Molecular epidemiology of white pine blister rust: recombination and spatial distribution

ABSTRACT Multilocus haplotypes (MLHs) were derived for the spermogonial (monokaryotic haploid) stage of Cronartium ribicola, the causal agent of white pine blister rust. Six random amplified polymorphic DNA loci and three single-strand conformational polymorphism markers were analyzed for 246 rust samples collected from two heavily infected white pine plantations. All cankers sampled were spatially located within the plantations. The hypothesis that spores are not locally disseminated was supported by the absence of any spatial clustering in the distribution of the MLHs. A large number of MLHs was found at both sites and the haplotypic diversity was close to the maximum (one) in both populations. All measures of recombination were not different from expectations under a scenario of sexual recombination. Genetic differentiation between the two sites was very low (theta = 0.023), yet it was significantly different from zero (P < 0.01). This analysis is in agreement with a scenario of extensive sexual recombination followed by some long-distance dispersal.     

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.     

White pine blister rust in north america: past and prognosis

ABSTRACT After a full century in North America, the blister rust epidemic has yet to stabilize, continuing to spread into warmer and drier areas previously considered climatically inhospitable. The disease apparently has no environmental limits wherever white pines and Ribes spp. cohabit and will eventually become pandemic. Although much timber value has been lost, more severe long-term damage is disruption caused to ecosystems by altered patterns of natural succession. During the last half of the century just past, development of genetic resistance superceded other direct control measures-mainly Ribes spp. eradication and antibiotics-which proved ineffective and/or unfeasible in large areas of the white pine range, especially in the West. Several mechanisms of complete (major gene) and partial resistance are common to at least several white pine species. Although North American populations of rust have low genetic variability overall, rust genotypes with specific virulence to major resistance genes exist in some local demes at high frequencies. The challenge will be to package and deploy resistance genes in ways that will dampen sudden increases in rust races of wide virulence. New introductions of blister rust from its gene center in Asia remain the gravest threat to genetic improvement programs.     

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.     

Genetic specificity in the white pine-blister rust pathosystem

ABSTRACT Four of eight white pine species native to western North America surveyed for resistance to white pine blister rust by artificial inoculation showed classical hypersensitive reactions (HR) at frequencies ranging from very low to moderate. Mendelian segregation, indicating a single dominant allele for resistance (Cr3), was observed in southwestern white pine (Pinus strobiformis), as it was previously in sugar pine (P. lambertiana, Cr1) and western white pine (P. monticola, Cr2). HR was present at a relatively high frequency (19%) in one of five bulk seed lot sources of limber pine (P. flexilis), and was also presumed to be conditioned by a single gene locus, by analogy with the other three species. HR was not found in whitebark pine (P. albcaulis), Mexican white pine (P. ayacahuite), foxtail pine (P. balfouriana), or Great Basin bristlecone pine (P. longaeva), but population and sample sizes in these species may have been below the level of detection of alleles in low frequency. When challenged by (haploid) inocula from specific locations known to harbor virulence to Cr1 or Cr2, genotypes carrying these alleles and Cr3 reacted differentially, such that inoculum virulent to Cr1 was avirulent to Cr2, and inoculum virulent to Cr2 was avirulent to Cr1. Neither of these two inocula was capable of neutralizing Cr3. Although blister rust traditionally is considered an exotic disease in North America, these results, typical of classic gene-for-gene interactions, suggest that genetic memory of similar encounters in past epochs has been retained in this pathosystem.     

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.     

Association of a novel Pinus monticola chitinase gene (PmCh4B) with quantitative resistance to Cronartium ribicola

Multiple families of pathogenesis-related (PR) proteins are believed to contribute to plant quantitative resistance to various pathogens. Along with other host PR proteins, PR3 chitinase is one protein component participating in genetic resistance of western white pine (Pinus monticola) to the white pine blister rust (WPBR) pathogen (Cronartium ribicola). In the present study, we characterized a novel P. monticola class IV chitinase gene (PmCh4B) and further analyzed its nucleotide variations in the open-pollinated seed families of diverse geographical distribution and variable levels of quantitative resistance to C. ribicola infection. PmCh4B showed high haplotype diversity (Hd=0.94) and nucleotide diversity (π=0.00965), similar to those of other conifer genes related to environmental stresses. A low level of intragenic linkage disequilibrium (LD) (but most of the levels with statistical significance) was found within a distance of ≈800 bp. Based on PmCh4B haplotype frequency, moderate to high levels of population structure were observed among P. monticola seed families currently used in breeding programs for WPBR resistance (average FST=0.163, P<0.001). Association analysis revealed that allelic variants and multiple single-nucleotide polymorphisms of PmCh4B were significantly associated with quantitative levels of P. monticola resistance against C. ribicola. This work represents the first association study for quantitative resistance in western white pine pathosystem and provides a potential for marker-assisted selection in white pine breeding.     

