Variability of cuban and international populations of <emphasis type="italic">alternaria solani</emphasis> from different hosts and localities: aflp genetic analysis

European Journal of Plant Pathology 110: 399–409, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.
Variability of Cuban and international populations of Alternaria solani
from different hosts and localities: AFLP genetic analysis
Sim´on P´erez Mart´ınez1, Rod Snowdon2 and J¨orn Pons-K¨uhnemann2,∗1Dpto, Fitopatolog´ıa-Centro Nacional de Sanidad Agropecuaria (CENSA), Carretera de Tapaste yAutopista Nacional, San J. de las Lajas, Habana, Cuba (E-mail: sperez@censa.edu.cu);2Institut f¨ur Pflanzenbau und Pflanzenz¨uchtung, Justus-Liebig-Universit¨at, Heinrich-Buff-Ring 26-32,D-35392 Giessen, Germany; ∗Author for correspondence (Phone: +49 641 37542; Fax: +49 641 37549;E-mail: joern.pons@agrar.uni-giessen.de) Key words: Alternaria solani, genetic variability, AFLP analysis, host specificity Abstract
As causal agent of early blight disease in tomato and potato, Alternaria solani is an internationally importanthorticultural pathogen. Genetic variability was surveyed by amplified fragment length polymorphism analysis ina total of 112 isolates from potato and tomato, representing pathogen populations from different Cuban provincestogether with isolates from the USA, Brazil, Turkey, Greece and China. Also included in the analysis were isolatesfrom catenulated Alternaria spp. from Brazil, Canada, Greece and Russia, along with single isolates of Alternariaporri, Alternaria alternata and a Curvularia sp. UPGMA clustering revealed a differentiation between the isolatesof A. solani and all other species with the exception of A. porri which could not be distinguished from A. solani.
Among the isolates of A. solani, two distinct subclusters were formed, with high genetic significance revealed bybootstrapping, corresponding to a general subdivision based on the respective solanaceous host. The results arediscussed with regard to potential host specificity of A. solani on tomato and potato, and in terms of the comparativecontributions of regional and international genetic variability in populations of this ubiquitous plant pathogen.
Introduction
Several studies have shown that A. solani isolates differ in morphology, physiology, pathogenicity, Early blight disease caused by Alternaria solani is one genetic makeup and cultural properties. Indeed, iso- of the most important fungal diseases of tomato and lates can vary so much in their cultural characteristics potato. This pathogen is also the causal agent of col- that it is possible to find almost as many morpho- lar rot in tomato. A. solani is a well-known pathogen types as the number of isolates tested (Rotem, 1966).
of the genus Alternaria and is considered a good Isolates also vary in the production of phytotoxins.
example for the world-wide distribution of a species However, no correlation was found between the vir- (Rotem, 1994). The fungus was described for the first ulence of specific strains and their ability to produce time in potato late in the 19th century (Ellis and Martin, toxins (Stancheva, 1989). In some cases, strains 1882) and attacks on potato and tomato during the from leaf and potato tubers differed in their ability first three decades of the last century, on practically all to attack potato leaves and tubers, but no correla- the continents, were described by Neergaard (1945).
tion was observed between potato isolates and their The first report of A. solani in Cuba was in 1918, pathogenicity on leaves and tubers (Bonde, 1929; at which point early blight in tomato was among the most important crop diseases in Havana (Bruner, Genetic analysis with isozymes (Petrunak and Christ, 1992), RAPD (Sharma and Tewari, 1998; Weir et al., 1998; Roberts et al., 2000) and RFLP Materials and methods
markers (Adachi et al., 1996; Aradhya et al., 2001) hasshown great variability between and within Alternaria species. A significant genetic distance was observedbetween isolates of A. solani from tomato and potato Isolates of A. solani were collected from typical symp- (Weir et al., 1998), suggesting the possibility of toms of tomato and potato early blight plants in dif- a pathogenic specialization on solanaceous hosts.
ferent provinces in Cuba (Table 1). Additionally, inter- Generally, at the species level, the genetic variability national isolates were provided by other researchers: corresponds to variation in morphological characteri- Ten isolates originated from Turkey, eight from Brazil, stics (Roberts et al., 2000; Sharma and Tewari, 1998) in four from Greece, eight from the USA and one from Alternaria populations of fruits and crucifers, respec- China. One isolate of Alternaria alternata, one of tively. On the other hand, correlation of genetic markers Alternaria porri, seven of catenulated Alternaria spp.
with host, geographic origin or resistance to fungi- and one of Curvularia sp. were included in the analysis.
cides was not always evident (Adachi et al., 1996; Origins and hosts of the isolates are listed in Table 1.
