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Since 1911, dothistroma needle blight, caused by Dothistroma septosporum, has been recorded in most European countries. In the Czech Republic, the fungus has become an important disease of pines since 2000, especially Austrian pines in plantations of Christmas and ornamental trees. The aim of this study was to analyse the population structure, gene flow and mode of reproduction of this pathogen. Microsatellite and mating-type markers were analysed in a Dothistroma population in the southeastern part of the country using reference isolates from other European countries. The haplotypic diversity was high, with 87 unique and 13 shared haplotypes (probable clones) identified in 121 samples. Based on structure analysis, the isolates were divided into two populations, with an uneven distribution over the sampling sites. The grouping of the sites to the populations did not follow a geographical pattern because certain isolates that were sympatrically co-occurring at the same site were placed in different populations. Tests for random mating (the index of association and a parsimony tree-length permutation test) showed a significant clonal mode of reproduction in most cases, but the intrapopulation haplotypic diversity is unexpectedly high. Although a teleomorphic stage of D. septosporum has not been previously observed in the Czech Republic, the high intrapopulation haplotypic diversity can be explained by infrequent sexual reproduction consistent with the occurrence of both mating types.
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Dothistroma needle blight (DNB) is caused by two closely related species, Dothistroma septosporum and D. pini (the anamorphic stage of Mycosphaerella pini) and results in severe needlecast in pines and, to a lesser extent, other conifers. According to European and Mediterranean Plant Protection Organization (EPPO) and European law, DNB is a quarantine fungal disease in Europe. DNB is distributed worldwide and is reported in at least 63 countries (Watt et al., 2009), and more than 80 host species. DNB has emerged in Europe in the past 15–20 years and is currently widespread in most European countries (Watt et al., 2009). Although its European isolates belong primarily to Dothistroma septosporum, D. pini has been detected in samples from France (Ioos et al., 2010), Hungary, Russia and Ukraine (Barnes et al., 2011). Woods et al. (2005) consider DNB to be a disease supported by global climate change and emphasize the influence of locally enhanced precipitation on the spread of the disease throughout the temperate zone.
In the Czech Republic (CR), D. septosporum was first noted in 1999 on imported nursery stock of Pinus nigra from Hungary, and the first detection of DNB in a plantation was recorded the following year in 6-year-old Austrian pines in Christmas tree plantations (Jankovskýet al., 2004). The increasing number of new findings is explained by the substantial number of trees imported in the 1990s and by the use of infested stock from nurseries until 2000, when strict phytosanitary precautions were instituted throughout the CR. Even though DNB monitoring was conducted prior to the 1990s, it is possible that the disease occurred during that period. In the CR, DNB has been identified in 21 pine species, four spruce species and Douglas-fir (Bednářováet al., 2006); however, D. pini has not been recorded in the country to date (March 2012). Observations of the current DNB outbreak have been conducted throughout the CR, and the disease has been found mainly in Austrian pine plantations or on amenity trees in urban areas, whilst the infestation of native Scots pine stands is relatively rare (Jankovskýet al., 2004; Bednářováet al., 2006). Since 2008, new observations in Scots pine plantations and Scots and bog pine stands in protected areas have been reported; the occurrence of native pine populations is anticipated at such natural sites (Mikeska et al., 2008). Therefore DNB may be caused by an older population of the pathogen there, whereas Austrian pine plantations have been more recently infected by infested nursery stock.
