Genetic diversity of Phaeoisariopsis griseola in Argentina as revealed by pathogenic and molecular markers

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Physiological and Molecular Plant Pathology 68 (2006) 158–167 www.elsevier.com/locate/pmpp

Genetic diversity of Phaeoisariopsis griseola in Argentina as revealed by pathogenic and molecular markers Sebastia´n A. Stenglein1, Pedro A. Balatti,2 Instituto de Fisiologı´a Vegetal, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, CC327, La Plata 1900, Argentina Accepted 13 October 2006

Abstract Angular leaf spot, a disease of common bean produced by Phaeoisariopsis griseola, an imperfect (Deuteromycotina) fungus, causes significant yield losses in Argentina. The development of a strategy to control and/or reduce the impact of P. griseola requires a previous knowledge of the population structure. Therefore, the purpose of this work was to analyze diversity among 45 isolates of P. griseola collected within the production area of common bean in Northwestern Argentina. Pathotypes diversity was determined based on a set of bean differentials and genomic differences between isolates were determined by means of molecular markers. We confirmed that isolates belonging to Middle American and Andean groups coexist in Northwestern Argentina and the level of diversity between them was considerable and of similar level within each group. Even though the number of isolates analyzed was 45, among them 37 were Middle American and only eight were Andean. The number of haplotypes found based on ISSR and RAPD markers were 18 and as expected, they were unrelated with pathotypes. The wild bean species, Phaseolus vulgaris var. aborigineus, showed a high level of tolerance to most pathotypes of P. griseola except 63.63 and 63.23. This together with the fact that none of the bean differentials was resistant to all pathotypes led us to conclude that the range of pathogenic responses might be conditioned by multigenic interactions between the pathogen and the host. Our results not only provided basic information about the diversity of the causative agent of the disease but it will also help to develop cultivars with enhanced tolerance and/or resistance to the disease. r 2006 Elsevier Ltd. All rights reserved. Keywords: Genetic diversity; ISSR; Pathogenic variability; Phaeoisariopsis griseola; RAPD; Wild bean

1. Introduction Phaeoisariopsis griseola (Sacc.) Ferraris is the causative agent of angular leaf spot (ALS), a disease of common bean (Phaseolus vulgaris L.), that causes yield losses in many countries around the world [1–3]. P. griseola is an Abbreviations: ALS, angular leaf spot; AFLP, amplified fragments length polymorphism; AMOVA, analysis of molecular variance; CCC, cophenetic correlation coefficient; CTAB, cetyltrimethylammonium bromide; ISSR, inter simple sequence repeats; PVP, polyvinylpyrrolidone; RAPD, random amplified polymorphic DNA; SSR, simple sequence repeats; UPGMA, unweighted pair group method with arithmetic average Corresponding author. Tel./fax: +54 221 4233698. E-mail addresses: [email protected], [email protected] (P.A. Balatti). 1 Is a fellow of CONICET. 2 Is a member of CICBA. 0885-5765/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2006.10.001

imperfect (Deuteromycotina) fungus in the class Hyphomycete, order Moniliales, family Stilbaceae. Diseased plants are characterized by necrotic angular spots on the leaf surface, shriveling of pods and shrunken of seeds [1–3]. Based on the morphological and agronomical traits, on seed proteins and molecular markers, two pools of origin have been defined for common beans, namely Middle American and Andean. Whether symbiotic or pathogenic, microorganisms have been shown to coevolve with their hosts and it seems that this is the case with the isolates of Phaeoisariopsis griseola. Those isolates pathogenic on large seeded beans form a group called Andean and those isolates pathogenic on both small and large seeded beans are part of the Middle American group [4,5]. This clustering has been confirmed by DNA and isozyme marker analysis [6–9]. In Africa, some Andean isolates infect Andean and Middle American beans and have been

