Poster Abstracts, Novel Molecular Tools

Identification of Basidiomycetes by PCR Assay Using Primers Targeted on Specific Region of Nuclear 18S rDNA

Alekhina, Irina A., & S.A. Bulat. Fungal Biochemistry Lab., V.L.Komarov Botanical Institute, St etersburg, 197376, Russia

The objective of the research was to develop PCR-based method suitable for reliable identification of ectomycorrhizal fungi to study species composition of fungal symbionts of forest-forming trees. The idea was to elaborate a method allowing the identification of ectomycorrhizal basidiomycetes using DNA extraction from rootlets and PCR with primers targeted on basidiomycete specfic sites of 18S rDNA. Two primers of our design were found to be able to specifically amplify ca 260 bp long fragment in basidiomycete 18S rDNA. Using this fragment digested with Sau3A and Taql enzymes it was possible to divide basidioma taxa and mycorrhizae samples into two groups. One large group included majority (12 of 16) of mycorrhizae as well as Russula taxa, Lactarius rufus, and Amanita citrina, while another one was consisted of three mycorrhizae samples as well as Leccinum vulpinum, two Amanita taxa, Cordnarius, Laccaria, Tricholoma and Hygrophorus taxa. However, the restriction analysis of the fragment chosen did not allow the reliable identification of all fungal spruce symbionts studied. For this purpose we used dd fingerprinting technique, which allowed us to reliable distinguish near all mycorrhizal samples and to find their counterparts.

Plasmid Partitioning in Aspergillus nidulans. Now in Full Colour

A. Aleksenko*, A. J. Clutterbuck+ *GNII GENETIKA, 1 Dorozhny 1, Moscow 113545 Russia +Division of Molecular Genetics, IBLS, University of Glasgow, Glasgow G 1 1 5JS, UK

A system has been devised which allows easy monitoring of behaviour and maintenance of autonomously replicating plasmids in a fungal colony. It consists of the yA gene from A. nidulans cloned on an AMA l -containing plasmid along with the selective marker gene argB. Distribution of vector molecules in the growing fungal colony is easily observed as green-coloured areas on the backgound of yellow-conidial recipient phenotype. The system has made it possible to demonstrate that, although typical phenotypic stability of replicative transformants on selective media does not exceed 60%, this figure does not reflect the frequency of mitotic loss. Mitotic loss of plasmid seems to be an infrequent event, but portions of mycelium lacking the plasmid remain viable due to cross-feeding with plasmidbearing portions.

The system was used to create a genomic library for screening sequences which affect behaviour of transforming DNAs in the fungus. It was demonstrated that, contrary to a common belief, unstable transformants are generated by a genomic library at a relatively high frequency. Experiments aimed at isolating sequences from the A. nidulans genome which affect plasmid replication and partitioning are currently under way.

Insertion of a Human Telomere at the End of a Chromosome of the Filamentous Fungus Podospora anserina Leads to the Formation of a Functional Telomere

C. Barreau, P. Wilson, V. Roques-Duflo & J. P. Javerzat. IBGC,- CNRS-UPR9026, Laboratoire de Genetique Moleculaire des Champignons Filamenteux; 33077 Bordeaux France

The repeat T2AG3 has been recently identified at telomeric locations on the chromosomes of the fungus Podospora anserina. About 200 bp of uniform repetitions of T2AG3 are present only at the ends of the chromosomes. The same telomeric motif is observed in other filamentous fungi N. crassa, F. oxysporum, C. fulvum as well as in most eucaryotes including humans.

A human telomeric sequence of 600 bp (HTEL) containing mainly T2AG3 repeats has been shown to be able to function as telomeres when inserted into chromosomes of human cell leading to the breakage of the chromosomes. When inserted at both ends of a linear vector, this sequence makes the plasmid behaving as an unstable minichromosome in the fungus P. anserina suggesting that the telomeric function is conserved.

In order to demonstrate that the human sequence can fullfil all the telomeric function in the fungus P. anserina, the HTEL sequence has been introduced by transformation at the end of a chromosome. The results presented show that in the transfon-ned strains, the HTEL sequence became functional leading to the breakage of the chromosome.

In such strains, the selectable ura5 marker used for transformation is now located very close to the telomere. In yeasts, it has been shown that the expression of genes placed in the direct viccinity of a telomere is strongly repressed. This is known as the telomere position effect (TPE) and attributed to the invadment of the gene by proteins of the telosome. By studying the expression of the ura5 gene, it will be investigated if such a telomeric position effect is observed in the fungus P. anserina.

