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Centro de Investigação das Ferrugens do Cafeeiro, Instituto de Investigação Científica Tropical, Quinta do Marquês, 2784-505 Oeiras, Portugal, and Departamento de Biologia, Centro de Estudos da Macaronésia, Universidade da Madeira, 9000-390 Funchal, Portugal
Ana Ribeiro
Vítor M.P. Várzea
Carlos J. Rodrigues, Jr.
Centro de Investigação das Ferrugens do Cafeeiro, Instituto de Investigação Científica Tropical, Quinta do Marquês, 2784-505 Oeiras, Portugal
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Random amplified polymorphic DNA (RAPD) was used to assess the genetic structure of Hemileia vastatrix populations. Forty-five rust isolates with different virulence spectra and from different hosts and geographical regions were analyzed. Out of 45 bands, generated with three RAPD primers, 35 (78%) were polymorphic and scored as molecular markers. Cluster analysis exhibits unstructured variability of this pathogen with regard to physiological race, geographical origin or host. The genotypic diversity (H') inferred from Shannon’s index was higher than gene diversity (Ht), suggesting that diversity is distributed among clonal lineages. Estimates of gene diversity in Africa and Asia populations were higher in total (Ht) as compared to within population diversity (Hs). Genetic differentiation was considerable among coffee rust isolates from Africa (Gst = 0.865) and Asia (Gst = 0.768) but not among isolates from South America (Gst = 0.266). We concluded that genetic diversity in H. vastatrix was moderately low and that the genetic differentiation among populations shows that asexual reproduction is likely to play an important role in the population biology of this fungus. This should be taken into account for the development of breeding programs.
Key words: coffee rust, DNA polymorphism, genetic variability, molecular markers, population structure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The disease exists in the tropics where coffee has green foliage all year. Urediniospores penetrate coffee leaves through stomata and develop powdery orange pustules on the abaxial surface, resulting in impaired photosynthesis, premature defoliation and decreased crop yield (Brown et al 1995). Disease is most severe on Coffea arabica L., which is responsible for about 75% of the world coffee supply. C. canephora Pierre accounts for the remaining 25% of coffee production and in general is considered to be tolerant to rust (Bettencourt and Rodrigues Jr. 1988).
H. vastatrix life cycle is understood incompletely. The fungus survives as urediniospores, uredia and mycelia, whereas the majority of rusts usually have five spore stages and two hosts. Despite the occasional production of teliospores and basidiospores, under cool dry conditions, no alternate host has been discovered. The basidiospores germinate in vitro but do not infect coffee leaves. Because basidiospores do not have a known function, it has been thought that physiological races arise as a result of mutation rather than genetic recombination (Rodrigues Jr. et al 2001).
Disease control with fungicides is possible but inconvenient, marginally effective and environmentally hazardous (Thurston 1998). Therefore efforts have been directed toward breeding for resistance in coffee plants. Despite breakthroughs achieved with the production of varieties derived from Híbrio de Timor (HDT) like Catimor, Sarchimor, Colombia and derivatives of these varieties, new virulent races of the pathogen continue to arise (Rodrigues Jr. et al 2001, Várzea et al 2001).
Evidence for variation in pathogenicity among H. vastatrix isolates was reported in the early 1930s (Rodrigues Jr. et al 1975). Further evidence of pathogenic variability has been recognized by distinct phenotypes among Coffea sp. plants used as differentials (Bettencourt and Rodrigues Jr. 1988, Várzea et al 2001). The differentials comprise a collection of coffee germplasm on which different rust isolates are classified into physiological races, based on individual strains virulence. These cumbersome screening methods prompted us to carry out molecular studies because direct analysis of DNA polymorphisms is a more reliable approach to detect genetic variation in organisms, especially those with limited morphological characters such as H. vastatrix, and little is known about the diversity of this fungus.
Standard molecular methods such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and microsatellite analysis have been used to estimate genetic diversity and give insights into the population structure of important rusts such as Puccinia recondita (Kolmer et al 1995), P. striiformis (Steele et al 2001, Justesen et al 2002), Melampsora epitea (Pei et al 1997, Samils et al 2001), Cronartium ribicola (Hamelin et al 1998, Kinloch et al 1998), C. flaccidum (Moricca and Ragazzi 1998) and Peridermium pini (Hantula et al 1998, Moricca and Ragazzi 1998).
