02_JPP1053RP (Mariano)_237 - SIPaV

4 downloads 0 Views 5MB Size Report
ing from Australasia and division II from the Americas. Currently .... in water and lyophilized (Denny and Hayward, 2001). Three R. ... C, Vilber. Lourmat, France).
02_JPP1053RP (Mariano)_237

31-07-2013

14:25

Pagina 237

Journal of Plant Pathology (2013), 95 (2), 237-245

Edizioni ETS Pisa, 2013

237

CHARACTERIZATION OF RALSTONIA SOLANACEARUM CAUSING BACTERIAL WILT IN BELL PEPPER IN THE STATE OF PERNAMBUCO, BRAZIL A.L. Garcia1, W.G. Lima1, E.B. Souza2, S.J. Michereff1 and R.L.R. Mariano1 1Departamento

de Agronomia, Universidade Federal Rural de Pernambuco, 52171-900, Recife, Pernambuco, Brasil 2Departamento de Biologia, Universidade Federal Rural de Pernambuco, 52171-900, Recife, Pernambuco, Brasil

SUMMARY

Seventy-seven bacterial strains obtained from wilted bell pepper plants from the Agreste and Mata mesoregions of Pernambuco State (northeastern Brazil), were identified as Ralstonia solanacearum using multiplex PCR. The strains were further analyzed to characterize their biochemical, physiological, and molecular diversities. The biovars and biotypes were determined biochemically, and a molecular characterization of the strains was made using multiplex PCR, rep-PCR, and ISSR. The genotypic diversity was assessed, considering the number of genotypes observed and how they were distributed throughout the populations, for their differences in terms of richness, evenness and diversity. Strains of biovar 3, biotype 8 and phylotype I were predominant (97.40%), but biovar 1, biotypes 3 and 6 and phylotype II were also present. Rep-PCR analysis using REP and BOX primers, and ISSR showed similarities among the majority of the strains; however, these primers did not allow strain separation by biovars, biotypes, phylotypes or areas. Analysis of the genotypic diversity revealed a moderate diversity in the overall population, with a high variability in the strains from the same municipality.

INTRODUCTION

Ralstonia solanacearum (Smith) Yabuuchi et al. causes bacterial wilt in more than 450 species of host plants belonging to approximately 54 botanical families (Wicker et al., 2007). This bacterium is considered one of the most destructive plant pathogens in tropical, subtropical and temperate regions worldwide, with the exception of the Antarctic continent (Grover et al., 2006). In Brazil, bacterial wilt was initially reported in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) in 1922 in the state of Rio Grande do Sul (Takatsu and Lopes, 1997). Although bacterial wilt in bell peppers (Capsicum annuum) occurs in most Brazilian states, Corresponding author: R.L.R. Mariano Fax: +55.81.33206205 E-mail: [email protected]

it is most important in the main production areas of the northeast region and in the Rio de Janeiro and Espírito Santo States (Lopes and Quezado-Soares, 1997; Malavolta et al., 2008). This disease has been present in the state of Pernambuco (northeast region) for some time, though its importance has recently increased, probably due to the increased use of hybrid cultivars of bell peppers, particularly in the municipalities of Camocim de São Félix, São Joaquim do Monte, Sairé and Bonito. Symptoms in bell peppers begin with the wilting of the younger leaves, which progresses to the older leaves and ultimately leads to the death of the plant, without causing any change to its green coloration. However, the internal vascular system does darken (Lopes and Quezado-Soares, 1997; Momol et al., 2008). Due to its variability and adaptation to a wide range of hosts and environmental conditions, R. solanacearum has been classified at the infraspecific level into five races (He et al., 1983) and six biovars (Hayward, 1994). The introduction of molecular techniques has enabled the study of the phylogenetic and evolutionary relationships of the bacterium, in addition to its variability (Silveira et al., 2005). Using RFLP, Cook et al. (1991) and Gillings and Fahy (1994) were able to define 33 groups or genotypes of R. solanacearum, and their relationships indicated the existence of two genetically distinct divisions (I and II) that are strongly correlated with the geographical origins of the strains, with division I originating from Australasia and division II from the Americas. Currently, R. solanacearum is considered a complex species and is classified into phylotypes (I-IV) and sequevars (1-51) based on the sequence of the ITS region (16S-23S rRNA) and, hrpB and endoglucanase genes, in addition to the clonal lines and biotypes (Fegan and Prior, 2005; Xu et al., 2009). In the Agreste and Mata mesoregions of Pernambuco, the occurrence of bacterial wilt in bell peppers is of great relevance. Considering the absence of research on the variability of these R. solanacearum populations and the difficulty in controlling the disease, obtaining knowledge of the diversity of this pathogen is a priority. Therefore, this study aimed at assessing the diversity of R. solanacearum based on the determination of biochemical, physiological, and molecular characteristics.

