n was scored 30 days after inoculation
using a visual scale of 0 - 3, where 0 = No symptoms of
the disease; 1 = Small pin point necrotic spots without
chlorosis, covering <1% leaf area infected, and incapable
of producing sporulation in a humid chamber under the
laboratory conditions; 2 = Symptoms typically angular
with chlorosis measuring 2 - 3 mm, covering > 1% leaf
area infected, with or without apparent sporulation; 3 =
Symptoms typically angular, coalescing, covering > 25%
leaf area infected, with or without apparent sporulation,
with severe chlorosis and causing premature death of the
leaves. For the purpose of identification of phenotypical
differences, disease ratings between 0 and 1 were con-
sidered Resistant (Incompatible reaction), and ratings be-
tween 2 and 3 were considered Susceptible (Compatible
reaction).
DNA Extraction: Fungal cultures were grown in Petri
plates containing 15 mL of V8-juice agar for three weeks,
the fungal growth was scraped from the plates and the
total DNA was extracted as described by Raeder &
Broda [7]. DNA was quantified by a DyNa Quant 200
Fluorometer (Pharmacia) and RNA was eliminated by
RNase (10 µg·mL1).
ERIC- and REP-PCR: We used Enterobacterial Re-
petitive Intergenetic Consensus (ERIC) and Repetitive
Extragenic Palindromic Sequence (REP) PCR fingerprint-
ing, and the analysis of internal transcribed spacer of
rDNA to identify genetic variability of R. areola. Use of
the ERIC/REP-PCR was originally reported for genomic
fingerprints of phytopathogenic bacteria [8]. In the recent
years, ERIC/REP-PCR is also being used do detect ge-
netic variability among fungal pathogens of several crops
[9,10]. The sequences of the primers are ERIC1R—
5’-ATGTAAGCTCCTGGGGTTCAC-3’; ERIC2—5’-
AAGTAAGTGACTGGGGTGAGCG-3’; REP1R-1—5’-
IIICGICGICATCIGGC-3’; REP2-1—5’-ICGICTTAT-
CIGGCCTAC-3’. PCR reactions were performed in a
volume of 25 µL containing 10 mM Tris-HCl (pH 8.3),
50 mM KCl, 2 mM MgCl2, 200 uM dNTP, 1.3 uL of 1%
bovine serum albumin, 50 pmol of each primer, 100 ng
of genomic DNA, and 1 U of Taq polymerase (Invitro-
gen). Amplification was performed in a Thermal Cycler
(MJ research, Inc. Watertown, MA, USA) according to
Louws et al. [9] and the PCR products (25 µL) were
submitted to electrophoresis in 2.0% agarose gels and
stained with ethidium bromide.
ITS rDNA: The isolates were also assessed by the in-
ternal transcribed spacer of rDNA (PCR-RFLP), using
the procedure as initially used [10]. The amplification pro-
ducts were digested using randomly selected eight re-
striction enzymes (Alu I, Bam H1, Bgl II, Dra 1, Eco R1,
Hae III, Hind III, and Hinf 1). The products of digestion
were separated through the gel electrophoresis in 2%
agarose. The reaction was analyzed in a total volume of
20 µL containing 1.5 µL of restriction enzyme. DNA di-
gestion was performed according to the instructions of
the supplier. All the amplifications and digestions were
repeated at least once to make sure the repeatability of
the reactions.
3. Results and Discussion
In glasshouse inoculations virulence spectrum showed
phenotypic variation among some isolates. Although the
resistance of genotypes CNPA BA-2003-2059 and FMT
02102996 as identified in earlier studies is governed by
two different genes [2,4], these genotypes showed a sus-
ceptible reaction to three isolates 13.2, 17.5 and 58.4
originated from three different Brazilian States (Ta ble 1 ).
With the exception of isolates 22.3 and 42.7, genotype
FMT 701 was susceptible to all the 16 isolates of R. are-
ola. These results confirm the earlier findings [3].
The ERIC/REP primers revealed polymorphism among
the isolates. The number of bands varied from 4 to 15 and
their sizes ranged from 150 to 1000 bp (Figures 1 and 2).
The dendrograms showed almost a unique profile for both
RIC and REP analysis for most isolates (Figures 3 and 4). E
Copyright © 2013 SciRes. AJPS
Identification of Phenotypic and Genotypic Variability among the Isolates of Ramularia areola of Brazilian Cotton
Copyright © 2013 SciRes. AJPS
1895
Table 1. Origin of 16 Ramularia areola isolates of Gossypium hirsutum and their virulence pattern on three cotton genotypes.
Cotton genotype and its reaction to R. areola isolates*
Isolate of R. areola Location and State of origin FMT 701 FMT 996 CNPA BA 2003-2059
12.8 Moreira Sales, Paraná S MR R
13.2 Sto. Ant. da Platina, Paraná S S S
17.5 Riolândia, São Paulo S S S
18.4 Riolândia, São Paulo S R R
19.4 Riolândia, São Paulo S R R
22.3 Unknown, Bahia R R R
25.1 Unknown, Bahia S R R
26.1 Unknown, Bahia S R R
40.6 Novo São Joaquim, Mato Grosso S R R
41.4 Primavera do Leste, Mato Grosso S R R
42.7 Campo Verde, Mato Grosso R R R
44.1 Ipameri, Goiás S R R
46.4 Ipameri, Goiás S R R
54.1 Primavera do Leste, Mato Grosso S R R
58.4 Chapada do Sul, Mato Grosso do Sul S S S
63.3 Mineiros, Goiás S R R
*R = Resistant. No symptoms of the disease; MR = Moderately Resistant. Small necrotic spots along with some chlorosis capable of sporulating in humid
chamber, covering < 1% leaf area infected (LAI); S = Susceptible. Typical angular spots, 3 - 4 mm, with or without chlorosis and without sporulation, covering
> 1% LAI 30 days after inoculation.
