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© by PSP Volume 26 – No. 1a/2017, pages 906-912 Fresenius Environmental Bulletin
906
GENETIC STRUCTURE OF POWDERY MILDEW DISEASE
PATHOGEN BLUMERIA GRAMINIS F. SP. HORDEI IN THE
BARLEY FIELDS OF CUKUROVA IN TURKEY
Ahmet Zeybek1,2,*, Mohd Kamran Khan2, Anamika Pandey2, Aslihan Gunel2,3,
Oktay Erdogan4, Mahinur S Akkaya2
1 The University of Muğla Sıtkı Koçman, Department of Biology, 48000, Muğla, Turkey
2 The University of Middle East Technical, Department of Chemistry, Biotechnology Graduate Program, 06800, Ankara, Turkey
3 The University of Ahi Evran, Department of Chemistry, 42200 Kırşehir, Turkey
4 The University of Nevşehir Hacı Bektaş Veli, Department of Biosystem Engineering, 50300, Nevşehir, Turkey
ABSTRACT
This work was conducted in order to
investigate the frequency of virulence genes, gene
complexities, and pathotype frequencies of
powdery mildew (Blumeria graminis f. sp. hordei)
populations on two different barley production
fields in the Çukurova Region. For this purpose, the
barley leaves, prior to the pre-harvest period, which
were infected with pathogen at the sexual period
developmental stage as Cleistothecium were
collected in 2007 and stored in the laboratory at
room temperature. Pathogenic isolates were
obtained in laboratory conditions with the aid of
susceptible control variety (Bülbül-89) by
stimulating ascospore output from each foliar.
Every isolate was further purified through
multiplying from a single spore. A total of 138
Blumeria graminis f. sp. hordei single spores were
obtained. The differential isogenic lines of 25, each
possessing a single resistance genes were
inoculated with the single spore isolates obtained
from Cleistothecium. The aggressiveness of gene
frequencies and virulence gene complexities, and
pathotype frequencies of the isolates were
determined according to the scale 0-4, developed by
Welz. The pathotypes were established through the
formula developed by Habgood. Virulence gene
frequencies varied from 0 % to 94.33 % in powdery
mildew populations. Gene frequencies of Va1,
(Va7+Vk) were at 0.0 % in both populations. In the
populations of Adana and Hatay 47 and 56
pathotypes were obtained, respectively. The isolate
with Habgood pathotype index of 04667601
showed 14.8 % gene frequency in Adana
population.
KEYWORDS:
Barley, Blumeria graminis f. sp. hordei, Virulence gene
frequency, Pathotype frequency, Habgood analysis
INTRODUCTION
Barley (Hordeum vulgare L.) is the most
important crop following wheat, corn and paddy in
the world. Turkey possesses a significant position
by planting area 2.783.583 hectares of barley and
136.5 million tons of yield in the world [1].
Powdery mildew (Erysiphe graminis DC. f. sp.
hordei Em Marchal Synamorph, Blumeria graminis
DC. Golovin ex Speer f.sp. hordei) is one of the
primary diseases affecting yield and quality in
barley production. Conidia spores, as a disease
factor, may infect barley plants in all growth stages
of the vegetation. A number of investigators
reported that powdery mildew causes 25-30 % yield
losses, especially in Central Europe and, more than
50 % in USA if the climatic conditions are
pertaining to spreading of the disease [2, 3, 4, 5].
The infected field ratio varies from 25 % to 70 %
when the coastal belt areas of the Aegean and
Mediterranean Regions dominated by the
Mediterranean climate. On the other hand the
intensity of the infection was stated between 1-100
%. Several strategies were improved in the battle
against powdery mildew such as use of fungicides,
planting resistance varieties, mosaic planting
techniques, rotated planting practices and combined
fighting systems in Europe possessing moist and
cool climate conditions [6, 7]. A number of studies
were carried out on distribution of powdery mildew
and occurrence of physiologic race in order to
develop early warning systems to control crop
© by PSP Volume 26 – No. 1a/2017, pages 906-912 Fresenius Environmental Bulletin
907
diseases, therefore almost all barley cultivars
produced in Europe was tested in view of resistance
against to powdery mildew [8, 9, 10].
It is revealed that European barley cultivars
possess mostly two or three resistance genes and
rarely more than three [7, 11, 12, 13]. Emergence of
new physiologic races in powdery mildew
populations results in resistance-breaking in spite of
planting newly developed varieties in relatively
short time period [14]. At the same time use of
fungicides is also being ineffective due to increased
resistance by pathogen [6, 15, 16, 17].
