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Euphytica 116: 167–179, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
167
Inheritance of several sources of resistance to Phomopsis stem canker
(
Diaporthe helianthi Munt.-Cvet.) in sunflower (Helianthus annuus L.)
A. Vigui
´
e, D. Touvieille de Labrouhe & F. Vear
∗
INRA, GREAT Station d’Am´elioration des Plantes et de Pathologie v´eg´etale, 234 Avenue du Br´ezet, Clermont-
Ferrand Cedex 02, France; (
∗
author for correspondence; e-mail: vear@clermont.inra.fr)
Received 31 March 1999; accepted 21 March 2000
Key words: additive gene effects, artificial infections, general combining ability, quantitative resistance, rate of
mycelium growth, semi-natural attacks
Summary
The hybrids from 2 factorial crosses involving a total of 7 female lines and 11 restorers, representing the known
range of reaction of sunflower to Phomopsis stem canker and most of the resistance sources used in breeding
programmes, were studied in multilocational semi-natural attack trials and by artificial infections on leaves and
stems in 1996, 1997 and 1998. The results confirm that resistance is quantitative, with predominantly additive
effects, although there were some female × male interactions when individual trials were analysed. For lines used
in both crosses, relative general combining abilities were stable. Some lines not bred for Phomopsis resistance
showed good or intermediate levels of resistance. Lines from or including Yugoslavian, Rumanian and Russian
sources of resistance and interspecific hybrids all provided good levels of resistance. The majority of parental lines
showed the same relative reactions to semi-natural attack and artificial infections on leaves or stems indicating that
their resistance is characterised by a slow rate of mycelial extension. However, one genotype showed resistance to
passage of Phomopsis stem canker from the petiole to the stem and two genotypes appeared susceptible to tests
but resistant to semi-natural attack, indicating that their resistance is controlled by factors not measured by the
mycelium tests. The use of combinations of these resistance sources to give high levels of stable resistance to
Phomopsis stem canker is discussed.
Introduction
Phomopsis stem canker of sunflower is caused by the
Ascomycete Phomopsis / Diaporthe helianthi Munt.-
Cvet. (1981). It was observed for the first time in
Yugoslavia in 1981 (Mihaljevic et al., 1982). Infec-
tions of sunflower leaf tips by ascospores occur during
vegetative growth of the plant; mycelium then spreads
along the petiole to the stem. The fungus invades the
vascular tissue, and when lesions encircle the stem,
the upper part of the plant dries and lodging may oc-
cur. Yield losses of up to 40% have been observed in
France (Carré, 1993).
Vranceanu et al. (1983) observed large differences
in reaction to Phomopsis stem canker attack between
sunflower genotypes, and suggested that resistance
was controlled by a small number of genes. Skoric
(1985) identified some sunflower genotypes with high
levels of resistance to the disease, and more recently,
it has been shown that among cultivated varieties,
there is a complete gradation, from highly resistant to
highly susceptible (Carré, 1993). Skoric (1985) and
Vranceanu et al. (1994) again reported that a small
number of genes is involved, but the latter authors
reported some partial dominance and some additive
effects. Following analysis of trials over three years,
Vear et al. (1997) concluded that resistance was un-
der additive control, with no significant interactions
between male and females parents. They found that
observations on inbred lines were not always closely
correlated with those on hybrids and concluded that
it is necessary to determine general combining abil-
ity for resistance. Deglène et al. (1999) also reported
mainly additive control.
168
Table 1. Origin of the inbred sunflower lines (Helianthus annuus) used in factorial crosses
to study the reaction to Phomopsis stem canker (Diaporthe helianthi) (cross A = 1996 and
1998, cross B = 1997)
Code Origin Breeder Factorial cross
Females
2603 Moroccan population INRA A, B
HA821 Peredovik USDA B
CMS1.50 Yugoslavian material IFVC A
HA22 Yugoslavian material IFVC B
HA74 Yugoslavian material IFVC A, B
LC1004 Rumanian material ICCPT B
XRQ HA89 × Progress (Russian population) INRA A
Males
RHA274 Wild H. annuus × Peredovik USDA A
RHA801 Wild H. annuus × Peredovik USDA B
83HR4 Complex-cultivated sunflower cross INRA A, B
PSC8 Recurrent selection for resistance to
Sclerotinia sclerotiorum INRA A
DPH1 Yugoslavian hybrid ‘Helios’ INRA A
DPC4 INRA line × Yugoslavian hybrid ‘Condor’ INRA A
DPF2 83HR4 × Yugoslavian hybrid ‘Flower’ B
(NSH45) INRA A, B
90R18 Yugoslavian hybrid ‘Flower’ (NSH45) INRA B
DPN2 INRA line × Yugoslavian line INRA A
LR1 Cultivated sunflower line × H. debilis INRA A
LR2 Cultivated sunflower line × H. argophyllus INRA B
ICCPT: Rumania; IFVC: Yugoslavia; INRA: France; USDA: USA.
All these studies were on limited numbers of gen-
otypes, often with only one source of resistance, or
with coded genotypes with no indication of resistance
origins. Therefore,it was decidedto makea studywith
a wider genetical basis, crossing lines with all types
of reaction to Phomopsis stem canker and involving
all known sources of resistance. Two factorial crosses
were thus studied in multilocational semi-natural at-
tack trials and by artificial infections, in 1996, 1997
and 1998, in ordertodetermineinheritanceparameters
and whether different types of resistance exist.
