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The development of advanced molecular diagnosis for the critical toxigenic Fusarium species is considered in this review. The specific topics discussed are (1) isolation of mating type genes of Gibberella complex, (2) molecular detection of Fusarium-producing fumonisins, (3) molecular detection of Fusarium-producing trichothecenes and enniatins. Particular attention is given to the development of PCR assays for genes involved in the toxin biosynthesis that would permit the early detection of Fusarium species-producing toxins and potentially even reveal which particular toxin may be present within a food or feed product. Most of these data have been obtained within the 'De-Tox Fungi' project supported by the European Commission (QLK1-CT-1998-01380).

Abstract The genus Septoria contains more than 1000 species of plant pathogenic fungi, most of which have no known sexual stage. Species of Septoria without a known sexual stage could be recent derivatives of sexual species that have lost the ability to mate. To test this hypothesis, the mating-type region of S. passerinii, a species with no known sexual stage, was cloned, sequenced, and compared to that of its close relative S. tritici (sexual stage: Mycosphaerella graminicola). Both of the S. passerinii mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one (MAT-2) or two (MAT-1) putative introns. The putative products of MAT-1 and MAT-2 are characterized by alpha-box and high-mobility-group sequences, respectively, similar to those in the mating-type genes of M. graminicola and other fungi. The mating-type genes of S. passerinii and M. graminicola are evolving rapidly, approximately ten times faster than the internal transcribed spacer region of the ribosomal DNA, and are not closely related to those from Cochliobolus or other loculoascomycetes in the order Pleosporales. Therefore, the class Loculoascomycetes may be polyphyletic. Furthermore, differences between the phylogenetic trees may indicate separate evolutionary histories for the MAT-1 and MAT-2 idiomorphs. A three-primer multiplex-PCR technique was developed that allowed rapid identification of the mating types of isolates of S. passerinii. Both mating types were present in approximately equal frequencies and often on the same leaf in fields in Minnesota and North Dakota. Analyses with isozyme and random amplified polymorphic DNA markers revealed that each isolate had a unique genotype. The common occurrence of both mating types on the same leaf and the high levels of genotypic diversity indicate that S. passerinii is almost certainly not an asexual derivative of a sexual fungus. Instead, sexual reproduction probably plays an integral role in the life cycle of S. passerinii and may be much more important than previously believed in this (and possibly other) "asexual" species of Septoria.

Two Fusariumspecies, F. oxysporumf.sp. asparagiand F. proliferatum, are known to be involved in the root and crown rot complex of asparagus. We have investigated reports on the involvement of F. redolens, a third species, which until recently was considered conspecific with F. oxysporumbecause of morphological similarities. RFLP analysis of the rDNA internal transcribed spacer region and AFLP fingerprinting identified eight strains from asparagus unambiguously as F. redolens. Four of these were tested and found to be pathogenic to asparagus either in this study (two strains) or in a previous one in which they were classified as F. oxysporum(three strains). Disease symptoms and disease development were the same as with F. oxysporumf.sp. asparagiand F. proliferatum. Present data and literature reports identify F. redolensas a host-specific pathogen involved in root, crown and spear rot of asparagus. The pathogen is formally classified as F. redolensWollenw. f.sp. asparagiBaayen.

Isolates of Fusarium oxysporum from lily were screened for pathogenicity, vegetative compatibility and DNA restriction fragment length polymorphisms, and compared to reference isolates of F. oxysporum f.sp. gladioli and F. oxysporum f.sp. tulipae to justify the distinction of F. oxysporum f.sp. lilii. Twenty-four isolates from different locations in The Netherlands (18 isolates), Italy (4 isolates), Poland and the United States (1 isolate each) shared unique RFLP patterns with probes D4 and pFOM7, while hybridization did not occur with a third probe (F9). Except for a self-incompatible isolate, these 24 isolates all belonged to a single vegetative compatibility group (VCG 0190). Isolates belonging to VCG 0190 were highly pathogenic to lily, but not to gladiolus or tulip, except for a single nonpathogenic isolate. Six saprophytic isolates of F. oxysporum from lily were nonpathogenic or only slightly aggressive to lily, gladiolus and tulip, belonged to unique VCGs and had distinct RFLP patterns. Three pathogenic isolates previously considered to belong to F. oxysporum f.sp. lilii were identified as F. proliferatum var. minus; all three belonged to the same VCG and shared unique RFLP patterns. These three isolates were moderately pathogenic to lily and nonpathogenic to gladiolus and tulip. The reference isolates of F. oxysporum f.sp. tulipae were pathogenic to tulip, but not to lily and gladiolus; they shared a distinct RFLP pattern, different from those encountered among pathogenic and saprophytic isolates from lily, and formed a separate new VCG (VCG 0230). Reference isolates of F. oxysporum f.sp. gladioli belonging to VCG 0340 proved pathogenic to both gladiolus and lily, but not to tulip. These isolates, as well as isolates belonging to VCGs 0341, 0342 and 0343 of F. oxysporum f.sp. gladioli, had RFLP patterns different from those encountered among the isolates from lily or tulip. These findings identify F. oxysporum f.sp. lilii as a single clonal lineage, distinct from F. oxysporum f.sp. gladioli and f.sp. tulipae.

