y22 ff5 fs7 fc0 sc0 ls1 ws1">(Wauseon, Ohio).
2.2. P. capsici Isolates and Inoculum
Preparation
Three virulent isolates from each of the A1 and A2
mating types of P. capsici were used in the mass screen-
ing and subsequent inoculation tests (Table 1). A mixture
of zoospores from these isolates was used in inoculating
the test plants. The zoospores were produced aseptically
by transferring 10 agar plugs from the advancing portion
of 5-day-old cultures (25˚C, under dark condition) of P.
capsici in 5% (v/v) clarified V8 juice agar (Kuhajek et al.,
Table 1. Isolates of P. capsici used for the mass screening of
Capsicum annuum accessions.
Isolate Mating type Source
PC-F6S1 A1 Bell pepper (Tift County, Ga.)
PC-F6S3 A1 Bell pepper (Tift County, Ga.)
PC-1A1 A1 Squash (Tift County, Ga.)
PC-F1R3 A2 Bell pepper (Tift County, Ga.)
PC-F1R6 A2 Bell pepper (Tift County, Ga.)
PC-F1S12 A2 Bell pepper (Tift County, Ga.)
2003) to 100 × 15 mm Petri dishes (ca. 12 plates/isolate)
and 10 ml of clarified V8 juice were added thereafter.
After 24 h of incubation at 25˚C under dark condition,
the V8 juice in each plate was replaced with 10 ml sterile
mineral salt solution (MSS) [13] and incubated at 20˚C,
30 cm under two fluorescent lights (cool white, 20 W,
25˚C, 35 µmol·m2·s1 for 24 h). The MSS from each
plate were then replaced with the same volume of fresh
MSS and allowed to incubate for three more days.
Zoospores from each isolate were harvested separately.
To harvest the zoospores, the MSS was removed from
each plate and then washed twice with 10 ml of sterile
distilled water. After the second washing, 10 ml of sterile
distilled water was added to each plate and placed in the
refrigerator (1.3˚C) for 45 min. The plates were then
placed on top of a laboratory bench and monitored for
zoospore release. The zoospore suspension from each
Petri dish were then transferred very slowly to a 250 ml
graduated cylinder and left undisturbed for five min. The
upper 50 ml of the zoospore suspension was pipetted out
and transferred to a 50 ml conical centrifuge tube. The
tube was then inverted gently 2 - 3 times to distribute the
zoospores in the suspension. One ml of the suspension
was transferred to a 2 ml microcentrifuge tube with flat
cap and vortexed for 90 s to encyst the zoospores. The
zoospore concentration was determined by using a hem-
acytometer and standardized at 5000 zoospores per ml
for foliar [5] and 40,000 zoospores for stem inoculation.
Equal volumes of zoospore suspensions were then com-
bined for inoculation.
2.3. Screening for Foliar Resistance
A total volume of 100 µl zoospore suspension was
placed on the upper surface of a partially expanded leaf
of a six-week-old seedling [5]. The inoculated seedlings
(4 - 6 seedlings per accession) were placed inside a hu-
midity chamber made of 0.1 mm plastic sheets that was
also used to cover the bottom of the greenhouse benches.
A home-use humidifier provided a relative humidity of
100% at night. Foliar blight assessment was performed
Copyright © 2012 SciRes. OPEN ACC ESS
B. L. Candole et al. / Agricultural Sciences 3 (2012) 732-737
734
14 days after inoculation by using a 0 - 5 foliar blight
severity scale [14]: 0 = no visible symptoms, 1 = small
circular or irregular spots on upper leaves, 2 = leaf-
enlarged symptoms with brownish lesions beginning to
appear on stems and <25% of the plant wilted, 3 = leaves
defoliated with lesions on leaves covering half of a leaf
and 25% - 50% of the plant wilted moderately, 4 = leaves
defoliated or dried, with rapidly expanding stem lesions
and 50% - 70% of the plant wilted severely, 5 = plant
dead; where a foliar blight severity of 0 - 1 is resistant,
and a foliar blight of greater than or equal to 2 is suscep-
tible.
2.4. Screening for Stem Blight Resistance
Stems of eight-week-old plants (4 - 6 seedlings per
accession) were tied with sterile absorbent cotton yarn (3
mm in diameter) [9] in two different places 2 - 3 cm
apart. One hour before inoculation, the yarn was saturat-
ed with sterile distilled water and a 45 µl of zoospore
suspension was placed on the upper yarn, with the bot-
tom yarn used to prevent any inoculum from reaching the
soil. Stem blight assessment was performed 14 days after
inoculation by using a 0 - 5 stem blight severity scale
[14]: 0 = no visible symptoms, 1 = brownish lesion at the
inoculation point, 2 = stem lesion extending 1 - 3 cm
from inoculation point, 3 = stem lesion progression up to
half of the plant height, 4 = stem lesion progressing to-
ward the shoot apex, 5 = plant dead. Plants with severity
ratings of 0 - 2 were classified as resistant while those
with severity ratings of greater than two were susceptible.
