Ecology and Population Biology
The Genetic Structure of Field Populations of Rhynchosporium secalis from Three Continents Suggests Moderate Gene Flow and Regular Recombination S. Salamati, J. Zhan, J. J. Burdon, and B. A. McDonald First author: The Norwegian Crop Research Institute, Kvithamar Research Centre, N-7500 Stjørdal, Norway; second and fourth authors: Department of Plant Pathology and Microbiology, Texas A&M University, College Station 77843-2132; third author: Centre for Plant Biodiversity Research, CSIRO - Plant Industry, G.P.O. Box 1600, Canberra, A.C.T. 2601, Australia. Current address of J. Zhan and B. A. McDonald: Institute of Plant Sciences, Plant Pathology Group, Federal Institute of Technology, ETHZentrum, LFW, CH-8092 Zürich, Switzerland. Accepted for publication 5 May 2000.
ABSTRACT Salamati, S., Zhan, J., Burdon, J. J., and McDonald, B. A. 2000. The genetic structure of field populations of Rhynchosporium secalis from three continents suggests moderate gene flow and regular recombination. Phytopathology 90:901-908. Restriction fragment length polymorphism (RFLP) markers were used to compare the genetic structure of field populations of Rhynchosporium secalis from barley. A total of 543 isolates representing 8 field populations were sampled from Australia, California, Finland, and Norway. Gene and genotype diversity were high in all populations. Nei’s average gene diversity across seven RFLP loci was 0.513. Hierarchical gene diversity analysis showed that 9% of the total genetic variability was distributed among continents, 4% was distributed among fields within continents, and 13% was distributed among collection stations within a field. The majority (74%) of genetic variability was distributed
Leaf blotch or scald of barley (Hordeum vulgare L.), caused by the haploid imperfect fungus Rhynchosporium secalis (Oud.) Davis, is an economically important disease that is found on barley worldwide. This pathogen is spread by splash dispersal of conidia and survives in barley seeds and debris. Several studies suggest that a wide pathogenic variation exists in most populations of this fungus (1,15,21,24,26,40,44,46,48). However, pathogenic variation appears to be lower in Australia (8) and Finland (38). Pathogenicity, isozyme, colony color, ribosomal DNA, and, recently, genomic restriction fragment length polymorphism (RFLP) DNA markers have been used to study genetic variability in R. secalis populations (16,17,29,31,32). These studies confirmed that most populations of R. secalis are highly variable for many genetic markers despite the absence of a known teleomorph. Isolates secured from different scald lesions taken from the same plant, and even different spores isolated from the same lesion, can vary significantly for sporulation rate, virulence, isozymes, and RFLP markers (7,20,32,36). Approximately 86% of the total isozyme diversity in one California field population was distributed over a spatial scale of 12.5 m2 (31), while 76% of the total RFLP diversity was distributed over an area of ~1 m2 in Australian populations (32). An isozyme survey that included 150
Corresponding author: B. A. McDonald; E-mail address:
[email protected] Publication no. P-2000-0612-01R © 2000 The American Phytopathological Society
within collection areas of approximately 1 m2 within fields. Gene flow appears to be significant on a regional scale but more restricted among continents. Allele frequencies were significantly different at several RFLP loci. Genetic distances were small among populations within regions and large between regions. Pairwise comparisons of genotype diversity in the populations revealed significant differences among populations that were related mainly to differences in sampling strategies. Isolates from Norway and Finland showed a lower copy hybridization pattern with probe pRS26. This probe functioned as a fingerprint probe for the California and Australian isolates. Seven out of the eight populations studied were at gametic equilibrium for RFLP loci, suggesting that R. secalis populations in Norway, Finland, and Australia undergo regular recombination, although a teleomorph has not yet been recognized. Additional keywords: barley scald, genetic distance, population genetics.
isolates from three continents (Australia, Europe, and North America) suggested a moderate degree of population subdivision (GST = 0.16) among continents across eight isozyme loci (16). The populations described in this isozyme survey, however, consisted of isolates sampled over a 27-year period across a variety of spatial scales (Table 1 in Goodwin et al. [16]), making it difficult to determine the spatial scale over which genetic diversity was likely to be most concentrated. A hierarchical sampling strategy was needed to provide more precise information on the spatial distribution of genetic diversity within and among populations. The source of the high level of genetic diversity in R. secalis has been the subject of speculation for many years. Several possible sources of variation have been suggested, including frequency-dependent selection (16,29), a parasexual cycle (24,36), spontaneous mutation and migration (17), and sexual reproduction (32). Although the sexual stage has not been reported for R. secalis, a recent study of a geographically diverse collection of Australian isolates showed that most of the alleles at isozyme loci were in gametic equilibrium (9). The degree of gametic equilibrium can be used as an indirect measure of the significance of genetic exchange and recombination in a presumably asexual population (28). Our previous study of Australian populations, using RFLP markers and DNA fingerprints, provided additional evidence for recombination in Australian populations of R. secalis. We found many different genotypes within field populations at a spatial scale of 1 m2 (32). Additional field populations from other continents were needed to determine whether a population genetic structure consistent with recombination extended beyond Australia. Vol. 90, No. 8, 2000
901
