Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.     

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weaklyvirulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.     

The transformation of protoplasts of Leptosphaeria maculans to hygromycin B resistance

Summary Conditions are described for the efficient isolation and regeneration of protoplasts of a fungal pathogen of brassicas, Leptosphaeria maculans. Treatment of the protoplasts with DNA of the plasmid pAN7-1 (containing an E. coli hygromycin phosphotransferase gene with Aspergillus nidulans expression signals) and plating under selective conditions resulted in the formation of hygromycin 13-resistant colonies. Southern blot analysis of resistant colonies indicated that single copies of the plasmid had integrated into different sites in the genome. In twelve of the transformants analysed so far, the integration is stable through mitosis. The demonstration of efficient transformation is an essential first step in the molecular analysis of pathogenicity of this commercially important pathogen.     

Electrophoretic karyotype of cellulolytic Penicillium janthinellum strains

Summary The genome of the cellulolytic fungus Penicillium janthinellum BIOURGE was resolved completely by rotating field electrophoresis. The gel pattern revealed 8–10 different chromosomes. On the basis of data for yeast chromosome size standards from Schizosaccharomyces pombe and Saccharomyces cerevisiae, chromosome sizes in the range from 2.0 to 8 mb were estimated. By Southern hybridization with heterologous probes, the chromosomal locations of rDNA, the elongation factor EF1, actin and ubiquitin have all been mapped. In addition, first results with heterologous and homologous probes for cellulolytic genes from Trichoderma reesei and P. janthinellum are presented. In contrast to the other probes, signals obtained with cellulase genes are clearly distributed over several chromosomes.     

Identification and characterization of polymorphic minisatellites in the phytopathogenic ascomycete Leptosphaeria maculans

Leptosphaeria maculans causes phoma stem canker, the most serious disease of oilseed rape world-wide. Sexual recombination is important in the pathogen life cycle and increases the risk of plant resistance genes being overcome rapidly. Thus, there is a need to develop easy-to-use molecular markers suitable for large-scale population genetic studies. The minisatellite MinLm1, showing six alleles in natural populations, has previously been used as a marker to survey populations. Here, we report the characterization of five new minisatellites (MinLm2–MinLm6), of which four were identified by a systematic search for tandemly repeated polymorphic regions in BAC-end sequencing data from L. maculans. Of 782 BAC-end sequences analysed, 43 possessed putative minisatellite-type repeats and four of these (MinLm3–MinLm6) displayed both consistent PCR amplification and size polymorphism in a collection of L. maculans isolates of diverse origins. Cloning and sequencing of each allele confirmed that polymorphism was due to variation in the repeat number of a core motif ranging from 11 bp (MinLm3) to 51 bp (MinLm4). The number of alleles found for each minisatellite ranged from three (MinLm4) to nine (MinLm2), with eight, five and six for MinLm3, MinLm5 and MinLm6, respectively. MinLm2–MinLm6 are all single locus markers specific to L. maculans and share some common features, such as conservation of core motifs and incomplete direct repeats in the flanking regions. To our knowledge, L. maculans is the first fungal species for which six polymorphic single locus minisatellite markers have been reported.Electronic Supplementary Material Supplementary material is available for this article at     

Genetic structure of a population of the fungus Leptosphaeria maculans in a disease nursery of Brassica napus in Australia

Microsatellite, minisatellite and mating type markers were used to determine the genetic structure of the fungus Leptosphaeria maculans within a disease nursery, where Brassica napus lines were screened for resistance to blackleg disease under high inoculum pressure. Fungal isolates were collected from pseudothecia in infected stubble and pycnidia within cotyledon lesions on seedlings within the nursery. Genetic diversity was high with gene diversity at H=0.700 across four polymorphic loci, and genotypic diversity at D=0.993. Among the 159 isolates analysed, 102 multilocus genotypes were identified. The even distribution of mating type idiomorphs MAT1-1 and MAT1-2 and gametic equilibrium within the population provided further evidence of random mating. Genetic diversity was distributed on a very fine scale in the disease nursery. The majority of genetic diversity (67%) was distributed among conidia within a lesion or among ascospores from a piece of stubble, while the remainder (33%) was distributed within lesions on seedlings or different stubble pieces. There were no among-group differences between samples from stubble and seedlings. This is consistent with the low level of genetic differentiation between the ascospore and conidia samples (F _ST=0.017) indicating that all isolates of L. maculans from the disease nursery most likely belong to one population, and that ascospores form the primary inoculum in the disease nursery.     

