Plant Breeding 125, 343—346 (2006)
2006 The Authors
Journal compilation 2006 Blackwell Verlag, Berlin
Search for partial resistance against Puccinia hordei in barley landraces from the
Fertile Crescent
M . J . Y . S h t a y a 1 , , J . C . S i l l e r o 2 and D . R u b i a l e s 1
1
Institute of Sustainable Agriculture, CSIC, Apdo. 4084, 14080 Córdoba, Spain, E-mail: mshtaya@yahoo.com; 2CIFA Alameda
del Obispo, IFAPA, Apdo. 4042, 14080 Córdoba, Spain
With 2 figures and 4 tables
Received May 26, 2005/Accepted January 30, 2006
Communicated by G. Fischbeck
Abstract
A collection of 111 barley landraces from the Fertile Crescent was
screened for resistance to barley leaf rust in the field and under
controlled conditions. Large variation was observed for disease
severity under field conditions. Accessions with high resistance because
of hypersensitivity were identified. Also segregation was observed in
some accessions, with individual plants showing hypersensitive reactions (IT £ 6). Partial resistance due to a reduction of infection in spite
of a compatible infection was commonly found (19%). Resistance of
12 accessions selected for their low disease severity and high IT, was
shown to be due to a prolonged latency period and increased
percentage of early aborted colonies not associated with host cell
necrosis. A high correlation was observed between the microscopic and
macroscopic components of partial resistance.
Key words: Hordeum vulgare — Puccinia hordei — barley —
landraces — leaf rust — histology
Barley leaf rust caused by the fungus Puccinia hordei Otth, is an
important foliar disease in temperate regions throughout the
world. The use of resistant barley cultivars has been an effective
method to control the disease and to reduce yield losses, which
may reach 40% in the susceptible cultivars (Griffey et al. 1994).
Evaluations of the Hordeum gene pool (H. vulgare, and its
wild progenitor H. vulgare ssp. spontaneum) have resulted in
the identification of 19 major race-specific resistance genes
named Rph1-Rph19 (Weerasena et al. 2004). However, only a
few of these genes have been deployed in commercial cultivars.
Genes Rph2, Rph3, Rph4, Rph7 and Rph12 have been used in
Europe (Dreiseitl and Steffenson 2000), whereas, Rph2, Rph6,
and Rph7 have been deployed in the United States (Steffenson
et al. 1993). The resistance controlled by these genes is not
durable and is assumed to operate on a gene-for-gene basis
with avirulence factors in the pathogen population. Virulence
to Rph3 and Rph9 has been identified in Europe, South
America and the Middle East (Brooks et al. 2000). Rph7 was
believed to be effective in Europe (Niks et al. 2000), but
virulence to it has recently been identified in Spain (Shtaya,
unpublished) and has been reported in Morocco (Parlevliet
1976), Israel (Golan et al. 1978, Brodny and Rivadeneira
1996), and the United States (Steffenson et al. 1993).
This short life of resistance has caused breeders to look for
other types of resistance such as partial resistance which
appears to be more durable and race-non-specific (Parlevliet
and Van Ommeren 1975).
The objectives of this study were to screen a collection of
barley landraces from its centre of origin (the Fertile Crescent)
looking for new sources of durable resistance to barley leaf
rust and to study their components of resistance.
Materials and Methods
Plant materials: Seed samples of 111 Hordeum vulgare landraces from
the Fertile Crescent were kindly provided by the International Centre
for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria,
and United States Department of Agriculture (USDA), USA
(Table 1).
Inoculum: A monosporic isolate (CO-01) with virulence/avirulence
factors Rph1,2,4,6,8,9,12/3,5,7 was derived from a Puccinia hordei
population collected at Córdoba, Spain on barley fields. The isolate
was maintained in liquid nitrogen, and multiplied on susceptible barley
line L94 before being used across all the experiments.
Field experiment: Field testing was performed at the CIFA experimental farm at Córdoba, Spain during the growing season 2003–2004.
Accessions were sown in November 2003 in three complete randomized blocks. Each accession was represented by 25–30 seeds in a single
row, 1 m long per replicate. A spreader row, of the very susceptible line
L94, was sown in the alleyways, perpendicular to the tested accessions.