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classified as Afro-Andean [10]. Based on molecular markers these Afro-Andean isolates were indistinguishable from representatives of the Andean group [10,11]. Phaeoisariopsis griseola pathotype diversity and structure might be related with their compatibility with the genotypes of cultivated beans. Whereas most commercial cultivars are susceptible to ALS, cv. KGM4, 2A7 and Mexico 54 have been identified as valuable sources of resistance [12,13]. Several studies demonstrated that the level of variability among and within populations of P. griseola is considerable, even though the sexual form of the fungus has not been found [14]. Previous studies demonstrated variation in pathogenicity of fungal isolates, as 53, 13 and 50 different pathotypes have been described among 54, 30 and 112 isolates from Africa, Brazil and Central America, respectively [12,13,15]. Although ALS has been found in Argentina many years ago, the pathotype structure of the fungus is unknown and probably, as a consequence of this, no commercial cultivars either with tolerance or resistance to the disease has been developed. Argentina, a major producer of common beans, cultivates an area of approximately 280,000 ha mainly concentrated in three Northern provinces, Jujuy, Salta and Tucuma´n and exports 98% of the production of 320,000 t of Andean and Middle American beans. Recently, ALS has become one of the most important causes of yield losses of common bean and information on both pathogen and plant genotypes, as sources of tolerance/resistance, are lacking [14]. Studies upon the diversity of P. griseola whether based on pathogenicity and/or molecular markers might provide the basic knowledge for a rational hypothesis about the population structure and the mechanisms involved in the evolution and diversification of the causative agent of ALS [16,17]. The advent of PCR technology led to important achievements in genome analysis. Several molecular methods have been used to analyze diversity of plant pathogens at the genome level, such as RAPD, AFLP, SSRs and ISSR [8,11,18–22]. RAPD has been one of the most widely used methods [8,9,21], but another method used to analyze diversity within and among fungi is ISSR [19,20]. SSR are tandem repeat motifs composed of one to six nucleotides, which are ubiquitous, abundant and highly polymorphic in most eukaryotic genomes [24]. ISSR consists in the amplification of DNA sequences between SSR by means of anchored or non-anchored SSR homologous primers [23]. Unlike SSR, ISSR does not require a previous knowledge of the sequence and generates specific and reproducible patterns due to the high stringent conditions of the reaction [25]. The purpose of this work was to collect representatives of P. griseola within the production area of bean in Northwestern Argentina and analyze their diversity based on both the interaction of the isolates with a set of 12 bean differentials and at the genetic level, by means of two alternative molecular methods such as RAPD and ISSR.

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2. Materials and methods 2.1. Fungal isolation and inoculum production A total of 45 monosporic isolates of P. griseola were used in this study. Leaves with ALS symptoms were collected from plants growing in different locations within the main area of common bean production in Argentina, i.e., Tucuma´n, Salta and Jujuy (Table 1, Fig. 1). Isolates were collected from young bean leaves between flowering and fruiting. Sampled plants were representives of seven bean cultivars that are grown along the sampled area TUC390, TUC500, TUC510, Camilo INTA, NAG12, Alubia Cerrillos and Overito. In some cases we obtained one isolate from a leaf. However, occasionally we succeeded in obtaining two to six isolates from the same leaf, which allowed us to evaluate if one leaf can host two pathotypes and/or haplotypes at a time. P. griseola conidia from sporulating lesions were picked with the tip of a sterilized dissecting needle and were streaked onto potato dextrose agar plates and were incubated at 24 1C for 48–72 h. In order to obtain monosporic cultures of each P. griseola isolate, mycelium from individual germinated conidia were transferred to V8 juice agar or potato dextrose agar and cultured under continuous darkness at 24 1C. 2.2. Inoculation and virulence phenotype determination Greenhouse screenings were performed by inoculating a spore suspension, which was prepared by scraping the surface of 14-day-old P. griseola cultures supplemented with approximately 2 mL of distilled water. After filtering the conidial suspension, it was adjusted to a concentration of 2  104 conidia mL1 with a hematocytometer. Pathogenicity was assessed on a set of 6 Andean and 6 Middle American bean cultivars [5] (Table 2). Bean differentials were sprayed on the adaxial and abaxial surfaces of the first trifoliolate leaf with a conidial suspension, until run off. After inoculation the plants were kept for 48 h in a growth chamber under a 12 h photoperiod, 24 1C and 95–100% RH. Two susceptible cultivars, TUC 500 (Middle American gene pool) and Alubia Cerrillos (Andean gene pool), were included as positive controls within each inoculated series. To assess pathogenicity of Phaseolus vulgaris var. aborigineus (Burkart) Baudet, a wild relative of cultivated common bean, accession 6370 was also included within each inoculated series. Disease symptoms were evaluated 18 days after inoculation by means of a 1–9 symptom scale [26], described as follows: 1, plants with no symptoms; 3, plants with 5–10% of the area with non-sporulating lesions; 5, plants with 20% of the area with lesions and sporulation; 7, plants with up to 60% of the leaf area with lesions and sporulation, associated with chlorosis and necrotic tissues; 9, 90% of the leaf area with lesions, frequently associated with early loss

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Table 1 Identification, origin and pathotype nomenclature of Phaeoisariopsis griseola isolates used in this study Isolatea