UP-PCR (RAPD Like) and Virulence Analysis of Host Specificity in Phytopathogenic Fungus Cochliobolus sativus

Bulat S., Nina Mironenko. Eukaryote's Genetic Laboratory, Petersburg Nuclear Physics Institute, Leningrad region, Gatchina, 188350, Russia

The objective was to evaluate at the genome and the virulence levels whether C. sativus is specialized with respect to its hosts. The 8 fungal populations were sampled from barley, wheat and wild barley and analyzed by the UP-PCR technique. Each population was found to be characterized by one major type of UP-PCR patterns. Of 7 types observed in total, two polymorphisms proved to be characteristic of wheat, one type was specific for wild barley and four others were barley specific. The phylogenetic analysis of polymorphisms showed the close relatedness of polymorphisms specific for wheat but not for barley.

The virulence analysis revealed that the isolates of two main UP-PCR polymorphisms from wheat population are more virulent to wheat than barley, while the isolates from barley population were of equivalent virulence regarding these two hosts.

Thus., C. sativus possesses the wheat specificity which is based on genome differences as well as pathogenicity variation.

Multiplex PCR Reaction for the Detection of Potential Aflatoxin and Sterigmatocystin Producing Fungi

Rolf Geisen. Federal Research Centre for Nutrition, Institute of Hygiene and Toxicology, Engesserstr. 20, 76131 Karlsruhe, Germany

A multiplex PCR reaction was developed to amplify the aflatoxin biosynthetic genes: norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1) and sterigmatocystin O-methytransferase (omt-A). The reaction gives a triplet banding pattern with aflatoxin producing strains of Aspergillus flavus, A. parasiticus and also with sterigmatocystin p -producing strains of A. versicolor. The pattern of aflatoxin negative A. flavus strains varied. One strain showed no signal. In one strain the band for the gene of the omt-A gene is missing but in a third strain the triplicate pattern is complete. A. oryzae and A. sojae, which do not produce aflatoxin, but are closely related to A. flavus and A. parasiticus apparently carries sequences homologous to the ver-I and the omt-A gene, but the primer set specific for the nor-1 gene gave no signal indicating sequence variation or deletion of that gene. Most other food related strains tested showed negative results for all genes. The exception was Penicillium roqueforti which showed two bands. One with an identical fragment length as that of the nor-1 PCR product of A. parasiticus and one with an increased length compared to the ver-I PCR signal. The multiplex PCR reaction therefore seems to be specific for potential aflatoxin and sterigmatocystin producer.

Use of Internal Transcribed Spacers (ITS) for Detection of Penicillium by the Polymerase Chain Reaction

Lars Hagsholm Pedersen, Pernille Skouboe, Marianne Boysen and Lone Rossen. Biotechnological Institute, building 227, Anker Engelundsvej 1,DK-2800 Lyngby, Denmark.

Rapid identification of filamenentous fungi is becoming increasingly important in food myclogy, both for monitoring a production process and for the identification of food spoilers.

A 610 bp region spanning the ITS regions and 5.8 S gene of 72 strains belonging to 28 Penicillium subgenus Penicillium species was sequenced and the data used for phylogenitic analysis and identification of specific PCR primers. The region is very conserved with only 29 individual positions differring in one or more species. The largest number of differences was found for P. rogueforti and P. carneum, as 13 nucleotide differences separated these two species from the rest of the penicillia. Bootstrapped parsimony analysis showed that the penicillia investigated could be allocated to three main groups.

The primer sets were tested by performing PCR on DNA extracted from a range of Penicillum subgenus Penicillium, other Penicillium and non Penicillum species. A 336 bp fragment was specifically amplified from Penicillium subgenus Penicillium DNA by the primers ITS212d/ITS549 and a 300 bp fragment was specifically amplified from P. rogueforti and P. carneum DNA by the primers ITS183/ITS401. In addition we tested the primer sets and PCR conditions on cheese extracts. Fungal hyphae or spores were partially purified from blue veined or camenbert cheese by aqueous two-phase polymer partitioning and DNA was extracted by CTAB after proteinase K digestion. The Penicillium subgenus specific fragment was amplified from both DNA extracts and the P. roqueforti / P. carneum specific fragment was amplified from DNA extracted from the blue veined cheese only.

Mutational and Differential Display Analyses of the Yeast to Hypha Transition in Mucor circinelloides

J.L Blasco, A. Velayos and E. A. Iturriaga. Area de Genetics, Departamento de Microbiologia y Genetica. Universidad de Salamanca. 37007 Salamanca Spain

Two different approaches are being employed to analyze the dimorphic transitions in Mucor circinelloides.