Few studies have investigated the genetic structure of D. septosporum populations. In New Zealand, no genetic diversity was detected in the pathogen using RAPD and RAMS DNA markers (Hirst et al., 1999). Microsatellite markers for D. septosporum were developed by Barnes et al. (2008) but have not been used in any population analysis. Dale et al. (2011) recently used AFLP and mating type markers to reveal a high haplotypic diversity, a mixed mode of reproduction, and an equal mating-type segregation ratio in D. septosporum populations in British Columbia, Canada. A population with a high genetic variation has a strong evolutionary potential, and it is probable that such a population can rapidly adapt to new conditions (McDonald & Linde, 2002). Gene flow among populations and recombination are two crucial sources of new allelic and genotypic variation that can increase the genotypic or haplotypic diversity of a population (McDonald & Linde, 2002). In pleomorphic ascomycetes, the role of propagation by asexual (conidia) or sexual (ascospores) diaspores is crucial for genotypic and haplotypic diversity. However, the role of ascospores for infections in situ is unclear. In Europe, DNB is usually considered a human-mediated disease, and D. septosporum is hypothesized to have short-lived conidia with limited dispersal abilities (Gadgil, 1970; Peterson, 1973). However, the precise data for the CR region are incomplete.
The aim of this study was to analyse the population structure, gene flow and mode of reproduction of D. septosporum in the CR using microsatellite (SSR) and mating-type markers.
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All eight of the microsatellite markers were polymorphic, and 81 different alleles were detected at the loci analysed. The eight preselected markers were determined to be sufficient for the population genetic analysis because the plot of genetic diversity versus the number of loci showed that six of the markers explained 99% of the observed variation. In all, 100 haplotypes were found. Of these, 87 were unique in the entire collection (i.e. present in only one copy); of the remaining 13 shared haplotypes, nine were found only within sites, and four were shared within and between sites. The population allele frequencies are shown in Table 2.
Table 2. Dothistroma septosporum population allele frequency based on eight microsatellite markers
|Locus||Population allele frequency|
structure analysis was performed in order to assign individual isolates to populations. Accordingly, samples were divided into two populations based on K parameter (Fig. 1), each with 29 sampling sites. Therefore, the two populations sympatrically co-occurred at 12 of the sampling sites. The placement of the samples into populations is shown in Table 1. Both populations have a similar proportion of polymorphic loci, and the percentage of variation is almost three times higher within populations than between them. The population statistics and characteristics are shown in Table 3. The observed haplotypic diversity was high, and the values of Nei’s gene diversity were 0·49 (Pop1) and 0·64 (Pop2). The FST and predicted Nm values between these populations are 0·2939 and 1·20124. These numbers, supported by results of a Mantel test, are indicative of prevalent range-limited spread by conidia and significant differentiation between two populations with very low numbers of migrants. Assessment of Nm is susceptible to violation of the underlying model for FST computation (Whitlock & McCauley, 1999) but nevertheless the computed number indicates very limited exchange of migrants between populations. The exact tests (arlequin v. 220.127.116.11) performed for each of the populations using the clone-corrected data set did not indicate that any of the loci are in linkage disequilibrium.
Figure 1. Delta K plot of the structure analysis, showing K = 2 as the most probable number of populations in the data set.
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Table 3. Population genetic statistics for Dothistroma septosporum populations
|Full data||CC||Locia||No. of haplotypesb||Haplotypicc||Nei’s gened|
In Pop1, including Czech and Austrian samples, significant correlation between the genetic and geographic distances was evidenced by a Mantel test conforming to assumed spatially-limited conidial spread. In comparison, Pop2 contained Czech and the remaining foreign samples from distant localities which were genetically similar. In the 2-D local spatial analysis, the sites with the highest number of samples in Pop1 (Sádek, Rožnov p. Radhoštěm - Písečná and Nová Pec sites) showed significant correlations with their two nearest neighbours as follows: four of five samples from Rožnov, all of the samples from Nová Pec and one of six from Sádek showed correlations. In the analysis of the three nearest neighbours, three of seven of the samples showed correlations at Rožnov, and three of four samples at Nová Pec. No correlations were found for Sádek. In the Multiple Dclass analysis, the correlation was the highest at the shortest distances and decreased as the distance increased, but it remained significant, even at the maximum distance tested.
Tests for random mating were performed using the index of association and a PTLPT analysis (Table 4) and generally showed a significant clonal mode of reproduction, even in the clone-corrected data set. However, the IA index for Pop1 suggested random mating. With the exception of Pop1, the PTLPT tests showed results that were consistent with the IA values, even in the clone-corrected data set.
