Random amplified polymorphic DNA (RAPD) analysis of Phytophthora infestans isolates collected in Canada during 1994 to 1996

Mating type, glucose-6-phosphate isomerase ( Gpi ) allozyme banding patterns, response to the fungicide metalaxyl and random amplified polymorphic DNA (RAPD) markers were used to characterize genetic variability among 141 Canadian isolates of Phytophthora infestans collected between 1994 and 1996. Multiple correspondence analysis of RAPD profiles separated isolates into 21 groups that were not correlated to groups defined by mating type, Gpi allozyme banding patterns or response to metalaxyl. Population subdivision analysis showed that 97% of the total genetic variation was found among individuals within populations, compared with 3% among populations. The average similarity coefficient among isolates was 80%. No significant differences in haplotypic diversity were observed among the years under study, but levels of genetic diversity among local populations of P. infestans were high (0.76). All classes of response to the fungicide metalaxyl were observed, with 55% of isolates displaying moderate levels of insensitivity. The high level of genetic diversity detected within populations indicates that migration and sexual recombination probably play important roles in the population biology of P. infestans in Canada.     

Genetic Variability of Canadian Populations of the Sapstain Fungus Ophiostoma piceae

ABSTRACT Genetic diversity was studied in seven Canadian populations of Ophiostoma piceae, the most prevalent sapstain fungus in Canadian softwoods. A total of 239 single-spore isolates were recovered following a systematic survey of sapstain fungi in logs and lumber at seven selected sawmills in six Canadian provinces (British Columbia, Alberta, Saskatchewan, Ontario, Québec, and New Brunswick). Sampling was carried out on five commercially important softwood species: balsam fir (Abies balsamea), white spruce (Picea glauca), black spruce (Picea mariana), jack pine (Pinus banksiana), and lodgepole pine (Pinus con-torta var. latifolia). The A and B mating types occurred at equal frequency (MAT A/ MAT B = 1.00:1.13) over all populations. Pseudo-allelic frequencies were estimated at each of 24 putative genetic loci by scoring for presence or absence of random amplified polymorphic DNA fragments generated by five primers. A total of 237 haplotypes were found among the 239 isolates, revealing a high level of genotypic diversity among isolates. Total gene diversity (H(T) = 0.414) was mostly attributable to diversity within populations (H(S) = 0.369). Thus, only 11.2% of the total variability was attributable to frequency differences among populations. An analysis of molecular variance revealed that most genetic variability occurred within subpopulations within mills (84.3%; P < 0.001), whereas low but statistically significant levels of genetic differentiation were also observed among subpopulations within populations (5.4%; P < 0.001) and among populations (10.3%; P < 0.001). Estimates of Nei' genetic distances were not correlated with geographic distances among sampling locations (r = -0.092; P = 0.310), although principal component analysis indicated that subpopulations located east of Saskatchewan were grouped on the same side of the second principal component axis. Overall, results suggest moderate genetic differentiation of O. piceae in Canada, which is consistent with the observation that sexual reproduction is frequently observed in this fungus.     

Genetic Diversity and Structure of the Apiosporina morbosa Populations on Prunus spp

ABSTRACT Populations of Apiosporina morbosa collected from 15 geographic locations in Canada and the United States and three host species, Prunus virginiana, P. pensylvanica, and P. padus, were evaluated using the sequence-related amplified polymorphism (SRAP) technique to determine their genetic diversity and population differentiation. Extensive diversity was detected in the A. morbosa populations, including 134 isolates from Canada and the United States, regardless of the origin of the population. The number of polymorphic loci varied from 6.9 to 82.8% in the geographic populations, and from 41.4 to 79.3% in the populations from four host genotypes based on 58 polymorphic fragments. In all, 44 to 100% of isolates in the geographic populations and 43.6 to 76.2% in populations from four host genotypes represented unique genotypes. Values of heterozygosity (H) varied from 2.8 to 28.3% in the geographic populations and 10.2 to 26.1% in the populations from four host genotypes. In general, the A. morbosa populations sampled from wild chokecherry showed a higher genetic diversity than those populations collected from other host species, whereas the populations isolated from cultivated chokecherry, P. virginiana 'Shubert Select', showed a reduction of genetic diversity compared with populations from wild P. virginiana. Significant population differentiation was found among both the geographic populations (P < 0.05) and populations from different host genotypes (P < 0.02). In the geographic populations, most of populations from cultivated and wild P. virginiana were closely clustered, and no population differentiation was detected except for the populations from Morris, Morden, and Winnipeg, Manitoba, Canada. Furthermore, the populations from P. virginiana in the same geographic locations had higher genetic identity and closer genetic distance to each other compared with those from different locations. Four populations from P. virginiana, P. pensylvanica, and P. padus, were significantly differentiated from each other (P < 0.02), except there was no differentiation between the Shubert Select and wild chokecherry populations (>P> = 0.334). Indirect estimation of gene flow showed that significant restricted gene flow existed between populations from different regions and host species. Gene flow rates (Nm) varied from <1 to 12.5, with higher gene flow rates among population pairs from the same host species (P = 1.000). The analysis of molecular variance revealed that a major genetic variance source came from the genetic variation among isolates within populations regardless of the origin and host genotype of the population. Although some locations had a limited number of isolates, the results of this study clearly showed that the genetic diversity and population differentiation of A. morbosa were closely associated with host genotypes and geographic locations, but mostly with the former.     