Morris et al., 2000; Aradhya et al., 2001). A high levelof genetic differentiation by host and/or pathogeni- Table 1. Hosts and origins of fungal isolates used in AFLP city, consistent with a hypothesis of specialization, has been observed in Alternaria spp. from Citrus(Peever et al., 1999). In Cuba, the study of the vari- ability of the causal agent of early blight has been limited to tomato, for which 12 races were found in Havana province (Izquierdo, 1981). More recently, we observed pathogenic (Mart´ınez et al., 2002) and genetic diversity in some Cuban isolates using RAPD analysis Amplified fragment length polymorphisms (AFLP; Vos et al., 1995) represent a powerful, highly repro- ducible, PCR-based DNA-fingerprinting technique for DNA of any origin or complexity. Because a large number of polymorphic loci can be investigated in a single experiment, the AFLP technique has become one of the major methods of choice for studies of genetic diversity, particularly in species where mark- ers requiring genomic sequence information are not available. The robustness of the AFLP procedure on fungal genomic DNA was corroborated by Pei and Ruiz (2000). The highly polymorphic nature of AFLP markers make them especially useful for differentiat- ing clonal lineages of fungi that reproduce asexually (McDonald, 1997). AFLP markers have been used to study genetic diversity in 18 isolates of Alternaria brassicicola (Bock et al., 2002). However, a large- scale international survey of genetic variability in Alternaria has not been published. The objectives of the present study were to use AFLP analysis to examine genetic differentiation within and between populations from diverse tomato and potato growing regions in Cuba and other countries, and to determine whether the influence of the host could be observed at the Curvularia Curvularia∗∗ Tomato 1999 Letter codes of the Cuban isolates are explained in the key to Figure 2. Total numbers of isolates are given in brackets. A double asterisk indicates monosporic isolates, whereas single aster- isks show isolates that were not monosporic but where spores were observed during the experiments. No spore production wasobserved in isolates without asterisks.
Isolation and identity of fungal cultures standard electrophoretic conditions suggested by themanufacturer.
Surface-sterilized sections from the leading edges of A 50–350 bp DNA length standard (LI-COR. Inc., lesions were placed in water agar. Small tufts of Nebraska, USA) was used as molecular weight marker mycelia that emerged around lesion borders were trans- for allele size calling. DNA from the isolate C-74 was ferred to potato dextrose agar and finally multiple- included as a reference marker in all gels, and all frag- conidial isolates were purified. Single-conidial isolates ments were scored against the two standards to ensure were dissected directly from seeding spores from the accurate designation of bands to their respective loci infected leaf tissue, under a stereo-microscope. Cri- over all genotypes. Isolate genotypes were screened teria for morphological identification of Alternaria for presence or absence (1–0) of all AFLP frag- isolates were based on Ellis (1971). Alternaria spp.
ments between 50 and 350 bp using the electrophoresis isolates from international sources were received as analysis software RFLPscan v2.1 (Scananalitics-CSPI, purified cultures. All isolates were stored in the culture Billerica, MA, USA). Lanes and bands were tracked manually and a binary data matrix was generateddescribing the presence or absence of bands at allscored loci for the three primer pairs.
Growth of mycelium and DNA extraction Mycelia were produced in 250 ml Erlenmeyer flasksfilled with 40 ml of potato dextrose broth. Flasks were Similarity matrices (DICE, JACCARD and SIMPLE) inoculated with tufts of mycelium and incubated in the produced using the WINDIST software (I.W. Yap, dark at 27 ±0.5 ◦C for 4 days in still culture. Mycelium University of Washington, USA) were used to clus- was harvested, vacuum-filtered and freezed at −80 ◦C ter the data with three algorithms (Single linkage, until use. DNA from each isolate was extracted from UPGMA and Complete linkage) using the SAHN mod- 0.3–0.6 g of freeze-dried mycelium based on the extrac- ule of NTSYSpc (v2.01, Exeter Software, Setauket, tion method of Sambrook et al. (1989). The DNA NY, USA). Cophenetic values were calculated using the samples were re-extracted once to reduce discoloura- MXCOMP module. Dendrograms were constructed tion and RNA was degraded by addition of 1 µl RNAse with the SAHN module using the similarity matrices (10 mg ml−1). After quantification using a fluorometer, from the three primer combinations considering all samples were diluted to 50 ng µl−1 in TE buffer and 542 polymorphic fragments, and alternatively with a reduced data set of 317 fragments in which loci forwhich all isolates were identical were pooled.