The aim of modern agriculture is to produce
permanently and securely within the means of
minimally polluting the environment with
fungicides. Therefore, the use of resistant varieties
through plant breeding or the implementation of
chemical control methods is considered inadequate.
In developing more effective combat systems, the
in-depth studies in the interactions of host-parasite
systems and means of integrated cross-disciplinary
should be conducted. The early warning systems in
the control against diseases, a systematic and
collaborative virulence spectrum of pathogens
world-wide should also be determined. To this end,
powdery mildew virulence surveys are carried out
in barley crop fields in Europe and in the different
regions of the world every year [12, 18, 19, 20, 21,
22, 23, 24]. However, to this date a periodical
survey for investigating the virulence pattern of
barley powdery mildew has not been implemented.
The purpose of this research is to determine
virulence spectrum in spore population of powdery
mildew in the sexual period, Cleistothecia, on the
barley fields of Çukurova Region. The data
gathered will help facilitate protection strategies in
the region.
MATERIALS AND METHODS
Plant materials. Differential test varieties of
25 isogenic lines are used to identify virulence
genes in Blumeria graminis f. sp. hordei
populations, developed by Kolster et al. [25],
widely used in Europe and a Turkish isogenic line
carrying no resistance (Table 1). Isogenic lines
were amplified at the Adnan Menderes University
farms, which were obtained from ICARDA (The
International Center for Agricultural Research in
the Dry Areas). Barley leaves with Cleistothecium,
from the barley fields in the Çukurova Region were
collected prior to harvest and stored at 4oC.
TABLE 1
Differential test varieties for the virulence of Blumeria graminis f. sp. hordei collected in the Çukurova
Region.
Isogenic Lines*
Parent
Origins-owned
Resistance Gene
Pallas-01
Iso 1R
CI 1637 Mla1, +?
Mla1, +?
Pallas-02
Ricardo
CI 6306
Mla3
Pallas-03
Iso 20R
CI 16151
Mla6, Ma14
Pallas-04a
Nordal
Calsberg
Mla7, Mlk, +?
Pallas-04b
Nordal
Calsberg
Mla7, +?
Pallas-05
Filler
Calsberg
Mla1
Pallas-06
Iso 10R
CI 16147
Mla7, Ml(LG2)
Pallas-07
Mona
Svalof
Mla9, Mlk
Pallas-08a
Senat
Svalof
Mla9, Mlak
Pallas-08b
Senat
Svalof
Mla9
Pallas-09
Iso 12R
CI 16149
Mla10, Ml(Du2)
Pallas-10
Emir
Cebeco
Mla12
Pallas-11
RuPl
Svalof
Mla13, Ml(Ru3)
Pallas-12
Hor 1657
Hor 1657
Mlc
Pallas-13
Hor 1402
Hor 1402
Ml (1402)
Pallas-14
W.41/145
WeihenstePhen
Ml(41/145)
Pallas-15
RuPee
Svalof
Ml(Ru2)
Pallas-19
Iso 5R
CI 16145
MlP
Pallas-20
Atlas
CI 4118
Mlat
Pallas-21
Deba
Abed
Mlg, Ml (CP)
Pallas-22
Riso 5678
CI 15219
mlo5
Pallas-23
Lofa
Abed
Ml (La)
Pallas-24
Iso 3R
CI 16141
Mlh
Pallas-30
Filler
Svalof
Mla1
Pallas
Pallas
Svalof
Mla8
Bülbül-89
Bülbül-89
Turkish cultivar
none known genes
*obtained from ICARDA
© by PSP Volume 26 – No. 1a/2017, pages 906-912 Fresenius Environmental Bulletin
908
Method. Pathogen isolates were obtained by
stimulating ascospore formations on each leaf of
the susceptible variety, Bülbül-89, in laboratory
conditions. Each isolate was purified from a single
spore [26]. Virulence tests were carried out on the
26 differential test lines (Table 1) grew under
controlled laboratory conditions at the 1-2 leaf
periods according to Pons [6]. The leaves of
isogenic lines were cut in a 3 cm length, placed in 9
cm in diameter Petri dishes containing BA
(benzimidazol agar) (5 ppm) in the
counterclockwise direction with the order in Table
1, where the bottom surface of the leaves were in
contact with the feeding environment. As a control,
variety Bülbül-89 leaf particles were placed at the
center of the Petri dishes.