A program was designed to isolate mutants affected in the yeast-to hypha transition. Nitrosoguanidine mutagenesis of M. circinelloides spores led to the isolation of several mutants which can grow only as yeasts under aeorobic conditions. Characterization of the mutants and complementation analyses are now in progress. We have designed also a procedure to isolate mutants affected in the hypha-to-yeast transition. The differential display technique is a powerful tool to detect and isolate genes which are differentially expressed in two cell types, two stages of development, or, as it is the case two alternative growth models (Liang, P. and Pardee, A.B, (1992). Science 257:967). We have displayed mRNAs from yeasts, hyphae, and the yeast-to-hypha transition. Total RNA was obtained from yeasts grown for 16 hours under a nitrogen atmosphere (yeasts), germlings grown for two days (mycelium), and yeasts grown for 16 hours under a nitrogen atmosphere and then allowed to Initiate the hyphal growth for 4 hours aerobically (transition). A number of cDNA fragments have been isolated which are differentially expressed during the transition as compared to yeast or mycelial growth. One of these cDNA clones shows high sequence similarity (about 70% at the amino acid level) with ornithine decarboxylase genes from several organisms. ODC activity was previously shown to increase 40-fold during the yeast-to hypha transition in Mucor- spp. (Calvo-Mendez C., Martinez-Pacheco, M. and Ruiz Herrera, J. (1987). Exp Mycol 11:270-277). Other cDNA clones specfically expressed during the phase shift show no relevant homology to any DNA or protein sequences in the databases and their function remains unknown.

These preliminary data suggest that analysis of M. circinelloide dimorphic transition can effectively be carried out by means of the display technique. Mutants altered in the phase shift can also contribute to elucidate the genes involved in the dimorphic response.

Restriction Enzyme-mediated DNA Integration in Coprinus cinereus

Jose Granado, Ussula Kues, Katerina Kertexz-Chaloupkova and Markus Aebi. Institut fur Mikrobiologie, ETH Zurich, Schmelzbergstr. 7, CH-8092 Zurich

Tagging genes by insertion of foreign DNA is an elegant way to create mutations for genetical studies. In filamentous fungi, the use of transposons is up till now not possible but recently an alternative technique (restriction enzyme-mediated DNA integration, REMI) has been introduced. By action of restriction enzymes added to the transformation mix, linear DNA is inserted into the host genome. Here we describe effects of restriction enzymes on transformation in the basidiomycete fungus Coprinus cinereus. It is our aim to establish an efficient system for tagging developmental genes in this fungus.

First we constructed pPAB2, a vector for REMI which has a number of unique restriction sites in poly-linker regions and which is able to complement pab1 mutations in C. cinereus. Two different strains and three different enzymes (BamHl, EcoRl, Pstl, 20-180 units per transformation sample) were used. We observed that transformants appear earlier when restriction enzymes were present and that transformation rates are strongly influenced by the amounts of enzyme added. Each enzyme has a specific concentration where the highest number of transformants is obtained. Transformation rates rapidly decrease with further increase of enzyme.

We isolated about 7500 transformants of C. cinereus and determined mutation rates in fungal development and mode of plasmid integration in relation to enzymes and enzyme units used. In line with the effects on transformation, mutation rates are lowest with optimal transformation rates and increase with higher enzyme doses. Plasmid integration varies in type and number. True REMI events and single integrations are preferentially found with lower enzyme concentrations. High enzyme concentrations can cause multiple insertion events and may accumulate mutations created by incorrect repair of DNA damage induced by added restriction enzyme.

Based on our experiences we therefore propose to use those enzyme concentrations for REMI mutagenesis which generate the highest amount of transformants and show the lowest rates of mutation. Under such conditions, the probability seems to be highest for a specific mutation induced by a single, restriction enzyme mediated integration of the selection marker.

Specific Detection of Phytophthora nicotianae Using the Polymerase Chain Reaction and Primers Based on the Sequence of its Elicitin Gene ParA1

L Lacourt, J. M. Duncan, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.

Primers based on the sequence of the elicitin gene ParAl of Phytophthora nicotianae were used to detect specifically the fungus by the polymerase chain reaction (PCR). Six primers from flanking and coding regions of the ParAl gene, were tested in various combinations. One combination, EL7/IL8, with EL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others from a variety of hosts. The sequence of the primer product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParAl gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.

Gene Targeting in Schizophyllum commune

Lengeler, K.B. and Kothe, E. Department of Biology, Molecular Genetics Philipps-University, Karl-von-Frisch Str. 35032

Marburg, FR-Germany

To aid site-directed integration of transforming DNA two gene targeting vectors have been constructed for S. commune. The vectors, pUT1 and pUT2, allow the investigation of gene expression excluding the positional effects of integration sites observed with ectopically integrating constructs.

Both vectors, pUT1 and pUT2, contain 31-truncated ural genes missing the last 162 bp or 312 bp, respectively. These constructs are not sufficient to complement the mutant ural- phenotype upon ectopic integration. only homologous recombination within the ural locus yields active ural+ genes and therefore can be detected by screening for uracil prototrophy.

Transformation efficiency of both pUT1 and pUT2 is approximately 500-fold reduced in comparison to transformation with a wild-type allele.

Molecular analysis of the transformants obtained with pUT1 confirmed the homologous integration event expected for gene targeting. The pUT2 transformants revealed properties of an replacement event instead.