Due to the small genome of A. solani, a modified AFLPtechnique was applied to ensure amplification of suffi- Population analysis was performed with the soft- cient polymorphic bands. Genomic DNA from each ware AMOVA (v1.5, Excoffier, University of Geneva, isolate was restricted with EcoRI and MseI and ligated Switzerland) using the reduced data set of 317 frag- to PCR adapters using the AFLP kit of Gibco, BRL ments. AMOVA input files were prepared using (Gaithersburg, MD, USA). A two-step PCR procedure AMOVA-PREP v1.01 (Mark P. Miller, University of was adopted for selective amplification. In the first Northern Arizona, USA). Pair-wise comparisons of the step (pre-amplification), primers with a single selective variation between populations were analysed by calcu- nucleotide extension (A) were used for both the MseI st values, which represent the proportion of the and the EcoRI fragment ends. In the second ampli- total variance that is partitioned between populations fication, MseI and IRD800-labelled EcoRI primers and measure the genetic differentiation of subpop- with one and two selective nucleotides, respectively, were used in combination (M-A and E-AT/-AC/-AG).
by non-parametric permutation procedures (Excoffier AFLP products were separated in polyacrylamide gels et al., 1992). The robustness of the phenogram was with 1 × TBE buffer on a LI-COR 4200 fluorescent determined by bootstrap analysis of 1000 bootstrapped DNA analyser (LI-COR Inc., Nebraska, USA) using samples using WINBOOT (Yap and Nelson, 1996).
A 70% bootstrapping frequency was used as the lowerlimit for recognition of robust clusters.
An example of typical AFLP variation is shown inFigure 1. A total of 542 fragments were observed in allisolates using the three selected primer combinations(Table 2). All primer combinations showed 100% poly-morphism for the complete set of isolates, whereas thedegree of polymorphism ranged from 89.2% to 98.6%in different isolate subsets. The primer combinationE-AT detected more fragments for all subsets of iso-lates, but was less polymorphic than E-AC and E-AGin the isolate subsets.
Dendrograms constructed using three similarity co-efficients (DICE, JACCARD and SIMPLE) and threeclustering methods (UPGMA, Single linkage and Com-plete linkage) were examined and compared to evaluatethe goodness of fit of the resulting phylogenetic treeswith respect to the reliability and stability of theinferred relationships (Table 3). High cophenetic values(ranging from 0.94 to 0.99) were found with all com-binations, both with all 542 fragments and also withthe reduced data set of 317 independent loci. In generalr > 0.9 indicates a very good fit, indicating no majorvariation among the dendrogram patterns or the threedistance calculation methods.
To assess the usefulness of AFLPs as phenetic markers, a similarity matrix based on the SIMPLE co-efficient and UPGMA cluster method was constructedto estimate the level of relatedness among the A. solani,A. porri, Alternaria spp. and Curvularia sp. isolates.
The resulting dendrogram (Figure 2) formed two mainclusters with extremely high bootstrap values that Figure 1. Example of AFLP fingerprints in 14 isolates of clearly distinguished the species, with the exception Alternaria solani using the primer combination M-A/E-AT with of the single A. porri isolate which cosegregated with nucleotide (A) and two selective EcoRI nucleotides (AT), respectively. Details of isolates are given in the A. solani isolates. All A. solani isolates grouped together in the first cluster (Cluster 1), whereas theother cluster grouped all Alternaria spp. together withthe A. alternata and Curvularia sp. isolates (Cluster 2).
being assigned to subcluster 1.1 and the majority of Within Cluster 1, two distinct branches were con- the potato isolates grouping in subcluster 1.2, with a sistently formed in 100% of the 1000 bootstrapped similarity coefficient of around 0.76 between the two trees. These subclusters revealed a general cluster- host-associated clusters. Subcluster 1.1 represents a ing by host, with most A. solani isolates from tomato large branch containing 87 genetically similar isolates Table 2. AFLP fragments amplified in isolates of A. solani and related species with different EcoRI primerselective nucleotides. In all cases the MseI primer included one selective nucleotide (A) a Total number of fragments observed.
b Percentage of total fragments in a given species that were polymorphic.