Cultivar

Seed colour/Bean groupb

Collection date and site

Pathotypec

T1 T2 T3 T4 T5a T5b T5c T6a T6b T7a T7b T8a T8b T8c S1 S2a S2b S3a S3b S4a S4b S4c S5a S5b S5c S6a S6b S6c S6d S6e S6f S7a S7b S8 J1a J1b J1c J1d J2a J2b J2c J2d J3a J3b J3c Pg1 Pg15 Ecu3 Ecu6

TUC 390 TUC 500 TUC 390 Camilo INTA TUC 510 TUC 510 TUC 510 TUC 500 TUC 500 TUC 500 TUC 500 TUC 500 TUC 500 TUC 500 NAG 12 NAG 12 NAG 12 Overito Overito NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 NAG 12 Alubia Cerrillos Alubia Cerrillos Alubia Cerrillos Alubia Cerrillos Alubia Cerrillos Alubia Cerrillos Alubia Cerrillos Alubia Cerrillos NAG 12 NAG 12 NAG 12 DOR 500 DOR 500 PVA 773 COS 16

Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Cranberry/A Cranberry/A Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M Black/M White/A White/A White/A White/A White/A White/A White/A White/A Black/M Black/M Black/M Black/M Black/M A A

12/01, Trancas 12/01, Trancas 12/01, Vipos 12/01, Vipos 12/01, Trancas 12/01, Trancas 12/01, Trancas 05/02, Trancas 05/02, Trancas 05/03, Za´rate 05/03, Za´rate 05/03, Za´rate 05/03, Za´rate 05/03, Za´rate 06/02, R. Lerma 05/02, Meta´n 05/02, Meta´n 04/03, Cerrillos 04/03, Cerrillos 04/03, La Merced 04/03, La Merced 04/03, La Merced 04/03, Chicoana 04/03, Chicoana 04/03, Chicoana 04/03, Cerrillos 04/03, Cerrillos 04/03, Cerrillos 04/03, Cerrillos 04/03, Cerrillos 04/03, Cerrillos 04/03, R. Lerma 04/03, R. Lerma 04/03, La Silleta 04/03, El Quemado 04/03, El Quemado 04/03, El Quemado 04/03, El Quemado 04/03, El Quemado 04/03, El Quemado 04/03, El Quemado 04/03, El Quemado 04/03, Palpala´ 04/03, Palpala´ 04/03, Palpala´ Trancas Trancas Pinhanpiro Pinhanpiro

63.39 63.15 63.15 63.15 63.7 63.7 63.7 63.47 63.47 63.63 63.63 63.23 63.23 63.23 63.55 31.39 31.39 31.0 31.0 63.47 63.47 63.47 63.7 63.7 63.7 63.31 63.31 63.31 63.31 63.15 63.15 39.7 39.7 63.15 14.0 14.0 63.15 63.15 14.0 30.0 14.0 14.0 63.39 63.39 63.39 63.39 63.15 30.0 31.0

a The letters identify the province: T (Tucuma´n), S (Salta), J (Jujuy); the numbers identify an infected leaf from a genotype; and the small letters identify different spots on the same leaf. Pg1 and Pg15 are Argentinean isolates collected and pathotype characterized by CIAT. Ecu3 and Ecu6 are isolates from Ecuador collected and pathotype characterized by CIAT. b Bean group classified by host gene pool: A, Andean; M, Middle American. c Pathotype designation as defined in Table 2.

of the leaves and plant death. All the scale points correspond to a range of affected leaf area and it is important to consider that plants with scores higher than 3 were all considered susceptible (compatible). The set of differentials does not include fully resistant cultivars therefore any cultivar with scores lower than 3 were

considered tolerant (incompatible). Pathotypes were determined by means of a binary value as described by PastorCorrales et al. [5]. Briefly, in pathotype 63.7 (Table 2), the number before the dot is the result of summing the numbers given to the susceptible Andean cultivars a, b, c, d, e, f; 1+2+4+8+16+32 ¼ 63, while the number after the

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chloroform: isoamyl alcohol (24:1 v/v) was added, vortexed and centrifuged at 10 000 g for 5 min. The aqueous phase, containing the DNA, was transferred to a new tube and precipitated overnight by adding isopropanol. The DNA was pelleted by centrifugation at 10 000 g for 10 min, washed with 10 mM ammonium acetate-75% ethanol (10 000 g, 10 min) and then with 70% ethanol (10 000 g, 10 min). Then, the pellet was dried and dissolved in 80 mL of TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0). The DNA concentration was estimated by electrophoresing the DNA samples in a 0.7% (w/v) agarose gel and was compared with a molecular marker 1 kb DNA ladder of known concentration (Promega Biotech. Corp.).