Molecular Data Suggest Further Re-classification of the Genus Trichoderma

Lieckfeldt, E., Kuhls, K., Daniel, H.-M., Borner, T. Humboldt-Universitat zu Berlin, Inst. for Biologie /Genetik, Invalidenstr. 43, 10115 Berlin, Germany

The genus Trichoderma introduced by PERSOON (1794) and later revised by RIFAI (1969) and BISSETT (1984, 1991) consists of deuteromycetous Trichoderma species as well as anamorphs of the ascomycetous genus Hypocrea. From comprehensive morphological studies on this genus two taxonomical keys are provided to differentiate Trichoderma species into 9 aggregates (RIFAI 1969) or 5 sections (Trichoderma, Longibrachiatum, Saturnisporum, Pachybasium, Hvpocreanun; BISSETT 1984). A taxonomy in terms of phylogenetic relatedness of the genus is still unresolved.

We have used a series of molecular methods (RFLP and sequence analysis of the internal transcribed spacers ITS-1 and ITS-2 of the rRNA gene complex, RAPD assay) differing in their sensitivity with respect to the characterization of relationships within the genus Trichoderma at different taxonomic levels (inter/intrageneric, inter/intrasectio, inter/intraspecies). We analyzed 117 strains belonging to the different species of section Longibrachiatum. All species within this section were found to be closely related. In order to evaluate these data with respect to "taxon" definition and taxon borders we compared ITS sequence data from the type species and related strains of the remaining sections of Trichoderma. Special emphasis was put on the inclusion of a broad spectrum of.strains representing anamorphs of Hypocrea spec. The molecular data strongly suggest a re-classification of the Trichoderma taxonomy according to BISSETT (1984, 1991): I. The type species of section Saturnisporun clearly falls into section Longibrachiatum. II. There is no clear. separation between sections Trichoderma and Pachybasium. III. Part of the Hypocrea species is clearly related to distinct Trichoderma species, i.e. we found new anamorph-teleomorph connections.

Heterologous and Homologous Transformation of Acremonium crysogenum

Jorg Nosek, Renate Radzio, Esther Schmitt, Ines Westphal & Ulrich Kuck. Lehrstuhl fur AIlgemeine Botanik, Ruhr- Universitat Bochum D-44780 Bochum

The filamentous fungus Acremonium chrysogenum is the most important producer of the -lactam antibiotic cephalosporin C. Many attempts have therefore been made to achieve a detailed understanding of the biosynthesis of this compound. The pcbAB/pcbC and cefEF/cefG genes, both of which are involved in this process, have been cloned and characterized. It has been shown, that pcbAB and pcbC are closely linked and transcribed in opposite directions, as are the cefEF and cefG genes (for review, see [1]). To study potential controlling sequences, which are expected to be located in the intergenic regions, reporter gene constructs have been made. The non translated sequences of both pairs of genes have been translationally fused to the lacZ reporter gene derived from plasmid pSI8.8 [2]. Using both constructs, a set of promoter deletions was transformed into A. chrysogenum for measuring the coresponding -galactosidase activities.

In previous work, the pcbC promotor turned out to mediate relatively strong activity [2]. To identify additional sequences with similar activity but different expression pattern, we used the reporter vector SI8.8 to screen a library of A. chrysogenum DNA fragments. Such sequences are suitable to express heterologous genes in the eukaryotic host A. chrysogenum.

A system which would allow the targeted integration of DNA into the genome of A. chrysogenum would be desirable. For this reason the tubulin gene of the fungus has been cloned. By introducing a point mutation into the gene it has been altered in such a way as to confer resistance to the fungicide benomyl [3]. This is the first step in developing a vector system, that allows targeted integration and positive selection of transformants in this organism.

[1] Martin JF, Gutierrez S, Fernandez FJ, Velasco J, Fierro F, Marcos AT & Kosalkova K (I 994) Antonie van Leeuwenhoek 65: 227-243

[2] Menne S, Walz M & Kuck, U (1994) Appl Microbiol Biotechnol 42: 57-66

[3] Nowak C & Kilck U (1994) Curr Genet 25: 3440

The Phylogeny of the Genus Claviceps

Sylvie Pazoutovai,IrenaHamplova, Marek Linka and Miroslav Flieger. Laboratory of the Physiology and Genetics of filamentous Fungi. Inst. Microbiology CAS, Videnska 1083, 142 20 Prague, Czech Republic

Claviceps species colonize wide variety of grasses and sedges. C. purpurea producing peptide ergot alkaloids exhibits the broadest host spectrum. Other Claviceps species are restricted to smaller host taxa: C. fusiformis (Panicoidea) producing clavine alkaloids, C. paspali (Paspalum) producing clavines and simple lysergic acid amides and C. gigantea (varieties of Zea mays), where we found not only clavines but also simple lysergic acid amides. Other strains used in this study were C. grohii (sedge), C microcephala (pearl millet), C. viridis (Oplismenus ) and C.phalaridis (Phalaris). In the past, two major reclassifications were suggested: the separation of C. purpurea isolates to different taxa (cf Loveless 197 1) and placement of C paspaii, C gigantea and C grohii into a new genus Mothesia (Oddo and Tonolo 1967). The above species were used for phylogenetical study based on RFLP and RAPD DNA fingerprinting and the analysis of ITSI and ITS2 DNA sequences.

