Physiological and Molecular Plant Pathology (1996) 48, 209–215
Temperature induced susceptibility to Phytophthora
sojae in soybean isolines carrying different Rps genes
M. G",#*, T. MG",#, M. B$ and R. B%
"Agriculture Canada, 1391 Sandford Street, London ON Canada N5V 4T3, #the Department of Plant Sciences,
University of Western Ontario, London ON, Canada, $The Samuel Roberts Noble Foundation, PO Box 2180, Ardmore
OK, U.S.A. 73402 and %Agriculture Canada, Harrow ON, Canada N0R 1G0
(Accepted for publication NoŠember 1995 )
Resistance of soybean plants to specific races of Phytophthora sojae is conditioned by a series of host
resistance (Rps) genes. Soybean cultivars that are resistant to certain races of the pathogen may
nonetheless be susceptible to infection at elevated temperatures. The objective of this study was
to determine if certain resistance genes are invariably associated with temperature induced
susceptibility. Nine different resistance genes (Rps1-a, Rps1-b, Rps1-c, Rps1-k, Rps2, Rps3-a, Rps4,
Rps5 and Rps6 ) were tested for temperature induced susceptibility in Williams isolines and 10
different resistance genes (Rps1-a, Rps1-b, Rps1-c, Rps1-d, Rps1-k, Rps2, Rps3-a, Rps4, Rps5 and Rps6 )
in Harosoy isolines. Three other soybean cultivars or lines carrying the Rps1-c gene were also
examined. Resistance or susceptibility was determined by inoculating 7-day-old etiolated
seedlings with a zoospore suspension isolated from P. sojae race 1, and incubating the inoculated
plants at 25 or 33 °C. The results suggest that temperature induced susceptibility is generally
consistent for specific Rps genes, regardless of genetic background. However, for soybean plants
that carry more than one Rps gene, temperature induced changes in host-pathogen compatibility
may not be readily predictable. We also show that a short pre-inoculation treatment of 44 °C may
induce susceptibility in plants that are not considered to be temperature sensitive. This indicates
that temperature induced changes in disease resistance may occur through different mechanisms,
depending on the severity of the temperature stress.
# 1996 Academic Press Limited
INTRODUCTION
The development of resistance and susceptibility is influenced by temperature in many
host-pathogen interactions. It has been commonly observed that an increase in
temperature of incubation may render normally resistant plants susceptible to certain
pathogens [2, 5, 8, 12 ]. Conversely, resistance may be enhanced at higher temperatures,
although this effect seems to be less common [9 ]. In fact, plant resistance to a specific
pathogen may either be heightened or lessened with increasing temperatures depending
upon the resistance gene present in the host. For example, in the interaction between
wheat (Triticum aestiŠum) and the wheat rust organism (Puccinia graminis), resistance
conferred by gene Sr15 at 18 °C is attenuated at 26 °C, while resistance gene Sr14 is
more effective at the higher temperature [9 ].
Temperature induced susceptibility of soybeans to Phytophthora sojae was first
described by Chamberlain and Gerdemann [5 ]. In their study, seedlings of Harosoy 63
*To whom correspondence should be addressed : Mark Gijzen, Agriculture Canada London Research
Centre, 1391 Sandford Street, London, Ontario, Canada N5V 4T3.
0885–5765}96}030209­07 $18.00}0
# 1996 Academic Press Limited
210
M. Gijzen et al.
(Rps1-a) were immersed in a water bath of 44 °C for 1 h immediately prior to
inoculation. This heat treatment caused the plants to become susceptible not only to
P. sojae, but also to other fungal species not normally pathogenic on soybean [5 ].
The effect of temperature on the interaction of P. sojae and soybean has also been
examined by incubating inoculated plants in environments of different ambient
temperature [1, 7, 10, 13, 16 ]. These studies have shown that a threshold temperature
may be reached, near 30 °C, beyond which resistance breaks down and plants become
susceptible [7, 16 ]. As in other host-pathogen interactions, this temperature induced
susceptibility may vary depending upon the host resistance gene and the pathogen race
[3, 11, 14 ]. However, it remains unclear what influence the genetic background of the
host may have on this phenomenon. It is also not known whether susceptibility induced
by a severe heat pre-treatment of plants prior to inoculation is similar to susceptibility
induced by a moderate increase in the temperature of incubation after inoculation.
