Journal of Experimental Botany, Vol. 54, No. 385, pp. 1305±1311, April 2003
DOI: 10.1093/jxb/erg129
RESEARCH PAPER
Resistance to broomrape (Orobanche spp.) in sun¯ower
(Helianthus annuus L.) is temperature dependent
H. Eizenberg1,3, D. Plakhine1, J. Hershenhorn1, Y. Kleifeld1 and B. Rubin2
1
ARO, Newe Ya'ar Research Center, Ramat Yishay, Israel
2
Faculty of Agriculture Food and Environmental Science, The Hebrew University of Jerusalem, Rehovot, Israel
Received 26 June 2002; Accepted 14 January 2003
Abstract
The effects of various temperature regimes in the
range 29±17/21±9 °C day/night on each stage of the
parasitism process of Orobanche cumana and
O. aegyptiaca on sun¯ower were studied under controlled conditions in polyethylene bags. The
response of the resistant sun¯ower variety `Ambar'
was expressed as the degeneration of the parasite
tissues after its establishment in the plant roots, and
this stage was found to be temperature dependent.
The degeneration rate of Orobanche tubercles in the
resistant sun¯ower variety was also found to be temperature dependent and was about ®ve times as
great as that in the sensitive variety in the highest
temperature regime tested of 29/21 °C day/night. The
ability to reject the parasite by causing its degeneration and death is the main factor that determines
the resistance. As the temperature rises, more tubercles degenerate and die, that is the sun¯ower plant
expresses higher levels of resistance.
Key words: Correlation, Orobanche aegyptiaca, Orobanche
cumana, resistance, sun¯ower, temperatures.
Introduction
The various broomrape (Orobanche spp.) species are
obligatory chlorophyll-lacking root parasites, which parasitize broad-leaf plants and cause severe damage to
vegetables and other ®eld crops, both in Israel and abroad.
The sun¯ower broomrape (O. cumana Wallr.) and
Egyptian broomrape (O. aegyptiaca Pers.) are the two
species of Orobanche, which parasitize sun¯ower
(Helianthus annuus L.) in Israel and cause severe
economic losses (Parker and Riches, 1993).
Both susceptible and resistant sun¯ower varieties
stimulate the germination of Orobanche seeds, but the
germ tubes fail to develop after penetration into a resistant
host (Eizenberg et al., 2003). Oil sun¯ower varieties,
which were recently developed in Eastern Europe and
introduced into commercial cultivation, were damaged by
O. cumana after a number of growing seasons (Antonova,
1994). In Bulgaria, between 1927 and 1992, Encheva and
Shindrova (1994), identi®ed ®ve different races of O.
cumana (O1±O5) which parasitized sun¯owers. In Spain,
an additional two races (O6±O7) of O. cumana were
reported recently (Alonso, 1998).
In resistant vetch (Vicia atropurpurea), the parasite
haustorium was blocked at the host root endodermis layer
(Goldwasser et al., 2000). No correlation between temperature and vetch resistance to O. aegyptiaca, was
observed (Goldwasser et al., 1997). Labrousse et al.
(2001) found that the resistance of several wild sun¯ower
species was due to their ability to increase wall deposition,
vessel occlusion or broomrape cellular disorganization.
Several researchers indirectly examined the effect of
temperature on Orobanche parasitism by changing the
sowing dates of the host, which were legumes in most of
these studies. It was found that under the Mediterranean
climate, delaying the sowing date of broad beans or lentils
from autumn to winter reduced O. crenata and O. aegyptiaca
infection, probably because of the delay in the development of the parasite in the prevailing low winter temperatures. In most of these studies no temperature data were
given (Linke et al., 1991; van Hezewijk, 1994; MesaGarcia and Garcia Torres, 1986; Kukula and Masri, 1984;
Kukula et al., 1985; ter Borg, 1986). Castejon-Munoz et al.
(1993) reported that early sowing of sun¯ower reduced
3
Present address and to whom correspondence should be sent: Department of Crop and Soil Science, Crop Science Building 331B, Oregon State University,
Corvallis, OR, 97331. Fax: +1 541 737 3407. E-mail: [email protected]
Journal of Experimental Botany, Vol. 54, No. 385, ã Society for Experimental Biology 2003; all rights reserved
1306 Eizenberg et al.
Orobanche infection. Ish-Shalom-Gordon et al. (1994)
reported a reduction of O. cumana infection on the
resistant `Sunbred-254¢ oil sun¯ower line when it was
sown in the winter rather than in the spring. Sukno et al.