2.5. Replicated Inoculation Tests
A total of 10 root rot-resistant accessions were se-
lected based on resistant reaction against root rot from
replicated greenhouse tests [12] and availability of seeds
were tested for foliar blight resistance in replicated (ran-
domized complete block design) greenhouse inoculation
tests. Each seed was sown in 8.9 cm square Kord green
pots (Kord Products, Toronto, Canada). Each accession
was replicated five times with three seedlings per repli-
cate. The test was performed twice. The Kord 18-pocket
tray containing the pots with seedlings were placed in an
F1020 flats with drainage holes. The inoculation proce-
dure and disease severity scales were the same as de-
scribed above for mass screening for foliar blight resis-
tance.
Planting, experimental design, and incubation tech-
nique for stem inoculation were the same as in foliar
inoculation as described above. The inoculation tech-
nique and the stem blight severity scale used were the
same as described above for mass screening for stem
blight resistance.
2.6. Data Analysis
Means, medians, modes, standard deviation, and Spear-
man correlation coefficients were calculated using Mini-
tab statistical software with a significance threshold of
0.05.
3. RESULTS
Not all of the accessions selected for mass screening
for stem blight or foliar blight resistance germinated in
sufficient quantity for testing. A total of 780 (56%) and
732 (53%) accessions germinated well enough to pro-
duce four to six plants for mass screening against stem
blight and foliar blight, respectively. 69% of the acces-
sions tested were resistant to stem blight (Figure 1), and
71% of the accessions tested were resistant to foliar
blight (Figure 2). Accessions with four or more plants
screened for root rot resistance [12], stem blight resis-
tance, and leaf blight resistance were used to determine
the correlation between these forms of resistance (Table
2). All three forms of resistance were positively corre-
lated, but the amount of variation explained was low.
In order to determine the usefulness of various root
rot resistant selections in breeding for stem and foliar
0
50
100
150
200
250
300
350
0 0.511.52 2.53 3.5 4 4.55
Mean stem blight severit y
Frequency
Figure 1. Frequency distribution of mean stem blight severity
derived from a mass screening of Capsicum annuum accessions
with Phytophthora capsici. Only accessions with four or more
scored plants are included in the distribution. Stem blight as-
sessment was performed 14 days after inoculation and was
based on a stem blight severity scale ranging from 0 (no symp-
toms) to 5 (dead plant).
Table 2. Spearman correlation coefficients between mean se-
verity scores of Capsicum annuum germplasm screened for root,
stem, and foliar resistance to Phytophthora capsici.
Mean stem
blight severity
Mean foliar
blight severity
Mean root rot severity 0.187** 0.110**
Mean stem blight severity 0.240**
**Significant at P < 0.001.
Copyright © 2012 SciRes. OPEN ACC ESS
B. L. Candole et al. / Agricultural Sciences 3 (2012) 732-737
Copyright © 2012 SciRes.
735
0
50
100
150
200
250
300
350
00.511.522.533.544.55
Mean f oli ar bl ight sever i t y
Frequency
severity was 0 for all lines, but lines PI 201237 (1), PI
566811 (2), PI 593572 (3), PI 593573 (3), PI 640532 (3),
and PI 640588 (1) were the only selections where all
tested plants responded within the resistant range (foliar
blight severity 1). The average stem blight severity
rating of these lines ranged from 0 to 0.4, with a median
of 0 (Table 4). The only line whose responses ranged
from resistant (0.4) to susceptible (3) was PI 593573.
4. DISCUSSION
In order to be most effective, phytophthora blight re-
sistant pepper cultivars should have resistance to all
phases of blight. However, research has demonstrated
that foliar blight resistance, stem blight resistance, and
root rot resistance are inherited independently in at least
one commonly used resistant line [6,9]. Breeding pro-
grams must, therefore, screen resistant selections to all
three phases of the disease before determining which will
be most useful to a breeding program. Initial testing of
the accessions resulted in the discovery of a large num-
ber of lines showing resistance to stem and foliar phases
of the disease. This may suggest that the testing protocols
were not stringent enough, however, susceptible controls
were consistently rated as highly susceptible (data not
shown) and many accessions were rated as highly sus-
ceptible (Figures 1 and 2). Since these forms of the
disease are less commonly encountered than phytoph-
thora root rot in the field, they may simply represent less
virulent syndromes of the disease and resistance to them
Figure 2. Frequency distribution of mean stem blight severity
derived from a mass screening of Capsicum annuum accessions
with Phytophthora capsici. Only accessions with four or more
scored plants are included in the distribution. Foliar blight
assessment was performed 14 days after inoculation and was
based on a foliar blight severity ranging from 0 (no symptoms)
to 5 (dead plant).