Leaf rust epidemic was initiated by artificial inoculation of the
spreader rows at growth stage DC 43 (Zadoks et al. 1974) by dusting a
mixture of urediospores and talcum powder. Disease severity (DS) was
at the end of the growing season as the percentage of leaf area covered
by the rust uredinia.
Seedling studies: In a first experiment, DS and infection type (IT) of all
111 accessions were studied in the seedling stage under controlled
conditions. About 12–15 seedlings per accession were grown in
7 · 7 · 11 cm pots in three replicates. The inoculation was carried
out by dusting fresh urediospores of isolate CO-01, diluted 10 times
with talcum powder, over the seedlings when the second leaf of the
seedlings had emerged. After inoculation, seedlings were incubated
overnight in complete darkness and at a relative humidity of 100%.
Seedlings were then transferred to a growth chamber at 18–22C and
white fluorescent light (12 h light/12 h dark).
Twelve days after inoculation, DS was estimated as the percentage
of the first leaf area covered by the rust uredinia. Infection type (IT) on
a 0–9 scale (McNeal et al. 1971) was also recorded on the first leaf.
In a second experiment, components of resistance of 12 selected
accessions were determined. Selected accessions were those that
showed a disease severity <15% in the field and compatible
interaction (IT ‡ 7) in the seedling stage (first experiment). Seeds of
the selected accessions were sown in soil in plastic trays
(35 · 20 · 8 cm) with three replicates of three plants each. In each
tray, eight accessions plus ÔVadaÕ (high level of partial resistance) and
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S h t a y a , S i l l e r o and R u b i a l e s
344
Table 1: Origin and source of the barley landraces used in this study
Origin
Israel
Jordan
Lebanon
Palestinian Territory
Syria
No. accessions
Source
4
29
15
23
40
USDA
ICARDA + USDA
ICARDA
ICARDA + USDA
ICARDA
L94 (very susceptible) checks were included. Eleven days after sowing,
the first leaf of each plant was placed in a horizontal position with the
help of metal staples and inoculated with isolate CO-01 of P. hordei.
The inoculation was carried out in a settling tower by dusting a
mixture of freshly collected spores and talcum powder (1 : 10, v/v).
Each tray was inoculated with 3 mg of spores that resulted in about
200 spores/cm2 deposition (Niks and Rubiales 1994). The inoculated
plants were kept in an inoculation chamber for 12 h at 20C with a
relative humidity of about 100% and in darkness. Plants were then
transferred to a growth chamber at 18–22C and white fluorescent light
(12 h light/12 h dark).
The components of resistance measured in this experiment were:
infection type (IT), latent period (LP) and infection frequency (IF). IT
was recorded 12 days after inoculation following the 0–9 scale
(McNeal et al. 1971). LP was determined by daily counting the
number of uredia visible in a marked area (2–3 cm2), using a 6· pocket
lens. The LP50 was calculated as the time from the inoculation to the
time at which 50% of the uredia had appeared (Parlevliet 1975). The
final number of uredia was used to determine the IF calculated as the
number of uredia/cm2. The actual LP and IF were converted into
relative latency period (RLP) and relative infection frequency (RIF),
taking the LP and IF of line L94 as 100% in each box.
Histological studies: Five days after inoculation, a central leaf segment
of nearly 2 cm2 per plant was collected. Leaves were fixed and cleared
by boiling for 1.5 min in lactophenol/ethnol (1 : 2, v/v) and stored
overnight in this mixture at room temperature. Segments were then
washed once with 50% ethanol for 30 min, once with 0.05 M NaOH
for 30 min, rinsed three times in water (10 min each), and soaked in
0.1 M Tris/HCl buffer (pH 8.5) for 30 min. They, then were stained
with a 0.1% solution of Uvitex 2B in the same buffer. This was
followed by rinsing four times with water. Segments were then
immersed in a solution of 25% glycerol for a minimum of 30 min (a
few drops of lactophenol were added to the solution to prevent
deterioration by fungi) and stored until observed. Leaf segments were
examined at 100 · with Leica epifluorescence equipment (DM LB,
330–380 nm wave length transmission). At least 100 infection units
were evaluated per leaf segment, and classified according to their stage
of development (Martı́nez et al. 2001). Early aborted colonies were
defined as individuals that formed a primary infection hypha and not
more than six haustorial mother cells. Those colonies that formed
more than six haustorial mother cells were classified as established
colonies. Colony size (CS) was estimated by calculating the length (L)
and the width (W) of 20 colonies. These colonies were randomly
chosen. CS was calculated using the formula:
CS ¼
Fig. 1: Frequency distribution of the accessions according to the
disease severity of Puccinia hordei in the field
accessions being even more susceptible than L94. DS was
<15% in 21 accessions. Nine of these accessions with low DS
in the field, showed incompatible infection (low IT). However,
12 accessions showed compatible infection (high IT) and low
severity in the seedling test and in the field (Table 2).