Inoculation of single spore isolates on test
varieties was implemented with the aid of home-
made inoculation tower in a sterile cabinet.
Inoculated pieces of leaves on Petri dishes were
incubated at 17oC / 14-hour dark and 10-hour light
periods in a climate chamber for 8-10 days. The test
plates were assessed for full inoculation and
sufficient colony formation and on the Bülbül-89
susceptible variety according to the 0-4 scale
developed by Welz [27]. Following the tests
determining the genetic composition of pathotypes,
the virulence gene frequencies and the level of
complexities in virulence genes were determined
using MS-Excel version 5.0 developed by Habgood
[28]. The identification of powdery mildew isolates
were determined using the software by Hermann et
al. [29]. According to this method, the 0-4 scale
results in evaluating the test sets were turned into a
binary code system. As a result, two-type outcomes
were obtained; the scale values of 0, 1, 2, were
translated as 0 (resistant), the values of 3 and 4
were translated as 1 (sensitive). The following
formula by Habgood [28] was used to determine the
pathotypes.
PTi kpi
nr
nr
1
25 1
2( ) .
(1)
Pti: Pathotype index, nr: order # of differential lines
(Table 1), kpi: binary code # of differential lines (0
or 1).
The values of the gene frequency aggression
were divided into 5 different groups according to
this style of grouping, if virulence gene frequency
in population is bigger than 90 %, the population is
considered fixed. If it is between 60 % and 90 %, it
is high; if it ranges between 30 % to 60 %, it is
medium; if the frequency is between 10 % and 30
%, it is very low degree and if it is smaller than 10
%, it is called recessive. Virulence complexity is a
number of genes in a single isolate. The number of
virulence genes was determined per isolate.
Afterwards, the number isolates having the same
complexity values as percentages are determined as
complexity frequencies.
RESULTS AND DISCUSSION
Virulence genes frequencies and complexities
obtained from powdery mildew population in
barley field of Çukurova Region are presented in
Table 2 and Table 3, respectively, according to
which, both of the populations here called as Adana
and Hatay locations, had 22 virulence genes. The
similarities were observed in both populations in
fixed and recessive genes. The Va8 gene frequency
was over 90 % in both of the populations indicating
the gene is fixed, the genes; Va1, Va7+Vk,
Va9+Vk, and Va9 were recessive in both
populations. The crucial differences were observed
in Vp and Vh genes, while frequency of Vp gene
was 81.64 % in Adana, it is 32.34 % in Hatay, on
the other hand the frequency of Vh virulence gene
was 18.30 % in Adana, it was 67.62 % in Hatay.
The lowest virulence gene frequencies were found
in Va1, Va7+Vk, Va9+Vak virulence genes (0.00
%) and detected in Adana population, whereas the
highest one was in Va8 gene (94.33 %). Virulence
gene frequencies of Va1+?, Va7+Vk, Va7, Va1,
Va9+Vk, Va9+Vak, Va9, Va13+(V(Ru3), Vo5,
Va1 genes were found to be recessive (<10%),
virulence gene frequencies of Va3, Va7+V(LG2),
Vc, V(Ru2), Vg+V(cp), Vh genes were low (10-30
%); Va6+Va14, Va12, V(41/145) genes were
medium (30-60 %); Va10+V(Du2), Va(1402), Vp,
Vat, V(La) genes (60-90 %) were high in the same
population. In the Hatay population, virulence gene
frequencies of Va1+?, Va7+Va14, Va1 genes were
0.00 %, virulence gene frequency of Va8 was 91.14
%. It is detected that Va7, Va1, Va7+V(LG2),
Va9+Vak, Va9, V(41/145), V(CP) virulence genes
were found to be recessive with having less than 10
% gene frequencies. The frequencies of Va3,
Va6+Va14, Va9+Vk, Va13+V(Ru3), Vc, V(Ru2),
Vo5 virulence genes were low (10-30 %),
frequencies of Va12, Vp were medium (30-60 %),
and frequencies of Va10+V(Du2), Va(1402), Vat,
V(la), Vh genes were high (60-90 %).
© by PSP Volume 26 – No. 1a/2017, pages 906-912 Fresenius Environmental Bulletin
909
TABLE 2
The virulence gene frequencies.
Virulence Genes
Virulence Gene Frequencies (%)
Adana
Hatay
Va1, +?
