Clonal Spread of Sclerotium cepivorum in Onion Production with Evidence of Past Recombination Events.

Three field populations of Sclerotium cepivorum from Ontario and Que-bec in Canada, one field sample from the North Island of New Zealand, as well as isolates from other onion- and garlic-growing areas were classified in mycelial compatibility groups (MCGs) and further characterized by DNA fingerprints and DNA sequence polymorphisms at six genomic regions. MCGs in the population samples were genetically distinct clones. Members of a clone belonged to a unique MCG with an associated multi-locus genotype and DNA fingerprint. The structure in each field population was predominantly clonal based on the repeated sampling of genotypes over a wide geographic area. Only three clones were identified within each of the population samples. The hypothesis that these clones were introduced to the three agricultural production areas is supported by their recovery from additional locations. Some evidence for past recombination was detected when genotypes from the three population samples and from other locations were pooled.

Phylogeography and Genotype-Symptom Associations in Early and Late Season Infections of Canola by Sclerotinia sclerotiorum.

ABSTRACT Both typical late season stem infections and atypical early season rosette infections of canola, a relatively new crop in the southeastern United States, were caused by Sclerotinia sclerotiorum. The 51 DNA fingerprints (from 71 isolates) did not match any fingerprints from previous studies of canola or other crops. Single locus haplotypes from nuclear DNA sequences included 18 in the intergenic spacer (IGS) of the rRNA repeat, four in 44.11, six in translation elongation factor 1alpha, three in calmodulin (CAL), and two in chitin synthase 1. Contingency permutation testing for associations of infection type with DNA fingerprint, single- or multilocus haplotype, or hierarchically nested clades based on single locus haplotypes found significant association of haplotype with mycelial compatibility group and DNA fingerprint for all loci except CAL. Significant association of IGS haplotypes with symptom type was detected in one pathogen population. Southeastern U.S. canola was infected by both recently evolved, geographically dispersed pathogen genotypes and older, indigenous genotypes (Carbone and Kohn, 2001. Mol. Ecol. 10:947-964). Indigenous haplotypes are infection-type generalists, and the most frequently isolated from rosette infections. In contrast, haplotypes from the most recently evolved, dispersed population were associated one-to-one with infection type, with only the most recently evolved haplotypes infecting rosettes.

Clonality in Sclerotinia sclerotiorum on Infected Cabbage in Eastern North Carolina.

ABSTRACT Eighty-four isolates of Sclerotinia sclerotiorum from four cabbage production fields in North Carolina and 16 isolates from an experimental cabbage field plot in Louisiana were DNA-fingerprinted and tested for mycelial compatibility. In a comparison with 594 unique DNA fingerprints of S. sclerotiorum from Canadian canola, no fingerprints were shared among Canadian, North Carolina, and Louisiana populations. DNA fingerprints from the North Carolina sample were distinctive from those of the Canadian and Louisiana samples, with significantly more hybridizing fragments in the 7.7- to 18-kilobase range. Forty-one mycelial compatibility groups (MCGs) and 50 unique DNA fingerprints were identified from the North Carolina sample. Three MCGs and three fingerprints were identified from the Louisiana sample. From the North Carolina sample, 32 MCGs were each associated with a unique fingerprint; of these, there were 11 clones (i.e., cases in which two or more isolates belonged to the same MCG and shared the same DNA fingerprint). Six clones sampled from two or more fields represented approximately 29% of the total sample (24 of 84 isolates), with six clones recovered from fields 75 km apart. There were 10 cases in which one MCG was associated with more than one DNA fingerprint and two cases in which one DNA fingerprint was associated with more than one MCG. The small sample from Louisiana was strictly clonal. The North Carolina sample had a clonal component, but deviated from one-to-one association of MCG with DNA fingerprint to an extent consistent with more recombination or transposition than the other two populations sampled.

Genetic Interactions Between Glycine max and Sclerotinia sclerotiorumUsing a Straw Inoculation Method.

Genetic interactions for disease response between cultivars of Glycine max and isolates of Scle-rotinia sclerotiorum were evaluated in controlled-environment inoculations of five soybean cultivars with four genetically unique isolates of S. sclerotiorum. The objective of this study was to identify host-pathogen interactions using isolates of the pathogen which had different geographical and crop-wise distribution as well as a different DNA-based fingerprint. To do so, 4-week-old soybean plants were inoculated with individual isolates of S. sclerotiorum using a straw inoculation method. Inoculated plants were incubated for 48 h in continuous leaf wetness and rated for disease severity 1 and 2 weeks after inoculation. Significant differences in disease severity were detected among the soybean cultivars, and NK S08-80 consistently had the lowest disease severity among the five cultivars tested. No significant differences in disease severity were observed among pathogen isolates and no significant interactions were detected between soybean cultivars and pathogen isolates. These results suggest the following interpretations: (i) either the clonal genotype of the four pathogen isolates as determined by mycelial compatibility and DNA fingerprint was not associated with level of virulence on the five soybean cultivars or (ii) the soybean cultivars themselves were not capable of revealing any differences in virulence among isolates that would be related to their genetic fingerprint or regional distribution. The results of this study are consistent with the practice of considering different isolates of S. scle-rotiorum sampled from soybean in the same geographical region as equivalent for the evaluation of soybean cultivars for resistance to Sclerotinia stem rot.