In seedling tests, most of the accessions (92.8%) displayed
compatible interaction (IT ‡ 7). Segregation was observed in
eight accessions, with individual plants showing low IT. The
susceptible check L94 showed 33% DS and the partially
resistant cv. ÔVadaÕ 15% (Fig. 2).
Table 2: Macroscopic components of resistance to leaf rust (Puccinia
hordei) isolate CO-01 in selected accessions of barley landraces from
the Fertile Crescent in the seedling stage and in the field
Seedlings in growth chamber
Accessions
IT1
RLP2
RIF2
DS2
Adult plants in
the field (DS)
IG31396
IG32710
IG32733
IG32747
IG110861
IG110870
IG110887
IG110906
IG115774
IG125768
IG125775
PI186425
ÔVadaÕ
L94
9
9
9
9
9
8
9
8
9
9
9
9
9
9
117ab3
122ab
118ab
122ab
124ab
131a
126ab
127ab
114b
117ab
118ab
118ab
123ab
100c
46bc
70abc
77ab
69abc
52bc
28c
58abc
28c
67abc
53bc
68abc
61abc
51bc
100a
15bc
20b
16bc
16bc
20b
17bc
9c
19b
19b
20b
22b
21b
15bc
33a
14b
11b
10b
13b
13b
12b
5b
4b
9b
14b
10b
8b
4b
47a
1
IT on a scale of 0–9 (McNeal et al. 1971).
Relative latency period (RLP) and relative infection frequency (RIF)
referred to L94 ¼ 100%. The actual values for L94 were LP ¼ 141h
and IF ¼ 70 pustules/cm2. DS ¼ disease severity.
3
Data with the same letter per column are not statistically different
(Duncan-test, P ¼ 0.05).
2
pLW
:
4
Data analysis: Analysis of variance (ANOVA) was calculated by using
PROC GLM in an SAS program (SAS Institute 1988). Comparisons
between lines were made by the Duncan-test.
Results
Under field conditions, the susceptible check L94 showed 47%
DS and the partially resistant cv. ÔVadaÕ only 4% DS (Fig. 1).
High susceptibility was common in the collection with seven
Fig. 2: Frequency distribution of the accessions according to the
disease severity of Puccinia hordei in climatic room (seedlings)
Partial resistance against Puccinia hordei in barley
345
Table 3: Microscopic components of resistance to Puccinia hordei in
selected accessions of barley landraces in the seedling stage
% of early
aborted colonies
% of established
colonies
Accessions
EA+1
EA)1
EST+1
EST)1
Colony
size (CS)1
IG31396
IG32710
IG32733
IG32747
IG110861
IG110870
IG110887
IG110906
IG115774
IG125768
IG125775
PI186425
Vada
L94
2a2
0.8abc
0c
0.5bc
0c
0c
0.7abc
0c
0c
0c
1.7ab
0c
0c
0c
44ab
22b
29ab
27ab
48a
28ab
26ab
37ab
23b
29ab
28ab
39ab
42ab
2c
0.3b
0.8 b
0b
0b
0b
9a
1b
0b
0b
0b
0b
0b
0b
0b
53.7bc
76b
71bc
72.5bc
52c
63bc
72.3bc
63bc
77b
71bc
70.3bc
61bc
58bc
98a
0.076bcd
0.080bc
0.072bcd
0.081bc
0.041e
0.068bcde
0.059cde
0.061bcde
0.091b
0.065bcde
0.078bc
0.060bcde
0.048de
0.132a
1
Expressed are percentage of early aborted colonies associated with
host cell necrosis (EA+), percentage of early aborted colonies without
host cell necrosis (%EA)), percentage of established colonies with host
cell necrosis (EST+) and percentage of established colonies without
host cell necrosis (% EST)) and mean colony size in mm2 (CS).