Materials and methods
Sunflower genotypes
The origins of the inbred lines usedin the two factorial
crosses are presented in Table 1. The plan denoted
cross A, studied in 1996 and 1998, was 4 females × 7
males, and cross B, studied in 1997, was 5 females ×
6 males. Two female lines (2603 and HA74) and two
male lines (83HR4 and 90R18) were common to both
factorial crosses. The cultivated varieties Viki and Ag-
risol were used as susceptible and resistant controls in
1996 and 1998.
D. helianthi isolates
For the semi-natural attack trials, the infections were
causedby thelocalpopulationsofD. helianthi.Forthe
artificial mycelial infections, isolate 96001 (obtained
from sunflower seed used in official French checks,
but whose origin was not available) was used in 1996
and 1997. In 1998, the isolate was 95066 (obtained
from a sunflower stem in Southwest France). These
isolates were chosen for their aggressiveness.
Semi-natural attacks
Cross A was studied in 7 trials in 1996 (T1 to T7)
and 3 trials in 1998 (T15 to T17), cross B was studied
in 7 trials in 1997 (T8 to T14). All locations were in
Southwest France, within 100 km of Toulouse. The
169
hybrids were sown in randomised block design trials
with plots of 50 plants and two replications. Infections
were obtained by the system described by Tourvieille
(1994), with infected crop debris showing D. helianthi
perithecia placed regularly through the trials and ir-
rigation to provide the necessary humidity during the
contaminationperiods.Floweringdates were noted for
each plot. Reaction to Phomopsis stem canker was
measured as the percentage of plants showing either
lesions > 5 cm on the stem or lesions encircling the
stem at the beginning of maturation, according to the
severity of attack: when there were few stem encirc-
ling lesions, lesions > 5 cm were counted; in some
cases both observations were available. In 1996, in
one trial, there were infections on capitula, which were
also counted.
Artificial infections
In the same years as the semi-natural attack trials, the
two factorial crosses were also planted at Clermont-
Ferrand, in randomised block design trials with 20
plants per plot and two replications. Leaf infections
were made in 1996 on 10 plants per plot, and in
1997 and 1998, both leaf and petiole infections were
made, each on 5 plants per plot, following the methods
described by Bertrand & Tourvieille (1987), with a
mycelial explant placed on the leaf-tip or cut end of
petiole, and then covered with aluminium foil to hold
it in place and stop drying. The leaves were infected
about three weeks before flowering, at the star bud
stage, the petioles at the beginning of flowering. In-
fections were made on 2 leaves per plant or 1 petiole.
After infection, irrigation was provided to maintain
the necessary humidity. Lesion lengths were measured
at a single date for each trial, 2 to 4 weeks after in-
fection, according to weather conditions. For leaves,
percentage success of infections was calculated and
then mean lesion lengths were calculated excluding
the zero values, since these were taken into account
in the first value. For the petiole infections, since
percentage success was almost 100%, as reported by
Viguié et al. (1999), only lesion lengths are presented.
Flowering dates were also recorded.
Statistical analyses
Analyses of variance were carried out to determine
treatment and replication effects and factorial analyses
to determine parental effects. The general combining
ability (GCA) variance / specific combining ability
(SCA) variance ratio for each trial was calculated fol-
lowing the method of Robert et al. (1987). Missing
values were calculated when possible to obtain com-
parable mean general combining ability values for the
parental lines for each observation or test. Correlation
coefficients from linear regressions were calculated
between the different measurements of phomopsis re-
action, the different trials and years and between
flowering dates and phomopsis reactions.
Results
Semi-natural attacks
For cross A (in 1996), 3 of the trials (T5-T7) showed
low levels of attack and high variation coefficients,
such that the results were not retained. The observa-
tions of stem encircling lesions were retained for T1,
T2 and T3, and those of lesions > 5cmforT1and
T4 (Table 2). The symptoms on capitula observed in
T3 are also presented in Table 2, for information, as
they are quite rare, but there were no significant gen-
otypic differences so no further analyses were made.
In 1998, 2 of the 3 trials were destroyed by storm
damage and the third had too low a mean level of
attack for analysis. In the trials presented, the mean
level of stem encircling lesions was 14.5% (40.5% on
Viki and 7.5% on Agrisol) and for lesions > 5cm,
27.8%. In the factorial analysis the hybrids with male
parents DPC4, 90R18 and LR1 were not included be-
cause of missing values in some trials. There were
significantdifferences between hybrids and significant
parental effects. In all cases, female × male inter-
actions were significant except in trial T4, but the
GCA variance/ SCA varianceratio was generally well
above 1.0, with a mean of 1.79 for observations of
stem encircling lesions and 1.93 for percentagelesions
> 5 cm. This indicates predominance of additive con-
trol of resistance, although some dominance effects
were detectable in some conditions.
For cross B, 4 of the 7 trials in 1997 gave us-
able results, the others having a low level of attack
(T12 and T13) or very irregular presence of inoculum
(T14). Stem encircling lesions were analysed in trials
T8 and T9 and lesions > 5 cm in T8, T10 and T11.
The mean percentage of stem encircling lesions was
32.5% and for lesions > 5 cm, the mean was 39.8%.