(2001) found high resistance in sun¯ower varieties only
when they were grown at 27 °C; this resistance was not
found at lower temperatures of 23±15 °C day/night, but no
details of the parasitism process were reported.
Numerous reports have shown that Orobanche germination is enhanced at high temperatures, with a concomitant
increase of the infection level, whereas low temperatures
delay the parasite development (Kasasian, 1973;
Sauerborn, 1989; Foy et al., 1991; van Hezewijk, 1994).
Kebreab and Murdoch (1999) found that maximum
germination of non-conditioned O. aegyptiaca seeds
occurred in a temperature range of 21±15 °C day/night.
It has been found that the sun¯ower variety `Ambar' is
resistant (R) to O. cumana only at high temperatures,
whereas the highly susceptible variety `Adi' (S) was
parasitized by O. cumana and O. aegyptiaca at temperatures of 29±9 °C day/night (Eizenberg et al., 2003).
Field and controlled-environment experiments have
shown that tomatoes are resistant to O. crenata only at
high temperatures (spring-summer) and are sensitive under
low temperatures (Eizenberg et al., 1998). Carrot was
found to be resistant both to O. crenata and to O. aegyptiaca
at high temperatures and susceptible at low temperatures
(Eizenberg et al., 2001). In the last two reports, the parasite
germinated and succeeded in attaching to the roots of host
plants at all temperatures tested.
The purposes of the present study were to elucidate the
temperature-dependent response of the resistant confectionery sun¯ower variety `Ambar' to O. cumana and
O. aegyptiaca, and to characterize the stage of the
parasitism process in which the resistance response takes
place.
Materials and methods
The experiments were conducted under controlled conditions in
polyethylene bags and Petri dishes. Experiments in polyethylene
bags were intended to test the effects of various temperature regimes
on O. cumana and O. aegyptiaca seed germination and development
in the presence of resistant and sensitive sun¯ower plant roots.
Experiments in Petri dishes were intended to test the effects of
different temperature regimes on the rate of O. cumana and
O. aegyptiaca seed germination in the presence of the arti®cial
stimulant GR-24. (A synthetic Strigol analogue germination stimulant (Hassan-Ali, 1983). Aqueous solution of GR-24 supplied by the
laboratory of A Johnson, School of Molecular Science, University of
Sussex, Brighton BN1 9QG, UK.)
Plant material
Sun¯ower seeds of the resistant (R) variety `Ambar' and the
susceptible (S) variety `Adi' were kindly provided by Dr Baruch
Retig of the Agriculture Research Organization, Israel. Both
sun¯ower varieties were selected from the variety D.Y. 3 that was
developed in Israel.
O. aegyptiaca and O. cumana in¯orescences were collected from
plants parasitized on tomatoes and sun¯owers, respectively. Seeds
were separated from the dry in¯orescences on 300-mesh sieves, and
stored in the dark at 4 °C until use. A germination test under standard
conditions (25 °C; 10 ppm of GR-24 applied after 12 preconditioning days), performed before each experiment, resulted in germination rates of 80% and 88% for O. aegyptiaca and O. cumana,
respectively.
Temperature regimes
Temperature regime experiments were conducted in a phytotron
under natural lighting, with an average of 14 h of daylight. The day/
night temperature regimes were ®xed throughout the experiment and
were: 17/9 °C; 20/12 °C; 23/15 °C; 26/18 °C; and 29/21 °C day/
night.
Cultivation in polyethylene bags
The polyethylene bags (PEB) were prepared according to Parker and
Dixon (1983) and modi®ed as required for this study (Fig. 1). This
method allowed the host roots to be observed and the various stages
of Orobanche development to be monitored during the parasitism
process: seed germination, attachment of the germ tubes to the host
root and the appearance of parasite spikes.
Orobanche seeds were surface-sterilized for 60 min in 1% sodium
hypochloride mixed with 0.01% non-ionic surfactant (DX±alkaryl
polyether alcohol 800 g l±1).
