Sequences of the intergenic spacer between the 28S and the 5S rDNA genes (IGS 1) were compared for three European isolates belonging to three Puccinia striiformis f. sp. tritici races, of different geographic origins and virulence spectra. Two PCR products (IGS 1-1 and IGS 1-2, of 1.3 and 1.1 kb, respectively) were recovered from all spore multiplications. The analysis of IGS 1-1 or IGS 1-2 showed that a low mutational polymorphism existed between the three isolates (from 0.7 to 1.1%) and that each IGS 1 type was characterised by a succession of repetitions. The comparison of IGS 1-1 and IGS 1-2 to each other showed that the polymorphism between the two types of IGS was due to a length polymorphism consisting of a 216 bp insertion/deletion located in the repeated region. Two hypotheses can be proposed to explain the coexistence of two different IGS 1 types within one isolate: either each nucleus of this dikaryotic fungus contains one type of IGS 1 or there are two discrete ribosomal units resulting from recombination in the same nucleus.

A microsatellite-enriched library was constructed from genomic DNA of Phialophora gregata f. sp. sojae, the causal agent of soybean brown stem rot. Specific oligonucleotide primer pairs were designed to flank individual microsatellite loci, and used in PCR to detect polymorphisms among 118 contemporary and archival isolates of P. gregata from several countries, which represent two distinct, but sympatric populations delineated by rDNA genotypes. In general, low levels of genetic variation were observed and manifested in two ways. First, the number of polymorphic loci was low. Among 24 microsatellite loci screened, four polymorphic loci were detected, and two of them were located on the same clone. Second, the number of alleles in the polymorphic loci was low. Only two alleles were observed in each polymorphic locus. Alleles of the polymorphic microsatellite loci were invariant within, but different between, the two populations of P. gregata. From all evidence available, P. gregata f. sp. sojae exhibits several genetic features characteristic of clonal genetic structure, namely widespread distribution and stability of identical genotypes, absence of recombinant genotypes, and correlation of independent genetic traits including three sets of molecular markers, defoliating and nondefoliating pathotypes, and differential pathogenic fitness.

Genetic variation within and between eighteen isolates of Alternaria brassicicola, five isolates of A. alternata, and a single isolate of Rhynchosporium secalis was compared using Amplified Fragment Length Polymorphisms (AFLPs). The AFLPs consistently distinguished between the three species. AFLPs thus provide a reliable tool for identifying A. brassicicola. The analysis of eighteen isolates of A. brassicicola from different sites also indicated at least moderate levels of genetic diversity within A. brassicicola along the New South Wales coast. Of 43–66 markers scored per primer combination, 16·7–27·9% were polymorphic. On the basis of an unweighted paired group method of arithmetic averages analysis using data from four primer combinations, most isolates were identified as separate genotypes. However, multiple isolates from particular locations tended to cluster together, implying the potential for population structure, and there was a significant positive correlation between Nei's measure of genetic distance and physical distance (r = 0·5486, P<0·001). Despite the absence of an identified sexual stage, A. brassicicola would appear to have a means for generating and maintaining significant variation.

Gaeumannomyces graminis var. tritici is the aetiologic agent of take-all disease of wheat and barley. Heavily-melanized variants of a lightly pigmented, virulent wild-type strain were isolated and characterized. These variants were phenotypically similar to Phialophora sp., the proposed anamorph of Gaeumannomyces graminis var. tritici. Unlike the wild-type G. graminis strain, Phialophora-like variants produced conidia and lost their ability to reproduce sexually after serial transfer. Some ascospores from initial self-crosses of the Phialophora-like variants regained the G. graminis phenotype, and these derivatives could again produce Phialophora-like variants. As with Phialophora isolates from the field, Phialophora-like variants produced in this study were non-pathogenic and produced less extracellular protein.

Coniothyrium canker, caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus in subtropical parts of South Africa. A Coniothyrium associated with similar symptoms to those in South Africa was observed on E. camaldulensis in 1996 in Thailand. It was previously thought that C. zuluense was restricted to South Africa. This study compares South African isolates of C. zuluense with isolates of the Coniothyrium from Thailand. Results of morphological comparisons indicate that the South African and Thailand isolates are the same. This was further confirmed when all Coniothyrium isolates associated with stem cankers on Eucalyptus spp. grouped together in a single major clade for both rDNA sequence data and AFLP analysis. This clade was distant from isolates of other Coniothyrium spp. included for comparative purposes. Although the Coniothyrium isolates from South Africa and Thailand resided in two separate clades, these were closely related and, we believe that the isolates from Thailand represent C. zuluense. This is the first record of the important Eucalyptus stem canker pathogen outside South Africa.