Multilocus nested haplotype networks extended with DNA fingerprints show common origin and fine-scale, ongoing genetic divergence in a wild microbial metapopulation.

Nested haplotype networks for three loci in a haploid, fungal plant pathogen, Sclerotinia sclerotiorum, in two natural, Norwegian populations of the woodland buttercup, Ranunculus ficaria, were extended with DNA fingerprints to determine fine-scale population divergence. To preserve the cladistic structure in the network for both nonrecombinant and postrecombinant haplotypes in highly recombinant clades, recombinant events were not removed ('peeled off'), but instead were examined in alternative (marginal) networks. Fungi from both sampling locations share a common origin with subsequent genetic divergence, consistent with expectations for metapopulation structure. Evidence for divergence includes (i) lack of shared fingerprints between the two locations, (ii) evolution of new fingerprints, via transposition and recombination, within 2 years on a fine spatial scale within one sampling location, and (iii) increase in the size of the intergenic spacer (IGS) in both sampling locations. Sites of microsatellite repeat expansion and of an insertion were consistent with the boundaries of two recombination blocks in the IGS. Both alternative networks based on the recombination blocks were essential to finding all associations of DNA fingerprints with IGS size, sampling site, sampling year and mycelial compatibility group. Variation in the elongation factor 1alpha and calmodulin loci supported the topologies and the recurrent, ongoing polarity of change in fingerprints and IGS size inferred from the IGS.

Electrophoretic and immunological comparisons of developmentally regulated proteins in members of the sclerotiniaceae and other sclerotial fungi.

The fungal stroma is a distinct developmental stage, a compact mass of hyphal cells enveloped by a melanized layer of rind cells which is produced from vegetative mycelium. Two types of stromata that are characteristic of members of the Sclerotiniaceae but are also produced in a wide range of other fungi, i.e., the determinate tuberlike sclerotium and the indeterminate platelike substratal stroma, were compared in these studies. Developmental proteins found in determinate sclerotial and indeterminate substratal stromata, but not in mycelia, were characterized and compared in 52 isolates of fungi, both ascomycetes (including 18 species in the Sclerotiniaceae and 5 species of Aspergillus) and the basidiomycete Sclerotium rolfsii. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of mycelial, stromatal initial, and stromatal extracts demonstrated that all members of the Sclerotiniaceae produced proteins unique to stromatal extracts within a molecular weight range of 31,000 to 39,500 which composed 13 to 58% of the total protein in stromata. Proteins unique to the sclerotial stage were also produced in Sclerotium rolfsii and the Aspergillus species but within a generally lower-molecular-weight range of 11,000 to 30,000. The proteins were then characterized by two-dimensional electrophoresis to determine the number and isoelectric point of polypeptides composing each protein. Polyclonal antibodies were raised to the major 36-kDa sclerotial protein of Sclerotinia sclerotiorum (Ssp). Immunoblots demonstrated that all sclerotial proteins from species in the Sclerotiniaceae cross-reacted with anti-Ssp antibodies, while no cross-reaction was observed with proteins from substratal stromatal species in the Sclerotiniaceae, sclerotial species of Aspergillus, or Sclerotium rolfsii. Results of discriminant analysis of the data from competitive inhibition enzyme-linked immunosorbent assays were consistent with the results of immunoblotting. Three groupings, sclerotial species in the Sclerotiniaceae, substratal stromatal species in the family, and sclerotial species outside the family, were delimited on the basis of relative decreasing ability to compete for anti-Ssp antibody. These data demonstrate that stromatal proteins differ among different taxonomic groups of fungi and suggest that the Sclerotiniaceae may include two distinct lineages of genera.

Electrophoretic Karyotypes of Sclerotinia sclerotiorum.

Electrophoretic karyotypes (EKs) of 83 isolates were variable within agricultural and natural populations of Sclerotinia sclerotiorum, as well as among S. sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum. Variation in EKs was not observed within six mitotic or three meiotic lineages of isolates. EKs of 8 to 10 chromosome-sized DNAs were observed. Homologous and heterologous probes hybridized to four linkage groups.

Random association among alleles in clonal populations of Sclerotinia sclerotiorum.

Multiple loci identified in DNA fingerprints were used to test for random association in two agricultural populations of S. sclerotiorum. In linkage disequilibrium tests among pairs of loci with frequencies between 0.1 and 0.9, 44.5 and 80.5% of pairs of loci were consistent with random association in the clone-corrected samples of the Canadian canola and the North Carolina cabbage populations, respectively. In estimates of corrected (Bonferroni) P value, 70.66 and 98.89% of pairs of loci were in random association. All four possible genotypes for each pair of loci were observed in the Canadian canola sample, consistent with random association among loci. In multilocus association tests across all loci, however, significant association was observed in both populations. In the Canadian canola population, 40 possible heterokaryons were identified. Our data suggest that populations of S. sclerotiorum are predominantly clonal and that occasional genetic exchange and recombination, and not mutation alone, may be a source of new genotypes.