Table 4. Random mating test of Dothistroma septosporum on full data set and clone-corrected (CC) data set
|Population||Index of association (IA)a||PTLPTb|
| IA || P value||L||L*|| P value|
|Pop1||0·07102|| 0·145||73 (1)||86||<0·0001|
|Pop1 CC||0·0492321||0·5639||75 (2)||71|| 0·002985|
|Pop2 CC||0·286235||0·0005||104 (1)||116||<0·0001|
|Total CC||0·33665||<0·0001||176 (2)||199||<0·0001|
Both mating types were found in the Czech sampling sites; in contrast, all of the Fennoscandian samples included only mating type 2. Mating type 2 occurred more frequently than mating type 1 in both Pop1 and Pop2. However, the ratio of mating type 1 to mating type 2 including samples from both populations did not differ significantly from a 1:1 segregation (χ2 = 0·54, P = 0·462), and a chi-square analysis of the clone-corrected data yielded similar results (χ2 = 0·87, P = 0·349). Nevertheless, both of the mating types were detected within a sampling site in 10 cases (21·7% of the sampling sites; Bynina – Christmas tree plantation, Jakule, Karolínka – medical centre, Luhačovice, Mštenovice, Pernek, Valašské Klobouky, Vídeň at Velké Meziříčí, Zubří– sawmill and Zubří– poultry farm). Among these sites, five had samples that all belonged to Pop1 (Bynina – Christmas tree plantation, Jakule, Karolínka – medical centre, Mštenovice and Valašské Klobouky) and one site had samples that belonged to Pop2 (Zubří– sawmill). The four remaining sites included samples that fell into both populations.
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The genetic separation of Dothistroma samples into two populations by structure analysis does not correspond to their geographical location. The highly geographically separated samples from Austria, Finland, Norway and Slovenia were placed into Pop2, as were 23 of the Czech sites. This result could be an artefact of the analysis resulting from insufficient sampling of these sparsely sampled distant locations. Alternatively, the Pop2 sites could represent descendants of the same former colonization event. However, samples placed in both populations occurred sympatrically at 12 of the Czech sites. For example, three sites in the town of Rožnov pod Radhoštěm (library, Písečná and Rybníčky), separated by a distance of 2 km, included samples from both populations. Moreover, the site sampled in detail (Písečná) included samples from both populations. The occurrence of both populations is primarily limited to urban greenery and arboretums, where the occurrence of the pathogen is strongly influenced by human activities and where the disease was introduced by the planting of infected nursery stock. The effects of human-mediated haplotype transfer are misleading in terms of the population assignment. It is probable that the conidia are dispersed only over short distances (maximum 150 m) by rain or splashing water (Peterson, 1973) and that the unreleased conidia in acervuli lose viability during the 4–6 months on the ground (Gadgil, 1970). Therefore, the role of the conidia in long-distance dispersal is limited. These assumptions were partially corroborated by the spatial analyses that showed a significant genetic correlation between the samples in Pop1 located in close proximity. The FST and Nm values indicate limited ability of the fungal diaspores to disperse over long distances as they show significant differentiation between populations, and marginal numbers of migrants between populations. The haplotypes shared among the samples (presumably clones) were distributed primarily within the sampling sites, and only four such haplotypes occurred at two different sites. Nová Pec and Pernek, sharing two common haplotypes, are approximately 4 km apart. The first possible clone was recorded only on Pinus ponderosa at both of the sites, whereas the second possible clone was detected on two pine species, Pinus mugo and P. uncinata subsp. uliginosa. The other example of identical haplotypes was detected at Lhotka nad Bečvou and Jasenice (5 km apart): both of the occurrences were found on P. nigra, and it is possible that these pines shared a common origin. These findings of shared haplotypes are explainable by human-mediated haplotype transfer. Nevertheless, one example of a shared haplotype occurred in Havlíčkův Brod and Křtiny, 90 km apart, a result that is surprising.