Morphological characters and genetic relationships were studied among 36 Verticillium isolates derived from different host species. Conidial length varied from 3.5 μm to 12 μm and the DNA content ranged from 0.028 pg to 0.078 pg with a bias towards large conidia and higher DNA content among the Swedish and German isolates from Brassica napus. Ability to produce nitrate nonutilising (nit) mutants was also assayed. Of all 36 isolates tested, eight V. dahliae isolates from non-Brassica hosts and two from B. napus, V. nigrescens from flax and V. albo-atrum from tomato generated nit mutants. To facilitate disease diagnosis, a species-specific PCR primer pair was developed, aiming towards a PCR-based analysis to confirm Verticillium wilt infection in Brassica plants. To identify useful DNA templates for the PCR primer design, random genomic clones from isolates VD12 and VD17 were used as probes in RFLP analysis. The RFLP results were also utilized in phenetic analysis and data from 29 isolates revealed three major groups. The first group consisted of seven non-Brassica V. dahliae isolates and the V. nigrescens isolate. The second group contained the two V. albo-atrum isolates and the V. dahliae isolate from red clover. Finally, the third group contained all the 18 isolates of Verticillium from B. napus. In the third group, two subgroups were identified, separating the three French isolates from the German and Swedish isolates. The dendrogram indicated that the isolates in the latter subgroup were closely related to each other. Based on the above mentioned criteria, we confirm that the isolates of Verticillium from B. napus in Sweden and Germany should be classified as V. longisporum.

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes, causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes, and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

Compared with other Armillaria species, natural melanized rhizomorphs of A. tabescens are rarely observed. Growing in autoclaved Vitis stem segments, A. tabescens isolates from the Ozark Mountains in the central USA formed fully melanized rhizomorphs, thinner and shorter than those observed for other Armillaria species under field conditions, and only under conditions of both high oxygen availability, [ges ]2 μg cm−2 min−1 and moisture near saturation. Conducive conditions were used to compare the rhizomorph generation capacities of A. tabescens, A. gallica, and A. mellea, which have overlapping host and geographic ranges in central North America. While the rhizomorphs of A. tabescens were significantly shorter than those of the other two species, A. gallica and A. tabescens produced similar numbers of rhizomorph initials. Finally, we demonstrated the ability of melanized A. tabescens rhizomorphs to span woody food bases and thereby establish viable infections by penetration of intact bark of Vitis stem segments. We hypothesize that A. tabescens rhizomorphs form under conditions of periodic saturation which promote rapid water movement through naturally occurring lacunae in the soil. Thus, the role of A. tabescens rhizomorphs in the spread of the fungus should be re-evaluated.

The mycelial fatty acid profiles of several Pleurotus strains and their application in intrageneric differentiation were investigated. In the lipids produced by strains of Pleurotus abalonus, P. calyptratus, P. columbinus, P. cornucopiae, P. cystidiosus, P. ostreatus, P. pulmonarius, P. sajor-caju and P. sapidus, the predominant fatty acid was linoleic (33–68% total lipids), while in P. eryngii strains the major fatty acid was oleic (43–46%). In all strains studied, oleic and palmitic acids were present in significant concentrations (>12%), whereas stearic acid was found in lower ones. By using the ratios C18[ratio ]1/C18[ratio ]0, C18[ratio ]2/C18[ratio ]1 and ΣC18/C16[ratio ]0 as variables, Pleurotus strains were clustered into six groups. Group I (P. ostreatus); group II, divided in three sub-groups (IIa: P. columbinus, IIb: P. eryngii, IIc: P. cornucopiae), group III, divided in two sub-groups (IIIa: P. sajor-caju, IIIb: P. pulmonarius), group IV (P. sapidus), group V (P. abalonus) and group VI divided in two sub-groups (VIa: P. cystidiosus and VIb: P. calyptratus). It is concluded that the mycelial fatty acid composition of Pleurotus can be used in the intrageneric taxonomy of the genus.