phytophthora blight resistance, 10 phytophthora root rot
resistant lines [12] were selected for replicated testing
against foliar and stem blight. Based on their overall
mean blight severity scores, all ten lines were resistant to
both stem and foliar blight (Tables 3 and 4).
The mean foliar blight severity ratings for the 10 lines
ranged from 0 - 0.8, and the most commonly-observed
response was 0 (Table 3). The median foliar blight
Table 3. The response of selected root rot-resistant Capsicum annuum accessions to foliar blight caused by a mixture of zoospores from
six Georgia isolates of Phytophthora capsici.
Foliar blight severityb
Accession/Varietya
Mean Range Median Mode St. Dev.
Grif 9109(3) 0.8 0 - 3 0 0 1.1
PI 201237(1) 0.1 0 - 1 0 0 0.3
PI 224438(4) 0.3 0 - 2 0 0 0.7
PI 439273(1) 0.2 0 - 2 0 0 0.5
PI 566811(2) 0.0 0 0 0 0.0
PI 593572(3) 0.1 0 - 1 0 0 0.3
PI 593573(3) 0.0 0 0 0 0
PI 640532(3) 0.0 0 0 0 0
PI 640581(3) 0.5 0 - 2 0 0 0.8
PI 640588(1) 0.0 0 0 0 0
Camelot (susceptible control) 4.6 0 - 5 5 5 1.0
Criollo de Morelos 334 (resistant control) 0.0 0 0 0 0
aNumbers in parentheses after the accession numbers denote test plant number; bFoliar blight severity was based on a scale ranging from 0 (no symptoms) to 5
(dead plant). Means were based on five replicates with three plants per replicate.
OPEN A CCESS
B. L. Candole et al. / Agricultural Sciences 3 (2012) 732-737
736
Table 4. The response of selected root rot-resistant Capsicum annuum accessions to stem blight caused by a mixture of zoospores from
six Georgia isolates of Phytophthora capsici.
Stem blight severityb
Accession/Varietya
Mean Range Median Mode St. Dev.
Grif 9109(3) 0.2 0 - 1 0 0 0.4
PI 201237(1) 0 0 0 0 0.0
PI 224438(4) 0.1 0 - 1 0 0 0.3
PI 439273(1) 0.2 0 - 1 0 0 0.4
PI 566811(2) 0.0 0 - 1 0 0 0.2
PI 593572(3) 0.1 0 - 1 0 0 0.3
PI 593573(3) 0.4 0 - 3 0 0 0.7
PI 640532(3) 0 0 - 1 0 0 0.2
PI 640581(3) 0.4 0 - 1 0 0 0.5
PI 640588(1) 0.1 0 - 2 0 0 0.4
Camelot (susceptible control) 3.4 2 - 4 4 5 0.7
Criollo de Morelos 334 (resistant control) 0.1 0 - 1 0 0 0.4
aNumbers in parentheses after the accession numbers denote test plant number. bStem blight severity was based on a scale ranging from 0 (no symptoms) to 5
(dead plant). Means were based on five replicates with three plants per replicate.
may be more common.
Unfortunately, there were low correlations between
root, stem, and foliar resistance. Thus resistance to one
form of the disease was a poor predictor of resistance to
other forms, confirming that resistance to each form of
the disease needs to be analyzed independently. Given
the relatively common occurrence of resistance to stem
and foliar blight in the accessions, and the rarity of root
rot resistance [12], and the fact that root rot resistance is
the primary goal of our resistance breeding program, it
was determined that it would be more productive to
screen for root rot resistance initially, and then screen
resistant lines for resistance to stem and foliar blight.
One line from each of ten root rot resistant accessions
was chosen for testing for levels of stem and foliar blight
resistance. These ten lines were chosen based on their
consistent high levels of resistance to root rot [12], their
uniqueness from other lines, and their ability to set seed
in greenhouse culture. Mean blight severity scores
ranked all lines as resistant to both stem blight and foliar
blight. This is advantageous to the breeding program, as
it means a single resistance source may be used to breed
for all three forms of the disease. Even if inheritance to
the three forms of the disease is inherited independently,
as it is in CM-334 [6,9], it is not surprising that resis-
tance to all forms of the disease was found together in
single lines since environments where the disease is en-
demic will expose the entire plant to infection and thus
resistance would be needed in each plant organ for the
plant to survive to seed set.