dot is the result of adding the values of the susceptible Middle American cultivars g, h, i ¼ 7 [14]. 2.3. DNA extraction Two hundred and fifty milligrams of mycelia were frozen in liquid nitrogen and were ground in a mortar into a fine powder, which was mixed with 800 mL of CTAB extraction buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 0.2% (w/v) b mercaptoethanol)+200 mL CTAB 10%+1% (w/v) PVP. DNA was extracted by heating the slurry at 60 1C for 30 min. Then, one volume of 64°

68°

2.4. RAPD analysis

Jujuy

Twenty 10 bp oligonucleotide primers were evaluated for their ability to amplify polymorphic DNA fragments among P. griseola isolates. Seven RAPD primers (Table 3) (DNAgency, Malvern, Pennsylvania, USA), that yielded consistent polymorphic banding patterns were selected and used to generate fingerprints of all fungal isolates. Amplifications were carried out in 15 mL reaction mixtures containing 10–20 ng of genomic DNA, 10X reaction buffer (2 mM Tris-HCl pH 8.0, 10 mM KCl, 0.01 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% Tweens 20, 0.5% Nonidets P40), 7.5 mg BSA, 0.4 mM of primer, 200 mM of each dNTPs (Promega Biotech. Corporation), 1.2 mM MgCl2, 0.5 units of Taq DNA polymerase (Higway Molecular Biology-InBio-UNICEN-Tandil). Reaction mixtures were overlaid with 20 mL of mineral oil. DNA amplification was performed in a thermal cycler MJ Research (PTC-100) with an initial denaturing step at 92 1C for 5 min, followed by 45 cycles at 92 1C for 45 s, 34 1C

11

24°

10 Salta

9 6 4 7 8 5 31

161

2

Tucumán 28°

Fig. 1. Location of the places of collection of the isolates of Phaeoisariopsis griseola. 1 ¼ Trancas; 2 ¼ Vipos; 3 ¼ Zarate; 4 ¼ Rosario de Lerma; 5 ¼ Meta´n; 6 ¼ Cerrillos; 7 ¼ La Merced; 8 ¼ Chicoana; 9 ¼ La silleta; 10 ¼ El Quemado; 11 ¼ Palpala´.

Table 2 Response of the set of differential bean cultivars to inoculation with 45 isolates of Phaeoisariopsis griseola collected in Argentina Andean groupa

Middle American groupb

a 1

b 2

c 4

d 8

e 16

f 32

g 1

h 2

i 4

j 8

k 16

l 32

  + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + +  + + + + + + + +

 + + +  + + + + + + + +

    + + + + + + + + +

   + + + + + + + + + +

   + + + + + + + + + +

   + + + + + + + + + +

      +  +  +  +

       + +   + +

   +      + + + +

a,b

Pathotypec

Number of isolates

14.0 30.0 31.0 31.39 39.7 63.7 63.15 63.23 63.31 63.39 63.47 63.55 63.63

5 1 2 2 2 6 8 3 4 4 5 1 2

Andean groups included cultivars: (a) Don Timoteo; (b) G 11796; (c) Bolon Bayo; (d) Montcalm; (e) Amendoin; (f) G 5686. Middle American group included cultivars: (g) Pan 72; (h) G 2858; (i) Flor de Mayo; (j) Mexico 54; (k) BAT 332; (l) Cornell 49–242. c Pathotype designation is based on the sum (binary values) of bean cultivars with43 scale value. (+), Compatible reaction; (), Incompatible reaction [14]. All pathogenicity tests included three replicates per isolate.

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Table 3 Sequences and annealing temperatures of RAPD and ISSR primers used to fingerprint the genome of Phaeoisariopsis griseola Primer

Sequence (50 to 30 )

Annealing temperature (1C)

Reference

RAPD OPA 02 OPA 09 OPA 10 OPA 11 OPA 14 OPA 18 OPL 17

tgccgagctg gggtaacgcc gtgatcgcag caatcgccgt tctgtgctgg aggtgaccgt agcctgagcc

34 34 34 34 34 34 34

[9] [9] [9] [8] [8] [8] [12]