In the present study we used RAPD markers to examine molecular polymorphisms among isolates of H. vastatrix from different coffee growing regions and assess broader levels of genetic diversity. This is the first time that RAPDs have been applied to population genetic studies of this species. The purpose was to increase our knowledge about the pathogen variability and differentiation that might be used to investigate pathogen evolution and to design strategies for disease management as well as for developing new cultivars.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). From these, 38 isolates had been identified as different physiological races by means of inoculations on Coffea spp. differentials. List of isolates, physiological races, hosts and geographical origins are given (TABLE I) (Rodrigues Jr. et al 1975, Várzea et al 2002).
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procedure. Concentrations of DNA were estimated by measuring absorbance at A260 nm with a Lambda EZ201 UV/Vis spectrophotometer (Perkin Elmer, USA). Typically, 0.2–0.8 µg of DNA were obtained per mg of fresh urediniospores. DNA samples were diluted to working solutions of 25 ng/µL, confirmed by agarose gel electrophoresis and stored at 4 C until use.
RAPD analysis.— –
Standard arbitrary 10-mer oligonucleotides (Operon Technologies Inc., USA) were tested for RAPD analysis using the method of Williams et al (1990). Reactions were carried out in a final volume of 25 µL containing 20 mM Tris-HCl pH 8.4, 50 mM KCl, 3 mM MgCl2, 0.2 mM dNTPs, 0.3 µM primer, 50 ng of genomic DNA and 1 U of Taq DNA polymerase (Invitrogen). Amplifications were performed in a PTC-100 thermocycler (MJ Research Inc.) programmed for an initial denaturation cycle (2 min at 94 C) followed by 40 cycles of denaturation (1 min at 94 C), annealing (1 min at 35 C) and extension (2 min at 72 C) with a final extension of 5 min at 72 C. Each reaction was performed at least twice to assess the consistency of the band profiles. The reproducibility of the technique was calculated Si2= n/2t (Sneath and Johnson 1972), where Si2 represents the variances of a primer set between replicate isolates, n the number of isolates with discrepancies and t the total number of duplicate isolates. Reaction products (10 µL) were resolved by electrophoresis in 1.2% agarose gels stained with ethidium bromide in 1x TAE buffer at 90 V for 140 min. All reactions included negative controls in which DNA or primer was omitted in the amplification reaction mixture. Because the urediniospores in this study were obtained from greenhouse-grown infected Coffea differentials, and in such conditions, contamination with the hyperparasitic fungus Verticillium may occur, a positive control with Verticillium sp. DNA also was included.
Data analysis.— –
Differences in fingerprinting patterns between isolates were assessed visually. Polymorphisms including faint bands that could be scored unequivocally were included in the analyses. Presumed homologous bands were scored as present (1) or absent (0) to create a binary matrix. The binary matrix was analyzed with the computer program NTSYSpc version 2.02h (Applied Biostatistics Inc., USA). An unweighted pair group arithmetic mean method (UPGMA) cluster analysis was performed based on the DICE’s similarity coefficient SD= [2a/(2a + b + c)], where a is the number of bands common for isolate x and y, b is the number of bands present only in isolate x, and c is the number of bands present only in isolate y. Dendrograms were generated with the tree display option (TREE). Goodness of fit of the cluster analysis was determined by computing a cophenetic value matrix. A cophenetic correlation of r > 0.9 was considered a good fit. Clustering and statistical analyses were performed on data generated from each primer set separately and on the combined data primer sets. The ability of the most informative primers to differentiate between isolates was assessed by calculating their Resolving power (Rp) (Prevost and Wilkinson 1999). Rp was calculated with the formula Rp = 
Ib, where Ib (band informativeness) takes the value of 1–[2x (0.5–p)], and p is the proportion of isolates containing the band. The genetic variability was quantified in two ways: (i) the percentage of polymorphic loci (representing polymorphic bands) and (ii) Shannon information index (H'—denotes the diversity of RAPD markers) (Lewontin 1972). Calculations of these two diversity estimates were performed with Popgene version 1.31 (Yeh et al 1997). Correlation coefficients were determined with the XLSTAT program version 6.1.8. The occurrence of any population structure was studied by calculating total gene diversity (Ht) of the polymorphic loci, mean gene diversity for each RAPD marker within populations (Hs) and genetic differentiation (Gst) across populations as a proportion of total diversity (Nei 1973), using the Popgene program.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) that consistently generated polymorphic amplicons were retained for the molecular analysis. These primers allowed the scoring of 45 bands among the amplified fragments of the 45 isolates (TABLE I
), of which 35 (77.8%) were unambiguously reproducible and polymorphic (TABLE II), suggesting high genetic variability. Multiple bands common to all H. vastatrix isolates were observed in RAPD experiments, while the Verticillium control produced RAPD patterns lacking discernable similarity with H. vastatrix patterns (FIG. 1). Hence we concluded that electrophoretic profiles obtained were due to genetic differences among H. vastatrix isolates and not to a putative contamination with Verticillium. Reproducibility of these polymorphisms was assayed in at least two independent experiments with independent DNA extractions and ranged from 88 to 95%.