All the hybrids were present in all the locations so
the factorial analysis was complete (Table 3). Hybrid
and parental effects were highly significant except the
170
Table 2. Percentage of sunflower plants infected by Diaporthe helianthi in trials with semi-natural attack in 1996 (factorial
cross A)
Hybrids % stem encircling lesions % stem lesions > 5 cm % symptoms on capitula
Female parent Male parent T1 T2 T3 T1 T4 T3
Agrisol 0.0 7.5 0.0 1.0 3.3 9.8
Viki 29.1 51.6 – 55.2 ––
2603 × REA274 28.6 51.8 70.2 56.6 100.0 4.2
2603 × 83HR4 28.0 52.8 61.7 75.0 47.6 6.8
2603 × DPH1 13.4 15.0 7.6 26.8 37.4 20.7
2603 × DPC4 25.7 – 70.9 52.3 100.0 5.3
2603 × 90R18 – 43.3 32.5 – 48.0 20.8
2603 × LR1 – – 53.6 – 57.9 14.9
2603 × PSC8 17.8 57.7 26.0 35.7 – 25.3
XRQ × RHA274 20.2 14.3 18.0 39.4 66.3 20.5
XRQ × 83HR4 17.3 8.3 3.3 36.4 18.3 10.4
XRQ × DPH1 5.8 12.6 1.5 9.8 21.3 9.3
XRQ × DPC4 – 9.8 1.7 – 7.1 1.8
XRQ × 90R18 1.2 12.2 0.0 3.6 26.3 18.5
XRQ × PSC8 10.7 7.0 2.0 17.4 12.0 8.3
HA74 × RHA274 7.7 12.4 2.2 17.7 38.3 7.7
HA74 × 83HR4 2.9 4.2 0.0 5.9 10.6 2.6
HA74 × DPH1 3.0 9.3 0.0 5.0 7.1 0.0
HA74 × 90R18 0.0 ––1.0 23.4 –
HA74 × LR1 3.2 7.2 0.0 7.4 6.5 0.0
HA74 × PSC8 1.3 8.3 1.9 2.5 3.2 3.1
CMS1.50 × RHA274 6.9 8.3 7.7 7.9 15.8 11.9
CMS1.50 × 83HR4 2.8 5.6 0.0 6.6 14.8 0.0
CMS1.50 × DPH1 25.9 16.3 0.0 31.9 31.5 0.0
CMS1.50 × DPC4 1.9 8.8 0.0 2.8 – 0.0
CMS1.50 × 90R18 1.0 11.6 – 1.0 0.0 –
CMS1.50 × LR1 3.0 4.6 0.0 6.0 3.2 0.0
CMS1.50 × PSC8 4.9 12.1 1.3 6.9 25.8 0.0
Mean 12.3 18.5 12.7 23.9 31.7 8.2
F hybrid 4.46
∗∗
18.02
∗∗
26.92
∗∗
7.51
∗∗
5.04
∗∗
2.31ns
F female parent 11.44
∗∗
66.05
∗∗
82.00
∗∗
22.51
∗∗
11.83
∗∗
F male parent 1.72ns 3.37
∗
21.03
∗∗
4.39
∗
7.84
∗∗
F female × male 3.05
∗
6.89
∗∗
10.53
∗∗
3.5
∗
1.84ns
LSD hybrids 13.5 12.8 12.8 23.0 33.7
CV 51.6 32.5 47.3 45.2 49.9 91.4
GCA var / SCA var 0.86 2.36 2.15 1.93 4.79
Correlations / flowering –0.473
∗
–0.161ns –0.028ns –0.485
∗
–0.36ns –0.176ns
∗∗
: p<0.01;
∗
: p<0.05; ns: not significant; –: not tested.
Figures in italics were not included in the factorial analyses because of missing hybrids.
171
Table 3. Percentage of sunflower plants infected by Diaporthe helianthi in trials with semi-natural attacks
in 1997 (factorial cross B)
Hybrids % stem encircling lesions % stem lesions > 5cm
Female parent Male parent T8 T9 T8 T10 T11
2603 × 83HR4 90.3 5.1 98.1 13.2 25.3
2603 × 90R18 96.3 17.7 97.2 12.4 36.5
2603 × DPF2 97.1 15.7 100.0 6.7 55.1
2603 × DPN2 98.2 5.1 100.0 10.3 64.6
2603 × RHA801 100.0 35.9 100.0 6.5 76.0
2603 × LR2 98.1 6.6 99.1 29.2 64.9
HA821 × 83HR4 70.1 0.0 86.4 9.4 35.7
HA821 × 90R18 20.0 0.0 39.7 1.1 28.4
HA821 × DPF2 25.5 1.5 49.1 5.4 47.6
HA821 × DPN2 70.6 0.0 95.6 2.0 56.8
HA821 × RHA801 68.0 20.7 75.3 2.0 79.7
HA821 × LR2 51.4 1.5 74.2 0.0 25.2
HA22 × 83HR4 31.3 0.0 58.5 1.1 37.0
HA22 × 90R18 23.3 2.5 49.4 0.0 24.4
HA22 × DPF2 34.8 2.5 59.9 3.1 37.7
HA22 × DPN2 25.2 1.5 45.8 1.1 38.0
HA22 × RHA801 86.8 6.6 91.0 10.9 58.8
HA22 × LR2 49.5 0.0 71.6 1.0 19.3
HA74 × 83HR4 42.1 0.0 70.9 4.1 18.8
HA74 × 90R18 70.5 1.5 91.6 1.0 30.0
HA74 × DPF2 31.5 0.0 62.0 0.0 14.1
HA74 × DPN2 59.7 6.6 75.7 2.0 37.3
HA74 × RHA801 82.3 5.6 88.8 0.0 42.0
HA74 × LR2 36.2 0.0 57.5 1.0 18.8
LC1004 × 83HR4 61.9 0.0 95.2 1.0 27.5
LC1004 × 90R18 24.6 0.0 42.8 2.2 15.3
LC1004 × DPF2 55.2 0.0 73.1 3.5 17.2
LC1004 × DPN2 62.0 0.0 82.2 5.2 13.6
LC1004 × RHA801 78.0 14.1 84.2 2.2 64.1
LC1004 × LR2 58.5 0.0 92.5 5.5 24.7
Mean 60.0 5.0 76.9 4.8 37.8
F hybrid 5.54
∗∗
4.27
∗∗
5.99
∗∗
5.55
∗∗
2.71
∗∗
F female parent 21.76
∗∗
10.49
∗∗
17.78
∗∗
20.25
∗∗
6.13
∗∗
F male parent 6.72
∗∗
10.70
∗∗
6.10
∗∗
1.74ns 7.20
∗∗
F female × male 2.00
∗
1.42ns 3.61
∗∗
0.91ns
LSD hybrids 32.3 11.5 23.0 7.4 33.6
CV 26.3 111.8 14.6 75.7 43.5
GCA var / SCA var 4.22 7.94 1.10 –23.40
Correlations / flowering 0.446
∗
–0.548
∗∗
0.260ns 0.118ns –0.663
∗∗
∗∗
: p<0.01;
∗
: p<0.05; ns: not significant.