Table 3. Comparison of cophenetic values obtained from three similarity coefficients andthree clustering used for analysing the AFLP data a Cophenetic values considering all 542 detected fragments.
b Cophenetic values considering only the 317 unequal fragments.
(similarity coefficient >0.86), including 74 isolates hand, not all isolates from Brazil clustered together, from tomato and 12 from potato. Statistical support although they all came from the Lavras region. Three for minor subgroups containing isolates of different Turkish isolates (T-4, T-5 and T-6) from tomato formed geographic origin was poor (bootstrap values <60%), a separate subgroup in subcluster 1.2 with a higher however the A. solani isolates from more exotic sources genetic distance to the other isolates, albeit with a (China, Greece, Turkey) generally tended to group lower statistical support of only 63%.
separately from the Cuban and Brazilian A. solani Cluster 2 is comprised of nine isolates from tomato, isolates, with some exceptions. The Cuban isolate from different substrates and with different geograph- C-240 appears to be genetically distinct from the rest ical origins. These included the airborne isolate of of subcluster 1.1, as it grouped separately from all A. alternata, six isolates of Alternaria spp. with small other isolates with 93% confidence. The isolates form- catenulated spores and the Curvularia sp. isolate. The ing subcluster 1.1 remained almost unchanged when dendrogram revealed great genetic dissimilarity within the phenogram was generated using other similarity this cluster, with high statistical support for the sub- indices. The A. porri isolate from onion also clustered groups. The three isolates from Russia were almost identical, as were the two isolates from Canada. The The other major branch within Cluster 1 (subclus- Curvularia and A. alternata isolates were genetically ter 1.2) comprises 26 A. solani isolates, including 21 from potato and 5 from tomato. Of all 34 isolatesoriginating from potato, 65% grouped within thissubcluster (Figure 2). Isolates from different origins within the USA, Greece and Turkey clustered sepa-rately to isolates originating from Cuba and Brazil, AMOVA was used to estimate and partition the total with a similarity coefficient of 0.80. All isolates from genetic variance into within- and between-subgroup the USA clustered together in a single subgroup within subcluster 1.2 at confidence level of 100%, although were made separately between different populations, they originated from different states. On the other considering the species, hosts and countries of origin.
Figure 2. Phenogram showing genetic similarity among 112 isolates of Alternaria solani, seven isolates of catenulated Alternaria spp.
and one isolate each of A. porri, A. alternata and Curvularia sp., revealed by UPGMA cluster analysis based on AFLP genetic fingerprintsobtained with three primer combinations. Numbers shown above branches represent percentage confidence limits obtained by bootstrapanalysis with 1000 bootstrap repetitions. Confidence limits below 70% are not shown. The isolates are separated into two highly significantclusters with a similarity coefficient of around 0.60, and the larger cluster is further divided into two subclusters with a similarity coefficientof less than 0.80.
Of the 112 A. solani isolates, 105 formed different hap- were found between the population representing USA lotypes with the three primer combinations tested, indi- isolates and seven of the other eight populations anal- cating a great genetic diversity. Considering A. solani ysed. The Cuban populations from tomato and potato (together with the A. porri isolate), all catenulated showed a moderate but significant genetic difference; Alternaria isolates and Curvularia sp. as separated the same was observed between Turkish isolates, populations, most of the total variability resided among whereas between the isolates from Greece the differ- ences were quite large ( st = 0.9591, P < 0.0001).
54.15% of the total variability) and the rest within pop- A pair-wise comparison between the domestic Cuban ulations. After re-configuring the data to represent six and foreign tomato and potato isolates, respectively, different populations consisting of A. solani isolates revealed highly significant (P < 0.0001) genetic dif- from Cuba, Brazil, USA, Greece, Turkey and China, ferences between the Cuban isolate populations and the 46.35% of the total genetic variability was found to reside between countries ( st = 0.4635). Considering Genetic variability among the Cuban provincial A. solani isolates from tomato and potato as two differ- populations ( st = 0.1289) was considerably lower ent populations, the genetic distance among them was than among populations from different countries 0.330 with high statistical significance (P < 0.0001).