These uncertainties have practical and theoretical implications.
In this investigation we tested many different resistance genes for temperature
induced susceptibility when expressed in either Harosoy or Williams genetic
backgrounds. The results suggest that the identity of the resistance gene itself has a
greater influence on temperature dependent effects than the genetic background of
expression. Additionally, we show that plants carrying resistance genes not considered
to be subject to temperature induced susceptibility may nonetheless become susceptible
when exposed to high temperatures immediately prior to inoculation.
MATERIALS AND METHODS
Soybean [Glycine max (L.) Merr.] cultivars and isolines were from the collection of the
Harrow Research Station. The L lines originated from R. L. Bernard (University of
Illinois, U.S.A.). Plants were grown in vermiculite in darkness with a 16 h 25 °C and
8 h 19 °C temperature cycle.
Phytophthora sojae Kaufmann and Gerdemann (syn. Phytophthora megasperma f. sp.
glycinea Kuan and Erwin) race 1 was isolated in 1981 and race 5 in 1979, from Essex
County, Ontario, by T. Anderson (Agriculture Canada) ; race 25 was obtained in 1985
from K. Athow (Purdue University, Indiana, U.S.A.). Cultures were retrieved from
cryogenic storage and maintained on V8 agar with incubation at 25 °C in the dark.
Zoospores were produced from 5-day-old cultures by repeatedly flooding the colonies
with sterile distilled water [17 ]. The zoospore density was estimated using a
haemocytometer, and was adjusted to 10& ml−" by dilution with water. Etiolated
seedlings were harvested 7 or 8 days after sowing and placed in glass trays before
inoculation with a single 10 µl droplet of zoospore suspension [17 ]. The trays were then
transferred to incubators at 25 or 33 °C. For plants subject to pre-inoculation heat
treatment, the etiolated seedlings were immersed to the level of the cotyledons in a
water bath of 44 °C for 12 min. Plants were then placed in trays and inoculated as
above [17 ].
Disease severity was rated 48 h after inoculation, as follows : R, fully resistant ; Rs,
mostly resistant but with spreading lesions ; Sn, mostly susceptible but with necrosis ; S,
fully susceptible [17 ].
Temperature induced susceptibility of soybean to Phytophthora sojae
211
RESULTS
The ability of temperature to influence disease development in Williams isolines varied
considerably depending on the resistance gene (Table 1). Resistance genes found to be
T 1
Influence of temperature on resistance of Williams isolines inoculated with race 1 of Phytophthora sojae
Resistance
gene
Isoline
Rps1-a
L75-6141
Rps1-b
L77-1863
Rps1-c
L75-3735
Rps1-k
Williams 82
Rps2
L76-1988
Rps3-a
L83-570
Rps4
L85-2352
Rps5
L85-3059
Rps6
L89-158
Temp*
(°C)
Disease
rating†
25
33
25
33
25
33
25
33
25
33
25
33
25
33
25
33
25
33
Rs
Sn
Rs
Sn
Rs
Rs
R
R
R
R
R
S
R
Sn
Rs
Rs
R
Sn
*Etiolated seedlings were grown as described in Materials and Methods and incubated at
25 or 33 °C after inoculation.
†Disease symptoms were scored 48 h after inoculation of 10–20 hypocotyls with a zoospore
suspension. Disease symptoms of the plants were rated as : fully resistant (R), mostly resistant
but with spreading lesions (Rs), mostly susceptible but with necrosis (Sn), and fully susceptible
(S).
temperature sensitive included Rps1-a, Rps1-b, Rps3-a, Rps4 and Rps6. Temperature did
not greatly affect disease development in Williams isolines carrying Rps1-c, Rps1-k, Rps2
or Rps5.