Population Genetic Structure of Septoria passerinii in Northern Great Plains Barley

ABSTRACT The genetic structure of Septoria passerinii from nine field populations was examined at several scales (within lesions, among lesions in a leaf, among leaves in a field, and among fields in North Dakota and western Minnesota) by using amplified fragment length polymorphism (AFLP) markers. A total of 390 isolates were sampled from seven barley fields located in North Dakota and two barley fields located nearby in western Minnesota in 2003 and 2004. Based on 57 polymorphic AFLP markers, AFLP DNA fingerprints identified 176 different genotypes among 390 (non-clone-corrected) isolates in nine different fields. In two intensively sampled sites, ND16 (Williston, ND) and ND17 (Langdon, ND), only one to four different genotypes were found within a lesion. A higher level of genetic and genotypic diversity was found within a leaf in which six to nine different genotypes were found from lesions on a leaf. The genetic diversity within a leaf was similar to the genetic diversity within a field. The average genetic diversity (H) within a field across all AFLP loci was approximately 0.3, except at site ND12 (Carrington, ND) where it was 0.16. Genotypic diversity was high in all populations, and with the exception of ND15 (Rothsay, MN), very low multilocus linkage disequilibrium values ( r(d)) were found in all populations. The population differentiation, G(ST), was relatively high (G(ST) = 0.238) among the nine populations due to the high G(ST) in ND12, ND14 (Twin Valley, MN), and ND15. Population differentiation without those three populations was 0.09. A lack of correlation between geographical distance and genetic distance was found, suggesting the potential for a high level of gene flow between different geographical regions. The population genetic structure described in this study for S. passerinii in North Dakota and western Minnesota is consistent with that of a sexually reproducing fungus.     

Etiology and Population Genetics of Colletotrichum spp. Causing Crown and Fruit Rot of Strawberry

ABSTRACT Isolates of Colletotrichum spp. from diseased strawberry fruit and crowns were evaluated to determine their genetic diversity and the etiology of the diseases. Isolates were identified to species using polymerase chain reaction primers for a ribosomal internal transcribed spacer region and their pathogenicity was evaluated in bioassays. Isolates were scored for variation at 40 putative genetic loci with random amplified polymorphic DNA and microsatellite markers. Only C. acutatum was recovered from diseased fruit. Nearly all isolates from crowns were C. gloeosporioides. In crown bioassays, only isolates of C. gloeosporioides from strawberry caused collapse and death of plants. A dendrogram generated from the genetic analysis identified several primary lineages. One lineage included isolates of C. acutatum from fruit and was characterized by low diversity. Another lineage included isolates of C. gloeosporioides from crowns and was highly polymorphic. The isolates from strawberry formed distinctive clusters separate from citrus isolates. Evaluation of linkage disequilibrium among polymorphic loci in isolates of C. gloeosporioides from crowns revealed a low level of disequilibrium as would be expected in sexually recombining populations. These results suggest that epidemics of crown rot are caused by Glomerella cingulata (anamorph C. gloeosporioides) and that epidemics of fruit rot are caused by C. acutatum.     

