Identification of Phenotypic and Genotypic Variability among the Isolates of <i>Ramularia areola</i> of Brazilian Cotton

doi:10.4236/ajps.2013.49232

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American Journal of Plant Sciences, 2013, 4, 1893-1898

http://dx.doi.org/10.4236/ajps.2013.49232 Published Online September 2013 (http://www.scirp.org/journal/ajps)

Identification of Phenotypic and Genotypic Variability

among the Isolates of Ramularia areola of Brazilian Cotton

Larissa Girotto1, Mariana S. Marangoni1, Janaina N. Matos1, Rafael Galbieri2, Wilson P. Almeida1,

Yeshwant R. Mehta1*

1Instituto Agronômico do Paraná—IAPAR, Londrina, Brazil; 2Instituto Mato-Grossense do Algodão—IMA, Primavera do Leste,

Brazil.

Email: *[email protected]

Received July 1st, 2013; revised August 1st, 2013; accepted August 20th, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Genotypic and phenotypic variation among 16 isolates of Ramularia areola of Gossypium hirsutum collected from five

different geographical regions of Brazil was studied through virulence spectrum on three cultivars in the glasshouse and

through ERIC- and REP-PCR and ITS1-5.8S-ITS2 rDNA analysis. Difference in virulence spectrum and molecular

analysis of some isolates was observed. ERIC- and REP-PCR showed similar results and revealed a high level of diver-

sity among the isolates. A unique profile for both ERIC and REP was obtained for most isolates. On the other hand, the

ITS rDNA analysis did not show different PCR-RFLP patterns. While some isolates differed among each other consid-

ering genotypic and phenotypic reactions, no clear cut evidence was found about the existence of genetic lineages of R.

areola in Brazil. Identification of genetic variability among the R. areola isolates originated from different geographic

regions would permit screening of Brazilian germplasm and achieve sources with a wide spectrum of resistance. This is

the first report of the genotypic and phenotypic variability among the R. areola isolates originated from five cotton

growing regions of Brazil.

Keywords: Ramularia; False Mildew; Genotypic and Phenotypic Differentiation; Gossypium hirsutum

1. Introduction

Leaf blight of cotton caused by Ramularia areola is eco-

nomically important especially for Brazil, causing heavy

yield losses [1-4]. In Brazil, some years ago it was con-

sidered as a secondary and late season disease, however,

in recent years the disease is of principal concern espe-

cially because now it attacks cotton during the entire crop

cycle. In the Cerrado region of Brazil the yield losses

caused by this disease are estimated to be around 30%

but in severe cases they can be up to 75% in the State of

Mato Grosso [1,2,4]. Yield losses of over 60% are also

recorded in Madagascar [5]. At present the disease is

partially controlled in Brazil by 5 - 8 applications of fun-

gicides thereby adding to the cost of cultivation and at

the same time making the intensive use of fungicides

almost unsustainable in the course of time. Resistance of

some genotypes was detected under field conditions but

their resistance was not found to be consistent presuma-

bly because of the genetic variability among the patho-

gen population [1-4].

Although the perfect state of the pathogen is not

known to occur in nature, existence of “field strains” is

believed to be present in Brazil. So far, cultivars resistant

to different “field strains” of R. areola are not yet avail-

able. It is obvious that the disease can be controlled

through the use of resistant cultivars but for this it is nec-

essary to identify the phenotypic and genotypic variabil-

ity among the R . areola isolates.

Differential reaction of some cotton cultivars to four

isolates of R. areola was studied in the United States as

early as in 1976 [6]. Recently, variability among the 23

isolates of R. areola of Brazilian cotton originated from

different geographical regions was studied by Pezenti et

al. [3], using three Gossypium hirsutum genotypes under

glasshouse conditions. These authors demonstrated that

the three genotypes were useful in distinguishing pheno-

typic variability among some isolates. Genetic differen-

tiation among the isolates of R. areola has not been re-

ported so far either from Brazil or from elsewhere in the

world.

*Corresponding author.

Identification of Phenotypic and Genotypic Variability among the Isolates of Ramularia areola of Brazilian Cotton

1894

The objective of the present investigation was to verify

phenotypic and genotypic variability among 16 R. areola

isolates collected from different geographical regions of

Brazil, through their virulence spectrum in the glass-

house and through ERIC- and REP-PCR and ITS1-5.8S-

ITS2 rDNA. The objective was also to comprehend rela-

tionship between their phenotypic and genotypic varia-

tion.

2. Material and Methods

Fungal Isolates: Out of the 23 isolates of R. areo la stud-

ied earlier for phenotypic variation [3], 16 isolates were

used in this study for phenotypic and genotypic variation

since other isolates had lost their pathogenicity due to

constant culturing in artificial media. These isolates are

considered representatives of the pathogen occurring

across the cotton growing areas of Brazil and their origin

is presented in Table 1. Fungal isolates were grown in

Petri plates containing V8 juice agar for three weeks, the

growth was scraped from the plates with distilled water

and a few drops of Tween 20 was added to the suspen-

sion. Conidial concentration was adjusted to 106 conidia

ml−1. Twenty five days old plants of three cotton geno-

types (FMT 701, FMT 02102996, CNPA BA 2003-2059)

grown in the glasshouse were inoculated individually.

Twelve plants of each cultivar were inoculated by a hand

sprayer till run off and were incubated in dark for 24 h at

approximately 80% humidity, and later were transferred

to the glass-house bench [3].

Disease Rating: Leaf of each plant showing the high-

est level of infection 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·mL−1).

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

Identification of Phenotypic and Genotypic Variability among the Isolates of Ramularia areola of Brazilian Cotton

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

100

Figure 3. Dendrogram produced by UPGMA cluster ana-

lyses based on the primer ERIC for Ramularia areola.

25.1

22.3

100

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.

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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