In Harosoy isolines, temperature induced susceptibility was observed for Rps1-a,
Rps1-b, Rps1-d, Rps4 and Rps6, whereas Rps1-c, Rps1-k and Rps2 function was not
compromised by incubation at higher temperatures (Table 2). The results were
inconclusive for resistance genes Rps3-a and Rps5 in Harosoy isolines since susceptibility
occurred regardless of temperature.
Since Rps1-k differed from Rps6 in temperature induced susceptibility, it was of
interest to test a soybean line that carries both of these resistance genes. This was
studied by inoculating Conrad 94 plants (Rps1-k, Rps6) separately with three different
races of P. sojae ; race 1, avirulent on Rps1-k and Rps6 ; race 5, avirulent on Rps1-k and
virulent on Rps6 ; and race 25, virulent on Rps1-k and avirulent on Rps6 (Table 3).
Resistance to race 5, provided by the Rps1-k gene, was not affected by temperature
212
M. Gijzen et al.
T 2
Influence of temperature on resistance of Harosoy isolines inoculated with race 1 of Phytophthora sojae
Resistance
gene
Isoline
Rps1-a
Harosoy 63
Rps1-b
HARO 13
Rps1-c
OX682
Rps1-d
HARO 16
Rps1-k
HARO 1572
Rps2
L70-6494
Rps3-a
HARO 3272
Rps4
HARO 4272
Rps5
HARO 5272
Rps6
HARO 6272
Temp*
(°C)
Disease
rating†
25
33
25
33
25
33
25
33
25
33
25
33
25
33
25
33
25
33
25
33
R
Sn
Rs
Sn
R
R
R
Sn
R
R
Rs
Rs
Sn
S
R
Sn
Sn
S
R
S
*Etiolated seedlings were grown as described in Materials and Methods and incubated at
25 or 33 °C after inoculation.
†Disease symptoms were scored 48 h after inoculation of 10–20 hypocotyls with a zoospore
suspension. Disease symptoms of the plants were rated as : fully resistant (R), mostly resistant
but with spreading lesions (Rs), mostly susceptible but with necrosis (Sn), and fully susceptible
(S).
T 3
Influence of temperature on resistance of Conrad 94 (Rps1-k, Rps6) plants inoculated with different races
of Phytophthora sojae
Resistance
gene
Rps1-k­Rps6
Line
Phytophthora
race*
Temp†
(°C)
Disease
rating‡
Conrad 94
1
25
33
25
33
25
33
R
Sn
R
R
R
S
5
25
*Phytophthora sojae race 1 is avirulent on Rps1-k and Rps6 ; race 5 is avirulent on Rps1-k and
virulent on Rps6 ; race 25 is avirulent on Rps6 and virulent on Rps1-k.
†Etiolated seedlings were grown as described in Materials and Methods and incubated at
25 or 33 °C after inoculation.
‡Disease symptoms were scored 48 h after inoculation of 10–20 hypocotyls with a zoospore
suspension. Disease symptoms of the plants were rated as : fully resistant (R), mostly resistant
but with spreading lesions (Rs), mostly susceptible but with necrosis (Sn), and fully susceptible
(S).
Temperature induced susceptibility of soybean to Phytophthora sojae
213
whereas resistance to race 25, provided by the Rps6 gene, was temperature sensitive.
Surprisingly, resistance to race 1, provided by both genes, was also temperature
sensitive in Conrad 94.
To determine the effect of pre-incubation heat treatment on resistance conferred by
Rps1-c, etiolated seedlings were placed in a water bath at 44 °C immediately prior to
inoculation. Exposure for 12 min at 44 °C resulted in susceptibility (Table 4).
T 4
Influence of temperature on resistance of soybean lines carrying the Rps1-c gene to inoculation with race 1
of Phytophthora sojae
Cultivar or
line
Pretreatment*
Incubation
temp
(°C)
Disease
rating†
Williams 79
—
—
44 °C
—
—
44 °C
—
—
44 °C
25
33
25
25
33
25
25
33
25
R
R
S
R
R
Sn
R
R
S
Harovinton
Harosoy BC4
*Etiolated seedlings were grown as described in Materials and Methods. Plants were either
directly inoculated or were first pre-treated by immersion in water at 44 °C for 12 min
immediately prior to inoculation.