172
Table 4. Percentage infections and lesions lengths after artificial Diaporthe helianthi infections
on sunflower leaves and petioles (Clermont-Ferrand, factorial cross A)
Hybrids % infections Lesion lengths (mm)
Female Male on leaf on leaf on stem
parent parent 1996 1998 1996 1998 1998
Agrisol
a
45.0 60.5 61.7 58.8 204.5
Viki
a
– 95 – 173.4 216.0
2603 × RHA274 77.2 100.0 77.3 202.0 225.5
2603 × 83HR4 65.2 80.0 63.9 186.0 175.5
2603 × DPH1 66.7 73.5 89.4 166.3 210.5
2603 × DPC4 59.8 – 85.0 – –
2603 × 90R18 55.0 84.5 51.2 109.8 218.5
2603 × LR1 70.0 75.0 113.4 164.2 198.5
2603 × PSC8 77.1 – 74.3 – –
XRQ × RHA274 57.5 90.0 80.7 228.6 238.5
XRQ × 83HR4 35.0 95.0 62.0 130.0 174.5
XRQ × DPH1 79.1 83.0 72.8 155.5 263.0
XRQ × DPC4 83.5 – 62.3 – –
XRQ × 90R18 47.5 84.0 57.8 131.5 194.5
XRO × LR1 55.0 82.0 66.7 114.1 216.0
XRO × PSC8 56.5 70.5 61.5 116.1 181.5
HA74 × RHA274 60.0 94.0 60.8 110.6 201.5
HA74 × 83HR4 62.5 41.0 58.9 65.3 153.0
HA74 × DPH1 85.0 38.0 39.1 33.1 173.0
HA74 × DPC4 67.5 – 35.5 – –
HA74 × 90R18 79.0 13.0 36.7 77.5 165.5
HA74 × LR1 515 41.5 51.3 82.5 175.0
HA74 × PSC8 84.9 50.0 39.9 76.9 147.5
CMS1.50 × RHA274 55.0 73.5 76.3 103.8 226.0
CMS1.50 × 83HR4 40.0 70.5 56.4 94.7 172.8
CMS1.50 × DPH1 85.0 50.0 47.0 45.7 174.5
CMS1.50 × DPC4 57.5 – 45.5 – –
CMS1.50 × 90R18 56.7 26.5 38.6 45.0 157.5
CMS1.50 × LR1 70.0 65.0 60.3 104.7 188.0
CMS1.50 × PSC8 72.5 40.0 51.5 38.8 146.0
Mean 64.8 68.0 61.3 117.5 195.1
F hybrid 1.02ns 7.14
∗∗
4.30
∗∗
7.81
∗∗
15.16
∗∗
F female parent 23.37
∗∗
19.62
∗∗
31.30
∗∗
35.04
∗∗
F male parent 9.15
∗∗
4.95
∗∗
7.94
∗∗
29.58
∗∗
F female × male 2.42
∗
1.53ns 1.89ns 5.38
∗∗
LSD hybrids 27.3 25.3 56.8 22.3
CV 29.2 19.2 20.1 23.1 5.5
GCA var / SCA var 4.15 6.51 8.32 2.74
Correlations / flowering – 0.187ns – 0.133ns –0.075ns
∗∗
: p<0.01;
∗
: p<0.05; ns: not significant; –: not tested.
Figures in italics were not included in the factorial analyses because of missing hybrids.
173
male effect in T10. Female × male interactions were
significant in one of the 2 trials where stem encirc-
ling lesions were observed, and 1 of the 3 trials with
lesions > 5 cm. In all cases GCA variance / SCA vari-
ance ratios were above 1.0, indicating that control of
resistance is mainly additive.
Artificial infections
Observations of cross A in 1996 and 1998 are presen-
ted in Table 4. For the leaf test, although mean per-
centage infections were similar between years (1996:
64.8%; 1998: 68.0%), the percentage of successful
infections did not vary significantly between hybrids
in 1996 but it did in 1998, with significant parental ef-
fects and additive control of resistance. In some cases
hybrids that were not significantly different for per-
centage attack differed for lesion lengths. Mean lesion
lengths were 61.3 mm in 1996 and 117.5 mm in 1998;
this was certainly due to the fact that observations
were made 15 days after infection in 1996 and 27
days after infection in 1998. However, in both years,
lesion lengths showed significant differences between
hybridsand between parents and no female × male in-
teraction.For the petioletest carried out in 1998, mean
lesionlengthson the stemwas 195.1mm, 22daysafter
infection. The results showed significant differences
between hybrids and parental effects, with a signific-
ant female × male interaction but a GCA variance /
SCA variance ratio of 2.74, indicating predominance
of additive control.