ISSR B C D E F G H

(ag)8 tg (ag)8 cg (ag)8 ctc (gt)8 ct (ga)8 gag (caa)5 (gcc)5

48 48 48 48 54 48 66

This This This This This This This

paper paper paper paper paper paper paper

for 60 s, and 72 1C for 90 s, a final extension cycle at 72 1C for 7 min was included. 2.5. ISSR analysis Among 10 ISSR primers, seven yielded polymorphic banding patterns and were selected to analyze diversity among P. griseola isolates (Table 3). Amplifications were performed in a 25 mL final volume containing 12–15 ng of genomic DNA, 10X reaction buffer (2 mM Tris-HCl pH 8.0, 10 mM KCl, 0.01 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% Tweens 20, 0.5% Nonidets P40), 0.7 mM of primer, 200 mM of each dNTP (Promega Biotech. Corporation), 2.5 mM MgCl2, 1.25 units of Taq DNA polymerase (Higway Molecular BiologyInBio-UNICEN-Tandil). Reaction mixtures were overlaid with 20 mL of mineral oil. DNA amplification was performed in a thermal cycler MJ Research (PTC-100) with an initial denaturing step at 94 1C for 7 min, followed by 33 cycles at 94 1C for 60 s, 48 1C for 75 s, and 72 1C for 4 min and a final extension cycle at 72 1C for 7 min. The annealing temperature was modified according to the sequence of the primer used (Table 3). Each reaction was performed at least twice. Products from RAPD and ISSR reactions were examined by electrophoresis in 1.5% (w/v) agarose gels containing 0.2 mg mL1 of ethidium bromide at 80 V in 5X Trisborate-EDTA buffer for 4 h at room temperature. Fragments were visualized under UV light. The size of the DNA fragments was estimated by comparing the DNA bands with a 1 kb DNA ladder (Promega Biotech. Corp.). Gel images were photographed with a Polaroid Camera DS34 and film type 667. 2.6. Analysis of genetic dissimilarity A data matrix was generated based on pathogenicity ratings by considering incompatible interactions (rated

lower than 3 in the symptom scale used to assess pathotypes) as the absence of virulence (0) and compatible interactions (all those rated higher than 3 with the same rating scale) as the presence of virulence (1) on each differential cultivar. The DNA amplification pattern of the 45 isolates were compared with those of four controls, two Andean (Ecu 3 and Ecu 6) and two Middle American (Pg1 and Pg15), which were supplied by CIAT. Bands amplified by RAPD or ISSR were scored as present (1) or absent (0) and fragments of the same size were considered homologous. Only clear and reproducible amplified fragments were scored and then, the data was assembled in a matrix. Genetic similarities between all the pairs of isolates were computed using: S ¼ Jaccard coefficient [27]. Dissimilarities were computed as genetic distance ¼ 1S, and the data were used to construct a dendrogram using the UPGMA. The CCC was chosen to indicate the level of distortion between the similarity matrix and cluster analysis. Because the RAPD and ISSR markers are correlated (r ¼ 0.85), using MXCOMP analysis in the NTSYSpc, data from the two markers were combined and subsequently analyzed as a single data set. Correlation between pathogenicity ratings and RAPD-ISSR lineages was determined using Mantel test. NTSYSpc version 2.0 was used to perform these analyses [28]. 2.7. Analysis of molecular variance and genetic diversity AMOVA [29] was used to calculate the variance among and within groups of P. griseola formed based on molecular markers. This procedure is based on an analysis of variance using distances between haplotypes. The distance chosen was a Euclidean metric equivalent to the number of differences between two individuals in their multilocus profile. Gene diversity (expected heterozygosis) [30], or the average probability that two randomly chosen alleles at a locus are different, was estimated for the entire population, as well as for the two subgroups identified. Expected heterozygosis (H) corresponds to the probability that two alleles taken at random from a population can be distinguished using the marker in question. A genetic diversity of 1 indicates high genetic diversity, where any two alleles at a locus sampled from a population are different, while a genetically uniform population will have a diversity of 0, because any two individuals will be identical. Arlequin 2000 was the software used to perform these analyses [31]. 3. Results 3.1. Diversity of pathotypes of Phaeoisariopsis griseola Pathotypes of P. griseola can be identified based on pathogenicity on a series of 12 bean differentials [5]. Examination of pathotype diversity among 45 isolates of P. griseola collected from Northern Argentina revealed the existence of isolates highly pathogenic on both Andean and