Low genetic variability in Sclerotinia sclerotiorum populations from common bean fields in Minas Gerais State,Brazil, at regional,local and micro‐scales

Sclerotinia sclerotiorum, causal agent of white mould, is the most destructive and widely distributed soilborne pathogen of common bean during the autumn–winter season in Brazil. Nevertheless, little is known about the genetic structure of the pathogen population. Microsatellite (SSR) markers and mycelial compatibility groups (MCGs) were used to characterize 118 isolates collected from 20 bean fields located in the most important growing regions of Minas Gerais State (MG). Additionally, the genetic variability among 10 isolates obtained from a single sclerotium was investigated in 10 different sclerotia. Seventy SSR haplotypes and 14 MCGs were identified among the 118 isolates. The genetic differences within bean growing areas accounted for most of the genetic variation (72%). Despite the relatively high genotypic diversity, the SSR loci were at linkage disequilibrium. Moreover, 70% of the isolates were assigned to only two MCGs, and haplotypes of a given MCG were closely related. The discriminant analysis of principal components revealed five groups. There was strong genetic differentiation between isolates collected in one municipality in southern MG when compared to other regions. Common bean resistance to white mould should be assessed with representative isolates of the five genetic groups and, if possible, of the different MCGs detected in the present study. One to five haplotypes were detected among the 10 isolates obtained from a single sclerotium. Therefore, in order to ensure genetic identity of an isolate, hyphal tip or monoascosporic isolates should be used.     

Genetic Diversity in Relation to Sexual and Asexual Reproduction in Populations of Melampsora larici-epitea

Genetic diversity of Melampsora larici-epitea leaf rust from three cultivated stands of the willow Salix viminalis was studied using AFLP polymorphisms at 60 loci. One population was located in Northern Ireland and two in Sweden. Analysis of molecular variance (AMOVA) showed that most of the genetic variation was distributed on a fine scale within the field in all populations. Both Swedish populations displayed a very high genotypic diversity (normalized Shannon's indices of 0.95 and 1.00) and random association among loci. These results suggested that sexual reproduction had an important influence on the Swedish populations. The occurence of the alternate host (larch) adjacent to one of the Swedish rust populations did not affect the genetic diversity. However, severe rust attacks started earlier in the season in this population. The M. larici-epitea population in Northern Ireland was characterized by a low genotypic diversity (normalized Shannon's index = 0.54) and non-random association among loci was indicated by test of multilocus association and by pairwise tests among loci. These results suggested that asexual reproduction had a major effect on the genetic structure of this population, probably because of the overwintering of asexual spores and/or a population bottleneck associated with the annual sexual phase.     

Development of genetic SSR markers in Blumeria graminis f. sp. hordei and application to isolates from Australia

The barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh), exists in numerous haplotypes and displays significant differences in fungicide sensitivity. It causes considerable yield losses throughout the world. Microsatellite SSRs are useful tools to study the population level and biogeographic aspects of intraspecific diversity, but so far none have been defined for Bgh. Here, eight polymorphic microsatellite loci were identified and characterized. Primer pairs amplifying the loci were then applied to 111 isolates of Bgh from Australia. The number of alleles per locus ranged from 4 to 13, and Nei's genetic diversity ranged from 0·25 to 0·76. The microsatellite primers detected several clones among the isolates and defined 97 unique haplotypes. There was little evidence for regional genotypic subdivision, suggesting that gene flow may not be restricted among geographic regions. All data was consistent with high levels of genetic diversity, potentially resulting from random mating and spread within each region.     