The objectives of this study were to: (i) compare the genetic structure of field populations of R. secalis from Australia, Northern Europe, and North America to determine the spatial distribution of genetic diversity on local, regional, and global scales; and (ii) determine whether the genetic structure of these R. secalis populations is more consistent with sexual, parasexual, or asexual reproduction. MATERIALS AND METHODS Sampling methodology. Infected plant material was collected from naturally infected spring barley fields during the summer of 1996 from one location in Finland (Jokioinen, cv. Kymppi) and from two locations in Norway. Two areas in Norway were chosen: one in southeastern Norway (Buskerud, cv. Tyra), which experiences occasional epidemics of scald, and the other in central Norway (Stjørdal, cv. Tyra), which experiences annual epidemics of the disease. The collections from Finland and southeastern Norway were made using hierarchical sampling strategies described previously (32). Infected plant material from central Norway was collected using a fine-scale transect sampling method. Two parallel 10-m-long transects, separated by 2 m, were arranged within the field. Ten stops were made along each transect at 1 m intervals. At each stop, one infected leaf from each of four tillers was collected, and the leaves were placed in separate envelopes. A total of 80 leaves was sampled from the 20 points in this field. The collection from California was a representative subsample of a large, single-field collection made in 1988. This population was characterized previously using isozyme, RFLP, and virulence markers (29,31). Collections representing Australian populations were sampled from southeastern Australia during October and November 1996. Three collections were made in New South Wales: Wagga Wagga Agricultural Institute (cv. Clipper), Methul (cv. O’Connor), and Rannock (cv. Schooner). One collection represented Horsham, Victoria (cv. Arapiles). The hierarchical sampling method used for these barley fields was described previously (32). RFLP analysis. Isolation and culture of R. secalis strains and fungal DNA extraction procedures were as described previously (32). In addition to the eight probes used (32), probe pBD4 was assayed in the Norwegian and Finnish isolates, and pRS49 was assayed in all populations. pRS49 was a single-locus probe chosen from the R. secalis genomic library described previously (32). Probe pBD4 contains a complete ribosomal DNA (rDNA) repeating unit from Saccharomyces cerevisiae (4). We showed previously that pBD4 can produce RFLPs in the rDNA of R. secalis (29). Probe pRS26 (32) was used as a DNA fingerprint probe. Data analyses. Each probe-enzyme combination defined a different RFLP locus. DNA fragments or combinations of fragments with different sizes were treated as alleles at each RFLP locus. Each allele was assigned a unique number. A sevendigit (California), eight-digit (Australia), or nine-digit (Norway and Finland) numeric identifying code was formulated for each isolate by joining together the numbers identifying the allele present for each locus. These numeric codes were termed the multilocus haplotype (MLHT) for each isolate. For the pRS26 probe, each hybridizing fragment profile was treated as a DNA fingerprint. Isolates having identical MLHTs (Norway and Finland) or DNA fingerprints and MLHTs (Australia and California) were considered members of the same clone. Probe pRS26 did not generate informative hybridization profiles that could be used to identify clones in Norwegian or Finnish populations because too few DNA fragments hybridized with the probe in these isolates. The data were clone-corrected by including only one member of each clone to calculate allele frequencies in field populations. Hierarchical gene diversity analyses as proposed by Beckwitt and Charaborty (3), were used to determine the distribu902
PHYTOPATHOLOGY
tion of gene diversity within and among populations across all RFLP loci. Pairwise measures of genetic distance and identity and differentiation (GST) between populations were calculated according to Nei (35). Pairwise genetic distances were subjected to a cluster analysis using UPGMA (unweighted pair group method using arithmetic averages) and displayed in a phenogram using the SYSTAT software package (Version 8.0. 1988. SPSS Inc., Chicago). Allele frequencies across all collections were compared using the contingency χ2 test of Workman and Niswander (47). We assumed that an island model estimates the amount of gene flow (Nem) among populations as described previously (5,32). Genotype diversity in a population, based on comparisons of either multilocus haplotypes or DNA fingerprint patterns combined with multilocus haplotypes, was calculated using the measure proposed by Stoddart and Taylor (43). The maximum possible value for G$ , which occurs when each individual in the sample is unique, is the number of individuals in the sample. To compare G$ in collections with different sample sizes, we divided G$ from each collection by its sample size to calculate the percentage of maximum possible diversity that was obtained. A ttest was used for statistical comparisons between the normalized measures of G$ (11). The degree of gametic disequilibrium in each collection of isolates was evaluated using Brown's method (6) as described previously (49). RESULTS A total of 543 isolates was included in this study. Among these isolates, 366 distinct genotypes were identified. Isolates with the same genotype were often found within a 1 m diameter of a sampling station within a field. Exceptions to this pattern were seen in 10 shared MLHTs found at distances ranging from 10 to 40 m in the southeastern population in Norway (Buskerud) and the population in Finland (Jokioinen). Another exception was seen in the California collection, where some clones were found at a distance of up to 10 m apart. The population from central Norway (Stjørdal), which was collected using the fine-scale transect sampling method (sampling stations separated by 1 m), showed a relatively high degree of clonality (Table 1). This population shared one MLHT with the Finnish population. The California population also showed a relatively high degree of clonality compared with the other populations. Isolates in this collection were sampled every 0.5 m from two plots, each measuring 10 × 10 m (31). On average, the population from southeast Norway had 5.4 alleles per locus, the Finnish population had 5.0 alleles, and the population from central Norway had 4.9 alleles. We refer to these populations as the Nordic populations in the remainder of the paper. The Nordic populations possessed more private alleles (alleles found only in one location) than the other populations (Table 1). The fingerprint probe (pRS26) hybridized to many DNA fragments of different sizes in isolates from field populations in Australia and California (Fig. 1). We found that isolates with the same pRS26 hybridization pattern also had the same alleles at individual RFLP loci, suggesting that this probe was useful for direct differentiation of genotypes. But pRS26 did not exhibit a high degree of polymorphism in the Nordic isolates, hybridizing to fewer DNA fragments on average in the size range preferred for DNA fingerprinting (Figure 1). The average number of hybridizing fragments between 0.5 to 14 kb were 2.9 to 3.3 for the Nordic isolates and 5.5 to 6.1 for the Australian isolates. Allele frequencies were significantly different at most of the loci, both within and between regions (Table 2). Generally, all field populations showed high degrees of genetic variability. Nordic populations had nearly identical values of gene diversity (0.493 to 0.498; Table 1). Of the total gene diversity present in the Nordic