The diversity of Tricholoma matsutake basidiomata and their distribution across Japan was investigated, for the first time, by using PCR-RFLP and sequencing. Eight distinct IGS1 rDNA types were identified by using the restriction endonuclease Cfr13I. Four (A, B, G, H) were characterized by one IGS1 dominant sequence, which could then be recorded by direct sequencing of the PCR products. In contrast, the four other types (C, D, E, F) were characterized by up to more than four codominant IGS1 copies that needed to be cloned before sequencing. Among the copies, one was always exhibiting the RFLP pattern of type A basidiomata. Pairwise nucleotide variation among IGS1 spacer copies, recorded in RFLP types A, B, C, D, F, and H, was very low and never exceeded 1·9%. In contrast, type G emerged and its IGS1 spacer sequence exhibited at least 10·6% nucleotide variation compared with all other types. While the data recorded suggests a higher genetic diversity in the southern than in the northern part of Japan, no host/matsutake ribotype specificity could be evidenced. In Japan, the T. matsutake basidiomata belonging to RFLP type A were by far the most frequent. They were found at most sites and were usually the most abundant at each site. Furthermore, this predominance persisted over time during ca 50 years. The dominant RFLP type A may represent the original Japanese population while the other genotypes may have been more recently introduced.

The entomopathogenic hyphomycete Paecilomyces fumosoroseus is a promising candidate for biocontrol of economically important agricultural pests. Assessment of genetic relatedness of this species appears to be essential to gain insight into the monitoring of such biocontrol products. Intraspecific variation within the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was studied in 48 isolates of P. fumosoroseus. These strains were isolated from different geographical and biological origins, more particularly from the silverleaf whitefly, Bemisia tabaci-argentifolii (Homoptera: Aleyrodidae), a major insect pest in field and greenhouse crops. PCR amplification of the ITS1-5.8S-ITS2 rDNA region followed by restriction analysis of the PCR products underlined the overall highly variable nature of this region. Digestion with the six endonucleases AluI, HaeIII, Hin6I, HpaII, NdeII, and SmaI allowed the separation of the isolates into three distinct groups. The group 1, representing 25 isolates, is composed of strains isolated only from the host B. tabaci-argentifolii. By contrast, the group 3 is more diffuse as it included strains from various insect host and geographical origins. Phylogenetic analysis of rDNA-ITS sequence data strongly supported the conclusions of the PCR-RFLP analysis and recognized three monophyletic groups within the P. fumosoroseus complex. The high level of polymorphism within the P. fumosoroseus species is discussed in the light of their biological origin.

This paper describes, as a part of the resistance risk assessment, the genetic analyses which were performed to evaluate the resistance risk of the phenylpyrrole fungicide fludioxonil and the implications for cross resistance with the dicarboximide vinclozolin. Cross resistance between dicarboximides and phenylpyrroles, often found in the laboratory, has been extremely rare in the field. Over seven years of monitoring in Switzerland no fludioxonil field resistant isolates were found. Sexual crosses between a fludioxonil field resistant/vinclozolin field resistant strain isolated in France and a sensitive one showed an independent segregation of the resistance to the two fungicides, suggesting that different genes regulate field resistance to fludioxonil and vinclozolin. Sexual crosses between a fludioxonil field resistant B. fuckeliana strain and a fludioxonil laboratory resistant strain indicated that the two fludioxonil resistance phenotypes segregate independently. We concluded that fludioxonil resistance is influenced by at least two different genes. Genetic studies with laboratory strains resistant to both fungicides demonstrated a low segregation rate between fludioxonil and vinclozolin resistant phenotypes. These results let us hypothesize that fludioxonil and vinclozolin resistance induced in laboratory could be due to two genes closely linked. Finally, pathogenicity tests performed with progenies of sexual crosses showed a positive correlation between virulence and phenotypes.

Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The β-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro. In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley roots, and to reduce efficiently the severity of F. culmorum without affecting plant emergence. Quantification of GUS activity of IK726c5 in peat and vermiculite and on seeds was carried out. The GFP transformant, IK726d11, was visualized by epifluorescence and confocal scanning laser microscopy directly in soil, vermiculite, on carrot seed and roots, and on barley leaves. It was shown that C. rosea can thrive in very different niches. Conidia germination, colonization and conidiogenesis were demonstrated in vivo in all four environments. This is the first report on transformation of Clonostachys rosea with marker genes.

Cronartium flaccidum and Peridermium pini are rust fungi occurring on two-needle hard pines. According to previous molecular and morphological analyses, they are very closely related despite differences in their life-cycles. In this study we showed that although a low level of genetic differentiation occurs among populations of both P. pini and C. flaccidum, there is no overall differentiation between the two rusts, and in this respect they resemble a single taxon. We also observed evidence for linkage disequilibrium occurring between different alleles of separate loci of Peridermium pini suggesting that its population structure would be clonal. This suggests that strains of P. pini would have originated as asexual or self-fertilizing host range mutants of C. flaccidum.

The cell wall alkali-extractable water-soluble polysaccharide (F1SS) purified from the three species of Cephalotheca has been characterized. Two different structures have been deduced by chemical and structural analyses. The structure of the polysaccharide F1SS from C. purpurea and C. reniformis is similar, and composed of 3,6-di-O-substituted mannopyranose with terminal residues of rhamnopyranose. The polysaccharide F1SS from C. sulfurea contains 2,6-di-O-substituted mannopyranose and terminal residues of galactofuranose. Our results confirm the relatedness of C. purpurea and C. reniformis with Ophiostomatales and of C. sulfurea with Sordariales.

Culture media influenced the germination of conidia, appressorial development and mycelial growth of Metarhizium anisopliae. Although the addition of KCl to Sabouraud dextrose agar (SDAM) significantly reduced germination of the conidia of three isolates in vitro and lowered germination and appressorial development on the cuticles of Myzus persicae and Meligethes aeneus, conidia grown on SDAM and minimal medium (MM) were more aggressive than conidia derived from SDA or yeast extract agar. No relationship was found between either germination or appressorial development and LT50 values, suggesting that virulence depends on the speed of infection. Calcofluor staining and lectin binding showed that culture conditions also influenced the adhesion of Metarhizium anisopliae to insect cuticles through alteration of surface carbohydrates such as 1-4-β-glucans. Dark, pigmented conidia from MM bound less lectins and calcofluor whereas the paler conidia from the other media fluoresced more intensely. Conidia from SDAM fluoresced 20-fold brighter and adhered more readily to insect cuticle compared with conidia derived from MM. Although inocula from both media showed elevated pathogenic activity against aphid and pollen beetle, MM-grown conidia were more aggressive since high mortality was induced by comparatively fewer conidia. When fluorescent intensity of calcofluor-stained conidia was compared to the number of conidia adhering to the insect cuticles, the relationship was significant, indicating that this dye has practical implications for the assessment of fungal strains in screening programmes for commercial development.

Anthracnose caused by Colletotrichum is a severe foliar disease in rhododendron plantations in Sweden and Latvia. Isolates of this pathogen were collected and characterised based on morphological and molecular criteria. Out of 37 isolates examined, two with falcate spores were identified as C. dematium. The remainder of the isolates had straight cylindrical conidia, which were too variable to be reliably assigned to C. gloeosporioides (syn. C. azaleae) the reported causal agent of anthracnose of Rhododendron spp. These isolates were compared with the reference strains of C. gloeosporioides, with holo- and paratypes and with reference isolates of C. acutatum. The light and scanning electron microscopy examinations revealed that conidial shape was not a reliable criterion for separation of these two taxa and for the identification of our isolates. The two internal transcribed spacers (ITS1 and ITS2) and the 5·8S rRNA gene were PCR amplified and sequenced from all the rhododendron isolates and the reference strains, including the holo- and paratype materials. In addition, a segment of mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and a fragment of a β-tubulin gene from several representatives of isolates of rhododendron as well as the reference strains were amplified and sequenced. The sizes of the amplified fragments among the isolates studied were heterogeneous only for mtSSU rDNA, which allowed separation of C. acutatum from C. gloeosporioides. Parsimony analysis of the individual and combined nucleotide sequence data sets were concordant and indicated that all isolates originating from rhododendrons belonged to C. acutatum. The grouping of the isolates was further supported by bootstrap analysis. Thus, C. acutatum was identified as the causal agent of anthracnose of Rhododendron in Sweden and Latvia. To our knowledge, this is the first report of C. acutatum and C. dematium on Rhododendron. This study is also the first to determine the genetic status of the holotype of C. acutatum and also one of the paratypes on the basis of ITS sequences in comparison with world-wide living isolates of this taxon.