( st = 0.4635). This was consistent with the low Table 4 shows genetic differences explained by geographical origins of populations from tomato Table 5. Genetic distances among the Cuban and foreign isolates and potato, respectively. The greatest genetic dis- of A. solani from tomato and potato, respectively, calculated using tances among populations from tomato were observed between the isolates from Greece and China ( st =0.6923, P < 0.0001), and between the populations from Cuba and those from Greece and Turkey ( st = st = 0.4560, respectively; P < 0.0001).
Among potato populations, the greatest genetic dis- tances were found between the USA population and those from Cuba and Brazil. All genetic distances within this group showed highly significant differ- ences, with the exception of the populations from Numbers below the diagonal are measures of inter-population pair-wise populations comparison between different genetic distance ( st). Numbers above the diagonal are the prob- hosts revealed a great genetic differentiation. The st value will be greater than the observed value, and represent the significance of the observed Table 4. Genetic distances among nine populations representing different geographic origins and hosts of 112 A. solani isolates,calculated using three AFLP primer combinations Numbers below the diagonal are measures of inter-population genetic distance ( st). Numbers above the diagonal are theprobabilities that a random st value will be greater than the observed value, and represent the significance of the observed Table 6. Genetic distances among eight populations comprising 80 A. solani isolates from different provinces of Cuba, measuredusing three AFLP primer combinations Numbers below the diagonal are measures of inter-population genetic distance ( st). Numbers above the diagonal are the proba-bilities that a random st values will be greater than the observed value, and represent the significance of the observed bootstrap confidence levels for subcluster 1.1 in the of A. solani of 0.28, whereas Weir et al. (1998) revealed UPGMA cluster analysis (Figure 2). Results of a a distance of 0.23. A similar level of diversity was pair-wise comparison between the eight A. solani pop- observed in the present study, with a minimal genetic ulations from different Cuban provinces are shown in similarity coefficient of around 0.76 at polymorphic Table 6. The greatest genetic distance among popula- AFLP loci for A. solani isolates from Cuban and tions was observed between the samples from Pinar del Rio and Granma ( st = 0.5113; P < 0.0001), Phenetic and population analysis of the AFLP fin- however the Pinar del Rio sample consists of only one gerprint data revealed an association between genetic isolate. Considering only the significant distances at the background and host origin. A highly significant P = 0.01 level, Granma is the most distinct population genetic differentiation ( st > 0.1544; P < 0.0001) from the others with six significant distances ( st = was revealed among Cuban, Greek and Turkish iso- 0.1478–0.5113; P < 0.0001), followed by Camag¨uey lates from tomato and potato (Tables 4 and 5). Evidence ( st = 0.1007–0.2201; P < 0.0001) and Holgu´ın for host specialization of A. solani was apparent from ( st = 0.0757–0.1478; P < 0.0001) with four sig- the phenogram of 112 isolates revealed by UPGMA nificant distances each. Tunas showed three significant cluster analysis. Sixty-two percent of the isolates orig- distances ( st = 0.0757–0.1517; P < 0.0001) and the inating from potato and 86.7% from tomato clustered clearly in two separate branches in the dendrogram witha maximum bootstrapping confidence level (100%).
AMOVA analysis with no consideration of geographic Discussion
origin indicated a considerable genetic differentiation( st = 0.289) with high statistical significance (P < Alternaria solani is distributed all over the world where 0.0001) between the isolates representing populations tomato and potato are cultivated (Rotem, 1994). In this from tomato and potato. Franco et al. (2001) considered paper, we describe a large-scale survey of genetic vari- st values between 0.15 and 0.25 and above 0.25 to rep- ability in 122 isolates of A. solani and related species, resent moderately high and high genetic differentiation from tomato and potato hosts, using AFLP genetic fin- gerprinting. A modified AFLP protocol was applied, To date, evidence of pathogenic specialization optimized for the small size of the Alternaria genome of A. solani on tomato and potato has not been by the use of PCR primers with only one and two reported. However, the AFLP evidence presented selective nucleotides, respectively.
here is consistent with preliminary observations of The AFLP data corroborated the high genetic vari- pathogenicity in some of the A. solani isolates used in ability reported previously for A. solani populations this study. Isolates from potato were less aggressive using isozymes and RAPD markers (Petrunak and on different tomato genotypes than the tomato isolates Christ, 1992; Weir et al., 1998). Petrunak and Christ (data not presented). Information on the specialization (1992) estimated a genetic distance within a population of pathogen populations and detailed monitoring of their pathogenicity are essential for the development of reproduction is essential to the long-term viability of effective disease control strategies.
a species, because it provides phenotypic variation on In regard to pathogenic specialization, Simmons which selection may act (Gordon and Martyn, 1997).