populations, 96% was present within field populations. Genotype diversity measures (43) varied between 21 and 85% of the theoretical maximum for the Nordic populations, 48 and 97% for the Australian populations, and 25% for the California population (Table 1). Genotype diversity measures differed significantly among 15 of 28 pairs of populations (Table 3). Measures of population differentiation (GST) were 0.121 between continents (based on seven shared loci), 0.075 among the Nordic populations (based on nine shared loci), and 0.048 among the Australian populations (based on eight shared loci). Hierarchical gene diversity analyses showed that the total gene diversity across all eight populations was 0.513. Genetic diversity among the continents contributed 9% of the total diversity. Within a continent, 4% of the total diversity was distributed among fields. Of the total diversity, 87% was found within a field. Of this value, 74% of the global diversity was distributed within a collection station of approximately 1 m2. The genetic distances among field populations were relatively small within continents and large between continents (Fig. 2). With the exception of the population from Wagga Wagga, Australia, all of the field populations were in gametic equilibrium (Table 4). DISCUSSION Populations from Norway and Finland. RFLP markers used in this study revealed a high degree of genetic variability in the three Nordic populations. Higher genotype diversity was observed in the population from southeast Norway and the Finnish population than in the population from central Norway. This is likely due to the different sampling strategy used to collect the isolates from central Norway. In the central Norway population, 12 MLHTs were found five or more times. In each case, these clones were sampled from the same region of the field. The shorter distance between sampling points increased the likelihood of finding the same clone resulting from splash-dispersal of conidia. We had a similar result when using fine-scale transect sampling in Australian populations of R. secalis (32). Though allele frequencies were significantly different at five of nine loci, genetic distances were low. Estimated GST values suggest that gene flow between the populations from Norway and Finland is sufficient to prevent divergence due to genetic drift. Nem calculated from GST ranged from 5 to 13 among the nine RFLP loci. One MLHT (111132111, using the same order of loci shown in Table 2) was shared among two isolates sampled from the populations of central Norway and Finland. Although these isolates shared the same alleles at nine RFLP loci, we do not believe that they represent a widely distributed clone because they exhibited different amplification patterns for four of eight primers that we tested using RAPDs (Fig. 3). In our previous work with Mycosphaerella graminicola, we found similar cases where iso-
lates sampled from populations separated by 1,000 km shared the same multilocus haplotype but had different DNA fingerprints (5). The genetic distance and population differentiation between the two Norwegian populations was larger than the differences between the Norwegian populations and the Finnish population (Fig. 2, Table 3). We believe these differences are due mainly to geographic isolation between central and southern Norway and higher gene flow between central Norway and Finland. Central Norway is isolated from southeastern Norway by extensive, high mountain ranges. Central Norway is separated from Finland by Sweden and the Baltic Sea. If regional similarities are due to movement of wind-dispersed spores, then movement of spores may be more likely between southeastern Norway and Finland than between southeast and central Norway. It also is possible that the observed genetic distances are due to differences in the historical movement of seed between these areas. The barley varieties cultivated in central Norway have been selected to mature earlier than the cultivars grown in southeastern Norway and Finland. Some Norwegian barley cultivars, such as Arve, are grown on a significant scale in Finland and central Norway. In 1996 and 1997, 20% of the barley grown in Finland was Arve. Arve is grown in southeastern Norway for seed production and then shipped to Finland for further seed increase and sale to the Finnish market. In addition, several Swedish cultivars with mildew resistance are grown in both southeastern Norway and Finland but not in central Norway because they mature too late for this area. Comparison of the R. secalis populations across continents. The genetic structures of the R. secalis populations in our sample were quite similar, even though they originated from three continents. Common RFLP alleles were shared across continents (Fig. 1), and allele frequencies often were surprisingly similar (Table 2). Gene diversity was high in all eight populations, and genotype diversity was high in six of the eight field populations studied. These findings were very similar to those reported by Goodwin et al. (16), who used isozyme markers to compare collections of isolates from the same geographic regions. There were significant differences in the normalized measures of genotype diversity among populations, but six of the eight field populations exhibited relatively high degrees of genotype diversity. We believe that the low genotype diversity in the central Norway and California populations was due mainly to the smaller spatial scale over which infected leaves were sampled in these fields. Although the overall genetic structure was similar for all populations, significant differences were observed in the frequencies of alleles at individual RFLP loci. An approximately equal number of genotypes was sampled in Nordic (clone-corrected, N = 151) and Australian (clone-corrected, N = 189) populations, but populations from Norway and Finland, on average, had one or two extra alleles per locus for the seven shared RFLP loci assayed.