Both genome fingerprinting methods confirmed the higher intraspecific variability among C. purpurea isolates when compared with strains of C. paspali and C. fusiformis. However, their variability did not correlate with the host species or the geographic origin and therefore did not substantiate the sub-division of C purpurea species. The RFLP fingerprints of C purpurea and C. fusiformis were related as well as those of C. paspali and C. gigantea. The phylogeny tree obtained by the neighbor joining method from the comparison of ITSI and ITS2 confirned the close relatedness of C. purpurea and C. fusiformis as well as C. grohii. On the other side, C. paspaii and C. gigantea were closer to endophytic Balansiae. C.Paspali as well as Balansiae produce alkaloids on high phosphate media whereas C. purpurea and C. fusiformis are subjected to strong phosphate inhibition (Pazoutovai et al. 1983).The genetical and physiological distance of C. paspaii and C. gigantea from the "purpurea" group could substantiate their placement into a new genus .

Loveless AR (197 1) Trms. Br. Mycol. Soc. 56, 419434

Oddo N and Tonolo A (1967) Ann. Ist. Super. Sanita 3, 16-25

Pazoutovai S et al, (1983) Etir. J. Appl. Microbiol. Biotechnol. 16: 208-21 1,

Application of DDRT- PCR to the Analysis of Differential Gene Expression in a Plant-fungal Pathogen Interaction

Ernesto P. Benito, Theo Prins and Jan A.L. van Kan. Dept. of Phytopathology, Wageningen Agricultural University, P.O. Box 8025, 6700 EE Wageningen, The Netherlands.

Botrytis cinerea is pathogen to a wide range of host plants. A non-biassed approach is being carried out in order to detect those fungal genes which are differentially expressed during the interaction with the host plant. Among those genes specifically expressed in planta, one would expect to find fungal genes with a role in pathogenicity. To this end the expression pattern of B. cinerea during the pathogenesis process on tomato is being compared with its expression pattern during saprophytic growth by "differential display of mRNA" (DDRT-PCR). As a first step a standard inoculation procedure of B. cinerea on detached tomato leaves has been developed. Inoculation conditions have been optimized and high infection efficiencies have been achieved by inducing high germination rates on the leaves as well as synchronization of the infection process in different lesions. The infection process has been followed visually and microscopically. At the molecular level the progress of infection has been estimated by determining the proportion of fungal RNA in the total interaction RNA population in a time course experiment. Since in the interaction two organisms are present, by "differential display" cDNAs from both, the fungus and the plant, are detected. Most of these cDNAs represent fungal or plant genes constitutively expressed. Their origin can be discriminated by comparison of the expression pattern displayed in the interaction with the expression pattern of the fungus grown in vitro or the expression pattern of a non infected tomato plant. Interaction specific cDNAs are also detected which will represent either fungal in planta induced genes or plant defense genes induced in response to a pathogen. In order to discriminate these last two categories of cDNAs, samples from control infections of tomato leaves with two other pathogens (Phytophthora infestans and Tobacco Necrosis Virus) are being used.

The DDRT-PCR procedure has been adapted to the analysis of the interaction B. cinerea-tomato and the controls indicated above have been included. Several fungal interaction specific cDNAs have been isolated. A higher level of expression or a differential expression in planta for some of the corresponding genes has been demonstrated by Northern blot analysis.

RAPD Analysis of Trichophyton verrucosum Strains

Maridn Hajdlich1, Evzen Weigl1, Wadislav Raclavsky2,Vladimir Kotala1, Tomdg Michdlek1, Radomir Nemeek1

1 Department of Immunology and 2Department of Biology, Faculty of Medicine, Palacky University, Hnevotinska 3, Olomouc, CZ-775 15,Czech Republic

20 random primers (dekamers) were tested for their ability to amplify genomic DNA of Trichophyton verrucosum using random amplified polymorphic DNA (RAPD) analysis. Six of these primers were selected for further study aimed at discrimination of wild and vaccination strains of Trichophyton verrucosum. The estimated proportions of false positives and false negatives in the RAPD data were calculated from repetitive experiments to prevent PCR artifacts. Corrected values of Nei and Li's coefficient of similarity were then used for the comparison of typed strains.

The results indicate the ability of RAPD to distinguish strains of Trichophyton verrucosum. The method is even able to discriminate between the avirulent vaccination strains (Trichophyton verrucosum TVM-9 and Trichophyton verrucosum TV-M-310) prepared by UVmutagenesis originating in the standard wild strain Trichophyton verrucosum Straznice and the original wild strain Trichophyton verrucosum Straznice. These outcomes suggest new possibilities for epidemiological analysis, for discrimination between different vaccination strains and for studies of fungal population in infected host.