For cross B studied in 1997, the mean successful
leaf infection percentage was 87.2%, with significant
differencesbetweenhybrids(Table 5). Lesionslengths
on leaves and stems always showed significant hy-
brid and parental effects and small female × male
interaction, but with GCA variance / SCA variance
well above 1.0, indicating additive control. Following
the leaf infections, symptoms resembling those ob-
served after seminatural attack (percentage of plants
with lesions on stems > 5 cm and percentage of
plants with stem encircling lesions) were observed
after flowering (Table 5). As in the other analyses,
hybrid, parent and interaction effects were significant,
with predominantly additive control of resistance.
Correlations between trials
For a given type of observation, the results of semi-
natural attack trials in different locations were always
significantly correlated (Table 6). Table 7 presents
correlations between results of artificial infections in
each year (when significant differences were observed
betweenhybrids).Correlationsbetweenpercentagein-
fection on leaves and lesion lengths were significant
although at a low level in the cross B. The 3 observa-
tions on stems (lesion lengths from petiole infection,
% lesions > 5 cm and % stem encircling lesions
from leaf infections) were all significantly correlated
(r = 0.51 to 0.94). The lengths of leaf and stem le-
sions were correlated for the cross A but not for the
cross B. However the stem lesions from leaf infections
were correlated with leaf lesion lengths. Table 7 also
presents correlations between results of semi-natural
attack and artificial infections. Lesion lengths on both
leaves and stems are almost always significantly cor-
related with observations of field attack, but with a
closer relation for those on leaves (r = 0.59 to 0.74)
than those on stems (r = 0.33 to 0.60).
Flowering dates
Correlations between flowering date and infection
level are presented in Tables 2, 3, 4 and 5. In most
cases (14/19) there is no relation between flowering
date and severity of symptoms; in the 5 other trials,
the correlation is negative, later genotypes tending to
show less Phomopsis stem canker attack.
Analyses of trial means (Table 8)
Since the semi-natural attack trials and the observa-
tions of lesions > 5 cm or stem encircling lesions
on stems from artificial infections were significantly
correlated, factorial analyses were carried out on the
mean values of lesions > 5 cm and stem encircling
lesions in the trials presented in Table 2 for cross A
and in Tables 3 and 5 for cross B. Hybrid and par-
ental effects were always significant. It may be noted
that, in both crosses, the F values for the female par-
ents were at least double those of the male parents. In
contrast to the analyses of individual trials, female ×
male interactions were not significant for either cross
for stem lesions > 5 cm or for the stem encircling le-
sions in cross B. For cross A, the interaction was small
compared with main effects, the GCA variance / SCA
variance ratio being 3.32.
Analysis of the behaviour of parental lines
Table 9 presents the mean values for the female and
male parental fines in crosses A and B for leaf and
stem lesion lengths and percentage lesions > 5cm
and percentage stem encircling lesions. For the tests,
174
Table 5. Percentage infections and lesions lengths after artificial Diaporthe helianthi infections on
sunflower leaves and petioles (Clermont-Ferrand, factorial cross B)
Hybrids % infections Lesion length (mm) % stem % stem
Female parent Male parent on leaf on leaf on stem lesions encircling
> 5 cm lesions
2603 × 83HR4 85.9 109.3 89.0 55.6 40.4
2603 × 90R18 70.7 120.9 74.5 79.8 79.3
2603 × DPF2 90.4 141.0 81.2 100.0 100.0
2603 × DPN2 90.9 144.8 109.0 100.0 100.0
2603 × RHA801 95.5 143.5 120.0 100.0 100.0
2603 × LR2 80.3 165.7 71.9 90.4 90.4
HA821 × 83HR4 95.5 138.3 74.9 100.0 59.6
HA821 × 90R18 80.8 71.5 77.0 70.7 60.6
HA821 × DPF2 95.5 81.0 86.4 89.4 89.4
HA821 × DPN2 95.5 82.5 101.8 89.4 89.4
HAS21 × REA801 90.9 85.5 106.5 80.8 80.8
HA821 × LR2 100.0 103.5 77.5 45.5 30.3
HA22 × 83HR4 85.9 75.3 56.5 52.5 52.5
HA22 × 90R18 70.7 65.6 57.9 30.3 20.2
HA22 × DPF2 100.0 77.8 73.7 70.7 60.6
HA22 × DPN2 90.4 101.0 95.2 25.3 20.2
HA22 × RHA801 100.0 82.5 70.0 100.0 100.0
HA22 × LR2 85.9 97.3 65.0 22.2 11.1
HA74 × 83HR4 80.8 112.0 83.5 55.6 40.4
HA74 × 90R18 70.7 85.0 71.5 64.2 58.6
HA74 × DPF2 65.7 59.5 74.5 25.3 20.2
HA74 × DPN2 90.4 118.8 86.5 36.4 30.3
HA74 × RHA801 90.4 88.3 69.0 70.2 50.0
HA74 × LR2 100.0 112.5 74.7 10.1 10.1
LC1004 × 83HR4 80.8 83.1 75.0 71.7 34.9
LC1004 × 90R18 65.7 47.9 66.4 20.2 20.2
LC1004 × DPF2 95.5 70.8 75.5 60.6 60.6
LC1004 × DPN2 95.5 128.8 99.5 100.0 100.0
LC1004 × RHA801 85.4 74.5 89.2 89.4 78.3
LC1004 × LR2 90.4 120.5 44.5 20.2 20.2
Mean 87.2 99.6 79.9 64.2 56.9
F hybrid 1.74
∗∗
6.82
∗∗
3.71
∗∗
7.53
∗∗
4.78
∗∗
F female parent 1.47ns 22.89
∗∗
6.55
∗∗
18.40
∗∗
11.95
∗∗
F male parent 5.29
∗∗
10.73
∗∗
10.59
∗∗
12.75
∗∗
8.23
∗∗
F female × male 0.91ns 2.62
∗∗
1.42ns 4.05
∗∗
2.48
∗∗
LSD hybrids 22.5 32.3 24.6 31.0 40.8
CV 12.6 15.8 15.1 23.6 35.1
GCA var / SCA var – 3.08 – 1.35 1.84
Correlations / flowering 0.349ns 0.091ns 0.067ns 0.038ns –0.026ns
∗∗
: p<0.01;
∗
: p<0.05; ns: not significant.