TABLE 1. Information summary for eight populations of Rhynchosporium secalis from Norway, Finland, California, and Australiaa
No. of isolates No. of genotypes Average no. of alleles/locus No. of private alleles Gene
diversity c
Genotypic diversity (GD) d % of maximum GD a b c d
Southern Norway
Central Norway
Finland
California
Wagga Wagga
Methul
Rannock
Horsham
46 43 5.3 (5.4)b 6 (9) 0.46 (0.50) 39.2 85
140 45 4.3 (4.9) 4 (7) 0.45 (0.49) 29.4 21
82 63 4.4 (5.0) 2 (2) 0.46 (0.5) 51.7 63
52 26 3.1
66 49 2.6
57 51 3.1
61 60 2.6
39 29 2.5
1
1
1
0
1
0.55
0.55
0.52
0.51
13.0 25
31.7 48
45.6 80
59.2 97
0.51 21.5 55
Wagga Wagga, Methul, Rannock, and Horsham are Australian locations. Numbers in parentheses include restriction fragment length polymorphism data from probe pBD4, which was assayed only in the Nordic populations. Nei (35). Stoddart and Taylor (43). Vol. 90, No. 8, 2000
903
The higher allele diversity in Nordic populations compared with Australia may indicate that the Australian population was the result of a founder effect. Goodwin et al. (16) also found that Nordic populations had more diversity for isozyme loci than Australian populations. The relatively large number of private alleles suggests that gene flow has been restricted among continents. The average population differentiation (GST) among continents was moderate (0.12), providing additional evidence for restricted gene flow among continents. The Goodwin et al. (16) estimate of GST based on isozyme loci was slightly higher (0.16). The finding that probe pRS26 hybridized to fewer DNA fragments in Nordic isolates compared with isolates from Australia and
Fig. 1. Shared restriction fragment length polymorphism alleles in isolates of Rhynchosporium secalis originating from Australia, California, Norway, and Finland. Lamda HindIII size standards are shown in the final lane. A, Probe pRS37; B, Probe pRS47; C, Probe pRS2; and D, Probe pRS26, which hybridized to fewer fragments in the Nordic populations. 904
PHYTOPATHOLOGY
California (Fig. 1) is further evidence for restricted gene flow among continents. This finding also suggests that pRS26 may hybridize to a transposable element that is not active in the Nordic populations. The similarity among populations on a regional basis (within continents) suggests that gene flow is significant over spatial scales of at least several hundred kilometers. This hypothesis is supported by the finding that genetic distances among fields within regions were small, while genetic distances among fields from different regions were much larger (Figure 2). The mechanism for gene flow in this case could be infected seed, wind-blown straw, or ascospores from an undescribed teleomorph. A significant fraction of the total genetic variability was found within the smallest sampling area of approximately 1 m2. Only 9% of the total gene diversity was distributed among continents. We hypothesized previously (32), that pathogen populations exhibiting a genetic structure where a high percentage of genetic variability is distributed on a small spatial scale may adapt more quickly to changes in the environment, including introduction of new resistance genes. This hypothesis is consistent with the observed breakdown of major gene resistance to barley scald (13,22,25). Evidence for recombination in R. secalis populations. R. secalis has been classified as a deuteromycete. Despite many years of research by plant pathologists around the world, no one has identified a teleomorph for this pathogen. Other fungi that reproduce exclusively by asexual reproduction tend to have low genotype diversity arrayed as a limited number of clones or clonal lineages (e.g., Fusarium oxysporum [18], Colletotrichum graminicola, [39]) with corresponding high levels of multilocus associations (2,34). If R. secalis has a genetic structure typical of other asexual fungi, we would expect to find a significant degree of nonrandom association among unlinked loci (gametic disequilibrium) and a high degree of clonality within field populations (28,34). The observed genetic structure of the R. secalis populations in this survey are not consistent with an asexual pathogen. The primary infective propagules of R. secalis are thought to be conidia that are dispersed by rain splash (14). We expected that R. secalis populations would display a low level of genotype diversity within local populations and large degree of population differentiation among populations because of the limited potential for long-distance dispersal of conidia distributed by rain splash. We found little evidence for population subdivision over spatial scales of hundreds of kilometers. We considered two alternative hypotheses to explain our findings. One hypothesis is that genotypic diversity in R. secalis populations is due to regular cycles of parasexual recombination (36), and the genetic similarity among geographically separate populations is due to movement of infected seed or straw over distances of hundreds of kilometers (19,45). Parasexual recombination begins with the fusion of hyphae from two strains in the same vegetative compatibility group (VCG) forming a heterokaryon (37). The haploid nuclei from the two strains fuse to form a diploid nucleus. Homologous chromosomes in the diploid nucleus can undergo synapsis and recombination, leading to recombinant chromosomes if the homologs have different alleles. The diploid nucleus begins to lose chromosomes through cycles of mitotic divisions, forming an intermediate stage of partial diploidy for some chromosomes before eventually returning to the haploid state. During the Pontecorvo cycle, the heterokaryon and diploid or partially diploid nuclei represent intermediate stages that can be detected using codominant, neutral genetic markers, such as RFLPs and isozymes. In our initial round of data collection, we found 21 strains in the sample of 410 Australian isolates that had two different alleles for at least one RFLP locus, suggesting that these isolates were partial diploids. We prepared fresh single-spore cultures for each of these isolates and found that 16 of the isolates became regular haploids.