02_JPP1053RP (Mariano)_237

238

31-07-2013

14:25

Pagina 238

Characterization of pepper strains of Ralstonia solanacearum

MATERIALS AND METHODS

Bacterial suspensions for all experiments were prepared using sterile distilled water and strains cultured on tetrazolium chloride (TZC) medium (Kelman, 1954) at 28ºC for 48 h, unless otherwise noted. Bacterial concentrations were determined based on absorbance and were adjusted to A570 = 0.54 (5x108 CFU/ml) using a photocolorimeter (Analyser 500M, Brazil). All experiments and tests described were performed in duplicate. Isolation, pathogenicity and biochemical and physiological characterization of the strains. Bell pepper plants with bacterial wilt symptoms were collected from the main production municipalities of the Agreste (Bez-

Journal of Plant Pathology (2013), 95 (2), 237-245

erros, Camocim de São Félix, Caruaru, Garanhuns and Sairé) and Mata (Chã Grande) mesoregions of Pernambuco from January to October of 2008. The number of collected samples varied among the areas, depending on disease intensity. The isolation of R. solanacearum was performed in triphenyl tetrazolium chloride agar medium (TTC), with an incubation at 28°C for 48 h. Virulent colonies, which are irregularly round, fluidal, white, and pink-centered (Kelman, 1954), were selected for pathogenicity tests. Bell peppers (cv. Atlantis) were grown in pots containing 500 g natural soil plus Basaplant substrate (Base Agro Indústria e Comércio, Brazil) (1:3, v:v) for 21 days. Seedlings were inoculated by making a semicircular wound with the help of a stylus in the soil next to a plant stem, and depositing an aliquot of a

Table 1. Host, geographical origin, race, biovar, biotype and phylotype of the strains of R. solanacearum used in this study.

1

Strain1

Host

B5-4, B5-5, B5-7, B5-8, B5-10, B5-17, B5-18, B5-21, B5-22, B5-24, B5-27, B5-28, B5-29, B5-30 B5-32

Bell pepper

C1-7, C3-11, C6-22, C6-23, C6-25, CM10

Bell pepper

CAR2, CAR4, CAR5, CAR6, CAR11, CAR12,CAR15, CAR18, CAR19, CAR20, CAR21,CAR22, CAR24, CAR28, CAR29

Geographical origin2 Bezerros, PE

Race

Biovar

Biotype

Phylotype

1

3

8

I

Camocim de S. Félix, PE

1

3

8

I

Bell pepper

Caruaru, PE

1

3

8

I

CGM2, CGM3, CGM6, CGM8, CGM9, CGM10,CGM13, CGM14, CGM15, CGM17, CGM18, CGH3, CGH6, CGH8, CGH12, CGH16, CGH20, CGH21,CGH26, CGH28, CGH31, CGH32, CGH34, CGH41, CG2-2, CG2-17, CG2-27

Bell pepper

Chã Grande, PE

1

3

8

I

CGM11

Bell pepper

Chã Grande, PE

1

1

6

II

CGH36

Bell pepper

Chã Grande, PE

1

1

3

II

GH3, GH8, GH10, GH12, GH16

Bell pepper

Garanhuns, PE 1

3

8

I

SCN4, SCN5, SCN21, SCN23, SCN27,SCN28, SBV31

Bell pepper

Sairé, PE

1

3

8

I

RS18

Tomato

Manaus, AM

1

3

8

I

FIO181C2

Eggplant

Parintins, AM

1

1

3

II

IBSBF292

Tomato

E.U.A

1

1

3

II

All strains were obtained in this work except for RS18 (Embrapa Hortaliças, Brasília), FIO181C2 (Instituto Nacional de Pesquisa da Amazônia, Manaus) and the type strain IBSBF292 (= ICMP 1727, Coleção de Culturas de Fitobactérias do Instituto Biológico, Campinas). 2 All strains are from Brazil, except for the type strain IBSBF292 (= ICMP 1727) which is from the United States.

