibited significant differences in
pathogenicity towards different tobacco cultivars. Branched
broomrape from Karlsruhe parasitized all nine tobacco
cultivars but differences were observed in the susceptibil-
ity of the three different tobacco types (Fig. 2a). Virgin
tobacco types were most severely affected with between
114 and 169 mean Orobanche shoots per host plant. Cul-
tivars BO11 (Burley) and Badischer Geudertheimer (dark-
air cured) were least affected, with an average of 22 and
16 Orobanche shoots per tobacco plant, respectively.
The broomrape population from tobacco fields near
Speyer also colonized all tobacco cultivars tested, but to a
much lesser extent than the Karlsruhe population (Fig. 2b).
Additionally, no clear differences in susceptibility between
the tobacco types were observed. The Virgin tobacco cul-
tivar HyV8 was the most heavily parasitized, with a mean
number of 55 O. ramosa shoots per tobacco plant, whilst
Virgin-tobacco cv. Helena, the Burley-type cv. BO11 and
the dark air-cured tobacco cv. Z992 were the least suscep-
tible, with an average of 10, nine and two broomrape
shoots per plant, respectively.
The average seed germination rate of the two broom-
rape populations was very high (98%) when treated with
the synthetic germination stimulant GR24 (Linke et al.,
2001) but, as expected, was lower in both populations when
stimulated by tobacco alone in root-chamber experiments.
© 2005 BSPP Plant Pathology (2005) 54, 650– 656
 Pathogenicity of Orobanche ramosa on tobacco
Figure 2 Mean number (± SD, n = 6) of developing Orobanche ramosa
shoots in the Karlsruhe (a) and Speyer (b) populations on tobacco
Virgin tobacco cultivars (a, Helena; b, HyV8; c,= Golta), Burley
tobacco cultivars (d, HyB2; e, B971; f, BO11) and dark air-cured
tobacco cultivars (g, Z992; h, Adonis; i, Badischer Geudertheimer).
For example, the highly susceptible Virgin tobacco cv.
HyV8 and the less susceptible dark air-cured tobacco cv.
Z992 produced 78 and 74% germination (combined data
for both O. ramosa populations) on host roots. No signi-
ficant differences in percentage seed germination were
found between the two O. ramosa populations. However,
fewer tubercles and underground shoots of O. ramosa
developed on cv. Z992 in contrast to cv. HyV8 (Fig. 3).
Although the Orobanche populations parasitized the
tobacco cultivars to different extents (number of broom-
rape shoots produced), damage on field-grown tobacco
was similar or more severe in the Speyer region than in the
Karlsruhe region according to reports from regional farm-
ers. This observation can be explained by the comparison
of the biomass of both Orobanche populations on the same
hosts (Fig. 4). On most tobacco cultivars, the broomrape
shoots that developed from the less aggressive Speyer
seed material produced much higher biomass than their
relatives from the Karlsruhe area, which parasitized the
hosts, producing higher numbers of broomrape shoots.
Only on the tobacco cultivars Helena (Fig. 4, bar a) and
Z992 (Fig. 4, bar g) was the biomass of the shoots of
the Karlsruhe Orobanche population (per tobacco plant)
higher.
© 2005 BSPP Plant Pathology (2005) 54, 650– 656
653
The effect of parasitization by O. ramosa (Speyer
population) on the biomass production of the different
tobacco cultivars is summarized in Fig. 5. The compari-
son of nonparasitized controls and diseased plants illus-
trates that the organs of the hosts are affected to different
extents. The Virgin and Burley cultivars showed signifi-
cant reductions of biomass of leaves and/or stems. In the
case of Virgin cv. HyV8 and dark-air-cured cv. Badischer
Geudertheimer, there was also a reduction in root bio-
mass. The root biomass in cv. BO11 was much higher in
the parasitized plants than in the controls. Although O.
ramosa produced a relatively high biomass on the two
dark air-cured cvs Adonis and Badischer Geudertheimer,
the leaf biomass of these tobacco plants was not signifi-
cantly reduced as a result of infection. In all cases, the
number of flowers of parasitized tobacco plants was
reduced, with the exception of cv. Z992.
The German O. ramosa populations used in the present
experiments did not differ in either shoot size and habit
or flower size and colour (white). In contrast, the plants
of the Sudanese population used as an outgroup in mole-
cular studies are larger, have more branches and blue
flowers. Using the molecular fingerprinting technique
ISSR-PCR there was no variation within populations. Four
and seven polymorphisms were identified between the
German and the Sudanese O. ramose populations using
the primers (GATA)4 and (CTC)4RC, respectively. (Fig. 6a
and b). Seven of the ISSR-PCR primers used did not reveal
any polymorphisms for the two German broomrape
populations, but PCR-products of 452 and 546 bp for
primers (GATA)4 and (CTC)4RC differentiated the two
populations with two bands present in the Karlsruhe
population absent in the Speyer samples.
Discussion
To address specific questions on the biology and manage-
ment of this widespread agronomically important para-
sitic flowering plant, it is necessary to work with defined
populations. Benharrat et al. (2002) demonstrated that it
is possible to characterize Orobanche populations of the
section Orobanche (O. hederae and O. amethystea) by
means of ISSR-PCR, and the results presented here sup-
port ISSR-PCR characterization of populations of O.
ramosa belonging to the Orobanche section Trionychon.
In accordance with Benharrat et al. (2002) the use of non-
anchored ISSR primers revealed fewer polymorphic bands
than when anchored primers were used. The use of the
primers (GATA)4 and (CTC)4RC enabled even neighbour-
ing populations from southwestern Germany to be distin-
guished, although the number of polymorphisms was
very low. Even so, ISSR-PCR appears to be reliable and
superior to reports involving RAPD analysis and RFLP
(Nagaoka & Ogihara, 1997).
The need to characterize O. ramosa populations is
underlined by the present results on the susceptibility of
tobacco. Here, two European populations of O. ramosa
expressed the same virulence (host range) but differed in
aggressiveness towards nine tobacco cultivars. Virgin-type
654 H. Buschmann et al.