TABLE 2. Allele frequencies at restriction fragment length polymorphism (RFLP) loci in Rhynchosporium secalis field populations from Norway, Finland, California, and Australiaa Locus
Allele
Finland
pRS49
1 2 3 12 1 2 3 5 1 2 3 9 10 11 12 1 2 3 1 2 3 4 5 6 7 8 9 10 15 1 2 3 4 5 1 2 3 5 1 2 4 1 2 3 6 8 12 14
0.87 0.03 0.02 0.05 0.27 0.00 0.63 0.10 0.84 0.02 0.00 0.08 0.00 0.00 0.06 0.84 0.00 0.16 0.00 0.24 0.15 0.32 0.02 0.13 0.00 0.00 0.10 0.03 0.00 0.03 0.47 0.45 0.00 0.05 0.55 0.23 0.18 0.02 0.76 0.18 0.05 0.44 0.05 0.20 0.00 0.15 0.05 0.00
pRS37
pRS35
pRS47 pRS6
pRS36
pRS52
pRS2 PBD4
a b c
S.E. Norwayb 0.88 0.02 0.00 0.00 0.53 0.00 0.42 0.02 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.70 0.00 0.28 0.02 0.26 0.21 0.07 0.05 0.09 0.07 0.02 0.05 0.09 0.05 0.09 0.37 0.14 0.30 0.07 0.49 0.27 0.15 0.05 0.88 0.05 0.05 0.43 0.29 0.20 0.03 0.03 0.00 0.00
Central Norwayc
Wagga Wagga
Methul
Rannock
Horsham
California
0.31 0.58 0.02 0.00 0.38 0.00 0.62 0.00 0.89 0.00 0.00 0.02 0.00 0.00 0.09 0.87 0.00 0.13 0.00 0.13 0.07 0.18 0.02 0.22 0.13 0.00 0.07 0.16 0.00 0.00 0.62 0.31 0.00 0.07 0.76 0.16 0.04 0.00 0.73 0.24 0.00 0.32 0.27 0.14 0.09 0.05 0.02 0.05
1.00 0.00 0.00 0.00 0.68 0.00 0.32 0.00 0.42 0.42 0.00 0.02 0.09 0.05 0.00 0.47 0.21 0.32 0.00 0.00 0.00 0.61 0.00 0.16 0.00 0.23 0.00 0.00 0.00 0.00 0.53 0.37 0.00 0.08 0.07 0.62 0.31 0.00 0.47 0.53 0.00
1.00 0.00 0.00 0.00 0.75 0.00 0.25 0.00 0.45 0.46 0.05 0.00 0.02 0.00 0.00 0.56 0.07 0.37 0.00 0.04 0.09 0.39 0.00 0.33 0.00 0.16 0.00 0.00 0.00 0.05 0.36 0.54 0.00 0.05 0.11 0.65 0.24 0.00 0.21 0.79 0.00
0.97 0.03 0.00 0.00 0.82 0.00 0.18 0.00 0.57 0.33 0.05 0.03 0.02 0.00 0.00 0.49 0.11 0.40 0.00 0.00 0.00 0.51 0.00 0.16 0.00 0.33 0.00 0.00 0.00 0.08 0.25 0.67 0.00 0.00 0.07 0.62 0.32 0.00 0.59 0.41 0.00
1.00 0.00 0.00 0.00 0.56 0.00 0.44 0.00 0.65 0.12 0.00 0.03 0.18 0.00 0.00 0.54 0.00 0.46 0.00 0.03 0.00 0.15 0.00 0.21 0.00 0.62 0.00 0.00 0.00 0.03 0.45 0.53 0.00 0.00 0.05 0.62 0.33 0.00 0.68 0.32 0.00
0.87 0.07 0.07 0.00 0.46 0.27 0.27 0.00 0.92 0.08 0.00 0.00 0.00 0.00 0.00 0.58 0.04 0.38 0.67 0.07 0.07 0.07 0.07 0.04 0.00 0.00 0.00 0.00 0.00 0.73 0.23 0.04 0.00 0.00 0.92 0.04 0.04 0.00
Alleles with frequencies less than 2% are not shown. Wagga Wagga, Methul, Rannak, and Horsham are Australian locations. Southeastern Norway (Buskerud). Stjørdal
TABLE 3. Pairwise comparisons of genotype diversity a (above diagonal) and population differentiation b (below diagonal) among Rhynchosporium secalis populations from Norway, Finland, California, and Australia c,d S.E. Norwaye
Central Norwayf 5.56*** g (184)h
S.E. Norway
Finland 1.77 (125) 5.49*** (219)
California 4.91*** (96) 0.45 (190) 4.27*** (131)
Wagga Wagga 2.15* (106) 1.91 (200) 0.94 (141) 1.62 (112)
Methul 0.34 (101) 6.20*** (195) 1.59 (136) 5.27*** (107) 2.01* (111)
C. Norway
0.067
Finland
0.031
0.046
California
0.076
0.118
0.088
Wagga Wagga
0.115
0.164
0.096
0.162
Methul
0.142
0.191
0.122
0.156
0.034
Rannock
0.104
0.170
0.098
0.154
0.033
0.032
Horsham
0.084
0.140
0.069
0.171
0.070
0.064
Rannock 0.97 (105) 16.12*** (199) 4.26*** (140) 10.72*** (111) 3.51*** (121) 1.63 (116)
Horsham 1.95* (83) 3.03** (177) 0.54 (118) 2.56* (89) 0.42 (99) 1.79* (94) 3.64*** (98)
0.033
a b
Chen et al. (11). Nei (35). c Wagga Wagga, Methul, Rannock, and Horsham are Australian locations. d *, **, and *** indicate significance at P = 0.05, 0.01, and 0.001, respectively. e Southeastern Norway (Buskerud). f Stjørdal. g t test values. h Degrees of freedom. Vol. 90, No. 8, 2000
905
In these cases, we believe that the heterozygosity at RFLP loci was an artifact due to coculture of two genetically distinct strains that were present in the same mycelial tuft used to make the original isolation. The remaining five isolates were still heterozygous for at least one locus following the single-spore isolation procedure. This finding could be interpreted as evidence that parasexual recombination occurred in these five isolates and that we found an intermediate stage in the Pontecorvo cycle. This hypothesis requires further testing using pulsed-field gel electrophoresis to separate the chromosome complements of these five strains. If we confirm that these isolates are partial