02_JPP1053RP (Mariano)_237

31-07-2013

14:25

Pagina 239

Journal of Plant Pathology (2013), 95 (2), 237-245

15 ml suspension of the isolate (Felix et al., 2012). Only one pathogenic strain of each sample was selected for characterization. All pathogenic strains were preserved in water and lyophilized (Denny and Hayward, 2001). Three R. solanacearum strains were used as standards, FIO181C2 and RS18 race 1, biovars 1 and 3, from the Instituto Nacional de Pesquisa da Amazônia (INPA, Manaus, AM, Brazil) and from Embrapa Hortaliças (CNPH, Brasília, DF, Brazil), respectively, and the type strain IBSBF292 (= ICMP 1727) race 1, biovar 1 from the Coleção de Culturas de Fitobactérias do Instituto Biológico (IBSBF, Campinas, SP, Brazil) (Table 1). The strains of R. solanacearum under study were characterized using Gram staining, oxidase and catalase reactions, starch and gelatin hydrolyses, esterase activity (Tween hydrolysis), nitrate reductase activity, growth in medium containing 2% sodium chloride and a hypersensitive reaction in tobacco leaves. The biovar was determined according to a modified Hayward (1964) method. Briefly, freshly cultured R. solanacearum cells were stabbed into a soft agar tube of minimal medium containing 1% filter-sterilized lactose, maltose, cellobiose, mannitol, sorbitol and dulcitol and incubated at 28°C for 14 days. Positive cultures changed the culture medium from green to yellow. The biotype scheme generates a unique metabolic profile and may be used to predict the phylotype and sequevar to which the strains belong. The biotype was determined like the biovar using Ayers minimal medium containing maltose, mannitol, malonate, trehalose, inositol, and hippurate at 1% (Fegan and Prior, 2005). Molecular characterization. For the molecular characterization of the strains, total genomic DNA was initially extracted from cultures grown in NYDA (nutrient yeast dextrose agar) for 48-72 h (Silveira et al., 2005). The concentration of DNA was estimated by 0.8% agarose gel electrophoresis using a Low DNA Mass Ladder marker (Invitrogen, USA) and visualized with a photodocumentation system (DP-CF-011.C, Vilber Lourmat, France). The concentration was adjusted to 20 ng/µl and DNA was stored at 4°C until needed. Species and phylotype identification was achieved using multiplex PCR, i.e. the 759/760 primer pair (Bioneer Corporation, South Korea) for the species, and primers Nmult21:1F, Nmult21:2F, Nmult23:AF, Nmult22:InF, and the species-specific reverse primer Nmult22:RR for the phylotype (Fegan and Prior, 2005). The amplification reaction was performed using a thermocycler (PTC-100, MJ-Research, USA) and a program consisting of an initial denaturation at 96°C for 5 min, 30 cycles at 94°C for 15 sec, 59°C for 30 sec and 72°C for 30 sec and a final extension at 72°C for 10 min. PCR products were analyzed in 1.5% agarose gel, visualized and photodocumented after staining with SyBRGold (Invitrogen, USA). The sizes of the amplified fragments

Garcia et al.

239

were estimated by comparing them to the 100-bp DNA ladder molecular marker (Invitrogen, USA). Fingerprinting of the R. solanacearum strains was performed by amplifying the sequences based on their repetitive DNA (rep-PCR) and by the ISSR (Inter Simple Sequence Repeats) amplification technique. The rep-PCR of the R. solanacearum strains using the primers BOX-A1R, REP 1-I and REP2-I was subjected to the same protocol described by Louws et al. (1994). The amplification reaction in a final volume of 15 µl consisted of 1X Taq polymerase buffer (500 mM KCl, 100 mM Tris HCl), 1.5 mM MgCl2, 0.2 mM each dNTP, 2 µM primer and 1 U Taq polymerase. The reaction was performed using a thermocycler with the following protocol: an initial denaturation at 95°C for 8 min; 30 cycles at 94°C for 1 min, 52°C for 1 min (BOX-A1R) or 40ºC (REP2-I) and 65°C for 8 min; and a final extension at 65°C for 15 min. PCR products were analyzed using a 1.5% agarose gel, visualized, and photodocumented after staining with SyBRGold. The sizes of the amplified fragments were estimated by comparing them with the 100-bp DNA ladder molecular marker. Seventy strains were characterized by ISSR amplification using the primers GTG5 (5’GTGGTGGTGGTGGTG3’), GACA (5’GACAGACAGACAGACA3’) and 820 (5’GTGTGTGTGTGTGTGTC3’) (Invitrogen, USA) and the following protocol: an initial denaturation at 95ºC for 5 min; 30 cycles at 94ºC for 30 sec, 45ºC (820) or 52ºC (GTG5 and GACA) for 45 sec, and 72ºC for 2 min; and a final extension at 72ºC for 7 min. PCR products were analyzed in 1.5% agarose gel, visualized, and photodocumented after staining with SyBRGold. The sizes of the amplified fragments were estimated by comparison with a 100-bp DNA ladder. Genotypic diversity. The genotypic diversity of the R. solanacearum population from the Agreste and Mata mesoregions of Pernambuco and from each town sampled (Bezerros, Camocim de São Félix, Caruaru, Chã Grande, Garanhuns and Sairé) was studied using the band patterns obtained with the REP and BOX primers by rep-PCR. We used the results to evaluate the number of genotypes observed and how they were distributed in the population and varied in terms of richness, evenness and diversity (Grünwald et al., 2003). The Stoddart and Taylor (G = 1/Σpi2) and Hill (N1) genotypic diversity indices were calculated. The Hill index was calculated by the equation N1 = eH’, where H' refers to the Shannon-Wiener index, H' = {–Σi[pi × ln(pi)]}, and pi is the observed frequency of the ith genotype. N1 represents the number of equally common genotypes that produce the same diversity. The differences in the values of N1 and G among the populations were tested with bootstrapping, using 1,000 resamplings with a 95% confidence interval (Grünwald et al., 2003).