175
Table 6. Correlations between trials (T) in semi-natural attacks of sunflower by
Diaporthe helianthi for each observation on stem and for each cross
Observations: % stem lesions > 5 cm % stem encircling lesions
Trials: T1 T8 T10 T1 T2 T8
Cross A
T2 0.719
∗∗
T3 0.780
∗∗
0.883
∗∗
T4 0.780
∗∗
Cross B
T9 0.596
∗∗
T10 0.507
∗∗
T11 0.430
∗
0.366
∗
∗∗
: p<0.01;
∗
: p<0.05.
Table 7. Correlations between observations on sunflower hybrids following artificial infections and semi-natural attacks by Diaporthe
helianthi (for semi-natural attacks, cross A = 1996 and cross B = 1997; correlations were made using tile means of all trials [T]
retained)
Artificial infections Semi-natural attacks
% infections Lesion length Lesion length Stem lesions Stem lesions Stem encircling Symptoms on
on leaf on leaf on stem > 5cm >5 cm lesions capitula
Cross A
(1996 and 1998) 1998 1998 T1, T4 T1, T2, T3 T3
% infections on leaf 1998 0.620
∗∗
0.478
∗∗
0.615
∗∗
Lesion length on leaf 1996 – – 0.633
∗∗
0.610
∗∗
0.529
∗∗
Lesion length on leaf 1998 0.756
∗∗
0.744
∗∗
0.593
∗∗
0.626
∗∗
Lesion length on stem 1998 0.689
∗∗
0.686
∗∗
0.458
∗
0.325ns 0.599
∗∗
Cross B
(1997) T8, T10, T11 T8, T9
% infections on leaf 0.254ns 0.163ns
Lesion length on leaf 0.311
∗∗
0.608
∗∗
0.617
∗∗
Lesion length on stem 0.324ns 0.313ns 0.432
∗∗
0.431
∗
Stem lesions > 5 cm 0.317ns 0.373
∗
0.507
∗∗
0.722
∗∗
0.638
∗∗
Stem encircling lesions 0.310ns 0.363
∗
0.537
∗∗
0.941
∗∗
0.722
∗∗
0.637
∗∗
∗∗
: p<0.01;
∗
: p<0.05; ns: not significant.
Table 8. Overall factorial analyses of variance calculated for percentage stem lesions > 5 cm and per-
centage stem encircling lesions on sunflower plants infected by Diaporthe helianthi, in the factorial
crosses A and B under semi-natural attacks or artificial infections (means of individual trials were used
as replications)
Stem lesions > 5 cm Stem encircling lesions
Cross A Cross B Cross A Cross B
T1+T4 T8+T10+T11+Clermont-F
d
T1+T2+T3 T8+T9+Clermont-F
d
F hybrids 6.65
∗∗
3.85
∗∗
7.69
∗∗
3.37
∗∗
F female parent 19.60
∗∗
12.06
∗∗
26.88
∗∗
12.10
∗∗
F male parent 6.49
∗∗
6.30
∗∗
3.06
∗
5.57
∗∗
F female × male 2.38ns 1.60ns 2.83
∗
1.08ns
GCA var/SCA var 3.32
∗∗
: p<0.01;
∗
: p<0.05; ns: not significant.
176
Table 9. Mean values of the sunflower hybrids made with each of
the female and male parental inbred lines in the crosses A and B for
observations after artificial and semi-natural infections by Diaporthe
helianthi
Artificial infections Semi-natural attacks
Lesion lengths (mm) % plant attacked
Leaf Petiole Stem Stem
test test lesions encircling
> 5 cm lesions
Female inbred lines
Cross A
2603 120 199 54 51
HA74 59 168 13 4
CMS1.50 64 178 14 7
XRQ 104 211 26 12
Cross B
2603 138 91 63 65
HA74 96 77 37 30
HA22 84 70 38 29
LC1004 88 75 42 37
HA821 94 88 50 41
Male inbred lines
Cross A
90R18 69 184 17 12
83HR4 90 169 27 16
PSC8 75 160 20 14
DPH1 81 205 22 9
DPC4 81 – 25 –
LR1 95 195 21 –
RHA274 118 222 43 19
Cross B
90R18 78 70 37 33
83HR4 103 76 46 35
LR2 120 67 39 31
DPF2 86 78 44 40
DPN2 115 99 49 45
RHA801 95 91 61 61
the hybrids derived from DPC4 were not present in
1998, so for the leaf test, the 1996 values were cor-
rected by the 1996–1998 mean; for the petiole test,
no data with this parent were available. For the le-
sions > 5 cm in semi-natural attack trials, 3 hybrids
(CMS1.50 × DPC4, 2603 × PSC8) were absent from
certain locations, so their data were corrected from
the locations where they were present. However, for
stem encircling lesions, no GCA are presented for
LR1 and DPC4 as the hybrids between these lines and
2603 were not grown in certain locations and these
susceptible hybrids have a major effect on the means.