diploids, it would suggest that parasexual recombination is relatively rare but frequent enough to be a significant source of genotype diversity. A second hypothesis to explain the genotypic diversity within populations and similarity over regional spatial scales is that regular sexual recombination is occurring in these R. secalis populations and that ascospores from the unknown teleomorph are disseminated over distances of up to hundreds of kilometers. We consider this hypothesis to be more likely than the previous hypothesis for several reasons. First, we and other investigators, using codominant markers and large sample sizes, have not found other evidence for intermediate stages in the parasexual cycle. In a sample of 1,331 isolates from two California field populations (31), we did not find any partial diploids with four isozyme markers. Goodwin et al. also did not identify intermediate stages of the parasexual cycle in a sample of 1,025 isolates (17). If
parasexual recombination is common in nature and if recombinant parasexual strains produce stable intermediate forms, we would expect to find partial diploids in a significant fraction of the isolates we and others assayed previously. Another factor that decreases the likelihood for parasexual recombination is the fact that two isolates must be part of the same VCG to form a vegetative heterokaryon (this differs from the heterokaryon that occurs during regular sexual reproduction [27]). Such cases of vegetative compatibility are believed to be rare in most species under natural conditions (10) due to the occurrence of genetically determined vegetative incompatibility. Isolates with the same VCG possess matching alleles at several loci (27). Because it is unlikely for two randomly chosen isolates to have the same alleles at each of five to seven loci (unless the populations are largely clonal), isolates in the same VCG are often correctly assumed to be clones. However, our data show that few widespread clones exist in field populations of R. secalis, suggesting that only a small fraction of the R. secalis population is likely to have the same VCG. While we believe that anastomosis between strains with different RFLP alleles is not likely to occur at a high frequency, this hypothesis needs to be tested. There is now evidence that partial diploids may form during the process of meiosis in plant-pathogenic fungi. G. H. J. Kema (personal communication) found that a gene duplication was formed in the progeny of a cross between two Mycosphaerella graminicola isolates. This duplication probably was due to a translocation because probes for the alleles, which had identical DNA sequences, hybridized to different chromosomal bands on pulsed-field gel electrophoresis filters. McDonald and Martinez
Fig. 2. Phenogram derived by UPGMA (unweighted pair group method, arithmetic mean) based on Nei’s genetic distance (35) between eight populations of Rhynchosporium secalis from three continents.
TABLE 4. Multilocus associations among restriction fragment length polymorphism (RFLP) loci in Rhynchosporium secalis collections from Australia, Finland, Norway, and Californiaa Clone-correctedc Populationsb Wagga Wagga Methul Rannock Horsham Finland S.E. Norway C. Norway California a b c
No. of RFLP loci
N
(Sk2)
L
8 8 8 8 9 9 9 7
50 50 60 29 62 43 45 26
2.480* 2.044 2.098 2.514 1.260 0.293 1.072 0.566
2.328 2.231 2.243 2.565 2.560 2.239 2.594 2.669
Based on the measure of Brown et al. See reference 6. Wagga Wagga, Methul, Rannock, and Horsham are Australian locations; S.E. = southeastern; C. = central. N = population size. Sk2 = observed variance in number of heterozygous comparisons. L = upper 95% confidence limit. * indicates Sk2 exceeds L.
906
PHYTOPATHOLOGY
Fig. 3. Random amplified polymorphic DNA (RAPD) analysis showing different amplification patterns for isolates FF11 (Finland) and NKB43 (central Norway), which shared the same alleles at nine restriction fragment length polymorphism (RFLP) loci. Arrows indicate differences at four RAPD loci. Isolate FG41 differed from FF11 at six of nine RFLP loci but came from the same field. A, Amplicons based on Operon primer C8; B, amplicons based on Operon primer D2.