02_JPP1053RP (Mariano)_237

240

31-07-2013

14:25

Pagina 240

Characterization of pepper strains of Ralstonia solanacearum

The genotypic richness and evenness were also calculated. Richness is expressed by the number of genotypes expected in the sample and was evaluated using the rarefaction method (Grünwald et al., 2003). This method assumes that the number of expected genotypes in a random sample of n individuals out of a total sample of N individuals, where ni corresponds to the number of individuals per genotype is E(gn). The value of E(gn) is based on the sum of probabilities that each genotype is included in the sample. To contrast the richness of the populations of the different towns, N was estimated to be 5, which was the smallest sample size among the studied populations. The rarefaction estimates were obtained after compiling the algorithm (Grünwald et al., 2003). The evenness (E5) indicates how the genotype is distributed in a given sample and was calculated by the index of Ludwig and Reynolds (1988) using the formula: E5 = (G-1)/(N1-1). The evenness index is calculated based on the number of observed genotypes, the richness and the values of G and N1. Because the evenness increases in constant richness, the G and N1 indices also increase. The evenness index varies between 0 and 1, with a maximum at N1 = G = g = 1 (Grünwald et al., 2003). All indices were calculated using the R 2.10.1 software (R Development Core Team, Austria). Statistical analyses. The profiles obtained from the molecular characterization were analyzed by converting the restriction data (or fragments produced by repPCR) into binary data (1 = presence and 0 = absence of a fragment). Using binary data and the computer program NTSYSpc version 2.1 (Exeter Software, USA), pairwise comparisons of strains were performed to generate the Jaccard similarity coefficients, and a dendrogram was constructed with the unweighted pair-group method with arithmetic averages (UPGMA).

Journal of Plant Pathology (2013), 95 (2), 237-245

BF292 appeared to belong to biovar 1 (Table 1). Seventy-five strains were identified as biotype 8, like RS18. However, strain CGM11 was identified as biotype 6, and strain CGH36 as biotype 3, similarly to strains FIO181C2 and IBSBF292. Molecular characterization. All strains were amplified by the 759/760 primer pair and showed a band of approximately 280 bp, confirming the species as R. solanacearum. Of the 77 strains from Pernambuco, 75 were identified as phylotype I, having a band of approximately 144 bp (consistent with the phylotype) (Fig. 1). However, CGH36 and CGM11 from Chã Grande, were identified as phylotype II (372-bp), along with FIO181C2 and the type strain IBSBF292 (Table 1). The cluster analysis obtained by the combined data from primers REP and BOX allowed the division of the strains into four similarity groups, considering a coefficient of 75% (Fig. 2): (i) group I was composed of 73 strains, including CGM11 and FIO181C2; (ii) group II consisted of three strains: GH8, CGH36, and IBSBF292; (iii) group III comprised two strains from the town of Bezerros; (iv) group IV was composed of two strains from the town of Chã Grande. However, these primers did not allow the separation of the strains by the biovar, biotype, phylotype or sampling area. Results similar to the rep-PCR were obtained in the ISSR analyses using the 820, GTG5 and GACA primers alone: there was a high similarity and an inability to separate the strains by biovar, biotype, phylotype or sampling area. The 820 primer showed a greater similarity among the strains. The cluster analysis generated with the data from this primer showed the existence of three different groups at a 75% similarity level. The cluster analysis obtained by the combined data from primers 820, GTG5

RESULTS

Biochemical and physiological characterization. The 77 strains obtained from bell peppers in Pernambuco, in addition to the strains RS18, FIO181C2 and IBSBF292, behaved the same in the biochemical and physiological tests performed. All strains were Gram-negative and showed positive reactions for oxidase, catalase, esterase and nitrate reduction and all induced a hypersensitive reaction in tobacco leaves, with the exception of strain CGH34. The strains showed negative results for starch hydrolysis, did not grow in a medium containing 2% sodium chloride and hydrolyzed weakly gelatin. Of the 77 strains, 75 were identified as biovar 3, which is similar to RS18, whereas strains CGM11 and CGH36 from Chã Grande, and FIO181C2 and IBS-

Fig. 1. Multiplex PCR products for the identification of the species and phylotype of Ralstonia solanacearum using a 1.5% agarose gel. Lane M, 100 bp DNA ladder molecular marker; lane 1, type strain IBSBF292; lane 2, strain FIO182C2; lane 3, strain GH8; lane 4, strain CGH34; lane 5, strain CGH41; lane 6, strain CG2-27. Lanes 1 and 2, phylotype II; lanes 3 to 6, phylotype I.

02_JPP1053RP (Mariano)_237

31-07-2013

14:25

Pagina 241

Journal of Plant Pathology (2013), 95 (2), 237-245

Garcia et al.

241

Fig. 2. Dendrogram based on the UPGMA method according to the profiles generated by the combined REP and BOX-PCR analyses, showing the relationship between 80 strains of Ralstonia solanacearum: 77 obtained from different areas of bell pepper cultivation in the state of Pernambuco, Brazil, and the strains FIO181C2 from the Instituto Nacional de Pesquisa da Amazônia (Manaus, AM, Brazil), RS18 from Embrapa Hortaliças (Brasília, DF, Brazil) and the type strain IBSBF292 (= ICMP 1727) from the Coleção de Culturas de Fitobactérias do Instituto Biológico (Campinas, SP, Brazil).