Cloning and Characterization of a Putative Antifungal Peptide Gene (Pm-AMP1) in Pinus monticola

ABSTRACT We have been working on proteins that are involved in the defense response of western white pine (WWP) (Pinus monitcola) to the blister rust fungus Cronartium ribicola. Our objective was to identify candidate genes that could be used for improving resistance of WWP to this rust pathogen. During proteomic analysis of bark proteins extracted from WWP trees exhibiting slow-canker-growth (SCG) resistance, a 10.6-kDa peptide, termed Pm-AMP1, was found to be enriched at the receding canker margin. The cDNA encoding this peptide was cloned and characterized. A BLASTX search revealed that the Pm-AMP1 encoded by its cDNA has a 50% homology with MiAMP1, a broad-spectrum antifungal protein isolated from Macadamia integrifolia. Based on the deduced amino acid sequence, an antibody was produced against the Pm-AMP1. Immunochemical quantification of the Pm-AMP1 in bark samples of susceptible WWP trees revealed this protein to be barely detectable in the cankered tissues, but occurring in higher concentrations in healthy tissues away from canker margins. Foliage of SCG-resistant trees contained higher concentrations of the Pm-AMP1 than foliage from susceptible cankered trees. Both wounding and methyl jasmonate treatment of WWP needles induced the expression of this protein, further supporting its putative role as a defense response protein.     

Gene cloning of a thaumatin-like (PR-5) protein of western white pine (Pinus monticola D. Don) and expression studies of members of the PR-5 group

To understand the molecular defence response of western white pine to the blister rust pathogen Cronartium ribicola, we have endeavoured to isolate and characterise pathogenesis-related (PR) proteins from western white pine. A full-length cDNA (Pin mTLP) was isolated from a cDNA library constructed from inoculated foliage. BLASTX search results indicated that the sequence shared a high degree of identity with the thaumatin-like proteins (TLPs) of the PR-5 family, and in particular with those of the ‘small-TLP’ subgroup mainly identified in monocots. The mature protein predicted from the cDNA sequence has a molecular mass of 16 kDa and pI of 4.1. Southern blot analysis showed that Pin mTLP is a single-copy gene member of a multi-gene family.Two-dimensional Western blot analysis of total western white pine protein extracts probed with a Douglas-fir anti-TLP antibody detected a major and a minor protein spot of 23 and 17 kDa, respectively. Both 23 and 17-kDa proteins were identified as TLPs by Nanospray MS/MS analysis. The 23-kDa protein was shown to accumulate in canker margins in the bark of infected seedlings indicating that it is locally induced in response to fungal invasion. Both local wounding and methyl jasmonate treatments induced expression of the protein, whereas salicylic acid treatment did not. These results suggest that the 23-kDa TLP plays a role in the molecular defence response of western white pine to wounding and pathogen attack.     

Identification and Characterization of Random Amplified Polymorphic DNA Markers Linked to a Major Gene (Cr2) for Resistance to Cronartium ribicola in Pinus monticola

ABSTRACT DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.     

Evidence of Cytoplasmic Inheritance of Virulence in Cronartium ribicola to Major Gene Resistance in Sugar Pine

ABSTRACT Tests for Mendelian segregation of virulence and avirulence in Cronartium ribicola, causal agent of white pine blister rust, to a major gene (R) for resistance in sugar pine were made using haploid basidiospore progenies from single diploid telia as inoculum on resistant genotypes. The telia were sampled from a small deme in the Siskyou Mountains of northern California, where a few mature sugar pines known to be Rr genotypes had become infected after withstanding the chronic blister rust epidemic for several decades and where intermediate frequencies of virulence in the ambient basidiospore population were subsequently measured. Infection type on inoculated seedlings with R was qualitative: all progenies of 81 single telia tested over 3 different years were either virulent (compatible) or avirulent (inducing hypersensitive necrosis), never a mixture of both reactions. The complete absence of heterozygotes in the telia population is strong evidence that virulence is not controlled by a nuclear gene. The data are consistent with earlier tests showing that basidiospore inoculum derived from aeciospores isolated from infected Rr trees produced mostly (>90%) virulent reactions on R- seedlings. The evidence indicates that transmission of virulence is uniparental via the cytoplasm of aeciospores. Exchange of spermatia between haploid thalli does not appear to be involved.     