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Middle American bean differentials and because of this were considered as belonging to the Middle American group of the fungus. Other isolates were pathogenic only on Andean differentials and were considered members of the Andean group (Tables 1 and 2). Among 37 Middle American isolates 10 pathotypes were identified, which were highly pathogenic mostly on Middle American differential beans but also on all Andean differentials examined, except pathotypes 31.39 and 39.7, which had low disease ratings on Andean cultivars G5686 and Montcalm and Amendoin, respectively (Table 2). Eight other isolates were highly pathogenic only on Andean cultivars and therefore, were considered to belong to the Andean group, among them three pathotypes were identified (Tables 1 and 2). Some pathotypes occurred more frequently than others, however the importance of this observation is constrained by the haphazard collection. Pathotypes 63.15 and 63.7 were found in 8 and 6 leaf spots of leaves sampled in 3 and 2 different areas, respectively. Whereas pathotypes 14.0 and 63.31 were found in 5 and 4 leaf spots of leaves sampled within the same area, pathotype 63.55 was found in only 1 spot in a leaf collected from 1 place (Table 2). Different pathotypes coexisted in certain areas of production. P. griseola isolates collected at Trancas (Tucuma´n), such us T1, T2, T5 and T6, belonged to pathotypes 63.39, 63.15, 63.7 and 63.47, respectively. A similar situation was detected in Cerrillos and R de Lerma (Salta) (Table 1). Different spots on the same leaf can host pathotypes that may or may not belong to the same Middle American or Andean fungal group. Middle American pathotypes 63.31 and 63.15 and Andean pathotypes 14.0 and 30.0 were collected from different spots of a leaf, the former ones from cultivar NAG12 and the latter ones from cultivar Alubia Cerrillos. Also, Andean and Middle American isolates such as pathotype 63.15 and 14.0 were collected from distinct spots on the same leaf of Andean cultivar Alubia Cerrillos from El Quemado (Jujuy) (Table 1). Wild beans are potential sources of tolerance to ALS. Except for pathotypes 63.23 and 63.63, none of the isolates were highly pathogenic on P. vulgaris var. aborigineus. Thus, diseased leaves rating 3 or less were observed on leaves inoculated with all pathotypes except those inoculated with pathotypes 63.23 and 63.63, which according to the scale were rated 5. 3.2. Analysis of molecular markers Diversity among isolates of P. griseola was analyzed by means of RAPD and ISSR markers. All reactions were performed at least twice and only seven primers of each RAPD and ISSR markers yielded polymorphic banding patterns and were selected to analyze diversity of the fungus (Table 3). A total of 76 bands were amplified by the RAPD primers used (Fig. 2a) and among them 36 were polymorphic (47%). Fingerprints were used to build a

163

Fig. 2. Amplification patterns of eleven representative isolates of Phaeoisaripsis griseola. (a) RAPD primer OPA 10. (b) ISSR primer G. M ¼ molecular size marker.

dendrogram by cluster analysis of the dissimilarity matrix, which was created to analyze the genetic distances between isolates using the Jaccard coefficient (CCC ¼ 0.99). Cluster analysis of RAPD data defined nine haplotypes that clustered in two main groups, Andean and Middle American, showing 43% of genetic dissimilarity (data not shown). By means of ISSR primers, 93 DNA fragments were amplified, among them 63 (68%) were polymorphic. A representative amplification reaction performed with ISSR primer 50 gag(caa)530 is presented in Fig. 2b. The genetic distance between isolates was calculated based on the banding pattern generated by ISSR primers and a dendrogram was built based on Jaccard coefficient (CCC ¼ 0.97). Cluster analysis of ISSR data defined 17 haplotypes among P. griseola isolates, which were grouped in two main clusters, Andean and Middle American, with 56% of genetic dissimilarity (data not shown). The 50 anchored and the 30 -anchored primers were, based on the average of polymorphic bands amplified, more informative than non-anchored primers. The average of polymorphic markers generated by non-anchored, 50 -anchored and 30 anchored primers were 5, 11 and 10, respectively. RAPD

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and ISSR are both neutral markers that are distributed throughout the whole fungal genome and we found that they were correlated (r ¼ 0.85). Therefore, the data were combined and analyzed as a single set. Genetic distance between isolates was calculated and a dendrogram was built based on Jaccard coefficient (CCC ¼ 0.99). The isolates were grouped in two major groups Andean and Middle American (Fig. 3) with an average dissimilarity between groups of 0.50%. Although one of the clusters included 10 Andean representatives (eight isolates and two controls) and the other one, included the remaining 37 and two controls Middle American isolates, variability among each cluster was almost identical. Furthermore, within the Andean and the Middle American clusters isolates were mostly grouped according to their place of origin. The Andean group included subcluster 1 and 2 with 23% of genetic dissimilarity, subcluster 1 included the isolates from Salta, while subcluster 2 six Andean isolates from Jujuy. Regarding the Middle American cluster, it was divided in