The sexual system of Heterobasidion insulare was elucidated and 40 specimens originating from the mainland of China, Taiwan, and Japan were studied with the aid of mating tests. Most of the material was also investigated with DNA fingerprinting using random amplified microsatellite (RAMS) and M13 minisatellite primers. Denaturing gradient gel electrophoresis (DGGE) and sequencing were applied for detecting variation in the internally transcribed spacer (ITS) region of the ribosomal DNA cluster. H. insulare proved to be a species complex with bipolar mating system. Clamp connections occurred in both heterokaryotic and homokaryotic mycelia. Three intersterility groups were found: the ‘T group’ included specimens from Taiwan and Guizhou Province (southern China), the ‘N group’ specimens from northern China and Japan, and the ‘Y group’ specimens from Yunnan (south-west China). The T group seems to be totally intersterile with the two other groups, and was grouped separately in neighbour joining analysis based on DNA fingerprints. As concluded from mating experiments, groups N and Y are partially intersterile, but they are able to produce hybrid heterokaryons, no group specific DNA markers were found, and they were grouped together in neighbour joining analysis. The identity of H. insulare sensu stricto was studied from the type collections. It seems that none of the above-presented taxa can be referred to it, and we propose the term H. insulare sensu lato to be used in future studies.

Genetic variability of 95 isolates of Beauveria bassiana from different geographical regions and hosts was assessed using RAPDs and ITS-RFLP molecular markers in order to characterize its genetic variability. Clusterings were obtained using UPGMA and Principal Coordinates Analysis. Four groups were identified with RAPDs. A 930 bp fragment was amplified with primers ITS1 and PN16, and polymorphisms were observed with AluI and MspI, generating two main clusters. Results indicate low genetic variability present in the Colombian B. bassiana population, no association to host type or geographic locality, and a population structure that is not clonal. This characterization sets criteria for determination of bio-insecticide potentiality, introduction of foreign strains and improvement of entomopathogens within an IPM program.

RAPD amplifications of 119 Mexican Colletotrichum gloeosporioides isolates from wild native Stylosanthes plants, of two Australian (internal reference isolates of type A and type B) and of two African (previously demonstrated as different from the A and B types) allowed the generation of four polymorphic clusters. The RAPD cluster I contained the Australian type A and 103 Mexican isolates from other species than S. guianensis. The cluster II contained the Australian type B isolate and 14 Mexican isolates from S. guianensis. The clusters III and IV contained one African and one Mexican isolate each.

A phylogenetic study was performed using the ITS1 sequences from 32 representative Mexican isolates selected in the four RAPD clusters, from the two Australian and the two African used as references, from 13 C. gloeosporioides from various hosts and origins available in the EMBL databank and from three phylogenetically related species of Colletotrichum. Two clusters (S and VHO) were generated. Cluster S contained the 30 isolates clustered in RAPD I and II, and the type A and B isolates from Stylosanthes selected in EMBL. Cluster VHO contained the two isolates belonging to RAPD clusters III and IV as well as the remaining Colletotrichum spp. and C. gloeosporioides isolates from various hosts and origins selected in EMBL.

Severe type A lesions were observed on several Stylosanthes species, including S. guianensis, after inoculation with the 26 Mexican isolates selected in RAPD cluster I. The four Mexican isolates selected in RAPD cluster II induced extensive type B lesions only on S. guianensis genotypes. The virulence spectra of the isolates was not linked to variations in their ITS1 region sequence. A wide range of anthracnose lesions was induced by the Mexican isolates in interactions with the various Stylosanthes genotypes.

On the basis of results obtained from the molecular and pathogenic experiments in the present study, of morphological and molecular data from previous work, as well as from other authors, a taxonomic infraspecific differentiation was proposed, as C. gloeosporioides f. stylosanthis f. sp. stylosanthis and f. sp. guianensis.