and GACA allowed the separation of the R. solanacearum strains into five different groups at a 75% level of similarity (Fig. 3). Group I consisted of 28 strains, group III was composed of 39 strains, including CGH36 and FIO181C2, and groups II, IV and V consisted of one strain each: CGM15, CAR20 and CGM13, respectively. The molecular characterization data confirmed the similarities between most of the strains from the Agreste and Mata mesoregions of Pernambuco, as was also observed through the biochemical and physiological analyses. Genotypic diversity. There were 39 different genotypes (RS1 to RS39) in a total population of 77 R. solanacearum isolates from the two mesoregions of Pernambuco (Table 2). The genotypic diversity of the total population was 50.65% of the possible maximum in which each isolate represented one genotype. Twentyeight genotypes were only observed once in the total population, whereas the most frequent genotype was observed thirteen times. There were 11 genotypes with frequencies higher than 1. Among these, genotype RS1 was found in three towns (Bezerros, isolates B5-4, B5-8, B5-10, B5-29, and B5-30; Chã Grande, CG2-2, CG2-27, and CGH20; and Garanhuns, GH3), genotype RS6 was

found in four towns (Bezerros, isolates B5-21, B5-24, B5-27, and B5-28; Chã Grande, CGH3, CGH16, CGH26, CGH28, CGH32, and CGH41; Camocim de São Félix, C3-11 and C6-25; and Garanhuns, GH16) and genotype RS10 in four towns (Caruaru, isolate CAR6; Chã Grande, CG2-17; Garanhuns, GH10 and GH12; and Sairé, SCN21 and SCN28). The values for the Hill (N1) and Stoddart and Taylor (G) diversity indices, which form comparisons between 1 and the total of the evaluated sample, found moderate diversities of 33.22 and 20.65% (N1 = 25.58; G = 15.90), respectively, in the total populations of the two mesoregions (Table 2). However, there were high genotypic diversities within the towns of Sairé (n = 7; N1 = 4.71 or 67.29%; G = 4.45 or 63.57%), Camocim de São Félix (n = 6; N1 = 3.78 or 63%; G = 3.6 or 60%), Caruaru (n = 15; N1 = 10.98 or 73.2%; G = 9.78 or 65.2%) and Garanhuns (n = 5; N1 = 3.79 or 75.8%; G = 3.57 or 71.4%), with the latter showing the greatest diversity. Analyzing the evenness of the total population of R. solanacearum in the two mesoregions, it was found that the diversity (G = 15.90 and N1 = 25.58) and richness (g = 39.0) indices were dissimilar, such that the population showed little evenness, even though it had a moderate diversity (G/gobs = 0.408) (Table 2). In the evaluation by

02_JPP1053RP (Mariano)_237

242

31-07-2013

14:25

Pagina 242

Characterization of pepper strains of Ralstonia solanacearum

Journal of Plant Pathology (2013), 95 (2), 237-245

Fig. 3. Dendrogram based on the UPGMA method, with the profiles generated by the combined ISSR analysis, showing a relationship between the 70 representative strains of Ralstonia solanacearum obtained in different areas of bell pepper cultivation in Pernambuco State, Brazil, and the strains FIO181C2 from the Instituto Nacional de Pesquisa da Amazônia (Manaus, AM, Brazil) and RS18 from Embrapa Hortaliças (Brasília, DF, Brazil).

Table 2. Genotypic diversity of the strains of R. solanacearum from Pernambuco State, Brazil, calculated by the richness, evenness and diversity indices, with the band patterns obtained by rep-PCR using REP and BOX primers. Indices

Bezerrosf

Camocimf

Caruaruf

Chã Grandef

Garanhunsf

Sairéf

Total population

Sample n

15

6

15

29

5

7

77

6.06 (4.12-8.01) 4.79 (2.91-6.67)g

3.78 (2.55-5.01) 3.6 (2.42-4.78)

10.98 (8.47-13.49) 9.78 (7.27-12.29)

11.21 (8.67-13.75) 9.24 (6.69-11.80)

3.79 (2.49-5.10) 3.57 (2.30-4.84)

4.71 (3.30-6.12) 4.45 (3.05-5.85)

25.58 (21.0-27.93) 15.90 (11.18-20.61)

Richness gobsc E(gn)d

8 3.76

4 3.67

12 4.64

14 4.31

4 4

5 4.05

39 4.55

Evenness E5e G/gobs

0.75 0.60

0.94 0.9

0.88 0.82

0.81 0.66

0.68 0.89

0.93 0.89

0.606 0.408

Diversity N1a Gb

a

Hill’s diversity index (Grünwald et al., 2003) Stoddart and Taylor’s diversity index (Grünwald et al., 2003) c Number of observed genotypes d Expected number of genotypes estimated by the rarefaction method (Grünwald et al., 2003) e Ludwig and Reynold’s evenness index (Ludwig and Reynolds, 1988) f Areas of bell pepper cultivation in Pernambuco g Numbers in parenthesis indicates 95% confidence interval calculated by bootstrapping (1.000 resamples) using the accelerated bootstrap method (Grünwald et al., 2003). b

02_JPP1053RP (Mariano)_237

31-07-2013

14:25

Pagina 243

Journal of Plant Pathology (2013), 95 (2), 237-245

towns, it was observed that the evenness values were high within each town, between 0.82 and 0.90 (G/gobs), except for Bezerros and Chã Grande, which had evenness indices of G/gobs = 0.60 and 0.66, respectively. In the analysis of the confidence intervals of the diversity indices, it was found that the isolates from the towns of Bezerros, Camocim de São Félix, Garanhuns and Sairé did not differ because the intervals involving G overlapped, which did not occur with the Caruaru and Chã Grande isolates and the total population, indicating that they differed from the other towns.