Typing of Aspergillus fumigagus Isolates by the Random Amplified Polymorphic DNA Technique

Edit Rinyu and Janos Varga. Department of Microbiology, Atilla Jozsef University, P.O. Box 533, H-6701 Szegd, Hungary

Aspergillus fumigatus Fresenius is an opportunistic human pathogenic fungus, which may cause several diseases, such as allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Typing of these isolates is at the center of interest of epidemiological studies. We have tested the applicability of a PCR-based method for this purpose. Sixty-one isolates and collection strains of Aspergillus fumigatus were compared by the random amplified polymorphic DNA (RAPD) technique. Although the patterns of the strains were very similar for most of the primers, the application of three primers (OPC-06, OPC07, and OPC-10) made it possible to cluster the 61 A. fumigatus isolates into 47 groups. The results allowed the RAPD technique to be used more efficiently for typing A. fumigatus isolates than isoenzyme analysis, or restriction enzyme analysis of the mitochondrial DNA and the ribosomal gene cluster of these strains (1). The application of another primer (OPC-08) permitted the distinction of A. fumigatus from other potentially pathogenic aspergilli (A. niger, A. flavus and A. terreus) and also from closely related species (species of section Fumigati, e.g. Aspergillus fennelliae and A. thermomutatus). The only cross-reacting species was A. fischerianus, which is the species most closely related to A fumigatus.

(1) E. Rinyu, J. Varga and L. Ferenczy (1995) Phenotypic and genotypic analysis of variability in Aspergillus fumigatus. J. Clin. Microbiol. 33: 2567-2575

Comparison the Isolates of the Postharvest Pathogen Mucor piriformis Using Molecular Markers

Csaba Vagvolgyi, Tamas Papp, Agnes Nagy1, Zsuzanna Palagyi, Themis H, Michaikides2 and Lajos Ferenczy

Department of Microbiology, Atilla Jozsef University, P.O. Box 533, H-6701 Szegd, Hungary. 1Chemical Works of Gedeon Richter Ltd., P.O.Box 27 H1475, Budapest Hungary. 2 Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, Parlier, California, 93648 USA

The soilbome fungus M. piriformis Fischer is an important postharvest pathogen of different fruits and vegetables. This can cause the substantial decay of such agricultural products if they are stored or transported at low temperature for a prolonged time.

The genetic diversity of M. piriformis was studied by isozyme polymorphism and random amplified polymorphic DNA (RAPD) analyses. Six enzyme activities, catalase (CAT), -esterase (EST), glucosc-6-phosphate dchydrogenase (G6P), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) have been tested for 10 (+), 9 (-) and 10 neutral isolates. Some isozyme markers (EST, G6DH, and MDH) were useful to differentiate the strains with mating abilities from neutral ones.

The six different 10-bp primers used for RAPD analyses revealed different levels of diversity, three to nine different amplification patterns were detected. The results indicated a higher degree of variability than that found in isoenzyme studies. The dendrogram produced bv the unweighted pair group method from the unified data sets revealed a correlation between the two clusters obtained and the mating potency of the isolates. These results demonstrate that both isoenzyme and RAPD analysis provides an efficient approach for genetic studies of M. piriformis.

This work was supported in part by the Hungarian Scientific Research Fund (OTKA) # F/4 017677.

Highly Efficient Homologous Integration via Tandem Exo-6-1,3-glucanase Genes in Common Mushroom, Agaricus bisporus

Miranda van de Rhee, Odette Mendes, Marc Werten and Hans Mooibroek. Agrotechnological Research Institute ATO-DLO, P.O.Box 17, 6700 AA Wageningen, The Netherlands

Recently, a transformation system was developed for common mushroom, Agaricus bisporus. Plasmids carrying the hygromycin resistance gene controlled by regulatory sequences from either Aspergillus nidulans (pAN7-1) or A. bisporus were introduced into Agaricus protoplasts by electroporation. Approximately 1-100 transformants arose from 0.2-1 x 108 protoplasts using 10 ug linearized DNA. Linearization of plasmid DNA increased the number of transformants 2-5 fold. A random 3 kb genomic fragment from A. bisporus was cloned into plasmid pAN7-1, downstream of the A. nidulans terminator. The resulting plasmid pHAG3-1 was linearized within this homologous region (called AbGH3) before transformation. Transformation frequency was not enhanced by this plasmid, although it was found that homologous integration occurred in about 50% of the transformants. Homologous integration was found with pHAG3-1 linearized at three different positions within the AbGH3 sequence generating either blunt, 5'- or 3'-overhanging ends. Tandem integrations were also observed at the homologous position with restoration of the restriction sites used for linearization.