A microbial population-species interface: nested cladistic and coalescent inference with multilocus data.

Using sequence data from seven nuclear loci in 385 isolates of the haploid, plant parasitic, ascomycete fungus, Sclerotinia, divergence times of populations and of species were distinguished. The evolutionary history of haplotypes on both population and species scales was reconstructed using a combination of parsimony, maximum likelihood and coalescent methods, implemented in a specific order. Analysis of site compatibility revealed recombination blocks from which alternative (marginal) networks were inferred, reducing uncertainty in the network due to recombination. Our own modifications of Templeton and co-workers' cladistic inference method and a coalescent approach detected the same phylogeographic processes. Assuming neutrality and a molecular clock, the boundary between divergent populations and species is an interval of time between coalescence (to a common ancestor) of populations and coalescence of species.

Genotyping, gene genealogies and genomics bring fungal population genetics above ground.

As ubiquitous decomposers, symbionts and parasites, fungi build populations not easily accommodated by population genetic theory. Identifying and delineating individuals and populations is often difficult, and recombination can occur in complex and variable ways. Genotyping and gene genealogies provide the framework for identifying and delineating individuals and for detecting recombination in natural populations. Expanding genomic databases now make fungi ideal subjects for tracking mutation and expression in genes of adaptive importance in experimental populations.

Divergence in fitness and evolution of drug resistance in experimental populations of Candida albicans.

The dissemination and persistence of drug-resistant organisms in nature depends on the relative fitness of sensitive and resistant genotypes. While resistant genotypes are expected to be at an advantage compared to less resistant genotypes in the presence of drug, resistance may incur a cost; resistant genotypes may be at a disadvantage in the absence of drug. We measured the fitness of replicate experimental populations of the pathogenic yeast Candida albicans founded from a single progenitor cell in a previous study (L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn, J. Bacteriol. 182:1515-1522, 2000) and evolved in the presence, and in the absence, of the antifungal agent fluconazole. Fitness was measured both in the presence and in the absence of fluconazole by placing each evolved population in direct competition with the drug-sensitive ancestor and measuring the reproductive output of each competitor in the mixture. Populations evolved in the presence of drug diverged in fitness. Any significant cost of resistance, indicated by reduced fitness in the absence of drug, was eliminated with further evolution. Populations evolved in the absence of drug showed more uniform increases in fitness under both conditions. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or stationary-phase cell densities of the competitors in isolation, suggesting the importance of interactions between mixed genotypes in competitions.

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.

Life changes and urinary norepinephrine in myocardial infarction.

The relationship between norepinephrine and stress caused by life changes was assessed using urinary norepinephrine levels and responses to a 57-item stress questionnaire interview obtained from 21 post-myocardial infarction and 27 healthy control male and female subjects. High correlations between norepinephrine and duration of present stress and duration and severity of life changes, and moderate correlations between norepinephrine and anxiety and depression were found among the post-infarction group. No significant correlations were found in the control group. Post-infarction subjects with elevated norepinephrine had significantly higher scores on duration of present stress and duration and severity of life changes than did post-infarction subjects with normal norepinephrine levels. Control subjects with normal and elevated norepinephrine did not differ significantly on any of the comparison variables. Standard risk factors failed to add significantly to the prediction of norepinephrine in either group. However, 68.3 percent of the variance in norepinephrine was accounted for by a single predictor, the duration of present stress.

Multilocus genotyping indicates that the ability to invade the bloodstream is widespread among Candida albicans isolates.

Multilocus genotyping was used to compare populations of Candida albicans from oral mucosa and blood. No significant differences in allele frequencies between the two samples were detected, and in a dendrogram of genotypic similarities, genotypes from both types of samples were finely interspersed. This is evidence for widespread distribution of invasive potential.

Evolution of drug resistance in experimental populations of Candida albicans.

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.

Infrequent genetic exchange and recombination in the mitochondrial genome of Candida albicans.

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast Candida albicans. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of C. albicans from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of C. albicans, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.

Multilocus genotypes and DNA fingerprints Do not predict variation in azole resistance among clinical isolates of Candida albicans.

If variation in azole resistance is due to inherent differences in strains of Candida albicans, as a predominantly clonal organism, then correlation between multilocus genotypes and drug resistance would be expected. A sample of 81 clinical isolates from patients infected with human immunodeficiency virus in Toronto, Canada, plus 3 reference isolates were genotyped at 16 loci, distributed on all linkage groups, by means of oligonucleotide hybridizations specific for each of the alleles at each locus. These multilocus genotypes were significantly correlated with DNA fingerprints obtained with the species-specific probe 27A, indicating widespread linkage disequilibrium in the genome. There were 64 multilocus diploid genotypes and 77 DNA fingerprint types delineated in this sample. Neither the multilocus genotyping nor DNA fingerprinting alone identified all of the 81 types identified by the combination of these two methods. Multilocus genotypes were not predictive of fluconazole resistance, suggesting that resistance is gained or lost too quickly to be predicted by linkage with neutral markers.