Resistant and susceptible sun¯ower seedlings at the cotyledon
stage were mounted onto 11.5323 cm glass ®bre ®lter paper
(Whatman, GF/A, Whatman International Ltd., Maidstone, England)
onto which had been scattered sterilized Orobanche seeds. Two
sun¯ower seedlings were placed in each paper, and then the glass
®bre sheets were inserted into a clear polyethylene bag (25335
containing 20 ml sterilized half-strength Hoagland nutrient solution
(Hoagland and Arnon, 1950), with each root system on half of each
GF/A sheet, together with the Orobanche seeds. The PEB were
nourished twice a week from the top of the bag with 20±30 ml halfstrength Hoagland nutrient solution.
In each temperature regime the following treatments were carried
out in eight replications (each of which comprised one PE bag unit):
1. `Ambar' (resistant variety) plants inoculated with O. cumana
seeds; 2. `Ambar' plants inoculated with O. aegyptiaca seeds; 3.
`Ambar' plants without any Orobanche seeds (control); 4. `Adi'
(susceptible variety) plants inoculated with O. cumana seeds; 5.
`Adi' plants inoculated with O. aegyptiaca seeds; 6. `Adi' plants
without any Orobanche seeds (control).
Observations were carried out with a binocular microscope (DZ,
Zeiss, Germany) at 403 magni®cation, in order to detect and
monitor the events of seed germination and the successful continuation of the parasitism process. The microscope observations
included the area of the main sun¯ower root, where the numbers
of the all broomrape seeds, germinating seeds, tubercles, spiders,
spikes, and degenerated attachments were recorded (Fig. 1).
Orobanche germination in Petri dishes under various
temperature regimes
The germination tests were conducted according to Kasasian and
Parker (1971), under sterile conditions in 50 mm Petri dishes padded
with ®lter paper discs soaked with 1 ml of sterile water. Eight 10 mm
®lter paper discs were placed on the main ®lter paper in the Petri
dish, and 20±30 Orobanche seeds were placed on each small disc
after being sterilized for 60 min in 1% sodium hypochloride mixed
with 0.01% non-ionic surfactant (DX±alkaryl polyether alcohol 800
g l±1). After 12 d of preconditioning, the discs containing the
Orobanche seeds were transferred to another dish that contained a
water-soaked ®lter paper ring in order to maintain humidity; four
Temperatures affect sun¯ower resistance 1307
Fig. 1. O. cumana parasitizing S (a) and R (b) sun¯ower varieties in different stages in polyethylene bag, 40 d after penetration; growing at
29/21 °C day/night. a1: tubercles; a2: spider; a3: Orobanche shoot; b1: attachment.
discs were placed in each Petri dish, from which water had been
drained. To each disc was added 30 ml of 10 ppm GR-24 solution.
Orobanche seed germination was recorded 6 d after the addition of
the stimulant. The germination test was conducted under the same
temperature regimes as the PEB. Each experiment was conducted
twice and almost identical results were obtained.
calculated with the JMP software (version 4.0.3; SAS Institute
Inc., USA). Averages were separated by Tuckey Kramer Honestly
Signi®cant Difference (HSD) (P <0.05).
Statistical analysis
The two experiments were arranged in a full factorial model with
three factors. Linear and polynomial regressions were employed and
ANOVA and R2 were calculated. Analysis of variance was
The effects of temperature on the responses of `Ambar' (R)
and `Adi' (S) sun¯ower varieties to O. cumana and
O. aegyptiaca were examined. The PEB experiments
enabled the reactions to different temperature regimes of
Results
1308 Eizenberg et al.
the host plants, the parasite and its developmental stages,
to be monitored and the stages at which the parasite ceases
to develop and dies to be identi®ed.
No differences between O. cumana and O. aegyptiaca
seeds in the time to germinate could be observed for either
sun¯ower variety. Therefore, only the results obtained with
O. cumana are presented (Fig. 2). As expected, the time
needed for germination at low temperature was 22±24 d,
compared with an average of 15 d at temperature of 23/15
°C day/night or higher. The R and S sun¯ower varieties
supported similar levels of Orobanche seed germination,
which gradually increased with increasing temperature
(Fig. 3). However, O. cumana germination rates were
higher than those of O. aegyptiaca seeds at all the
temperatures tested.