It may firstly be noted that there are similar relative
levels of resistance of the hybrids made with the two
female lines, or with the two male lines, common to
the two factorial crossing plans, in the two series of ex-
periments. The female line 2603 always gave hybrids
that were more susceptible than those from HA74, al-
though, for semi-natural attack, the differences were
greaterin 1996, when infection levels were lower, than
in 1997. For the common male lines, in both series of
crosses 83HR4 appeared less resistant than 90R18 for
lesion length on leaves and percentage lesions > 5cm
buthybrids from these2 linesalways had similar mean
behaviours for lesion lengths on stems and % stem
encircling lesions.
The data in Table 9 show that, for 11 of the 18
inbred lines used, relative reactions to the 2 artificial
infection tests and in the 2 semi-natural attack ob-
servations were consistent. 2603 (crosses A and B),
RHA274 (A) and RHA801 (B) (except for leaf lesion
length) gave hybridsthat always appeared susceptible,
HA821 (B) (except for leaf lesion length), DPC4 (A)
and DPF2 (A) gave hybrids that always appeared in-
termediate and HA22 (B), HA74 (crosses A and B),
CMS1.50 (A), 90R18 (crosses A and B) and PSC8
(A) gave hybrids that generally appeared to be the
most resistant. The 7 other lines showed differences
according to the method used for the measurement
of resistance. LC1004 (B) appeared more resistant in
artificial tests than in semi-natural attacks, LR2 (A)
appeared to have a good level of resistance except in
the leaf test, DPH1 (A), in contrast, was susceptible
only to the petiole test. 83HR4 was in general interme-
diate, but with good resistance to the petiole test in the
A cross and a particularly low level of stem encircling
lesions in the B cross. The field resistance of 3 lines,
DPN2 (B), XRQ (A) and LR1 (A), did not appear to be
well expressed in artificial infections: these lines ap-
peared susceptible to leaf and petiole tests (especially
XRQ to the petiole test) but quite or highly resistant in
observations of semi-natural attacks.
Discussion
For semi-natural attack, the 3 years of trials show that,
although the methodology is well known, the rate of
success in obtaining sufficient, homogeneouslevels of
attack was about 50%. Thus, a large number of trials
in differentenvironmentaland pathologicalconditions
177
are necessary to allow general conclusions on heredity
to be drawn. For both of the factorial crosses, obser-
vations in different locations were alwayssignificantly
correlated but the specific behaviours of some hybrids
in some trials may be due to interactions with different
factors. Firstly, climatic conditions may be important,
the correlation between differences in flowering date
and differences in level of Phomopsis stem canker at-
tack was significant in some trials but not in others.
Secondly, the low level of attack in 1996 may have
been the cause of the irregular behaviours of some
hybrids in 1996. Thirdly, pathogen variation may
be involved. Viguié et al. (1999) showed that there
are considerable variations in agressiveness between
D. helianthi isolates and some small but significant
host × pathogen interactions. Vranceanu et al. (1983)
reported genotype × year interactions whereas Vear et
al. (1997) did not, but in the latter case mean infec-
tion levels were high (55%) and obtained over 3 years.
This possibility of involvement of environmental and
pathogeneffects may also be suggestedby the absence
of female × male interactions in factorial analyses on
mean data from several trials, but their presence in in-
dividual trials. For this reason also, several locations
are necessary to draw general conclusions as to the
GCA of any genotype.
Of the 2 artificial infection methods used in this
study, the one on leaves appears to be better correlated
with natural attacks. The petiole test appears to meas-
ure a different resistance factor, which is less common
in resistant sunflower genotypes, but present for ex-
ample in LR2, which is relatively susceptible to the
leaf test but relatively resistant according to all the
other observations. The leaf test measures resistance
to mycelial extension on leaves whereas the petiole
test measures resistance to passage on to the stem and
extension on and around the stem (Tourvieille de Lab-
rouhe et al., 1988). Vear et al. (1997) suggested that
the leaf test could be used to eliminate susceptible gen-
otypes in early generations of breeding programmes.
From the study of Viguié et al. (1999), it appears
necessary to take into account both the percentage
rate of successful infections and lesion length, which
may measure different resistance factors. The need for
several tests to measure sunflower resistance to Pho-
mopsis stem canker is comparable with resistance to
Sclerotinia sclerotiorum in sunflowers, for which at
least 2 or 3 tests are necessary (Castaño et al., 1993).
Since this study was made using most of the
currently known sources of Phomopsis stem canker
resistance used in sunflower breeding programmes,
the results should be generally applicable. They con-
firm the polygenic, quantitative nature of resistance,
with predominantly additive effects. Measurements
of general combining ability, in a number of trials,
should permit the choice of hybrid combinations to be
made. The frequent small female × male interactions,
on single trials, are in agreement with the results of
Vranceanu et al. (1983) and Deglène et al. (1999),
and maybedue to environmentalandpathogen effects,
with the result that certain hybrid genotypes show re-
actions in individual trials which are different from
their mean behaviour calculatedfromthe GCA of their
parental lines.