2
Data with the same letter per column are not statistically different
(Duncan-test, P ¼ 0.05).
Table 4: Correlation coefficients (r) among traits in the selected
accessions of barley landraces
Traits
DS
(seedlings)
DS
(field)
RLP
(seedlings)
RIF
(seedlings)
DS (seedlings)
DS (field)
RLP (seedlings)
RIF (Seedlings)
EA) (seedlings)
0.74*
)0.67*
0.95*
)0.35***
)0.75*
0.54**
)0.63*
)0.69*
0.56**
)0.74*
DS, disease severity; RLP, relative latency period; RIF, relative
infection frequency.
*P ¼ 0.001; **P ¼ 0.01; ***P ¼ 0.1
Macroscopic results of the selected accessions with low DS
in the field and high IT in seedlings are shown in Table 2. All
the selected accessions showed longer latency periods (higher
RLP) than the susceptible check L94, and were similar to the
partially resistant ÔVadaÕ. RIF of these accessions was lower
than that of L94, although not always significantly so, and
always similar to that of Vada. A strong negative correlation
was observed between RLP and RIF, and between RLP at the
seedling and DS in both seedlings and adult plants in the field
(Table 4).
Percentages of early aborted or established colonies associated with host cell necrosis was very low in all selected
accessions and checks. All selected accessions had a percentage
of early aborted colonies not associated with host cell necrosis
(%EA-), higher than the susceptible check L94 and similar to
the partially resistant ÔVadaÕ (Table 3). Also, all selected
accessions showed smaller colony sizes (CS) than the susceptible check. High correlation was observed between the EA- at
seedling stage and RLP at the seedling stage (Table 4).
Discussion
In most European countries, landraces of major crops exist
only in gene banks (Ceccarelli et al. 2000), but in some areas of
West Asia and North Africa farmers still rely on landraces for
major crops such as barley. Barley is a typical self-fertilizing
annual crop and landraces of such crops are expected to
consist of more or less homozygous plants. For barley, the
relatively high number of seeds needed per ha, makes the
conscious selection of specific plants by the farmer barely
possible. It also means that seeds from a very large number of
plants are used for the next crop. Under these conditions a
very low frequency of cross-pollination and incidental survival
of volunteer plants from another landrace of a previous sowing
could easily lead to the low level of within-landrace variation
observed in infection type (IT) seen here. Fekadu and
Parlevliet (1997) found large variation within and between
Ethiopian barley landraces for many quantitative traits,
including partial resistance.
Level of partial resistance in the collection is high since
most of the accessions had a degree of rust reduction,
whereas hypersensitive resistance was observed only in 8% of
the accessions. Levels of partial resistance in some of the
selected accessions were similar to those of ÔVadaÕ. However,
Niks et al. (2000) found that modern European barley
cultivars have a higher level of partial resistance than old
cultivars. This accumulation of partial resistance was due to
breeding programmes and selection methods for partial
resistance.
The high level of partial resistance of the selected accessions,
observed in the seedling stage, was due to a reduced infection
frequency and prolonged latency period. Microscopically, this
was found to be associated with a high level of early aborted
colonies not associated with cell necrosis (EA-) and reduced
colony size, in agreement with Parlevliet and Van Ommeren
(1975), Niks (1982) and Statler and Parlevliet (1987).
From our results we can conclude that barley landraces
from the Fertile Crescent represent a substantial reservoir for
partial resistance, which can be used in breeding programmes
for partial resistance accumulation. Additional genetic studies
should be conducted to determine the relationship between the
resistance gene(s) in the selected landraces, with low infection
type together with their relationships with other known major
resistance genes.
Acknowledgements
The authors acknowledge the International Centre for Agricultural
Research in the Dry Areas (ICARDA) and United States Department
of Agriculture (USDA) for providing the seed samples used in this
study. The Spanish Agency for International Cooperation (AECI) and
CICYT project AGL2005-01781 for financial support.
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