0.00
0.00
Va3
29.56
26.46
Va6, Va14
36.68
27.93
Va7, Vk
0.00
0.00
Va7
1.40
1.47
Va1
4.22
7.35
Va7, V(LG2)
12.67
4.41
Va9, Vk
1.40
14.70
Va9, Vak
0.00
1.47
Va9
7.04
7.35
Va10, V(Du2)
84.48
85.26
Va12
30.97
33.81
Va13, V(Ru3)
2.81
4.41
Vc
15.48
8.82
Va (1402)
83.07
82.32
V(41/145)
36.60
7.35
V(Ru2)
25.34
16.17
VP
81.64
32.34
Vat
83.07
79.38
Vg, V (CP)
19.71
8.82
Vo5
4.22
14.70
V (La)
74.62
79.38
Vh
18.30
67.62
Va1
1.40
0.00
Va8
94.33
91.14
TABLE 3
The virulence gene complexity values.
Virulence Genes
Virulence Gene Complexities (%)
Adana
Hatay
5
2.81
0.00
6
23.80
7.35
7
18.30
20.58
8
39.42
27.93
9
8.44
4.44
10
5.56
7.35
11
1.40
1.47
12
0.00
2.94
TABLE 4
The pathotype frequencies.
Pathotypes
Populations
No
Habgood number
Adana
Hatay
1
04667601
14.08
0.00
2
00467611
8.44
0.00
3
04667611
8.44
0.00
4
04667631
8.44
0.00
5
0667611
8.44
0.00
6
24667611
7.04
0.00
7
0067611
4.22
0.00
8
0067615
2.81
0.00
9
04667611
2.81
0.00
10
20067603
2.81
0.00
11
404776721
0.00
7.35
12
410676320
0.00
4.41
13
004276320
0.00
2.94
14
006756721
0.00
2.94
15
204774721
0.00
2.94
16
400676321
0.00
2.94
17
400676720
0.00
2.94
18
404776721
0.00
2.94
Total frequencies of other pathotypes (%)
49.28
70.56
Number of pathotypes
47
56
Number of isolates tested
71
68
© by PSP Volume 26 – No. 1a/2017, pages 906-912 Fresenius Environmental Bulletin
910
The virulence gene complexity in Adana
changed from 5 to 11, and varied from 6 to 12 in
Hatay (Table 3). The gene complexity values of this
study are in consistent with the other studies
conducted on the cultured barley fields [30, 31, 32,
33]. The pathotypes commonly seen in the
powdery mildew populations in the locations of
Adana and Hatay are presented in Table 4. The
numbers of pathotypes are 47 and 56 in from Adana
and Hatay locations, respectively. The pathotype
with the Habgood number 0466760 from the Adana
had the highest frequency 14.08 %. As for the
Hatay location, the pathotype 404776721 has the
highest frequency as 7.35 %.
A study conducted more than a decade and a
half ago by Braun et al. [15] and Zeybek [34]
showed 9 genes were fixed on wild type barley. The
fixed genes in this study are consistent with those
cited reports above. Since then, the frequencies of
the virulence genes Va1, Va9, Va13, and Vo5
decreased substantially as shown in this study, and
they became recessive by a negative selection.
CONCLUSION
The findings produced by the samples of
powdery mildew obtained from two different barley
fields in the Çukurova Region showed differences
regarding the virulence gene frequencies, virulence
gene complexities and pathotype frequencies. One
striking similarity is that Va8 virulence gene is
fixed in both of the population followed by Va10
with high degree appearance. Moreover, examining
the distribution and frequency of virulence genes
during the vegetation period each year will
contribute towards a better understanding and
dissemination of information regarding the genetic
structure of powdery mildew populations.
ACKNOWLEDGEMENTS
We gratefully acknowledge Dr. Mogens S.
Hovmoller and ICARDA for providing the
Blumeria graminis f. sp. hordei races and Barley
differential lines, respectively. We also thank to
Adnan Menderes University for using their
facilities. This work was funded by the DOSAP
Program of METU and State Planning Organization,
DPT (Project No. DPT2004K120750).
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© by PSP Volume 26 – No. 1a/2017, pages 906-912 Fresenius Environmental Bulletin
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Tarla Bitkileri Anabilim Dalı, Fen Bilimleri
Enstitüsü Müdürlügü, Adnan Menderes
Üniversitesi, Aydın, Türkiye. (in Turkish)
Received: 06.05.2016
Accepted: 12.12.2016
CORRESPONDING AUTHOR
Ahmet Zeybek
Muğla Sıtkı Koçman University
Department of Biology
4800 Muğla – TURKEY
E-mail: ahmetzeybek@hotmail.com