In both crosses there were greater differences
between the female lines than among the male lines
(the F values for females was always greater than the
F values for males), although the latter were more
numerous. This probably resulted from the choice of
lines. For example, among the females, 2603 was
always very susceptible whereas most of the other
lines showed good resistance. In contrast, among the
males there was a complete gradation from RHA274,
almost as susceptible as 2603, through the intermedi-
ate types to DPH1 and 90RI8, so that differences in
behaviour were less distinct. The intermediate lines,
obtained from Yugoslavian hybrids, probably retained
only some of the genes which give the best levels of
resistance as in DPH1 and 90R18.
Four lines used here were also used as part of the
factorial cross studied by Vear et al. (1997), the fe-
males 2603 and XRQ and the males RHA274 and
DPH1. These genotypes represent well differentiated
susceptibility and resistance and they gave the same
relativeGCAinthecrossesreportedhere. It is possible
to compare theresults of thisstudywith otherprevious
publications on the genetics of resistance only for 3
lines. Skoric (1985) reported HA74 and HA22 to give
hybrids with good levels of resistance. LC1004 was
used by Vranceanu et al. (1994) and they concluded
that resistance was predominantly additive and by De-
glène et al. (1999) who reported this line to have quite
good combining ability for Phomopsis stem canker
resistance.
As might be expected for quantitative resistance,
this study shows that several different sources of res-
istanceexist: firstly, that foundin Yugoslavian and Ru-
manian cultivated sunflower genotypes selected under
natural attack in these countries; secondly, material
bred from the Russian population Progress; thirdly,
material bred for resistance to S. sclerotiorum;and
fourthly, interspecific crosses with annual Helianthus
178
species, H. debilis and H. argophyllus (Griveau et
al., 1992; Besnard et al., 1997). In the semi-natural
attack trials reported here, all these sources of resist-
ance appeared to be of similar value and sufficient to
avoid economic yield loss under conditions of moder-
ate attack. However, this might not be the case under
very favourable conditions for disease development
and it appeared important to try to determine which
sources of resistance were really different and thus
might give the highest level and most stable resistance
in combination.
Analysis of GCA shows that the majority of the
parental lines with resistance of Yugoslavian origin
gave hybrids with relative reactions that were sim-
ilar for both tests and observations of semi-natural
attack. Resistance to semi-natural attack of all these
genotypes appears to be determined by resistance to
growth of mycelium in both leaves and stems, as
measured by the two artificial infection tests. It was
rather unexpected that the male parent PSC8 should
also belong to the same category, as it was bred for
resistance to S. sclerotiorum, and Castaño et al. (1993)
reported that for mycelium extension of that fungus,
it was only moderately resistant; its main resistance
being based on reduced probability of successful in-
fection by ascospores. It is not yet known whether
genes giving resistance to S. sclerotiorum may con-
tribute to Phomopsis resistance. The quite widespread
existence of factors contributing to Phomopsis stem
canker resistance is shown also by the parental lines
of intermediatevalue, HA821 and 83HR4, whichwere
both bred in complete absence of the disease.
The lines which gave hybrids with varying beha-
viours depending upon the observation method are
of particular interest, since they make it possible to
distinguish different types of resistance. The resist-
ance of the Rumanian line LC1004 appears to be
different from that of Yugoslavian origin since it has
particularly good resistance in the mycelium tests.
LR2, the line developed from H. argophyllus ap-
pears to have resistance to passage of pathogen from
leaves to the stem, in agreement with Langar et al.
(1997). It may be suggested that XRQ (Progress)
and LRI (H. debilis), which show susceptible reac-
tions to mycelium tests but which are resistant to
semi-natural attack, possess resistance to infection by
ascospores. It is clear that further work is necessary,
including the development of artificial infections us-
ing ascospores (Mihaljcevic & Muntanola-Cvetkovic,
1985), and mapping of Quantitative Trait Loci (QTL)
from the different resistance sources as was done for
resistance to S. sclerotiorum by Mestries et al. (1998).
In conclusion, it has been confirmed that resist-
ance to Phomopsis stem canker is quantitative, with
mainly additive effects, in hybrids using a wide range
of resistance sources. There appear to be differencesin
the resistance factors provided by these sources. Bet-
ter knowledge of the resistance factors should make it
possible to determine which combinations would give
the highest levels of resistance under a wide range of
conditions.
Acknowledgements
The authors acknowledge the general support of Pro-
mosol and CETIOM for this programme. They would
like to thank T. André, J.P. Andrieu, P. Bataillon,
H. Bony, O. Cottet, A. Estragnat, C. Etineau, J.F.
Fournes, R. Fuser, B. Gabriel, P. George, G. Joubert,
P. Jouve, O. Ladsous, J.L. Madeuf, A. Mezzaroba,
P. Pactat, J. Philippon, S. Roche, G. Sansas, F.
Serre,P.Teyssier,E.Vrancken,andP.Walserfor
their participation and Y. Griveau and H. Serieys
(INRA France), D. Skoric (IFVC Yugoslavia) and V.
Vranceanu (ICCPT Rumania) for providing material.
The first author specially thanks the French ‘Min-
istère de l’Education Nationale, de l’Enseignement
Supérieuret de la Recherche’ for support of her thesis.
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