The observed overall haplotypic diversity was high, which agrees with the results of Dale et al. (2011). If the hypothesis of a relatively recent introduction into Europe is considered, this unexpected outcome could be explained either by occasional genetic recombination, by the colonization of an array of genetically distinct individuals, or by the simultaneous colonization of different sites by genetically diverse diaspores. The most probable explanation would include a combination of all these hypotheses. The division of the data set into two almost-equal numbers of samples assigned to each population may be explained by two separate introductions of D. septosporum into Europe. This hypothesis could be tested by an analysis of the genetic similarity between isolates from potential native populations located in either Central America or Nepal (Evans, 1984; Ivory, 1994). The first European record of D. septosporum was published in 1911 by Doroguine in the boreal zone around St. Petersburg, Russia (Doroguine, 1911). During the 1950s and 1960s, occasional occurrences of the species were recorded in Austria (Petrak, 1961), the UK (Murray & Batko, 1962) and France (Morelet, 1967). Although the presence of D. septosporum was confirmed in the Czech Republic during 1999 on imported stock and during 2000 in plantations (Jankovskýet al., 2004), the fungus may have persisted in the country for a longer period of time, and the accumulation of the observed haplotypic diversity could have occurred. The biology of the fungus allows it to respond sensitively to weather conditions (Woods et al., 2005; Welsh et al., 2009), and it is probable that the recent fluctuations in climatic conditions provided DNB better opportunities to spread. In general, it is probable that forest trees respond slowly to disease, because the generation time of trees is much longer than the timescale on which such pathogens can spread (Shaw & Osborne, 2011).
Although the random mating tests performed on the data indicate a dominant clonal mode of reproduction, the high level of intrapopulation haplotypic diversity can be explained by infrequent sexual reproduction. This explanation is in agreement with Dale et al. (2011). A teleomorphic stage of D. septosporum has not been observed in the CR, and reports of a teleomorphic stage in other European countries are unusual (Butin, 1985; Karadžić, 1989; Kowalski & Jankowiak, 1998). The asci and ascospores have only rarely been observed, although an extensive observation was performed (Kowalski & Jankowiak, 1998). Nevertheless, the occurrence of a teleomorphic stage was occasionally recorded in two neighbouring countries, Germany and Poland, with similar environmental conditions. Additionally, Groenewald et al. (2007) confirmed the presence of both mating types in Austria and Poland. The results presented here confirm the occurrence of both mating types at 10 sampling sites (21·7% of all of the sampling sites) with the possibility for sexual reproduction of the pathogen to occasionally occur. Four of these sites (Luhačovice, Pernek, Vídeň at Velké Meziříčí and Zubří– poultry farm) included samples placed in both Pop1 and Pop2. Such localities may serve as local hotspots of genotype diversity. The current haplotype diversity within these sites can be explained by possible mating and subsequent recombination, resulting in mixed genotypes that tend to assign to either Pop1 or Pop2. In addition, the IA results for the clone-corrected data in Pop1 suggest random mating. Nevertheless, IA is known to be less sensitive to detecting clonal mode than PTLPT analysis (Taylor et al., 1999). However, according to previous findings (Jankovskýet al., 2004; Bednářováet al., 2006), the spread of the fungus results primarily from the movement of infected planting stock. In urban areas and arboretums, it is probable that the diversity of D. septosporum is increased by the introduction of diverse fungal diaspores of multiple origins. Apart from these instances of long-distance human-mediated transfer, the local genotypic structure is consistent with a hypothesis of predominantly conidial, range-limited spread. This hypothesis is supported by the results of the Mantel test and the spatial analyses.
The future spread of DNB can be predicted as a slow dispersal across short distances via conidia if human-mediated transfer is reduced. From the practical point of view, the control of nurseries and plantations is necessary to prevent epidemic dispersal of the pathogen. The respective phytosanitary precautions are currently applied in many EU countries. Phytosanitary authorities should pay extensive attention to imported coniferous planting stock to prevent increase of haplotypic diversity of the pathogen due to import of diverse genotypes of D. septosporum from foreign countries.