(30) also found a duplication and translocation at a RFLP locus in a natural field population of this fungus. M. graminicola is now known to undergo regular cycles of sexual reproduction in field populations (23,49). These findings suggest that the presence of partial diploids may not be sufficient evidence to support parasexual recombination. We consider it more likely that regular sexual recombination is the source of genetic diversity in field populations of R. secalis. R. secalis is known to produce microconidia (42), which act as spermatia or male gametes in some ascomycetes (12,41). Little attention has been paid to the function of this organ in the R. secalis life cycle. We believe it may play a role in sexual reproduction. We postulate that R. secalis has a teleomorph that has not yet been recognized and that this teleomorph plays a significant role in the epidemiology and population biology of this disease. A comparison of sequences from the internal transcribed spacer (ITS) region of the ribosomal DNA shows that R. secalis is most closely related to the Helotiales, suggesting that the perfect stage would form an apothecium (S. B. Goodwin, personal communication). The presence of a regular sexual cycle would explain our findings of low clonality and random associations among RFLP loci. If the ascospores of the putative teleomorph are dispersed by air, this could account for gene flow on a regional scale of tens or hundreds of kilometers. If these two aspects of the R. secalis life cycle are confirmed, this could explain why the genetic structure of R. secalis populations is so similar to the genetic structures of M. graminicola and Phaeosphaeria nodorum. The latter two fungi produce wind-dispersed ascospores and splash-dispersed conidia, and they have a genetic structure that is remarkably similar to R. secalis (33). The best way to test this hypothesis is through a field experiment to detect recombination between R. secalis isolates with known, rare alleles or to find the actual teleomorph in nature. ACKNOWLEDGMENTS S. Salamati received support from the Norwegian Research Council. Undergraduate students J. Bernard and F. Garcia contributed to the collection of RFLP data from Australia and California with support from National Science Foundation Grant DEB-9306377. R. Pettway supervised the undergraduate students with support from the Texas Agricultural Experiment Station. B. Read assisted with all collections made near the Wagga Wagga experiment station. D. Moody made field collections near Horsham. R. Heywood provided considerable technical assistance, and C. Bock offered useful advice. M. Jalli collected the sample from Finland. The Buskerud Extension Office (Buskerud forsøksring) provided the collection from southeastern Norway. S. Banke conducted the RAPD analysis of isolates sharing MLHTs. B. A. McDonald thanks the Australian Grains Research and Development Corporation for the support provided through a Visiting Fellowship (VF22). LITERATURE CITED 1. Ali, S. M., Mayfield, A. H., and Clare, B. G. 1976. Pathogenicity of 203 isolates of Rhynchosporium secalis on 21 barley cultivars. Physiol. Plant Pathol. 9:135-143. 2. Anderson, J. B., and Kohn, L. M. 1995. Clonality in soilborne, plantpathogenic fungi. Annu. Rev. Phytopathol. 33:369-391. 3. Beckwitt, R., and Chakraborty, R. 1980. Genetic structure of Pileolaria pseudomilitaris (Polychaeta: Spirorbidae) Genetics 96:711-726. 4. Bell, G. I., DeGennaro, L. J., Gelfand, D. H., Bishop, R. J., Valenzuela, P., and Rutter, W. J. 1977. Ribosomal RNA genes of Saccharomyces cerevisiae [yeasts]. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal. J. Biol. Chem. 252:8118-8125. 5. Boeger, J. M., Chen, R. S., and McDonald, B. A., 1993. Gene flow between geographic populations of Mycosphaerella graminicola (anamorph Septoria tritici) detected by restriction fragment length polymorphism markers. Phytopathology 83:1148-1154. 6. Brown, A. H. D., Feldman, M. W., and Nevo, E. 1980. Multilocus structure of natural populations of Hordeum spontaneum. Genetics 96:523-536.
7. Brown, J. S. 1985. Pathogenic variation among isolates of Rhynchosporium secalis from cultivated barley growing in Victoria, Australia. Euphytica 34:129-133. 8. Brown, J. S. 1990. Pathogenic variation among isolates of Rhynchosporium secalis from barley grass growing in South Eastern Australia. Euphytica 50:81-89. 9. Burdon, J. J., Abbott, D. C., Brown, A. H. D., and Brown, J. S. 1994. Genetic structure of the scald pathogen (Rhynchosporium secalis) in south east Australia: Implications for control strategies. Aust. J. Agric. Res. 45:1445-1454. 10. Caten, C. E., and Jinks, J. L. 1966. Heterokaryosis: Its significance in wild homothallic ascomycetes and fungi imperfecti. Trans. Br. Mycol. Soc. 49:81-93. 11. Chen, R. S., Boeger, J. M., and McDonald, B. A. 1994. Genetic stability in a population of a plant pathogenic fungus over time. Mol. Ecol. 3:209-218. 12. Dryton, F. L. 1934. The sexual mechanism of Sclerotinia gladioli. Mycologia 26:46-72. 13. Elen, O., 1987. Parallel development of scald virulence and distribution of varietal resistance in barley. Väkstskydrapporter: Jordbruk 48: 18-20. 14. Fitt, B. D. L., McCartney, H. A., and Walklate, P. J. 1989. The role of rain in dispersal of pathogen inoculum. Annu. Rev. Phytopathol. 27:241270. 15. Goodwin, S. B., Allard, R. W., Hardy, S. A., and Webster, R. K. 1992. Hierarchical structure of pathogenic variation among Rhynchosporium secalis populations in Idaho and Oregon. Can. J. Bot. 70:811-817. 