With the exception of a single report (Esquivel, 1984) tomatophila, within the well-known A. solani, report- the sexual stage in A. solani has been reported neither in ing that A. tomatophila is ‘the common and widely nature nor in vitro (Simmons, 1992). The genetic vari- distributed incitant of early blight of tomato’. All iso- ability may therefore arise from a parasexual cycle, as is lates but one from potato examined in that study were the case in other imperfect fungi. However, evidence of A. solani. Host specialization in A. solani was indi- natural parasexualism has not been obtained to date in cated by the general separation of tomato and potato A. solani, or in the well-known A. alternata pathogens isolates into subclusters 1.1 and 1.2 with a genetic (Akamatsu et al., 1999; Salamiah et al., 2001). Het- similarity coefficient of 0.7 (Figure 2). However, some erokaryons (Tsuge et al., 1987) and stable fusants have isolates were not consistent with a general host spe- been purified (Salamiah et al., 2001).
cialization hypothesis. Nine potato isolates from Cuba, Because of the low number of catenulated Alternaria two from Turkey and two from Brazil were grouped spp. isolates studied it is difficult to make conclusions among tomato isolates in subcluster 1.1, whereas one regarding their genetic similarity to other isolates.
Cuban and three Turkish tomato isolates grouped However, our results are consistent with earlier studies within the potato isolates in subcluster 1.2. We have that found high variability within catenulated isolates therefore classified all potato and tomato isolates (Morris et al., 2000; Roberts et al., 2000; Andersen in A. solani, according to Ellis (1971), until detailed et al., 2001). Moreover, in our experiment the catenu- microscopic and cultural examination according to lated isolates showed a low genetic similarity (0.61) in relation to the A. solani isolates. In other studies The results of the present study revealed an influ- A. solani and A. alternata were also clearly distin- ence of geographic origin on genetic variability among guishable from each other (Petrunak and Christ, 1992; the populations of A. solani. Isolates from the USA, Weir et al., 1998). Three Alternaria species pathogenic Brazil, Cuba and Turkey were genetically distant from to crucifers with small and large spores were also distinguishable using RAPD analysis (Sharma and tions within Cuba showed little significant genetic diversity (Table 6). This lack of significant geographic This study demonstrates the suitability of the AFLP differentiation among regional Alternaria spp. pop- technique for detailed analysis of genetic variation ulations suggests (1) widespread dispersal of fungal in A. solani and related pathogens. Cluster analysis spores and (2) weak host selection pressure within indicated a level of host specificity within A. solani.
and between populations (Sharma and Tewari, 1998; This may have important implications for effective early blight disease management in tomato and potato.
A high degree of morphological and DNA similar- It appears that the use of reproducible, highly poly- ity among A. solani and A. porri has been observed morphic AFLP markers has the potential to play a (Neergaard, 1945; Pryor and Gilbertson, 2000).
major role in the accurate taxonomic identification of The members of the porri species-group (A. porri, A. solani, Alternaria dauci, Alternaria macrospora andAlternaria crassa) exhibit a high degree of rDNA sim-ilarity, with no differences or only minor variation in Acknowledgements
mitochondrial small subunit or nuclear internal tran-scribed spacer sequences. Based on this evidence, an We are grateful to Drs. E. Demirci and S. Benlioglu extremely close relationship among species in this (Turkey), I. Vloutoglou and Y. Manetas (Greece), Y.H.
group has been suggested (Pryor and Gilbertson, 2000), Tong (China), C. Wharam (USA), T.A. Evstigneeva and this was clearly reflected by the inability of the (Russia), M. Tu (Canada), P. Nurmberg (Brazil), AFLP analysis to distinguish the A. porri isolate from M.A. Dita and R. Casta˜neda (Cuba) for providing iso- A. solani in the present study.
lates used in this study. We also thank Petra Theuer The origin of the variability present in A. solani for technical assistance. SPM was supported by a grant is unknown. It is commonly assumed that sexual from DAAD (German Academic Exchange Service).
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