Electrophoretic karyotype of Cercospora kikuchii

Classical genetic analyses are not possible with the phytopathogenic fungus Cercospora kikuchii since no sexual stage has been identified. To facilitate gene mapping and to develop an understanding of the genome organization of C. kikuchii, an electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis (CHEF). Eight chromosomes, two of which migrate as a doublet, have been separated into seven bands ranging from 2.0 to 5.5 Mb. Using this determination of chromosome number and size, the total genome size of C. kikuchii is estimated to be 28.4 Mb. In addition, genes encoding tubulin, ribosomal DNA, and four previously isolated light-enhanced cDNAs from C. kikuchii were assigned to chromosomes by Southern-hybridization analysis of CHEF blots.     

Electrophoretic karyotype of the ascomycete Podospora anserina

Summary Fractionation of intact chromosomes of the ascomycete Podospora anserina by contour-clamped homogenous electric field (CHEF) gel electrophoresis resulted in the resolution of five distinct chromosomal bands. Two of these bands migrated as doublets. Using chromosomal standards from Schizosaccharomyces pombe we estimated the size of the P. anserina genome to about 33.4 megabases (Mb). By heterologous hybridization of fractionated chromosomes the rDNA locus was identified on one of the two chromosomes migrating at about 4.9 Mb.     

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora     

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio – Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n = 20), Typhimurium (n = 4), Zanzibar (n = 2), Manhattan (n = 1), Bovismorbificans (n = 1), Amsterdam (n = 1), Saint Paul (n = 1), Kentucky (n = 1) and Muenster (n = 1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1–5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.     

Electrophoretic karyotypes and gene mapping in eight species of the Fusarium sections Arthrosporiella and Sporotrichiella

Pulsed-field gel electrophoresis was used to identify karyotypes for eight species of the Fusarium sections Arthrosporiella and Sporotrichiella. The total number of chromosome-sized DNA molecules varied from six to nine, depending on the species. The sizes of chromosomes ranged from 0.4 to approximately 6.5 Mb which gave estimates of genome size of between 27.0 and 29.9 Mb. When fractionated chromosomes of the eight species were probed with Tox5, a gene coding for the key-enzyme of trichothecene biosynthesis, strong hybridization signals developed in F. poae and F. sporotrichioides, suggesting that of the eight species examined only these two have the genetic potentiality to produce trichothecene mycotoxins. By using heterologous probes from Aspergillus different rRNA loci have also been mapped on Fusarium chromosomes.     

Vaccinia viruses isolated from cutaneous disease in horses are highly virulent for rabbits

Two genotypically distinct Vaccinia viruses (VACV), named P1V and P2V, were isolated from an outbreak of cutaneous disease in horses in Southern Brazil. We herein investigated the susceptibility of rabbits, a proposed animal model, to P1V and P2V infection. Groups of weanling rabbits were inoculated intranasally (IN) with P1V or P2V at low (10^2.5 TCID₅₀), medium (10^4.5TCID₅₀), or high titer (10^6.5TCID₅₀). Rabbits inoculated with medium and high titers shed virus in nasal secretions and developed serous to hemorrhagic nasal discharge and severe respiratory distress, followed by progressive apathy and high lethality. Clinical signs appeared around days 3-6 post-inoculation (pi) and lasted up to the day of death or euthanasia (around days 5-10). Virus shedding and clinical signs were less frequent in rabbits inoculated with low virus titers. Viremia was detected in all groups, with different frequencies. Viral DNA was detected in the feces of a few animals inoculated with P1V and P2V, low titer, and with P2V at high titer. Gross necropsy findings and histological examination showed diffuse interstitial fibrousing pneumonia with necrosuppurative bronchopneumonia and intestinal liquid content. Neutralizing antibodies were detected in all inoculated animals surviving beyond day 9pi. These results show that rabbits are highly susceptible to VACV isolated from horses, and develop severe respiratory and systemic disease upon IN inoculation. Thus, rabbits may be used to study selected aspects of VACV infection and disease.     