DISCUSSION

This is the first study on the population diversity of R. solanacearum in Brazilian bell peppers. The biochemical and physiological, molecular and genotypic analyses provided important information with regard to the variability of the pathogen in the different towns that produce bell peppers in the Agreste and Mata mesoregions of Pernambuco. The strains obtained from the plants displaying wilting symptoms all showed the same biochemical and physiological profiles, corroborating the results observed for R. solanacearum isolates from potato (Nouri et al., 2009). The absence of a hypersensitive reaction (HR) in tobacco to the CGH34 isolate may be explained by the fact that, although positive HR occurs in incompatible pathogen-host associations, some strains do not follow this pattern (Horita and Tsuchiya, 2001; Alfenas et al., 2006) and should be tested in other hosts (Romeiro, 2001). A high prevalence of R. solanacearum biovar 3, biotype 8 was observed in the populations analyzed. Similarly, Silveira et al. (1998) found a 78% predominance of biovar 3 in the R. solanacearum strains of tomato from the town of Camocim de São Félix. The presence of biovars 1 and 3 in this town had been previously reported by Mariano et al. (1997) in tomato strains. Considering that the pathogen inhabits the soil and its dissemination does not occur easily within one growth cycle, it is understandable that there has been a predominance of biovar 3 in the area since 1997 (Mariano et al., 1997). The predominance of a biovar in a certain area depends on its initial inoculum source, dissemination method and the climate of the region. In this sense, the Brazilian distribution of R. solanacearum follows the report of Hayward (1991). Biovar 1 exists in all regions of the country, biovar 2 is predominant in regions with mild climates, such as the south, southeast, and westcentral areas, and biovar 3 is predominant in the north and northeast, which have warmer climates (Reifschneider and Takatsu, 1985). Biovar 3 is also apparently more versatile in terms of its adaptation to diverse environmental conditions and is less influenced by edaphic fac-

Garcia et al.

243

tors (Kumar et al., 2004). Furthermore a biovar-specific relationship was evident from the higher incidence of bacterial wilt associated with biovar 3 in peppers (Capsicum spp.) and biovar 1 in tomato plants, in the state of Amazonas (Coelho Netto et al., 2003, 2004). The strains of R. solanacearum classified as biovar 3 were identified as belonging to Asian phylotype I, and the isolates of biovar 1 were identified as American phylotype II, confirming the classification by Fegan and Prior (2005), and indicating the possibility of using these biochemical tests for the preliminary identification of phylotypes. Most of the strains analyzed in this study belonged to Asian phylotype I. This was unexpected because it corresponds to Division 1 (Cook et al., 1991), which contains strains primarily isolated in Asia. Thus, the question remains of how to explain the predominance of phylotype I in the R. solanacearum populations of bell pepper (this report) and tomato (Silveira et al., 1998) in Pernambuco, and also in peppers in the state of Amazonas (Coelho Netto et al., 2003, 2004). Furthermore, it is unclear when and how these strains were introduced into Brazil. It is known that significant Asian immigration has occurred and that Japanese farmers were established in many towns across Brazil, including Pernambuco and Amazonas. However, transmission by seed is not considered important in bell pepper, even though R. solanacearum has been reported to be seed-transmitted in tomato, eggplant and peanut (Kelman et al., 1994; Singh, 1995; Momol et al., 2008). Interestingly, Xu et al. (2009) were also unable to explain how several strains belonging to American phylotype II, Division 2 (mainly isolated in the Americas) were introduced into China. It is likely that further research will better elucidate the correlation between the phylotype/division and geographical origin. Asian phylotype I was detected in the Agreste and Mata mesoregions of Pernambuco, which are characterized by rainy tropical climates with dry summers. This finding agrees with that obtained by Toukam et al. (2009) who detected phylotype I in the hot and humid lowlands of Cameroon. In this study, the distribution of R. solanacearum strains from the same sampling areas in different similarity groups, as shown by rep-PCR and ISSR, demonstrated the high variability among strains of different areas. However, it was not possible to separate the strains by biovar, biotype or phylotype using the described primers. Studies of strains from the Brazilian Amazon region using the BOX primer separated the strains of tomato biovars 1 and 3 and biovar N2, showing the homogeneity of the latter. However, there was no correlation between the genomic profile, collection location and ecosystem of origin of the strains (Costa et al., 2007). In the Philippines, the characterization by ERIC-PCR of R. solanacearum from eggplant did not disclose a correlation between the genotypic profiles