Pathogenic isolates were selected representing all known vegetative compatibility groups (VCGs) and races of Fusarium oxysporum sensu lato from Dianthus spp. On basis of differences in the internal transcribed spacer region of the ribosomal DNA, six VCGs were classified as F. oxysporum f.sp. dianthi and four as F. redolens f.sp. dianthi. All VCGs of F. oxysporum f.sp. dianthi were characterized by unique restriction fragment length polymorphisms (RFLPs), unique overall esterase profiles, and unique virulence spectra, supporting a clonal lineage concept. Two VCGs of F. oxysporum f.sp. dianthi nevertheless comprised more than one race, but races within the same VCG shared the same distinct overall virulence spectrum. VCGs belonging to F. redolens f.sp. dianthi also had unique RFLPs and unique virulence spectra, but had grossly identical esterase profiles. Three new races (9, 10 and 11) are described for F. oxysporum f.sp. dianthi, and four for F. redolens f.sp. dianthi. Two races previously considered lost were recovered; race 7 was identified as a member of VCG 0021 of F. oxysporum f.sp. dianthi while race 3 was identified as a distinct VCG and race of F. redolens f.sp. dianthi. A summary of races and VCGs in F. oxysporum f.sp. dianthi and F. redolens f.sp. dianthi is presented.

The role of hemolysin in the nephropathogenicity of Escherichia coli was studied in a hematogenous pyelonephritis model in mice. The nephropathogenicity of a nonhemolytic, avirulent E. coli strain was increased by simultaneous injection with its hemolytic, nephropathogenic parent. This helper mechanism could be attributed to hemolysin, since the simultaneous injection of partially purified hemolysin gave a similar enhancement of nephropathogenicity. Intraperitoneal injection of hemoglobin or iron sulfate before intravenous challenge with this avirulent strain also led to increased virulence. The nephropathogenicity-enhancing effect of hemolysin is therefore supposed to depend on increasing the level of available iron in the host. Under conditions of plentiful iron, hemolysin production was repressed, as shown by in vitro growth experiments in the presence of exogenous iron. These results suggest that the production of hemolysin is regulated by feedback inhibition.

The nephropathogenic Escherichia coli strain P673 was shown to harbor two plasmids with molecular sizes of 70 and 41 megadaltons, respectively. The 70-megadalton plasmid, pCW1, coded for tetracycline resistance, whereas hemolysin production was coded by the 41-megadalton plasmid, pCW2. Plasmid pCW1 proved to be self-transmissible, in contrast to pCW2. Transfer of the hemolysin character was associated with the appearance of a 110-megadalton plasmid, pCW3. The incompatibility of pCW3 with both native plasmids and restriction enzyme analysis led to the conclusion that pCW3 is a cointegrate of pCW1 and pCW2, pCW2, carrying the hemolytic determinant, is involved in the nephropathogenic character of strain P673, because (i) elimination of pCW2 from P673 was associated with a loss of virulence and (ii) the nephropathogenicity of the avirulent mutant could be restored by reintroduction of pCW2 DNA as part of a cointegrate structure.

A haemolytic E. coli strain is nephropathogenic for mice. Mutagenesis by transposon insertion resulted in mutants with altered haemolytic activity.

We have analysed DNA modification in a HapII site (CCGG) present in the major intron of the discontinuous rabbit beta-globin gene. In most somatic tissues, including erythroid and non-erythroid tissues, about 50% of the DNA is resistant to cleavage at this site by HapII, though 100% cleavage is found with the isoschizomer MspI. Since the former enzyme is unable to cleave CCGG sites if the internal C residue is 5-methyl C (and since methylation is the only form of CpG modification documented in animal DNA), while the latter enzyme cleaves DNA irrespective of methylation at this residue, we infer that 50% of the CCGG sites in the beta-globin gene intron are methylated in these tissues. The same site appears to be 100% methylated (judged by the same criterium) in sperm DNA and about 80% methylated in brain DNA. DNA from the rabbit SIRC cell line is entirely unmethylated at this site.

The hemolysin plasmid pCW2, which enhances the virulence of Escherichia coli, has been mapped. Comparison of the region coding for hemolysin with other hemolysin determinants reveals differences that could explain differences in the ability to confer virulence.

The cleavage of DNA by restriction endonucleases HpaII and HapII is prevented by the presence of a 5-methyl group at the internal C residue of its recognition sequence CCGG. MspI, an isoschizomer of HpaII available from New England Biolabs, cleaves DNA irrespective of the presence of a methyl group at this position. This enzyme cleaves DNA from Haemophilus parainfluenzae and Haemophilus aphrophilus readily while HpaII and HapII cannot degrade these DNAs. Practically all HpaII sites in mammalian sperm DNA are also protected by methylation at the internal C position since HpaII and HapII barely cleave this DNA (average molecular weight 40 kb). MspI, however, cleaves the DNA to an average size of about 5 kb.