Genetic Diversity and the Presence of Two Distinct Groups in Ophiostoma clavigerum Associated with Dendroctonus ponderosae in British Columbia and the Northern Rocky Mountains

ABSTRACT The sapstaining fungal pathogen Ophiostoma clavigerum is associated with the mountain pine beetle (Dendroctonus ponderosae), which is currently the most destructive forest pest in North America. The genetic diversity of O. clavigerum populations collected from five sites in Canada and two sites in the United States was estimated with amplified fragment length polymorphism (AFLP) analysis. Genomic DNA from 170 O. clavigerum isolates was digested with EcoRI and PstI and amplified with six primer sets. A total of 469 AFLP markers consisting of 243 monomorphic and 226 polymorphic loci were scored. The overall genetic diversity of the O. clavigerum population was low (Hs = 0.0531) and the differentiation of the seven O. clavigerum populations was moderate (Phi = 0.143). Genetic distances among the populations were not significantly correlated with geographic distance (r = 0.3235, P = 0.074). Two genetically distinct groups in the O. clavigerum populations were shown by unique AFLP profiles and the unweighted pair group method with arithmetic averages. Further work to characterize biological differences between the two groups will be needed to confirm whether cryptic species are present in the O. clavigerum population.     

Genetic Structure of Cronartium ribicola Populations in Eastern Canada

ABSTRACT The genetic structure of populations of Cronartium ribicola was studied by sampling nine populations from five provinces in eastern Canada and generating DNA profiles using nine random amplified polymorphic DNA markers. Most of the total gene diversity (H(t) = 0.386) was present within populations (H(w) = 0.370), resulting in a low level of genetic differentiation among populations in northeastern North America (F(st) = 0.062). A hierarchical analysis of genetic structure using an analysis of molecular variance (AMOVA) revealed no statistically significant genetic differentiation among provinces or among regions. Yet, genetic differentiation among populations within regions or provinces was small (AMOVA phi(st) = 0.078) but statistically significant (P < 0.001) and was several orders of magnitude larger than differentiation among provinces. This is consistent with a scenario of subpopulations within a metapopulation, in which random drift following migration and new colonization are major evolutionary forces. A phenetic analysis using genetic distances revealed no apparent correlation between genetic distance and the province of origin of the populations. The hypothesis of isolation-by-distance in the eastern populations of C. ribicola was rejected by computing Mantel correlation coefficients between genetic and geographic distance matrices (P > 0.05). These results show that eastern Canadian provinces are part of the same white pine blister rust epidemiological unit. Nursery distribution systems are controlled provincially, with virtually no seedling movement among provinces; therefore, infected nursery material may not play an important role in the dissemination of this disease. Long-distance spore dispersal across provincial boundaries appears to be an epidemiologically important factor for this pathogen.     

不同干扰生境中荒漠小灌木红砂种群遗传多样性研究

应用RAPD标记技术对荒漠小灌木红砂(Reaumuria soongorica)种群在不同扰动下的遗传多样性进行了分析。18条随机引物对红砂6个种群的120个个体进行扩增,共检测102个位点,其中多态位点99个。研究结果表明:红砂种群的多态位点比率(P)为96.86%,显示了不同生境中红砂种群内存在较高的遗传多样性。Shannon多样性指数(0.5007)、Nei基因多样性指数(0.3307)和基因分化系数(Gst=0.1952)揭示了红砂种群遗传变异多存在于种群内,种群间的遗传分化则较小。聚类分析表明:红砂种群遗传距离与地理距离之间存在一定相关性;遗传多样性水平与物种特性和不同干扰生境有关,与生态因子无相关性。     

Trichothecene profiling and population genetic analysis of Gibberella zeae from barley in North Dakota and Minnesota

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.     

Genetic variability of Colletotrichum lindemuthianum in wild populations of common bean

The genetic structure of wild populations of Colletotrichum lindemuthianum was evaluated for virulence and molecular markers. Forty-five isolates were collected from five wild common bean populations located in their South-Andean centre of origin. The five pathogen populations were monomorphic in their ITS regions, but 45 polymorphic markers were identified using RAPDs. Polymorphism for virulence was also observed; 15 pathotypes were characterized on an international set of 12 differentials. A molecular variance analysis ( AMOVA ) showed that a very large part of the total genetic variation was within populations. Statistical analysis showed that there was a weak though significant differentiation among the five populations for the RAPD and virulence markers. A positive and significant correlation was found between geographic distance and the distances from RAPD and virulence data, suggesting migration between adjacent populations along the Argentinian transect. Our results suggest that the Andean wild isolates of C. lindemuthianum do not reflect all the putative diversity found in the isolates from cultivated common bean.     