Diversity and differentiation in two populations of Gibberella circinata in South Africa

Gibberella circinata [anamorph Fusarium circinatum (=  F. subglutinans f.sp. pini )] causes pitch canker and is an important pathogen in South African pine nurseries. The initial outbreak of the pitch canker fungus was limited to a single nursery at Ngodwana in Mpumalanga Province. Subsequently, several other pine nurseries in South Africa became infected. Most of these outbreaks were relatively small except for the outbreak in the Klipkraal nursery (Mpumalanga Province). The genetic diversity, population differentiation and relative frequencies of the sexual and asexual cycles among two South African subpopulations were determined to establish whether immigration, mutation and/or recombination contributed towards population structure. The allelic diversity of the initial population (Ngodwana) was observed to be lower (0·16) than that of the more recent Klipkraal population (0·25). Approximately 4% ( G _ST = 0·04) of total gene diversity could be attributed to differences among the subpopulations. Furthermore, six new vegetative compatibility groups (VCGs) have been identified since the initial outbreak of G. circinata in South Africa 10 years ago. The relatively low allelic diversity and low level of genetic differentiation suggest restricted gene flow among subpopulations, and indicate that the pathogen has been introduced recently. However, the amount of allelic and VCG diversity suggests that multiple genotypes have been introduced into South Africa. The increases in effective population number, allelic diversity and new VCGs over the past 10 years suggest that sexual reproduction might be occurring.     

Genotypic variability and aggressiveness of Bipolaris oryzae in the Philippines

The genotypic diversity of a collection of 352 isolates of Bipolaris oryzae obtained from 11 locations in the Philippines was estimated. The isolates could be divided into 50 haplotypes based on variation in microsatellite DNA with a moderately high genotypic diversity value of 0.88. Thirty nine haplotypes were represented by three or fewer isolates, whereas 80 % of the isolates belonged to only eight haplotypes, each containing 10 to 88 isolates indicating the prevalence of clonality. AMOVA revealed that the greatest variation was associated with the brown spot isolates collected within provinces (50.81 %), among varieties within provinces (48.17 %) and within ecosystems (49.33 %). Intensive sampling from a single field showed that the population was mostly clonal with about 98 % of the isolates belonging to a single VNTR haplotype. However, isolates within this haplotype exhibited a continuous range of aggressiveness when inoculated onto susceptible rice variety IR72. Several types of lesions were observed in the field during sampling, but the isolates obtained from each type of lesion produced a range of different lesion types when inoculated onto leaves of IR72, indicating that the type of lesion observed in the field was not related to the genotype of the pathogen. These results show that rice fields across the Philippines may contain B. oryzae isolates with considerable genotypic diversity, but an individual field may have both clonal and unique genotypes.     

Molecular,morphological and pathogenic diversity of Sclerotinia sclerotiorum isolates from common bean (Phaseolus vulgaris) fields in Argentina

White mould, caused by Sclerotinia sclerotiorum, is one of the most threatening fungal diseases occurring across major bean production regions worldwide. In Argentina, under favourable weather conditions, up to 100% seed yield losses occur on susceptible common bean cultivars. The aim of this study was to characterize the diversity of S. sclerotiorum isolates from six dry bean fields in the main production area of Argentina by means of molecular, morphological (mycelium colour, number and pattern of sclerotia distribution) and pathogenic approaches. Among 116 isolates analysed, high genotypic and morphological variability was observed. A total of 52 mycelial compatibility groups (MCGs) and 59 URPs (universal rice primers) molecular haplotypes were found. All the MCGs were location specific, while only 12% of the URP haplotypes were shared among locations. The molecular analysis of variance revealed a significant differentiation among populations, with higher genetic variability within the populations analysed than among them. The aggressiveness of the isolates towards bean seedlings was assessed in the greenhouse. Most of the isolates were highly aggressive, while no variation among locations was observed. The information generated in the present study provides, for the first time, information on the variability of S. sclerotiorum associated with white mould in the main common bean production area in Argentina. In addition, the findings suggest the occurrence of both clonal and sexual reproduction in the populations analysed. This work contributes to the development of sustainable management strategies in bean production aimed to minimize yield losses due to white mould.     