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). The relatively low polymorphism and Rp value of primer OPF-06 rendered this primer the least useful for discriminating among H. vastatrix isolates (FIG. 1A).
The patterns obtained with primer OPD-05 for isolates of the same race suggested that this primer has the ability to produce fingerprints specific to particular physiological races. For example most bands generated from isolates 37, 395 and 1821, identified as race III, were similar to other H. vastatrix isolates but yielded a distinct band at 0.85 kb (FIG. 1B). Representatives of race XV, isolates 70 and 772 (FIG. 1B), yielded a prominent band about 1.8 kb. In contrast isolates belonging to different physiological races, such as 2531 (race XLII) and 2542 (race XLI), lacked a 1.2 kb band (FIG. 1B).
Multiple distinct DNA fragments were found among H. vastatrix isolates examined with primer OPA-09. Of the 19 bands analyzed 17 (89.5%) were polymorphic across all the isolates. Some DNA fragments were present exclusively in isolates from the same physiological race, which could be considered race specific. For example isolate 70 (FIG. 1C), from race XV revealed a polymorphic band of 1.1 kb. Three bands (2.6 kb, 1.5 kb, 0.7 kb) similarly were specific for race II (isolate 1065 and isolate 1126, data not shown), while one band of about 1.2 kb was present in isolates 166, 178 and 2571 from race VIII (data not shown).
Cluster analysis.— –
Cluster analyses performed with the RAPD data matrix generated by each primer revealed that only primer OPA-09 grouped some isolates according to physiological races, although several races could not be distinguished (data not shown). The dendrogram obtained with all RAPD markers (FIG. 2) showed that genetic relatedness of the 45 coffee rust isolates ranged from 0.7 to 1.0. The cophenetic correlation coefficient was 0.93, indicating the dendrogram was an excellent fit representation of the original data. Reproducibility of the RAPD technique averaged 92% and we used this value to set the level for isolate differentiation, so 12 (C1 to C12) main clusters were delineated. Judging from cluster analysis, isolate 2307 (cluster C12) is genetically unique, whereas extensive clonality was found in the larger collection from India (cluster C2). Isolates classified in different physiological races, such as 292 (XVII) and 292a (XXIII), were identical at the molecular level and were closely associated within the same cluster (C6, FIG. 2). Isolates 2537(XL) and 2191 (XXXVII) similarly were indistinguishable at the DNA level (FIG. 2, cluster C5). In several cases there was high genetic similarity between isolates from different geographical regions (e.g., cluster C1) and hosts (e.g., cluster C7). Few rust isolates were exceptions and could be grouped according to the indicated physiological race. However overall results of cluster analysis showed no correlation with physiological races (Sperman’s rank correlation coefficient: r = –0.127, P = 0.202), geographical origin (r = 0.159, P = 0.148) or host (r = –0.043, P = 0.390).
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), indicating that about 77% of the total genetic variation could be explained by RAPDs differences while the remaining 23% might be attributable to differences among isolates. These results indicate that genetic differentiation was high in H. vastatrix populations and genetic diversity was low within the total sample and individual populations.
In the C. arabica population the Shannon diversity index was superior to the HDT population (TABLE III). This variation was reflected by the higher polymorphism detected among isolates from C. arabica (64.4%) when compared with those derived from HDT (42.2%). A positive correlation was detected between the Shannon index and the percentage of polymorphism for the HDT population but not for the C. arabica population (Pearson rank correlation: r = 0.999, P = 0.011; r = 0.969, P = 0.080, respectively). We suggest that RAPD markers were unevenly distributed in the former population. In addition the coefficient of gene differentiation (Gst) was similar in rusts from C. arabica and HDT, indicating that these populations were highly different but isolates were relatively similar.