Gene and Genotypic Diversity of Phytophthora cinnamomi in South Africa and Australia Revealed by DNA Polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D_m=0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D_m South Africa=0.020 and D_m Australia=0.025 respectively), negative fixation indices, and significant deviations from Hardy–Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.     

Genetic variation in populations of the cacao wilt pathogen, Ceratocystis cacaofunesta

Ceratocystis cacaofunesta (=  Ceratocystis fimbriata ) causes a lethal wilt disease of cacao ( Theobroma cacao ) in Latin America. Polymorphic microsatellite markers, (CAT)₅ nuclear DNA fingerprints and Hae III mitochondrial DNA fingerprints were used to compare genetic diversity among isolates of C. cacaofunesta collected from populations in western Ecuador, Costa Rica, Colombia, and Rondônia and Bahia in Brazil. Microsatellite markers and nuclear DNA fingerprints separated Ecuadorian isolates from isolates of the other four populations, and these two major groups correspond to genetic lineages already identified from ITS-rDNA sequences and intersterility groupings. Mitochondrial DNA fingerprints also demonstrated substantial diversity and split the Ecuadorian isolates into two groups. All marker types showed limited variation in the Colombian, Costa Rican and Bahian populations, as might be expected for introduced populations that have gone through recent genetic bottlenecks. In contrast, the Rondonian and western Ecuadorian populations showed gene diversity values similar to natural populations of other Ceratocystis species. The Rondonian population was the only sampled population in the native range of T. cacao (the Upper Amazon), and the putatively introduced populations were more closely related to the Rondonian population than to the western Ecuadorian population. The Ecuadorian population is in an area with other native Theobroma species, which may serve as natural hosts.     

Genetic diversity and differentiation of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> populations in sunflower

Ninety-six isolates of sunflower Sclerotinia sclerotiorum (Lib.) de Bary from Inner Mongolia (IM) in China, from Canada and the United Kingdom (UK) were sampled to investigate the genetic diversity and structure using Sequence-Related Amplified Polymorphism. A total of 123 polymorphic bands were obtained, ranging in size from 100 to 500 base pairs. The five populations of S. sclerotiorum isolated from the three countries showed various levels of genetic variability. The percentage of polymorphic loci varied from 30.89% in the UK population to 97.56% in the Middle IM population. The values of Shannon index (i) varied from 0.1876 in the UK population to 0.5301 in the West IM population. The heterozygosity of the five geographic populations obtained by estimating allele frequency varied from 12.91% in the UK population to 35.44% in the West IM population. The genetic identity, as indicated by the Nei unbiased identity index, ranged from 0.9744 between populations from Canada and East IM to 0.6477 between populations from West IM and UK. UPGMA cluster analysis using Nei’s genetic distance gave distances ranging from 0.0259 to 0.4343. The rates of gene flow among five geographic populations ranged from 1.5406 between West IM and UK populations to 18.4149 between West IM and Middle IM populations. The four populations from West IM, Middle IM, East IM and Canada were clustered into one subgroup in which the isolates from West and Middle IM belonged to one population, whereas those from East IM and Canada essentially were another population. The isolates from the UK formed a population that was significantly distinct from other populations.     

Microsatellite and Minisatellite Analysis of Leptosphaeria maculans in Australia Reveals Regional Genetic Differentiation

ABSTRACT The population genetic structure of the fungal pathogen Leptosphaeria maculans was determined in Australia using six microsatellite and two minisatellite markers. Ascospores were sampled from Brassica napus stubble in disease nurseries and commercial fields in different sites over 2 years. The 13 subpopulations of L. maculans exhibited high gene (H = 0.393 to 0.563) and genotypic diversity, with 357 haplotypes identified among 513 isolates. Although the majority of genetic variation was distributed within subpopulations (85%), 10% occurred between the regions of eastern and Western Australia, and 5% within regions. F(ST) analysis of subpopulation pairs also showed the east-west genetic differentiation, whereas factorial correspondence analysis separated Western Australian subpopulations from eastern ones. Bayesian model-based population structure analyses of multilocus haplotypes inferred three distinct populations, one in Western Australia and an admixture of two in eastern Australia. These two regions are separated by 1,200 km of arid desert that may act as a natural barrier to gene flow, resulting in differentiation by random genetic drift. The genetic differentiation of L. maculans isolates between eastern and Western Australia means that these regions can be treated as different management units, and reinforces the need for widespread disease nurseries in each region to screen breeding lines against a range of genetic and pathogenic populations of L. maculans.     

Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa

Restriction fragment length polymorphisms (RFLP) of the intergenic spacer region (IGS) of rDNA and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability among 181 isolates of Sclerotinia homoeocarpa from Ontario and 10 isolates from Japan. RAPD and IGS-RFLP analyses revealed polymorphisms within and between populations of S . homoeocarpa , distinguishing 151 genotypes. Both types of markers gave similar results in phenetic analysis of genetic distances between populations. Cluster analysis showed that Japanese isolates of S. homoeocarpa were genetically distinct from Ontario isolates, demonstrating significant intraspecific differentiation. An average genetic similarity of 0.66 was found between Japanese isolates. Among Ontario isolates, average genetic similarity was 0.86, and genotypic diversity analysis showed that 49.3% of the total genetic variation observed within Ontario populations occurred among individuals within populations compared to 50.7% between populations. Gametic linkage disequilibrium analysis within Ontario populations revealed an average 15.6% significant nonrandom associations between putative RAPD loci, and that half of the populations showed signs of significant linkage disequilibrium. These results suggest that both clonal propagation and recombination events occurred in local populations of S. homoeocarpa . The high level of genetic similarity between populations and the low levels of intraspecific genetic variation may reflect a small founding population for southern Ontario isolates of S. homoeocarpa .     

Influence of host resistance on the genetic structure of the white pine blister rust fungus in the western United States

Cronartium ribicola, the causal agent of white pine blister rust, has been devastating to five-needled white pines in North America since its introduction nearly a century ago. However, dynamic and complex interactions occur among C. ribicola, five-needled white pines, and the environment. To examine potential evolutionary influences on genetic structure and diversity of C. ribicola in western United States, population genetic analyses of C. ribicola were conducted using amplified fragment length polymorphism (AFLP) molecular markers. The fungus was sampled at six sites. Collections for two of the six sites were from separate plantings of resistant-selected western white pine and sugar pine. Heterozygosity based on polymorphic loci among populations ranged from 0.28 to 0.40, with resistant-selected plantations at the extremes. Genetic differentiation was also highest between these two populations. Principal coordinates analysis and Bayesian assignment placed most isolates that are putative carriers of virulence to major-gene resistance into a discernable cluster, while other isolates showed no clustering by site or host species. These results indicate that C. ribicola in western North America is not genetically uniform, despite its presumed single site of introduction and relatively brief residence. Moreover, major-gene resistance appears to have imposed strong selection on the rust, resulting in reduced genetic diversity. In contrast, no evidence of selection was observed in C. ribicola from hosts that exhibit only multigenic resistance.     

Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses

Two molecular genetic screening techniques, RAPD (random amplified polymorphic DNAs) and ISSR (inter-simple sequence repeats), were applied to detect the level and pattern of genetic diversity of Monochoria vaginalis, a common weed of rice fields, in seven populations from southern China. Among these populations, 116 bands were amplified by 18 RAPD primers, of which 34 bands (29.31%) were polymorphic, and 14 ISSR primers produced 111 bands with 87 polymorphic bands (78.38%). Within each population, a relatively low level of genetic diversity was detected by both RAPD and ISSR analyses, with a mean genetic diversity (H) of 0.0348 and 0.0551 respectively. Analysis of molecular variance of the data from the RAPD and ISSR markers detected that the majority of total genetic variation existed among populations (73.50% and 76.70% respectively) and only minor genetic variation within populations (26.50% and 23.30% respectively). Cluster analysis divided the seven populations into two groups, indicating that the genetic relationships among populations have relatively low correlation with their geographical distribution (Mantel test; r = 0.45 and 0.48 respectively). Our results indicated that both RAPD and ISSR markers were effective and reliable for accurately assessing the degree of genetic variation of M. vaginalis. Comparing the two techniques, ISSR markers were more efficient than the RAPD assay. The Mantel test gave r = 0.16, suggesting no correlation between these two molecular markers.     