Figure 3 Tubercles (t) and underground shoots (s) of Orobanche ramosa (Karlsruhe population) developing in root chambers on the roots (hr) of
the highly susceptible Virgin tobacco cv. HyV8 and on the less susceptible dark air-cured tobacco cv. Z992.

Figure 5 Effect of Orobanche ramosa (Speyer population) on biomass

Figure 4 Mean dry weight (g) (± SD, n = 6) of all Orobanche ramosa
shoots per host plant of populations originating from Karlsruhe and
Speyer attached to the roots of tobacco. Virgin tobacco cultivars (a,
Helena; b, HyV8; c, Golta), Burley tobacco cultivars (d, HyB2; e, B971;
f, BO11) and dark air-cured tobacco cultivars (g, Z992; h, Adonis;
i, Badischer Geudertheimer).
production of tobacco. Virgin tobacco cultivars (a, Helena; b, HyV8;
c, Golta), Burley tobacco cultivars (d, HyB2; e, B971; f, BO11) and
dark air-cured tobacco cultivars (g, Z992; h, Adonis; i, Badischer
Geudertheimer). The mean dry weights (n = 6) of organs of tobacco
are described in paired columns. The left column shows the biomass
of broomrape-affected tobacco and the biomass of all Orobanche
shoots, while the right column shows the unaffected control.

numbers of developing tubercles (Fig. 3). A similar obser-

tobacco cultivars were more severely affected by the
Karlsruhe population of O. ramosa than Burley and dark
air-cured tobaccos, even though the different tobacco cul-
tivars stimulated the germination of O. ramosa seeds to
the same extent, suggesting that the production of germi-
nation stimulants (xenognosins) is not a factor in infectivity
and susceptibility. However, the root-chamber experiments
indicated that successful attachment of the Orobanche
radicle to the tobacco roots is reduced in the less susceptible
tobacco cultivars, e.g. cv. Z992, as suggested by lower
vation was described by Zehhar et al. (2003) for the
oilseed rape cv. Zenith which shows low susceptibility to
this parasite. In the case of tobacco cv. Z992, the tubercles
did not show associated signs of necrosis, a phenomenom
observed by Labrousse et al. (2001) for O. cumana para-
sitizing resistant Helianthus species or H. annuus hybrids.
Although different levels of parasitization (i.e. number
of tubercles, shoots) on tobacco were recorded in the two
German O. ramosa populations, the biomass produced
by the parasite was about the same or even higher in the

© 2005 BSPP Plant Pathology (2005) 54, 650– 656

 Pathogenicity of Orobanche ramosa on tobacco
Figure 6 Intraspecific variation between three populations of Orobanche ramosa revealed by the ISSR-PCR primers (GATA)4 (a) and (CTC)4RC (b).
Lanes: M, molecular marker; Sp, Speyer; K, Karlsruhe; Su, Sudan populations; L, control. The numbers show fragment sizes of the DNA marker (M)
expressed in bp.
less aggressive Speyer population. This indicates that the
broomrape plants from Speyer were efficient at taking up
nutrients from the host. Only on cvs Helena and Z992 did
the Speyer population produce less biomass than O. Ram-
osa from Karlsruhe. This underlines the hypothesis of
Hibberd et al. (1998) that a host plant can only support
a specific number of parasites. In the case of the more
aggressive Karlsruhe population, competition between
the Orobanche plants for the resources supplied by the
host may have resulted in reduced biomass for each
parasitic plant.
In seven of the nine tobacco cultivars, parasitization
reduced host biomass compared with nonparasitized con-
trols, with leaves and roots being most affected. Leaves
also showed severe wilting symptoms and, according to
Labrada & Perez (1988b), this can lower the quality of
these leaves. The only exceptions to biomass reduction
were cv. BO11 (Burley) and the dark air-cured tobacco cv.
Z992, which appeared to overcompensate for parasitiza-
tion by producing more root biomass. A similar increase
was shown by the low susceptible sunflower cv. LR1 para-
sitized by O. cumana (Labrousse et al., 2001).
Since efficient and affordable control methods for O.
ramosa are not currently available, new control approaches
are required. So far, natural resistance mechanisms or
sources of resistance in tobacco have not been identified.
The present results demonstrate that different tobacco
cultivars show varying susceptibility to O. ramosa popu-
lations, but resistant tobacco cultivars were not identified.
Similar differences in susceptibility, but without complete
resistance, were described by Zehhar et al. (2003) for dif-
ferent cultivars of oilseed rape. Screening host germplasm
© 2005 BSPP Plant Pathology (2005) 54, 650– 656
655
for resistance to broomrapes requires prerequisite charac-
terization of pathogen populations and genotypes. As this
study has shown, molecular markers, such as ISSR–PCR
products, can discriminate between even closely related
O. ramosa populations.
Acknowledgements
We thank Dr Billenkamp, Landesanstalt für Pflanzenbau,
Forchheim Karlsruhe, for providing us with seed material
of the tobacco cultivars. We are also grateful to the
tobacco growers in the Rhine Valley between Speyer and
Freiburg, Germany, for their support during our seed col-
lections of O. ramosa on their fields.
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