M1 M2 12.8 13.2 17.5 18.4 19.4 22.3 25.1 26.1 40.6 41.4 42.7 44.1 46.4 54.1 58.4 63.3 NC
Figure 1. Amplification products of the Ramularia areola isolates using ERIC primers. M1 = molecular maker 100-bp, M2 =
1KbDNA Ladder Thermo Scientific, NC = negative control.
Identification of Phenotypic and Genotypic Variability among the Isolates of Ramularia areola of Brazilian Cotton
1896
M1 M2 12.8 13.2 17.5 18.4 19.4 22.3 25.1 26.1 40.6 41.4 42.7 44.1 46.4 54.1 58.4 63.3 NC
Figure 2. Amplification products of the Ramularia areola isolates using REP primers. M1 = molecular maker 100-bp, M2 =
1KbDNA Ladder Thermo Scientific, NC = negative control.
18.4
17.5
19.4
13.2
12.8
63.3
46.4
58.4
41.4
44.1
42.7
40.6
25.1
22.3
54.1
26.1
30
40
50
60
70
80
90
100
Figure 3. Dendrogram produced by UPGMA cluster ana-
lyses based on the primer ERIC for Ramularia areola.
25.1
22.3
30
40
50
60
70
80
90
100
20
10
26.1
54.1
41.4
44.1
42.7
40.6
58.4
63.3
46.4
12.8
17.5
18.4
13.2
19.4
Figure 4. Dendrogram produced by UPGMA cluster ana-
lyses based on the primer REP for Ramularia areola.
Copyright © 2013 SciRes. AJPS
Identification of Phenotypic and Genotypic Variability among the Isolates of Ramularia areola of Brazilian Cotton 1897
The size of the amplified DNA was around 580 bp.
None of the restriction enzymes was informative. Only
the enzyme Hinf 1 was able to cut the DNA of all the pa-
thotypes in three parts but identical banding pattern was
observed for all the isolates (Figure 5). Lack of differen-
tiation between the isolates in this analysis perhaps indi-
cates that the minor genes for resistance are not involved.
While some isolates differed among each other con-
sidering genotypic and phenotypic reactions, no clear cut
evidence was found about the existence of genetic line-
ages of R. areola in Brazil. It is quite understandable that
genetically similar isolates may differ in their phenotypic
reactions and vice versa. There are some reports in the
literature about the use of molecular techniques to distin-
guish races or pathotypes of fungal and bacterial patho-
gens [9,11]. However, in general, in several other patho-
systems no relationship between phenotypic reaction and
genetic diversity was observed based on DNA sequences
[12].
Identification of existence of variability among the
isolates of the pathogen would orient the breeding pro-
cedures to create new cultivars with a broader spectrum
of resistance against this pathogen. Although we tested
only 16 isolates of R. areola, the results would serve as a
basis for future studies in this area. More detailed infor-
mation is required to comprehend the virulence fre-
quency and the existance of the genetic lineages if any,
using a wider range of cultivars and isolates originating
from different cotton growing areas of Brazil. It is possi-
ble that other molecular techniques may also show genetic
differences among the isolates and assist in identification
of distinct genetic lineages of R. areola in Brazil. This
100 bp 12.8 13.2 17.5 22.3 44.1 58.4 63.3
500bp
Figure 5. Sample gel showing amplification products of the
Ramularia areola isolates using restriction enzyme Hinf I.
100 bp = Molecular marker; 12.8 through 63.3 randomly
selected isolates of R. areola.
would assist screening cotton germplasm for resistance
against genetic lineages/pathotypes showing perhaps both
phenotypic and genotypic variability. Resistance sources
identified in this way would remain stable for a longer
period of time. Such studies would also assist in estab-
lishing differential set of cotton cultivars to identify field
strains of R. areola in Brazil. This is the first report on
the identification genotypic differentiation among the
Brazilian isolates of R. areola and its relationship with
the virulence spectrum.
4. Conclusion
Considering the UPGMA cluster analysis formed by
ERIC and REP-PCR, it may be concluded that the 16
isolates of R. areola fell into three major groups belong-
ing to broadly separated geographical regions. While there
existed genotypic and phenotypic variability among the
isolates, so far no clear indication was observed as re-
gards the existence of genetic lineages of R. areola in
Brazil. Results indicate the necessity of using different
isolates for screening the germplasm aimed at creating
new cultivars with broad spectrum resistance.
5. Acknowledgements
Genetic seed material of the cotton genotypes was pro-
vided by Camilo de Lelis Morello (Embrapa Algodão
Campina Grande, PB, Brazil) and Paulo H. Aguiar (Fun-
dação MT, Rondonópolis, MT, Brazil). The present re-
search was conducted under the financial support of IMA,
MT, Brazil. Thanks are also due to Bonnie Vieira and
Mariane Vieira for technical assistance.
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