Population Structure of Botryosphaeria dothidea from Pistachio and Other Hosts in California

ABSTRACT Genetic diversity was investigated among California populations of Botryosphaeria dothidea, causal agent of Botryosphaeria panicle and shoot blight of pistachio, with random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction (MP-PCR). We surveyed 120 isolates, 112 of which originated from the California Central Valley and included pistachio isolates (n = 52) and isolates from other plant species (n = 60). Out-group isolates (n = 8) were obtained from pistachio in Greece. There was a strong correlation (r = 0.99; P < 0.0001) between the RAPD- and MP-PCR dissimilarity data sets. Little genetic variation (haplotypic diversity [Hs] < 0.002) was detected among B. dothidea isolates collected from central and southern California pistachio plantings. We observed relatively high diversity for isolates from a northern California pistachio orchard (Hs = 0.0146), where the disease was first diagnosed, and from the Chico U.S. Department of Agriculture Germ Plasm Repository (Hs = 0.0726), where the first pistachio trees were planted in California in 1929. Isolates obtained from other hosts, especially those associated with the rare occurrence of the sexual stage of this fungus, showed the highest levels of diversity (Hs = 0.1689). Thirty-eight pistachio isolates (73.1%) had DNA fingerprints identical to 28 pycnidiospore-derived isolates (56.0%) obtained from other host species. Greenhouse inoculations demonstrated that all isolates obtained from other hosts were capable of infecting pistachio and produced characteristic disease symptomology. Thus, California populations of B. dothidea from pistachio are, for the most part, genetically uniform, with the sexual stage rare to absent. However, the rare occurrence of the sexual stage of B. dothidea on other hosts, and more importantly, the capacity of these isolates to infect pistachio, indicate that other host species may serve as sources of inoculum and genetic variation.     

Virulence and Molecular Diversity in Cochliobolus sativus

ABSTRACT Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is an important disease of barley in many production areas of the world. To assess genetic diversity in this pathogen, a worldwide collection of C. sativus isolates was evaluated for virulence on barley and DNA polymorphism. Three pathotypes (0, 1, and 2) were identified among the 22 isolates tested in this study and the 36 isolates characterized previously on three barley differentials (ND5883, Bowman, and NDB112) that differ in their resistance to C. sativus. Pathotype 2, which exhibits high virulence on cv. Bowman, was only found in North Dakota, whereas the other two pathotypes occurred in many other regions of the world. Genetic diversity of the 58 C. sativus isolates, together with isolates of three related pathogenic Cochliobolus spp. (C. heterostrophus, C. carbonum, and C. victoriae) was analyzed using amplified fragment length polymorphism (AFLP) markers. A total of 577 polymorphic AFLP markers were recorded among the 70 isolates of the four Cochliobolus spp. using eight primer combinations. Cluster analysis revealed distinct groups corresponding to the four different species, except in one case where race 0 of C. carbonum was placed in an outgroup that may belong to a different species. In C. sativus, 95 polymorphic AFLP markers were detected with the eight primer pairs used, and each isolate exhibited a unique AFLP pattern. Allelic diversity in the pathotype 2 group was lower (0.10) than in the pathotype 0 (0.23) and pathotype 1 (0.15) groups, indicating that pathotype 2 may have arisen more recently. Cluster analysis did not reveal a close correlation between pathotypes and AFLP groups, although two AFLP markers unique to pathotype 2 isolates were identified. This low correlation suggests that genetic exchange may have occurred through parasexual recombination in the fungal population. Some isolates collected from different regions of the world were clustered into the same AFLP group, suggesting that migration of the fungal pathogen around these regions has occurred.     

Identification of Microsatellite Markers and Their Application to Population Genetics of Venturia inaequalis

ABSTRACT Microsatellite markers of Venturia inaequalis were developed using genomic libraries of V. inaequalis enriched for the simple sequence repeats (TC)(n) and (AAC)(n). Seven markers, three with (TC)(n) repeats and four with (AAC)(n) repeats, were selected for the analyses of 350 isolates of V. inaequalis collected from 11 sites in Europe. Polymorphism in the (TC)(n) repeats was higher than in the (AAC)(n) repeats. Nei's expected genetic diversity (H(E)) varied between 0.52 and 0.96 for the microsatellites containing (TC)(n) stretches and between 0.09 and 0.36 for the microsatellites containing (AAC)(n) stretches. Within-population diversity (H(S)) was very high with values ranging from 0.28 to 0.49, whereas differentiation among all European populations (G(ST)) was low with an average of 0.07. In the population from Ahrensburg (northern Germany) where isolates were mainly collected from apple varieties carrying the Vf gene, usually resistant to V. inaequalis, we showed a bottleneck effect with reduced diversity and loss of alleles. The great advantages of microsatellite markers over random amplified polymorphic DNA and polymerase chain reaction-restriction fragment length polymorphism markers are their high specificity, high polymorphism, good reproducibility, and unambiguous scorability.