中国4省猕猴桃细菌性溃疡病菌的生物型检测和遗传多样性分析

由丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv. actinidiae (Psa)侵染引起的猕猴桃细菌性溃疡病(kiwifruit bacterial canker)是全球猕猴桃生产上最具毁灭性的细菌病害。为探明福建、安徽、四川和陕西4省Psa菌株的生物型和遗传多样性,用5对PCR特异性引物PsaJ-F/-R、PsaK-F/-R、Tac-F/-R、Con002-F/-R和avrRps4-F1/-R2检测Psa菌株的生物型;用4对PCR引物27F/1492R、PsaF1/PsaR2、gapA-Fps/Rps和rpoD+364s/-1222ps分别扩增16S rRNA、ITS、gapA和rpoD基因,进行多基因联合分析Psa菌株的遗传多样性。结果表明,特异性引物Tac-F/-R从47株Psa菌株中均能扩增出一条545 bp的特异条带,其他4对引物未扩增出任何条带,说明供试Psa菌株的生物型均为biovar 3。多基因联合分析表明,4省Psa存在丰富的遗传多样性,4个群体共检测出27个单倍型,单倍型多样性为0.955。安徽、福建、四川和陕西群体的单倍型数差异较大,分别为1、8、12个和12个。4个群体的多态性位点数、核苷酸多样性和平均核苷酸差异数差异极显著(P<0.01),其中福建群体的多态性最丰富,而安徽群体的多态性最低。AMOVA分析表明,3.6%的遗传变异来源于种群间,而96.4%的遗传变异来源于种群内,说明种群内变异是遗传变异的主要来源。遗传分化分析表明,安徽省Psa群体与其他3个群体间的遗传分化极高(Fst>0.175),福建、四川和陕西群体间的遗传分化水平较低(Fst<0.017)。研究结果有利于了解福建省Psa的来源,为阻断Psa的传播和猕猴桃细菌性溃疡病的长期可持续控制提供了理论参考。     

Measuring the Genetic Diversity of Xanthomonas axonopodis pv. manihotis Within Different Fields in Colombia

ABSTRACT Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis, is a widespread disease that affects cassava (Manihot esculenta). We collected 238 X. axonopodis pv. manihotis strains by intensively sampling single fields in four edaphoclimatic zones (ECZs) in Colombia. DNA polymorphism of different X. axonopodis pv. manihotis populations was assessed by restriction fragment length polymorphism (RFLP) analyses, repetitive sequence-based polymerase chain reaction (rep-PCR), and amplified fragment length polymorphism (AFLP) assays. Genetic diversity, phenetic relationships among strains, and the coefficient of genetic differentiation were determined. All strains were tested for aggressiveness on the susceptible cassava cv. MCOL 1522. Strains were also tested for virulence on cassava differentials adapted to the strains' respective ECZs. Our study showed that the Colombian X. axonopodis pv. manihotis population has a high degree of genetic diversity. The hierarchical analysis of diversity showed genotypic differentiation at all levels, among ECZs, among fields within ECZs, and among strains within fields planted to several cassava genotypes. New RFLP haplotypes were detected, leading to the characterization of a new pathotype. Dendrograms from AFLP were more robust than those from RFLP data. A close association between the strains' geographical origin and DNA polymorphism was obtained using RFLP and AFLP data. We suggest that the host played a role in causing pathogen differentiation.     

Genetic Structure of Melampsora Epitea Populations in Swedish Salix Viminalis Plantations

Amplified fragment length polymorphism (AFLP) was used to study the genetic structure of populations of the willow leaf rust, Melampsora epitea, in Swedish willow plantations. In total, 197 isolates collected from Salix viminalis clones in three locations in Sweden were analysed. AFLP profiles based on 83 markers were used to compute genetic distances between pairs of individuals. High levels of gene and genotypic diversity were detected in all populations, with 96% of the AFLP loci being polymorphic and with normalized Shannon's diversity indices ranging from 0.977 to 1.0. Analysis of molecular variance (AMOVA) showed small significant differences among locations, although most of the molecular variability was found within locations (97.5%). Five isolates from one willow clone in one location differed markedly from the common pattern. When these five exceptional isolates were excluded, no significant differences among willow clones were found with AMOVA. Sexual reproduction and spore migration appear to be important factors for the population genetic structure of this pathogen.