Figure 4 shows that the attachment percentages of the
germs of O. cumana and O. aegyptiaca that germinated on
the roots of the R `Ambar' and S `Adi' sun¯owers were
mostly similar to one another, regardless of the host type:
at the low temperatures of 17/9 and 20/12 °C day/night,
20±30% of the germinated seeds succeeded in attaching to
the roots; at the medium temperature of 23/15 °C day/
night, 45±55%; and at the highest temperature of 29/21 °C
day/night there was a decrease in O. cumana attachments
but not in those of O. aegyptiaca. Further development of
attachments by tubercles and spiders were blocked on the
roots of the R `Ambar' inoculated with either parasite, and
was not in¯uenced by increases in temperature. However,
the development percentage of O. cumana (and also of
O. aegyptiaca) spiders that developed from the attachments on the roots of the S `Adi' signi®cantly increased in
response to increases in temperature (Fig. 5).
Monitoring of the parasitism process in PEB revealed
that the Orobanche tubercles and spiders on the roots of the
R `Ambar' degenerated and that the degeneration of the
parasite increased with increasing temperature.
A signi®cant positive polynomial correlation (Y=
±0.0004x2+0.5299x±125.42; R2=0.957) between the num-
Fig. 2. Effect of day and night temperature on time required for
O. cumana germination; exposed to R and S sun¯ower roots in PEB.
Vertical bars represent the LSD value within each temperature regime
(P <0.05).
Fig. 4. Effect of day and night temperature on O. cumana and
O. aegyptiaca attachment rates; exposed to R and S sun¯ower roots in
PEB. Vertical bars represent the LSD value within each temperature
regime (P <0.05).
Fig. 3. Effect of day and night temperature on O. cumana and
O. aegyptiaca germination rates; exposed to R and S sun¯ower roots
in PEB. Vertical bars represent the LSD value within each
temperature regime (P <0.05).
Fig. 5. Effect of day and night temperature on O. cumana tubercles
and spider production rates; exposed to R and S sun¯ower roots in
PEB. Vertical bars represent the LSD value within each temperature
regime (P <0.05).
Temperatures affect sun¯ower resistance 1309
ber of degenerated tubercles of O. cumana and O. aegyptiaca
and the temperature was found among those on the roots of
R `Ambar', but not in those on S `Adi' (Fig. 6).
The degeneration rate of O. cumana tubercles on the
roots of the R sun¯ower variety were signi®cantly higher
than those recorded on the roots of the S variety (Fig. 7).
Signi®cant differences were observed between the slopes
of the regression lines for `Ambar' (a=15.76) and those for
`Adi' (a=3.25). These differences in the slope demonstrate
the ability of `Ambar' plants to induce tubercle degeneration rapidly, at a rate/ef®ciency 4.8 times greater than that
induced by S `Adi', and hence to prevent the continuation
of the O. cumana parasitism.
The temperature dependence of the germination capacity of the two Orobanche species was examined by
arti®cially inducing germination (without a host) by means
of the arti®cial germinator GR-24 in Petri dishes. The
highest germination percentages of O. cumana were
achieved at 23/15 and 26/18 °C day/night. The germination
rate gradually decreased to 55% at the lowest temperature
of 17/9 °C day/night, and was even lower (40%) in the
high-temperature regime, at 29/21 °C day/night. The
germination rates of O. aegyptiaca were similar to those
of O. cumana except in the high temperature regime, in
which O. aegyptiaca germinated at a rate of 70%, which
was signi®cantly higher than the germination rate of
O. cumana (Fig. 8). The germination rates described here
were only affected by the interaction between Orobanche
seeds and the temperature, regardless of host roots, which
were not present in this experiment. The results are
somewhat different from those of the bag experiments,
because of the effect of host root exudates in the latter.
Discussion
Under both ®eld and greenhouse conditions, the `Ambar'
variety exhibited high resistance to both O. cumana and
Fig. 6. Effect of day and night temperature on degeneration rates of
O. cumana and O. aegyptiaca parasites, exposed to R and S sun¯ower
roots in PEB. Vertical bars represent the LSD value within each
temperature regime (P <0.05).
O. aegyptiaca. This resistance was manifested most
strongly at 26/18 °C, day/night and above, partially
manifested at 23/15 °C day/night, and was transformed
to sensitivity at the low temperature of 20/12 °C day/night
and below.