02_JPP1053RP (Mariano)_237

244

31-07-2013

14:25

Pagina 244

Characterization of pepper strains of Ralstonia solanacearum

and biovar classifications (Ivey et al., 2007). There was also no association between the genotypic variability and aggressiveness of race 1 of tomato, as reported by Jaunet and Wang (1999), who concluded that a population analysis based only on neutral markers is insufficient to explain the diversity of R. solanacearum. The analyses of the genotypic diversity indices confirmed the variability of the strains of R. solanacearum within each area; however, when the total population was taken into account, the variability was moderate when compared to the total number of genotypes observed and the number of strains studied. This could be verified by analyzing the Stoddart and Taylor’s G index and the Shannon-Wiener’s H’ index, as these indices take into consideration the richness (number of genotypes in the population), evenness (how the genotype is distributed in the population) and total population number (Grünwald et al., 2003). The fact that the R. solanacearum populations studied had, in general, low to moderate diversities could be due to the pathogen dissemination from one location to another by the transportation of infected seedlings or infested soil. In the areas investigated, the rental or loan of agricultural machinery is very common among farmers, leading to dissemination by contaminated machines and soil. A low diversity of biovar 3 strains from ginger was reported by Kumar et al. (2004) and was correlated both with a minimal selective pressure due to the narrow genetic base of R. solanacearum and with the prevalence of pathogen transmission by the exchange of ginger rhizomes between locations, indicating that the population was clonally propagated and transmitted. The analysis of the R. solanacearum population obtained from bell pepper plants in the Agreste and Mata mesoregions of Pernambuco in northeastern Brazil suggests a low to moderate diversity, with a predominance of phylotype I, biovar 3 and biotype 8. This information can guide future research aimed at finding adequate management strategies for bacterial wilt in this region, particularly for the genetic improvement of the phylotype-specific resistance to the disease.

ACKNOWLEDGEMENTS

We thank CAPES for a scholarship and CNPq for the scholarship in Research Productivity awarded to the researchers E.B. Souza, S.J. Michereff and R.L.R. Mariano. In addition, we extend thanks to Cintia de Souza Bezerra for her help with the diversity analyses.

REFERENCES Alfenas A.C., Mafia R.G., Sartório R.C., Binoti D.H.B., Silva R.R., Lau D., Vanetti C.A., 2006. Ralstonia solanacearum

Journal of Plant Pathology (2013), 95 (2), 237-245

em viveiros clonais de eucalipto no Brasil. Fitopatologia Brasileira 31: 357-366. Coelho Netto R.A., Pereira B.G., Noda H., Boher B., 2003. Caracterização de isolados de Ralstonia solanacearum obtidos de tomateiros em várzea e em terra firme, no Estado do Amazonas. Fitopatologia Brasileira 28: 362-367. Coelho Netto R.A., Pereira B.G., Noda H., Boher B., 2004. Murcha bacteriana no estado do Amazonas, Brasil. Fitopatologia Brasileira 29: 21-27. Cook D., Barlow E., Sequeira L., 1991. DNA probes as tools for the study of host-pathogen evolution: the example of Pseudomonas solanacearum. In: Henneke H., Verma D.P.S. (eds). Advances in Molecular Genetics of Plant-interactions, pp. 103-108. Kluver Academic Publishers, Dordrecht, The Netherlands. Costa S.B., Ferreira M.A.S.V., Lopes C.A., 2007. Diversidade patogênica e molecular de Ralstonia solanacearum da região amazônica brasileira. Fitopatologia Brasileira 32: 285-294. Denny T.P., Hayward A.C., 2001. Ralstonia. In: Schaad N.W., Jones J.B., Chun W. (eds). Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed., pp. 151-174. APS Press, St. Paul, MN, USA. Fegan M., Prior P., 2005. How complex is the Ralstonia solanacearum species complex? In: Allen C., Prior P., Hayward A.C. (eds). Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex, pp.449-461. APS Press, St. Paul, MN, USA. Félix K.C.S, Souza E.B., Michereff S.J., Mariano R.L.R., 2012. Survival of Ralstonia solanacearum in infected tissues of Capsicum annuum and in soils of the state of Pernambuco, Brazil. Phytoparasitica 40: 53-62. Gillings M.R., Fahy P., 1994. Genomic fingerprinting: Towards a unified view of the Pseudomonas solanacerum species complex. In: Hayward A.C., Hartman G.L. (eds). Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas solanacearum, pp. 95-112. CAB International, Wallingford, UK. Grover A., Azmi W., Gadewar A.V., Pattanayak D., Naik P.S., Shekhawat G.S., Chakrabarti S.K., 2006. Genotypic diversity in a localized population of Ralstonia solanacearum as revealed by random amplified polymorphic DNA markers. Journal of Applied Microbiology 101: 798-806. Grünwald N.J., Goodwin S.B., Milgroom M.G., Fry W.E., 2003. Analysis of genotypic diversity data for populations of microorganisms. Phytopathology 93: 738-746. Hayward A.C., 1964. Characteristics of Pseudomonas solanacearum. Journal of Applied Bacteriology 27: 265-277. Hayward A.C., 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annual Review of Phytopathology 29: 65-87. Hayward A.C., 1994. Systematics and phylogeny of Pseudomonas solanacearum and related bacteria. In: Hayward A.C., Hartman G.L. (eds). Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas solanacearum, pp. 123-135. CAB International, Wallingford, UK. He L.Y., Sequeira L., Kelman A., 1983. Characteristics of strains of Pseudomonas solanacearum. Plant Disease 67: 1357-1361.