The responses to foliar blight and stem blight were not
as variable as the responses to root rot [12] both among
lines and among plants within a line. Susceptible plants
within an accession rarely occurred. This is in contrast to
the root rot resistance trial where susceptible plants
within resistant lines commonly occurred [12]. This
suggests that either these lines are more genetically ho-
mogenous in terms of leaf and stem blight resistance
genes than they are for root rot resistance genes, or that
root rot resistance is more easily overwhelmed by high
disease pressure conditions than are stem and leaf blight
resistance.
The identification of novel accessions with resistance
to, as reported here, may allow the identification of new
resistance genes to these diseases. This would be the first
step towards the pyramiding of multiple resistance genes
into adapted material to provide resistance to multiple
pathogen isolates. The genetic aspects of these resistance
sources needs to be verified and so that the most effec-
tive use of resistance genes can determined.
REFERENCES
[1] Boatright, S. and McKissick, J. (2008) Georgia farm gate
vegetable survey. Center for Agribusiness and Economic
Development, College of Agricultural and Environmental
Sciences, The University of Georgia, AR-08-01, 62-63.
[2] Ristaino, J. and Johnston, S. (1999) Ecologically based
Copyright © 2012 SciRes. OPEN ACC ESS
B. L. Candole et al. / Agricultural Sciences 3 (2012) 732-737 737
approaches to management of phytophthora blight of bell
pepper. Plant Disease, 83, 1080-1089.
doi:10.1094/PDIS.1999.83.12.1080
[3] Wang, D. and Bosland, P. (2006) The genes of Capsicum.
HortScience, 41, 1169-1187.
[4] Lefebvre, V. and Palloix, A. (1996) Both epistatic and
additive effects of QTLs are involved in polygenic in-
duced resistance to disease: A case study the interaction
pepper—Phytophthora capsici leonian. Theoretical and
Applied Genetics, 93, 503-511. doi:10.1007/BF00417941
[5] Alcantara, T.P. and Bosland, P.W. (1994) An inexpensive
disease screening technique for foliar blight of chile pep-
per seedlings. HortScience, 29, 1182-1183.
[6] Walker, S. and Bosland, P. (1999) Inheritance of phy-
tophthora root rot and foliar blight resistance in pepper.
Journal of the American Society of Horticultural Science,
124, 14-18.
[7] Oelke, L., Bosland, P. and Steiner, R. (2003) Differentia-
tion of race specific resistance to phytophthora root rot
and foliar blight in Capsicum annuum. Journal of the
American Society of Horticultural Science, 128, 213-218.
[8] Bosland, P.W. and Lindsey, D.L. (1991) A seedling screen
for phytophthora root rot of pepper, Capsicum annuum.
Plant Disease, 75, 1048-1050. doi:10.1094/PD-75-1048
[9] Sy, O., Bosland, P.W. and Steiner, R. (2005) Inheritance
of phytophthora stem blight resistance as compared to
phytophthora root rot and phytophthora foliar blight re-
sistance in Capsicum annuum L. Journal of the American
Society of Horticultural Science, 130, 75-78.
[10] Glosier, B., Ogundiwin, E., Sidhu, G., Sischo, D. and
Prince, J. (2008) A differential series of pepper (Capsi-
cum annuum) lines delineates fourteen physiological races
of Phytophthora capsici. Euphytica, 162, 23-30.
doi:10.1007/s10681-007-9532-1
[11] Ortega, G., Palazon-Espanol, C. and Cuartero-Zueco, J.
(1995) Interactions in the pepper—Phytophthora capsici
system. Plant Breeding, 114, 74-77.
doi:10.1111/j.1439-0523.1995.tb00763.x
[12] Candole, B., Conner, P. and Ji, P. (2010) Screening of
Capsicum annuum accessions for resistance to Georgia
isolates of Phytophthora capsici. HortScience, 45, 254-
259.
[13] Kuhajek, J.M., Jeffers, S.N., Slattery, M. and Wedge, D.E.
(2003) A rapid microbioassay for discovery of novel fun-
gicides for phytophthora spp. Phytopathology, 93, 46-53.
doi:10.1094/PHYTO.2003.93.1.46
[14] Kim, Y.J., Hwang, B.K. and Park, K.W. (1989) Expres-
sion of age-related resistance in pepper plants infected
with Phytophthora capsici. Plant Disease, 73, 745-747.
doi:10.1094/PD-73-0745
Copyright © 2012 SciRes. OPEN ACC ESS