subclusters 3 and 4 with 18% of genetic dissimilarity including, except for three isolates, fungi from Salta and Jujuy and isolates collected in Tucuma´n, S1, S2a and S2b, respectively. The combination of RAPD-ISSR polymorphisms defined 18 haplotypes. The AMOVA indicated that most of the variation resulted from genetic differences between Andean and Middle American groups (79.68%; Po0.001), rather than to differences within groups (20.32%). Analysis of genetic diversity using these molecular markers showed that P. griseola is a highly variable pathogen. The Middle American population had 25.28% of polymorphic loci, whereas the Andean population had 20.11%. Diversity of the entire population was estimated to be 0.933 (SD ¼ 0.018) showing that the majority (93.3%) of the genotypes present within the P. griseola population were different. Furthermore, genetic diversity of the Middle American and Andean groups were 0.908 (SD ¼ 0.025) and 0.857 (SD ¼ 0.108), respectively. S3a S3b J1a J1b J2a J2b J2d J2c Ecu3 Ecu6 S6a S6b S6c S6d S6e S6f S8 J3a J1d J3c J1c J3b S5a S5c S5b S7a S7b S4a S4b S4c Pg1 T6b S1 S2a S2b Pg15 T1 T2 T3 T6a T5c T5b T5a T4 T7a T7b T8a T8c T8b

1

Andean group 2

3

Middle American group

4

0.55

0.44

0.33

0.22

0.11

Genetic dissimilarity Fig. 3. Cluster analysis dendrogram of the 49 isolates of Phaeoisariopsis griseola using molecular data.

0.0

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In addition, pathotype diversity was unrelated with diversity at the DNA level (r ¼ 0.05) revealing the lack of congruency between RAPD-ISSR markers and pathogenicity. 4. Discussion Phaeoisariopsis griseola like other common bean pathogens coevolved with its host [8,9]. Therefore isolates of the fungus have been divided, based on pathogenic and molecular markers data, into Andean and Middle American, in correspondence with the two gene pools of origin of common bean [4–6,8,32]. Among 45 isolates from Argentina, 37 were highly pathogenic on small seeded Middle American and Andean beans and the remaining eight isolates were highly pathogenic only on Andean beans, indicating that 37 isolates belong to the Middle American group and eight to the Andean group, which was confirmed by RAPD and ISSR markers. These results were predictable considering that both Middle American and Andean beans are cultivated in Argentina [33]. The number of isolates belonging to the Middle American group was higher than that of the Andean group. Although it can be argued that the number of isolates is too small and also that these results might be due to an unevenly distributed survey, the isolates were collected from the most important bean producing areas cultivated with the most important commercial cultivars. Therefore, the organisms collected should be representative of both the area cultivated with Andean and Middle American cultivars and might also reflect the distribution of the isolates among the bean producing are in Argentina. Pathogenicity of the 45 isolates upon the series of bean differentials suggested the existence of pathogenic variability among P. griseola. Similar levels of variation have been reported for isolates from other countries using the same set of differentials, e.g., 50 pathotypes within 112 isolates in Central America, what means a different pathotype for every 2.24 isolates [15]. In this work, 13 pathotypes were detected among the isolates suggesting a relation of 1 pathotype every three isolates tested. In Africa higher levels of variability were found, such as 53 pathotypes among 54 isolates examined although this was achieved on a set of 29 bean differentials [12]. The size of the differential set increases the number of virulence patterns [34]. Therefore, variability in most places might be more complex than described and a better description might be achieved by means of a larger number of bean differentials. Genetic diversity within each group of the fungus in Argentina was considerable, such as reported in other places [15]. Genetic diversity at the DNA level among isolates of P. griseola based on RAPD and ISSR markers, defined 18 haplotypes while virulence defined only 13, which is not surprising considering that RAPD and ISSR are neutral markers unrelated with pathotype diversity. These results are consistent with other findings suggesting that isolates of the same pathotype are not necessarily

