Evaluation of phytophthora root rot-resistant <i>Capsicum annuum</i> accessions for resistance to phytophthora foliar blight and phytophthora stem blight

doi:10.4236/as.2012.35088

Paper Menu >>

Journal Menu >>

Vol.3, No.5, 732-737 (2012) Agricultural Sciences

http://dx.doi.org/10.4236/as.2012.35088

Evaluation of phytophthora root rot-resistant

Capsicum annuum accessions for resistance

to phytophthora foliar blight and phytophthora

stem blight

Byron L. Candole1, Patrick J. Conner1*, Pingsheng Ji2

1Horticulture Department, University of Georgia-Tifton Campus, Tifton, USA; *Corresponding Author: [email protected]

2Plant Pathology Department, University of Georgia, Athens, USA

Received 17 June 2012; revised 27 July 2012; accepted 3 August 2012

ABSTRACT

A mixture of six Georgia isolates of Phyto-

phthora capsici (Leon.), the causal agent of phy-

tophthora blight, were used for greenhouse

mass screening of over 700 accessions of Cap-

sicum annuum for both stem blight and foliar

blight. From this screening, it was determined

that resistance to both fo rms of the d isease were

relatively common in the germplasm, but resis-

tance to one form of the disease was not strong-

ly correlated to resistance to the other form. Ten

accessions previously shown to possess root

rot resistance were tested for resistance to stem

rot and leaf blight, and were found to also be

highly resistant to these forms of the disease. It

appears that single accessions have resistance

to foliar, stem and root rot caused by P. capsici,

which may simplify breeding for resistance to all

three forms of the dis ease.

Keywords: Pepper; Phytophthora Blight; Root Rot;

Stem Blight; Foliar Blight

1. INTRODUCTION

Bell pepper (Capsicum annuum) is one of the eco-

nomically important crops in the state of Georgia where

the total farm-gate value of bell pepper was $100 million

in the year 2007 [1]. Phytophthora blight, caused by the

oomycete Phytophthora capsici, is a serious threat to

production of peppers worldwide [2]. Phytophthora blight

is becoming a major disease constraint to bell pepper

production in Georgia, affecting the plants at all growth

stages (from seedling to adult plant) and multiple plant

parts such as the roots, leaves, stems and fruit [3]. P.

capsici attacks the roots at all developmental stages,

causing a sudden wilt and collapse of the infected plant

[4]. Foliar blight starts with small circular or irregular-

shaped lesions which later enlarge, dry, and bleach to a

light tan [5]. Phytophthora foliar blight can lead to seri-

ous crop losses when soil containing P. capsici contacts

leaves by rain splash or working in wet fields [6]. Stem

lesions lead to an aerial blight where a black girdling

lesion develops most commonly in the leaf and branch

axils and extends upward and downward.

The development of phytophthora-resistant cultivars is

key to an integrative approach to phytophthora-disease

management [7]. Ideally, resistant cultivars will be resis-

tant to infection in all plant organs, but resistance to in-

fection in one organ is not necessarily related to resis-

tance in other organs. The commonly used resistant line

Criollo de Morelos-334 (CM-334) is resistant to Phyto-

phthora capsici in roots, stems, and leaves [5,6,8].

However, Walker and Bosland [6] found that in proge-

nies derived from CM-334 foliar blight resistance and

root rot resistance were controlled by independently seg-

regating genes. Further work by Sy et al. [9] demon-

strated that stem blight resistance from CM-334 was

controlled by a single gene, Psr, when “Early Jalapeno”

was the susceptible parent, and that this gene was inher-

ited independently from those controlling foliar blight

and root rot resistance. Thus it appears that foliar blight,

stem blight, and root rot are separate disease syndromes

that need to be analyzed independently.

Multiple races of P. capsici have been demonstrated to

exist in commercial production regions [7,10,11]. The

presence of both mating types of P. capsici in some pro-

duction regions [10] requires that new sources of resis-

tance should be found and incorporated into adapted

germplasm as insurance against the development of new

pathogenic strains of P. capsici. Additionally, resistance

sources should be tested against multiple isolates from

the growing region for which the resistant cultivar is tar-

B. L. Candole et al. / Agricultural Sciences 3 (2012) 732-737 733

geted.

Previously Candole et al. [12] screened 2301 acces-

sions from the USDA, ARS Plant Genetic Resources

Conservation Unit for resistance to Phytophthora capsici

root rot. High levels of resistance were found in several

accessions using greenhouse and field screening proto-

cols. The objective of this study was to evaluate root rot

resistant accessions for resistance to the stem and foliar

phases of phytophthora blight caused by P. capsici. The

results of these experiments will provide information

useful to breeders searching for germplasm to breed for

resistance to P. capsici.

2. MATERIALS AND METHODS

2.1. Plant Material

Capsicum annuum accessions were obtained from the

USDA, ARS Plant Genetic Resources Conservation Unit

in Griffin, Ga. A total of 1392 accessions were randomly

selected for foliar and stem inoculations. This number

represented 45% of the total (3118) C. annuum acces-

sions available from this location. Seeds from each ac-

cession were sown in plastic cells of a multipot bedding

plant container (Com-Pack D806, Hummert International,

St. Louis, Mo.). Each cell measured 6 cm × 4 cm × 5.5

cm and contained Redi Earth plug and seedling mix (Sun

Gro, Bellevue, Wash). A total of 6 - 12 seeds were

planted for each accession at the rate of two seeds per

cell. The cells containing the seeds were then placed in

52.3 cm × 25.9 cm × 6.1 cm plastic trays with drainage

holes (F1020 flats, Hummert International, St. Louis,

Mo.). The test plants were watered twice daily and fertil-

ized twice a week with water-soluble fertilizer (24N-6P-

16K) diluted to provide 315 ppm nitrogen. Separate sets

of the same accessions were prepared for foliar and stem

tests and were maintained in the greenhouse. The air

temperature in the greenhouse before and during the in-

cubation process had a diurnal range of 13˚C - 30˚C.

Cultivars Camelot and CM-334 were used as the suscep-

tible and resistant controls, respectively, in all tests.

CM-334 was kindly provided by P. Bosland (New Mex.

St. Univ.) and “Camelot” was obtained from Rupp Seeds

(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·m−2·s−1 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

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

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.

B. L. Candole et al. / Agricultural Sciences 3 (2012) 732-737

735

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

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