The AbGH3 fragment was found to contain two open reading frames in tandem, which showed 60% similarity to exo-B-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The function of these enzymes is unknown, but they are thought to be involved in cell wall metabolism. The upstream gene (AbEXGI) encodes a polypeptide of 419 amino acids, whereas only the start of the second gene (AbEXG2) is present on the genomic fragment, encoding the N-terminal 159 amino acids. Both polypeptides contain a predicted signal peptide region. The genes are interrupted by numerous short introns at conserved positions. Expression at the mRNA level is low in vegetative mycelium and relatively high in fruitbodies. Exoglucanase mRNA was increased in vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position. Fruitbodies are currently grown of this transformant to study the effect of exoglucanase overexpression.

Isolation of Meiosis Specific Genes from Aspergillus nidulans

Diana van Heemst, Edu Holub, Klaas Swart, Henk van den Broek, Christa Heijting and Theo Goosen.

Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703HA Wageningen, The Netherlands

A first step towards a molecular analysis of meiosis in any organism, is the cloning of meiosis specific genes from that organism. In order to clone genes involved in meiosis in the filamentous fungus A. nidulans several different strategies can be followed. The strategy that will be discussed here, is transformation complementation of mutants defective in meiosis. In A. nidulans two classes of such mutants exist.

The first class consists of mutants that are both defective in the repair of DNA damage as well as in meiosis. Mutants in the A. nidulans uvsC gene have such a phenotype. We will describe the cloning of the uvsC gene of A. nidulans by transformation complementation of an A. nidulans uvsCII4 mutant. Sequence analysis of the smallest fragment still giving full complementation, revealed strong homology of the predicted protein sequence with all known RAD51 homologs.

The second class are meiosis specific mutants. Since these were not already available in A. nidulans, we have set up a screening to isolate such mei mutants. One of them, meiAl, has been cytologically characterized and seems to be blocked at one of the later stages of the first meiotic division. We will report the cloning and characterization of the corresponding wild type meiA gene, which we are currently undertaking.

Characterization of Phytophthora infestans Ham34::GUS-Transformants

Pieter van West, Anke J. de Jong and Francine Govers. Department of Phytopathology, Graduate School Experimental Plant Sciences, Wageningen Agricultural University, Binnenhaven 9, 6709 PD, Wageningen, The Netherlands.

A few years ago Judelson et al (1991, MPMI 4: 602-607) have established a DNA-transformation procedure for the oomycetous plant pathogen Phytophthora infestans. In this method, CaCl2 and polyethylene glycol are used to introduce DNA complexed with cationic liposomes into protoplasts which are made from germinating sporangia. After DNA uptake the protoplasts are allowed to regenerate on selective medium. With this procedure we obtained a variety of stable P. infestans transformants. Among those are transformants containing a gene construct in which the Ham34 gene promoter of Bremia lactucae is fused to the coding region of the GUS-reporter gene. The activity of the Ham34 promoter was analyzed by GUS staining of in vitro and in plants grown mycelium, sporangia and germinating cysts. Although the Ham34 gene promoter is thought to be constitutive in P. infestans, it appeared that mycelium of a number of Ham34::GUS-transformants contains blue and white sectors, and in some transformants not all sporangia or germinating cysts stain blue. In other transformants blue staining was found in all hyphae, germinating cysts and sporangia. These observations raised questions about the transformation procedure and urged us to investigate the transformants in more detail. Results of the characterization will be presented.

REMI-Mutagenesis in Claviceps purpurea

Thorsten Vo , Ulrike Mulier and Paul Tudzynski. Westfalische Wilheims-Universitat Schlo garten 3, 48149 Munster, Germany

The ascomycete Claviceps purpurea attacks young flowers of many grass- and cereal species, especially Secale cereale1. To interrupt and to tag genes, which are involved in pathogenesis, we generated mutants of a possibly haploid benomyl-derivate from Claviceps purpurea with the REMI-technique (restriction enzyme mediated integration). The transformation-rate was lower with REMI than with the established transformation-system in Claviceps purpurea, in contrast to the results in many other systems. We isolated 1031 REMI-clones, which were generated with different enzymes. Most of these clones have only one copy of the vector. In 12,3 % of the tested strains it was possible to recover the vector with the REMI-enzyme. So far 64 transformants were tested on rye-flowers with regard to their virulence. Six of these clones not substantially differing in growth and conidia-germination-rate to the wildtype, showed a clearly reduced pathogenity on plants, representing the first pathogenity mutants of Claviceps purpurea. From two of these strains vector-flanking sequences were isolated and sequenced.

1Tudzynski, P., Tenberge, K. B., Oeser, B. (1995) Claviceps purpurea, in: Pathogenesis and host specificity in plant diseases. (U. Singh, ed.), Vol. IV Eukaryotes. Pergamon Press, New York, pp 161-187.

Molecular Analysis for Virulence in Fusarium oxysporum F.sp. dianthi

Cees Waalwijk, Jacq de Koning, Robert Baayen & Hilde van Pelt. Fusarium Research Group, Department of Virulence and Resistance, IPO-DLO, PO Box 9060, 6700 GW Wageningen, The Netherlands.