02_JPP1053RP (Mariano)_237

31-07-2013

14:25

Pagina 245

Journal of Plant Pathology (2013), 95 (2), 237-245 Horita M., Tsuchiya K., 2001. Genetic diversity of Japanese strains of Ralstonia solanacearum. Phytopathology 91: 399407. Ivey M.LL., Gardner B.B.M., Opina N., Miller S.A., 2007. Diversity and geographic distribution of Ralstonia solanacearum from eggplant in the Philippines. Phytopathology 97: 1467-1475. Jaunet T.X., Wang J.F., 1999. Variation in genotype and aggressiveness diversity of Ralstonia solanacearum race 1 isolated from tomato in Taiwan. Phytopathology 89: 32-327. Kelman A., 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44: 693-695. Kelman A., Hartman G.L., Hayward A.C., 1994. Introduction. In: Hayward A.C., Hartman G.L. (eds). Bacterial Wilt: the Disease and its Causative Agent, Pseudomonas solanacearum, pp. 1-7. CAB International, Wallingford, UK. Kumar A., Sarma Y.R., Anandaraj M., 2004. Evaluation of genetic diversity of Ralstonia solanacearum causing bacterial wilt of ginger using REP-PCR and PCR-RFLP. Current Science 87: 1555-1561. Lopes C.A., Quezado-Soares A.M., 1997. Doenças Bacterianas das Hortaliças - Diagnose e Controle. EMBRAPACNPH, Brasília, Brasil. Louws F.J., Fulbright D.W., Stephens C.T., Bruijn F.J., 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas cultivars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology 60: 2286-2295. Ludwig J.A., Reynolds J.F., 1988. Statistical Ecology: a Primer on Methods and Computing. John Wiley & Sons, New York, NY, USA. Malavolta Jr., V.A., Beriam L.O.S., Almeida I.M.G., Rodrigues Neto J., Robbs C.F., 2008. Bactérias fitopatogênicas no Brasil: uma atualização. Summa Phytopathologica, Special Supplement, 34: 1-88. Mariano R.L.R., Silveira E.B., Michereff S.J., 1997. Studies on tomato bacterial diseases in Pernambuco, Brazil. Proceedings 1th International Symposium on Tropical Tomato Diseases, Recife, Brazil: 156-159.

Received January 4, 2012 Accepted July 26, 2012

Garcia et al.

245

Momol T., Pradhanang P., Lopes C.A., 2008. Bacterial Wilt of Pepper. Retrieved Dec 10, 2010, from EDIS, University of Florida IFAS Extension. http://edis.ifas.ufl.edu. Nouri S., Bahar M., Fegan M., 2009. Diversity of Ralstonia solanacearum causing potato bacterial wilt in Iran and the first record of phylotype II/biovar 2T strains outside South America. Plant Pathology 58: 243-249. Reifschneider F.J.B., Takatsu A., 1985. Pseudomonas solanacearum. Aspectos macro-epidemiológicos. Fitopatologia Brasileira 10: 213. Romeiro R.S., 2001. Métodos em Bacteriologia de Plantas. Editora UFV, Viçosa, Brazil. Silveira E.B., Gomes A.M.A., Michereff S.J., Mariano R.L.R., 1998. Variability of Ralstonia solanacearum populations causing wilt on tomato in Agreste of Pernambuco, Brazil. Bacterial Wilt Newsletter 15: 8-10. Silveira J.R.P., Duarte V., Moraes M.G., Oliveira A.M.R., Barni V., Maciel J.L.N., 2005. Caracterização de estirpes de Ralstonia solanacearum isoladas de plantas de batata com murcha bacteriana, por PCR-Rep e RAPD. Fitopatologia Brasileira 30: 615-622. Singh R., 1995. Seed transmission studies with Pseudomonas solanacearum in tomato and eggplant. ACIAR Bacterial Wilt Newsletter 11: 12-13. Takatsu A., Lopes C.A., 1997. Murcha-bacteriana em hortaliças: avanços científicos e perspectivas de controle. Horticultura Brasileira 15: 170-177. Toukam G.M.S., Cellier G., Wicker E., Guilbaud C., Kahane R., Allen C., Prior P., 2009. Broad diversity of Ralstonia solanacearum strains in Cameroon. Plant Disease 93: 1121130. Wicker E., Grassart L., Coranson-Beaudu R., Mian D., Guilbaud C., Fegan M., Prior P., 2007. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential. Applied and Environmental Microbiology 73: 6790-6801. Xu J., Pan Z.C., Xu. J.S., Zhang Z., Zhang H., Zhang L.Q., He L.Y., Feng J., 2009. Genetic diversity of Ralstonia solanacearum strains from China. European Journal of Plant Pathology 125: 641-653.

02_JPP1053RP (Mariano)_237

31-07-2013

14:25

Pagina 246