In all cases in which resistance was expressed, the
parasitism process began as in the S variety. However, in
the parasite establishment stage, during the formation of
spiders and spikes, a difference developed between the
parasitism processes in the S and R varieties: in the roots of
the S variety the parasite continued to develop at all the
temperatures tested, although the development was slower
at 20/12 °C day/night and below; in the R variety the same
process was observed at the same temperatures, but at
temperature above 20/12 °C day/night the parasite
degenerated and died.
Fig. 7. Degeneration rate of O. cumana parasitizing R and S
sun¯ower roots in PEB in 29/21 °C day/night temperature regime.
Vertical bars represent the LSD value within each temperature regime
(P <0.05).
Fig. 8. Effect of day and night temperature on germination rates of
O. cumana and O. aegyptiaca, exposed to GR-24 in Petri dishes. Bars
followed by the same letter do not differ signi®cantly within each
Orobanche species, (P <0.05). * Indicates signi®cant differences
between O. cumana and O. aegyptiaca within each temperature
regime (P <0.05).
1310 Eizenberg et al.
The ®ndings of the present study are in good agreement
with the results obtained in pots under various temperature
regimes in O. aegyptiaca and O. cumana parasitism in
sun¯ower (Eizenberg et al., 2003). Both set of results (pot
and PEB) show that a defence mechanism was activated
only after the penetration of the parasite and its establishment in the host root tissues. The expression of the defence
mechanism increased with increasing temperature, in a
polynomial correlation, causing the parasite to cease its
development, degenerate and die. The resistance mechanism described in the present study is different from those
described by Labrousse et al. (2001) and Goldwasser et al.
(1997).
Ish-Shalom-Gordon et al. (1994) and Sukno et al.
(2001) previously reported on temperature-conditioned
resistance in sun¯ower, but they presented no details nor a
description of the phenology of the parasitism process. In
the present paper, evidence is provided on the stage of
parasitism that was affected by temperature. These results
are also in agreement with those of Eizenberg et al. (1998)
who found, under both ®eld and controlled conditions, that
tomatoes are resistant to O. crenata during the summer and
sensitive during the cold winter. Eizenberg et al. (2001)
also found that carrots were resistant to both O. crenata
and O. aegyptiaca at high temperatures and sensitive at
low temperatures under 18 °C. In the last two cases the
parasites germinated and succeeded in attaching to the host
plant roots at the tested temperatures, but failed to form a
permanent association.
Early sowing (in winter) of broomrape-susceptible
sun¯ower in Spain decreased Orobanche infestation
(Castejon-Munoz et al., 1993). These results are in
agreement with the results obtained with the `Adi' (S)
variety that showed a decrease in Orobanche infestation at
low temperatures. In other studies (Linke et al., 1991; van
Hezewijk, 1994; Mesa-Garcia and Garcia Torres, 1986;
Kukula and Masri, 1984; Kukula et al., 1985; ter Borg,
1986), although no temperature data were given and no
details on the parasitism process were reported, it was
shown that delaying the sowing of broad beans or lentils
from autumn to winter also reduced O. crenata and
O. aegyptiaca infestation.
The ®ndings here agree with the results reported
previously (Kasasian, 1973; Sauerborn, 1989; Foy et al.,
1991; van Hezewijk, 1994), that high temperatures facilitate seed germination of broomrapes and increase their
infestation level, while low temperatures delay the parasite
development. This could be because unfavourable conditions for Orobanche germination occur at low temperatures. It is shown in the present study that the germination
rate of O. aegyptiaca seeds was maximal at 23/15 °C and
29/21 °C day/night, as reported also by Kebreab and
Murdoch (1999).
The resistance response of the sun¯ower variety
`Ambar' was more pronounced as the degeneration process
of the attached and established parasite proceeded. This
stage is temperature dependent and the degeneration of the
parasite was almost ®ve times greater than that observed
on the S variety `Adi'. Hence, it is suggested that this
ability to eliminate the parasite is the main factor in the
resistant response. The higher the temperature the more
Orobanche tubercles degenerate and die and the more
resistant the sun¯ower is.
Acknowledgements
The authors are grateful to Dr Baruch Retig, ARO, Israel for his
valuable advice and for the sun¯ower `Ambar' and `Adi' variety
seeds, and to Shmuel Golan for photography.
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