Isolates of Fusarium oxysporum f.sp dianthi show a large variation in virulence for different cultivars of carnation. Previously we have identified more than 10 races, which all appear to belong to different VCGS. However, within VCG 0022, two races (1 and 8) occur that evoke similar reactions on most differentials, with the exception of cv. Pallas. This cultivar is highly susceptible to race 8 and completely resistant to race 1. Both races can be considered as near-isogenic as they belong to the same VCG and on the basis of electro-karyotyping and isozyme, RFLP and RAPD analyses.

Since both races only reveal differences in pathogenicity assays, we are currently investigating whether the gene-for-gene theory is applicable in this plant-pathogen interaction. In this patho-system the avirulent race 1 would differ from the virulent race 8 by one or a few genes involved in specific recognition by cv. Pallas. Two approaches have been chosen to test this hypothesis.

Firstly, mRNAs from the infected xylem of stem inoculated cv. Early Sam, that is susceptible to both races are analyzed by differential display. In this cultivar both races form sufficient biomass whereas the avirulence factor(s) from race 1 are assumed to be expressed.

Secondly, genomic subtraction is being performed using the DNA from the avirulent race 1 as tester-DNA and the DNA from the virulent race 8 as subtractor-DNA. Genomic fragments supposed to be involved in the race specificity of race 1 isolates are currently being analyzed.

Successful Crosses and RAPD Analysis of Progenies Indicate a Heterothallic Bipolar Mating System in Mycosphaerella graminicola

Gert H.J. Kema', Els C.P. Verstappen', Maria Todorova2 and Cees Waalwijk'. 'DLO-Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, the Netherlands. 2 Plant Protection Institute, 2230 Kostinbrod, P.O. Box 238, Sofia, Bulgaria.

Monospore isolates of Mycosphaerella graminicola derived from ascospores that were considered to originate from a single ascus were studied by the polymerase chain reaction (PCR) with 33 RAPD primers. The results of these analyses indicated that 7 of these monospores were indeed derived from the same ascus. One monospore was crossed with the remaining six and three of these crosses were successful, directing to a bipolar mating system in M. graminicola. Three RAPD primers were used to study segregation of markers in random ascospore progenies of 162 isolates from these crosses. Mendelian segregation of RAPD markers was observed, and in one progeny individual genotypes were confirmed by hybridization analyses. These results prove the existence of a bipolar heterothallic mating system in M. graminicola, which has important epidemiological consequences, and provides an explanation for the vast genetic variation in this pathogen.

Phylogenetic Relationships of Fusarium oxysporum Isolates as Revealed by RAPD-PCR, Isozyme and rDNA RFLP Analyses

T.Y li Mattila & S. Paavanen-Huhtala, Lab. of Plant Physiology and Molecular Biology, Dept of Biology, Univ. of Turku, FIN-20500 Turku, Finland.

Differences in RAPD-PCR and isozyme polymorphisms between 26 isolates of Fusarium oxysporum were compared using native polyacrylamide gel electrophoresis and agarose gel electrophoresis. 13 of the isolates were collected from barley, while the rest of the isolates were collected from various cultivated dicotyledonous plants in Finland. The species designation of all isolates was verified by morphology.

Clear isozyme polymorphims was detected in five enzymes by which the isolates could be divided into six main groups by UPGMA analysis at the similarity level of 55 % . The phenotypes from RAPD-PCR analysis formed 7 main groups in UPGMA analysis at the similarity level of 60 % and the composition of these groups was somewhat different from that of isozyme analysis. The phylogenetic relationships between F oxysporum isolates were studied bv the heuristic search mode of PAUP 3.1.1. and by the Neighbor Joining and branch and bound analyses of PHYLIP 3.5. When the data matrices from isozyme and RAPD-PCR analyses were combined, the F oxysporum strains could be divided into six main clades in phylogenetic analyses. In the Neighbor Joining tree each of these six clades was supported by bootstrap values higher than 50 %. A correlation was found between the geographical origin and the phylogenetic relationship of isolates collected from barley.

Five of the F.oxysporum isolates representing five of the six main clades of phylogenetic trees were furthermore studied by rDNA RFLP analysis on polvacrylamide gel together with 6 isolates of F. avenaceum and one isolate of F. redolens, F. graminearum, and E equiseti. The isolates of all species formed their own groups in UPGMA and branch and bound analyses of PHYLIP, except for one isolate of F oxysporum, which was grouped to the same group with the only isolate of F. redolens. The bootstrap values of all six groups of branch and bound analysis were higher than 70 %. Thus it is possible that at least one of the isolates that was morphologically identified as F. oxysporum actually belongs to the species F.redolens, which is known to be closely related to F. oxysporum.

Return to the ECFG Program

Return to the FGSC Home page

Contact the FGSC

Last modified 8/13/96 KMC