†Disease symptoms were scored 48 h after inoculation of 10–20 hypocotyls with a zoospore
suspension. Disease symptoms of the plants were rated as : fully resistant (R), mostly resistant
but with spreading lesions (Rs), mostly susceptible but with necrosis (Sn), and fully susceptible
(S).
Treatments for 5 min or less did not affect resistance, whereas treatments for 20 min or
more resulted in the death of the seedlings.
DISCUSSION
In this study we observed that Rps genes may differ in their ability to express resistance
at elevated temperatures. Susceptibility could be induced by transferring plants to
33 °C immediately after inoculation, but only for certain resistance genes. This
variation is probably related to differences associated with the Rps genes themselves,
rather than the genetic background of their expression. However, these differences may
also be due to the presence of tightly linked introgressed regions near the Rps genes. The
results also demonstrate that two apparently allelic resistance genes, mapping to the
same genetic locus, may nonetheless differ in regard to temperature induced
susceptibility. In both Williams and Harosoy isolines, Rps1-a was temperature
susceptible while Rps1-c was not. The difference in the sensitivity to temperature of
Rps1-a and Rps1-c suggests that temperature has a very specific effect on the interaction
of host resistance determinants and pathogen avirulence products.
214
M. Gijzen et al.
Analysis of an Rps1-k, Rps6 line (Conrad 94) showed that these plants displayed
temperature induced susceptibility to race 1 and race 25 but not to race 5. Resistance
to race 25 is provided by Rps6. This gene is temperature sensitive in the Williams and
Harosoy isolines. Likewise, resistance to race 5 is provided by Rps1-k. This gene is not
temperature sensitive in the Harosoy or Williams lines. These results provide additional
evidence that temperature induced susceptibility is Rps gene-specific. However, the
Rps1-k, Rps6 line was also temperature sensitive to race 1. This was unexpected since
both Rps1-k and Rps6 provide resistance to this race. Thus, if Rps6 function was
abrogated by the higher temperature, one might expect Rps1-k to provide resistance
under these conditions. Temperature dependent effects of Rps gene action may not be
easily predictable when more than one Rps gene is operating. In this particular
combination of Rps genes it seems that temperature induced susceptibility caused by
the Rps6 gene is epistatic to temperature insensitive resistance conferred by Rps1-k. It
is possible there are other genes, tightly linked to the temperature sensitive Rps6 locus,
that play a role in temperature induced susceptibility. This implies that temperature
induced susceptibility may be different from the normal susceptible host response.
Cytological studies have also indicated that there are distinct differences in the host cell
responses between temperature induced susceptibility and susceptibility at 25 °C [15 ].
Previous experiments have shown that a pre-inoculation treatment of 33 °C of the
seedlings or the fungus does not affect the outcome of the interaction. Even when the
host plant and}or the pathogen were grown at 33 °C, inoculated hypocotyls were
susceptible when incubated at 33 °C and resistant when incubated at 25 °C [6, 7 ].
However, a pre-inoculation treatment of very high temperatures (" 40 °C) may
induce susceptibility in normally resistant plants [4, 5 ]. Results from the present
investigation show that susceptibility induced by such a treatment occurs in plants
carrying an Rps gene that is not considered to be temperature sensitive. A preinoculation exposure to 44 °C caused Rps1-c plants to become susceptible. This 44 °C
treatment, although short in duration, is more severe on the seedlings than a prolonged
incubation at 33 °C. The results suggest that susceptibility induced by short, high
temperature pre-inoculation treatments is fundamentally different from susceptibility
induced by post-inoculation incubation at moderately elevated temperatures.
These findings have practical implications for soybean breeding programs since
certain Rps genes may be more effective than others under conditions of heat stress.
Temperature induced susceptibility may also lead to insight into Rps gene function.
This phenomenon has been observed for many other resistance genes in a variety of
plant species, and thus it may indicate certain common modes of action.
We thank Dr E. W. B. Ward for critical reading of the manuscript and Aldona
Gaidauskas-Scott for technical assistance.
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