Genotypic diversity varied among geographical regions, based on Shannon indices (TABLE III). The diversity within regions ranged from 0.140 in African to 0.241 in South American populations. A significant association (P < 0.05) was found between the Shannon index and the percentage of polymorphism for Africa and South America populations but not for Asian population (Pearson rank correlation: r = 0.801, P = 0.204). This absence of correlation probably was due to the larger number of physiological races and isolates in Asian population. Compared to gene diversity (Ht) the higher genotypic diversity (H') observed suggested that genetic diversity is distributed mainly among clonal lineages. Genetic subdivision analysis revealed the lowest level of genetic differentiation within the South American region (Gst = 0.266). This value may reflect that the isolates originated from two countries and mainly from HDT derivatives or that low genetic differentiation possibly was due to a more recent origin. In addition total gene diversity (Ht = 0.170) was attributable mostly to diversity within population (Hs = 0.125), indicating that isolates were likely to differ genetically.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Hamelin et al 1998, Kolmer and Liu 2000, Li et al 2001, Steele et al 2001). In addition knowing species genetic diversity has important basic and applied implications, including understanding pathogen evolution.
Our results suggest that RAPD markers can be used to evaluate genetic diversity within the species H. vastatrix through estimates of variation at multiple loci (35 in this study) across the genome. The number of alleles per locus, a potentially more sensitive measure of genetic diversity, cannot be determined from our dominant marker data. Nevertheless dominant markers predictably can underestimate genetic diversity (Krutovskii et al 1999, Wu et al 1999), and if this is true for H. vastatrix then the true genetic diversity might be higher than found in this study.
The molecular variation yielded up to 78% polymorphic bands, which represents a high degree of polymorphism uncommon in rust fungi (Kinloch et al 1998, Park et al 2000, Li et al 2001, Steele et al 2001). The fingerprints obtained with primer OPF-06 identified polymorphic differences among isolates but generated only a limited pattern of one or two additional bands. Primers OPA-09 and OPD-05 conversely showed a higher resolution. Primer OPA-09 can be used to unequivocally discriminate races XV and VIII, whereas primer OPD-05 discriminates races III and XV. However, to be used as race-specific markers, further surveying is required of a larger number of isolates.
Comparison of the H. vastatrix isolates by means of RAPD (FIG. 2) showed that rust isolates classified in the same physiological race by means of conventional methods (e.g., isolates from races XXIII and XXXVII) seemed to differ substantially at the molecular level. This variability could indicate the occurrence of genetic changes independent of pathogenicity. Cluster analysis suggests a widespread occurrence of homogeneous isolates of H. vastatrix, probably resulting from the introduction of isolates between countries as a consequence of germplasm exchange or long-distance dispersal (Brown and Hovmoller 2002). Large dispersal with no apparent grouping, regardless of host or location, also was observed with isolates of Melampsora epitea (Samils et al 2001), Botrytis elliptica (Huang et al 2001) and Plasmodiophora brassicae (Manzanares-Dauleux et al 2001).
The level of uniformity and the partitioning of genetic diversity suggest a clonal reproduction in the H. vastatrix population studied. The fungus has a population structure consistent with an asexual reproduction. This agrees with the absence of biological evidence for sexual behavior under natural and greenhouse conditions (Rodrigues Jr. et al 2001). Considering the ability of long-distance dispersal (Brown and Hovmoller 2002) clones can be spread worldwide, undergoing differentiation via mutation, asexual recombination or other sources of genetic variation (mechanisms occurring in asexually reproducing populations and favoring genetic exchange). Important phytopathogenic fungi, such as Cronartium ribicola (Hamelin et al 2000), Melampsora spp. (Pei et al 2000), Puccinia recondita (Park et al 2000), P. striiformis (Hovmoller et al 2002, Justesen et al 2002), are known to have clonal lineages and little genetic diversity worldwide.
Substantial genetic differentiation was observed between the African and Asian populations, contrary to populations within the South American region. High genetic differentiation was observed in asexually propagated populations (Wyand and Brown 2003). The differences observed in our study could be related to population age and level of gene flow among populations. H. vastatrix urediniospores are thought to have spread by air currents from Angola, where coffee leaf rust first was reported in 1966, to Brazil in 1970 (Bowden et al 1971). In this study populations from South American region mainly were formed by isolates collected from HDT derivatives (78%). These isolates were introduced to Latin America about 30 years ago (Rodrigues Jr. et al 2001). Since 1990 Colombian plants, derived from HDT, started showing susceptibility to rust (Rodrigues Jr. et al 2001). The moderate degree of differentiation in South American populations is consistent with a relatively recent introduction of the pathogen and with recent observations concerning the susceptibility of cultivars derived from HDT to coffee rust (Rodrigues Jr. et al 2001, Várzea et al 2001). Moreover, considerable variation within South America populations suggests a high evolutionary rate of H. vastatrix. Such pathogen populations are more likely to overcome genetic resistance (McDonald and Linde 2002) and might explain the frequent loss of resistance to the rust among Coffea genotypes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: mgouveia{at}uma.pt
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