16. Goodwin, S. B., Saghai Maroof, M. A., Allard, R. W., and Webster, R. K. 1993. Isozyme variation within and among populations of Rhynchosporium secalis in Europe, Australia and the United States. Mycol. Res. 97:49-58. 17. Goodwin, S. B., Webster, R. K., and Allard, R. W. 1994. Evidence for mutation and migration as sources of genetic variation in populations of Rhynchosporium secalis. Phytopathology 84:1047-1053. 18. Gordon, T. R., and Martyn, R. D. 1997. The evolutionary biology of Fusarium oxysporum. Annu. Rev. Phytopathol. 35:111-128. 19. Habgood, R. M. 1971. The transmission of Rhynchosporium secalis by infected barley seed. Plant Pathol. 20:80-81. 20. Habgood, R. M. 1973. Variation in Rhynchosporium secalis. Trans. Br. Mycol. Soc. 61:41-47. 21. Hansen, L. R., and Magnus, H. A. 1973. Virulence spectrum of Rhynchosporium secalis in Norway and sources of resistance in barley. Phytopathology 76:303-313. 22. Houston, B. R., and Ashworth, L. J. Jr.. 1957. Newly determined races of the barley scald fungus in California. Phytopathology 47:525. 23. Hunter, T., Coker, R. R., and Royle, D. J. 1999. The teleomorph stage, Mycosphaerella graminicola, in epidemics of Septoria tritici blotch on winter wheat in the UK. Plant Pathol. 48:51-57. 24. Jackson, L. F., and Webster, R. K. 1976. The dynamics of a controlled population of Rhynchosporium secalis changes in race composition and frequencies. Phytopathology 66:726-828. 25. Jones, E. R. L., and Clifford, B. C. 1991. Rhynchosporium of barley. U.K. Cereal Pathogen Virulence Survey. 1990 Annu. Rep.:41-43. 26. Jørgensen, H. J. L., and Smedegaard, P. V. 1995. Pathogenic variation of Rhynchosporium secalis in Denmark and sources of resistance in barley. Phytopathology 79:297-301. 27. Leslie, J. F. 1993. Fungal vegetative compatibility. Annu. Rev. Phytopathol. 31:127-150. 28. Leung, H., Nelson, R. J., and. Leach, J. E., 1993. Population structure of plant pathogenic fungi and bacteria. Adv. Plant Pathol. 10:157-205. 29. McDermott, J. M., McDonald, B. A., Allard, R. W., and Webster, R. K. 1989. Genetic variability for pathogenicity, isozyme, ribosomal DNA and colony color variants in populations of Rhynchosporium secalis. Genetics 122:561-565. 30. McDonald, B. A., and Martinez, J. P. 1991. Chromosome length polymorphisms in a Septoria tritici population. Curr. Genet. 19:265-271. 31. McDonald, B. A., McDermott, J. M., Allard, R. W., and Webster, R. K. 1989. Coevolution of host and pathogen populations in the Hordeum vulgare-Rhynchosporium secalis pathosystem. Proc. Natl. Acad. Sci. USA 86:3924-3927. 32. McDonald, B. A., Zhan, J., and Burdon, J. J. 1999. Genetic structure of Rhynchosporium secalis in Australia. Phytopathology 89:639-645. 33. McDonald, B. A., Zhan, J., Yarden, O., Hogan, K., Garton, J., and Pettway, R. E. 1999. The population genetics of Mycosphaerella graminicola and Phaeosphaeria nodorum. Pages 44-69 in: Septoria on Cereals: A Study of Pathosystems. J.A. Lucas, P. Bowyer, H.M. Anderson, eds. CAB International, Wallingford, UK. 34. Milgroom, M. G. 1996. Recombination and the multilocus structure of fungal populations. Annu. Rev. Phytopathol. 34:457-477. 35. Nei, M. 1973. Analysis of gene diversity in subdivided populations. Vol. 90, No. 8, 2000
907
Proc. Natl. Acad. Sci. USA 70:3321-3323. 36. Newman, P. L., and Owen, H. 1985. Evidence of asexual recombination in Rhynchosporium secalis. Plant Pathol. 34:338-340. 37. Pontecorvo, G. 1956. The parasexual cycle in fungi. Annu. Rev. Microbiol. 10:393-400. 38. Robinson, J., Lindqvist, H., and Jalli, M. 1996. Genes for resistance in barley to Finnish isolates of Rhynchosporium secalis. Euphytica 92:295-300. 39. Rosewich, U. L., Pettway, R. E., McDonald, B. A., Duncan, R. R., and Frederiksen, R. A. 1998. Genetic structure and temporal dynamics of a Colletotrichum graminicola population in a sorghum disease nursery. Phytopathology 88:1087-1093. 40. Salamati, S., and Tronsmo, A. M. 1997. Pathogenicity of Rhynchosporium secalis isolates on 30 cultivars of barley. Plant Pathol. 46:416424. 41. Shurtleff, M. C., and Averre, C. W. III, 1997. Glossary of PlantPathological Terms. The American Phytopathological Society, St. Paul, MN. 42. Skoropad, W. P., and Grinchenko, A. H. 1957. A new spore form in
908
PHYTOPATHOLOGY
Rhynchosporium secalis. Phytopathology 47:628-629. 43. Stoddart, J. A., and Taylor, J. F. 1988. Genotypic diversity: Estimation and prediction in samples. Genetics 118:705-711. 44. Tekauz, A. 1991. Pathogenic variation in Rhynchosporium secalis on barley in Canada. Can. J. Plant Pathol. 13:298-304. 45. Thomas, A. G., Gill, A. M., Moore, P. H. R., and Forcella, F. 1984. Drought feeding and the dispersal of weeds. J. Aust. Inst. Agric. Sci. 50:103-107. 46. Williams, R. J., and Owen, H. 1973. Physiologic races of Rhynchosporium secalis on barley in Britain. Trans. Br. Mycol. Soc. 60:223-234. 47. Workman, P. L., and Niswander, J. D. 1970. Population studies on Southwestern Indian tribes. II. Local genetic differentiation in the Papago. Am. J. Hum. Genet. 22:24-49. 48. Xue, G., and Hall, R. 1991. Pathogenic variation in Rhynchosporium secalis from southern Ontario. Plant Dis. 75:934-938. 49. Zhan, J., Mundt, C. C., and McDonald, B. A. 1998. Measuring immigration and sexual reproduction in field populations of Mycosphaerella graminicola. Phytopathology 88:1330-1337.