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closely related based on DNA analysis [35,36]. It is interesting to mention that RAPD and ISSR together showed higher levels of diversity than pathogenicity. However if diversity based on pathogenicity is a direct function of the number of differentials used, a larger set of them might reveal similar or higher levels of diversity than DNA markers. Although, 37 isolates belong to the Middle American group and only eight to the Andean, diversity based on molecular markers, was almost similar suggesting that these levels of diversity might be an intrinsic characteristic of the fungus. Pathotypes differ in their pattern of distribution and frequency in the three provinces surveyed. Furthermore, the pathotypes able to infect most bean differentials were not the most frequent ones. A similar situation was described in Brazil, where pathotype 63.63 was the most virulent pathotype but not the most prevalent one [22]. These findings suggest that the distribution and frequency of pathotypes is the result of a complex interaction between genotypes and the effect of the environment and management rather than virulence alone. At one location, Trancas, four different pathotypes were found, two other locations Cerrillos and El Quemado host three pathotypes, two other places Zarate and R de Lerma host two pathotypes and the rest six locations hosted only one pathotype. Our findings suggest both that probably the areas surveyed evolved rather isolated and that all these pathotypes might have been introduced in the fields by seeds or any other way. In addition to this the observation that more than one pathotype are present in most places suggest that unspecific tolerance to ALS in commercial cultivars is probably the best alternative to control the disease. In spite of the deficiency derived from the P. griseola isolation survey and the number of isolates evaluated, the results highlighted the existence of great diversity of pathotypes in Argentina, some of which overcome tolerance of most bean differential genotypes. Early studies of the P. griseola–common bean pathosystem suggested that it behaved as a gene for gene relationship [37]. However, the responses described for isolates of ALS on the different bean genotypes may simply represent a range of pathogenic responses conditioned by multigenic interactions between the pathogen and host [14]. Moreover, on the pathogen side, little is known on the mechanism(s) of pathogenicity and there has been no characterization or evidence for pathogen avirulence genes, only inferences based on pathogenicity results on different common bean cultivars. Among the 12 differentials none contained resistance genes that recognized all the P. griseola isolates from Argentina, indicating allelic diversity of virulence genes among P. griseola isolates from Northern Argentina. An interesting result was the characterization of pathotype 63.63, which has also been reported in Brazil [13,22] Honduras and Nicaragua [15]. This pathotype overcame tolerance in all bean differentials. The origin of the wide molecular and pathogenic diversity in P. griseola is unclear as the fungus has no

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sexual cycle [14]. However, mechanisms such as mutations, recombination, whether sexual or asexual and migration could be responsible of P. griseola diversity [10]. Evidences that high levels of haplotypic diversity can be maintained in asexually reproducing fungi through parasexual reproduction have been shown [38]. Chromosomal diversions, deletions and segment chromosome loss have the capability to increase diversity in fungi, contributing in this way to high haplotypic diversity [39]. Alternatively the responses of beans to the fungus might be a function of environmental variables and therefore, organisms that were found to be identical at the genetic level might be interacting with beans in a different manner. Both Andean and Middle American bean genotypes might have angular leaf spots in the same leaf caused by different pathotypes. Furthermore, a single leaf can host an Andean and a Middle American representative of P. griseola [40]. These findings argue against an explanation of the number of Andean and Middle American isolates due to the area cultivated with Andean and Middle American beans. Furthermore, coexistence of Andean and Middle American isolates might have importance in the development of pathotype diversity, if genetic barriers such as vegetative incompatibility are lacking and might also have epidemiological implications, since the presence of two isolates on the same leaf may result in parasexual recombination between organisms. Carefully planned surveys might help to elucidate the effect of co-infection on P. griseola diversity. Among 351 wild and weedy Phaseolus vulgaris accessions screened using mixtures of Andean and Middle American pathotypes of P. griseola under field conditions, only 16 were found to be resistant [41]. The Argentinean wild bean included in our studies had high levels of tolerance to the disease. Given the level of tolerance of Phaseolus vulgaris var. aborigineus to both Andean and Middle American pathotypes of P. griseola, wild beans appear to be a good source of genes and should probably be used in breeding programs aimed not only at developing tolerance and/or resistance to ALS and to broaden the genetic base of commercial beans, but also to develop resistance to other pathogens, since P. vulgaris var. aborigineus also showed high levels of tolerance/resistance to Colletrotrichum lindemuthianum [42]. This study revealed that P. griseola in Argentina displays high pathogenic and genomic diversity, which were as expected, unrelated since pathotypes are the result of the environment, plant and pathogens genome interaction. Frequency and distribution of Andean and Middle American isolates of the fungus seemed to be related to the size of the cultivated area with Andean and Middle American beans. However, an argument against this last statement is supported by two observations. First, the finding of plants coinfected with isolates belonging to each group of origin and second diversity within eight Andean and 37 Middle American isolates was similar.

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