Genome analysis of imperfect fungi: electrophoretic karyotyping and characterization of the nuclear gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Curvularia lunata

Summary The gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes.     

Multilocus sequence typing of Candida albicans isolates from animals

Multilocus sequencing strain types of a panel of 43 Candida albicans isolates from animals, including mammals and avian species, were compared with strain types for human isolates. The clade distribution of the animal isolates was significantly different from that of the human isolates, in both a comparison involving a total of 1580 isolates from multiple geographical sources and a comparison restricted to 675 human isolates from the same geographical regions as the animal isolates. A nearest-neighbour analysis involving the 43 animal isolates and 67 human isolates, randomly selected to give a proportionate distribution of geographical sources, showed a strong statistical trend towards genetic selection of different C. albicans strain types adapted to non-human animal hosts, but without complete genetic separation.     

Electrophoretic karyotype of Mucor circinelloides

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis was used to separate chromosomal size DNA molecules of two Mucor circinelloides strains. Electrophoretic karyotypes revealed the presence of eight distinct bands for the M. circinelloides f. lusitanicus strain, and four, presumably multiple, bands for the M. circinelloides f. gryseo-cyanus strain. The approximate sizes of the resolved chromosomal DNA bands ranged from 2.3 to 8.1 Mb, giving estimated genome sizes of 38.7 and 32.6 Mb, respectively. Hybridisation techniques were used to assign the leuA gene to a chromosome.     

Difference of genotypic and phenotypic characteristics and pathogenicity potential of Photobacterium damselae subsp. damselae between clinical and environmental isolates from Japan

Photobacterium damselae subsp. damselae has been known as an opportunistic pathogen in fish and mammals. Human infectious cases are often very serious and occasionally fatal. We previously reportedtwo fatal cases caused by this subspecies where the patients developed multiple organ failure within 20-36 h after the onset of initial symptoms. Despite its ability to cause serious infections in humans, this subspecies has not been well studied because human infectious cases caused by this subspecies are very rare. However, this subspecies has been reported to be present in a wide range with high incidence rate in aquatic environments. Thus, we investigated the genotypic and phenotypic differences between clinical and environmental strains of Photobacterium damselae subsp. damselae. Using molecular typing methods, such as ribotyping, AFLP (Amplified Fragment Length Polymorphism), and PFGE (Pulsed-Field Gel Electrophoresis) and sequencing analysis, we determined that thetwo clinical strains were genetically similar yet distinguishable from environmental strains, but not significantly so. On the other hand, phenotypic differences were clear; moreover, mouse assay and hemolytic assay indicated strong pathogenicity of only clinical isolates. Based on these data, we concluded that there are differences in pathogenicity potential among isolates of this subspecies, and some environmental isolates have the potential to become highly pathogenic.     

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68–127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Electrophoretic karyotype of the zygomycete Absidia glauca: evidence for differences between mating types

Summary Experimental conditions for the separation of chromosomes from the model zygomycete Absidia glauca by rotating field electrophoresis were established. The sexually compatible strains of the mating type pair A. glauca CBS 100.48 (+) and CBS 101.48 (-) showed considerable differences in their electrophoretic karyotype. By Southern hybridization with homologous probes we have mapped the chromosomal locations for rDNA, the repetitive element rAg1, and the genes for actin and the elongation factor EF1. For the mapping of ubiquitin information we used a heterologous probe from U. maydis. The combination of electrophoretic karyotyping and Southern mapping proved to be a useful tool for characterizing mutant genotypes, which were induced by integrative transformation.