Molecular diagnosis

Many fungal parasites have been associated with blackleg disease on Brassica crops and other crucifers. Especially, Phoma lingam and its teleomorph Leptosphaeria maculans have been described by an important pathogen of oilseed rape (Brassica napus). The infection of oilseed rape with Phoma is often accompanied with a devasting invasion of further parasitic as well as saprophytic fungi, which infect their host plant as more or less secondary pathogens in a later stage of disease development. Species of the genera Alternaria, Fusarium and Verticillium as well as Sclerotinia sclerotiorum, Botrytis cinerea, Cylindrosporium concentricum or the complex of Rhizoctonia solani and Pythium belong to this mixture of infection agents in the bio- and rhizosphere of rapeseed plants.

Their morphological identification as fungal phytopathogens in the early stages of disease is often handicapped by missing symptomatical characteristics or sometimes unspecific (small lesions, chlorotic spots) symptoms during plant growth. However, after an unequivocal identification of the fungi in the final period of vegetative growth and pod maturity it is economically not worth to treat the crops with fungicides.

Early monitoring of potential rapeseed pathogens in the seed and seedling stages may force the diagnosis of such fungi against their epidemical distribution.

One possibility is the development of molecular markers, which are highly taxon-specific, by fingerprint techniques, which reveal polymorphisms at the DNA level, like the random polymorphic DNA (RAPD) approach. Using the restriction fragment length polymorphism technique RAPD markers were selected, which are specific for one of the fungi in the background of plant DNA and the DNAs of its fungal "neighbours". After sequencing of the polymorphic RAPD fragments by a combined method of Gilbert/Maxam and Sanger sequencing procedures taxon-specific primer pairs have been designed, which facilitate the fast screening for phytopathogens in polymerase chain reactions based on plant cell extracts as source of total genomic DNA.

Cellulolytic enzymes

The rape seed pathogenic ascomycete Leptosphaeria maculans secretes a variety of toxins and cell-wall degrading enzymes during its development on the host plant. These chemical compounds may be involved in the pathogenesis of this fungus by entering the structural barrier of plant cell walls.

One of the most important cell-wall degrading enzymes are cellulases. Their expression provides the fungus with the ability to lyse healthy plant cell walls, which consist mainly of cellulose. Especially in the hypocotyle area of the host plant, where the fungus cause severe necroses and damping off, the cellulolytic activities may be required.

To investigate their putative role in disease development genetic studies have been started, which monitor the differential expression of cellulases from primary infection till damping off as typical symptom of the very last stage in disease development.

Humanpathogens

Apart from genetic studies on phytopathogenic fungi, zygomycetous fungi have been selected, which may function as humanpathogens. These fungi, which are very common in all parts of the biosphere, especially in soil and rotten plant material, follow nearly a saprophytic life style.

Most of these fungi are able to tolerate "higher" temperatures up to 40°C till 55°C. This ability implies their putative importance as humanpathogens in case of a decrease in efficiency of the immunsystem. So, a variety of zygomycetes were described, which cause severe soft-tissue and cutaneous mycoses in immunsuppressive patients. Species of the genera Pneumocystis (P. carinii), Rhinosporidium (Rn. seeberi), Rhizomucor (Rh. pusillus), Rhizopus (R. oryzae and R. microsporus), Cunnighamella (C. elegans), Conidiobolus (Co. incongruus, Co. coronatus), Absidia (A. corymbifera) and Mucor (M. ramosissimus, M. mucedo, M. hiemalis) were reported as causative agents of either localized, disseminated or systemic infections with pulmonary portal of entry in leukemic or diabetic patients, preterm infants as well as transplant recipients. While the development of these mycoses largely depend on the immunological status of the patient the infections are often AIDS-related by viral immunsuppression.

To identify these zygomycetes in a one-day-approach molecular markers will be developed, which recognize species-specific DNA sequences in any probe.

The evolution of microscopic fungi with emphasis on zygomycetes: Towards an establishment of a natural system by multi-gene approaches and investigation of the putative role of horizontal gene transfer

Fungi (Mycota) are heterotrophic (exclusively osmotrophic) organisms which comprise chytridiomycetes, zygomycetes, asco- and basidiomycetes. In order to study the evolution of microscopic fungi in multi-gene approaches, we determine sequences for the nuclear-encoded genes for the small subunit ribosomal RNA (18S), for actin as well as for beta-tubulin, for the translation elongation factor EF1-alpha and for the cell division cycle proteinkinase CDC2/28. These sequence data are applied in concatenated analyses of tree constructions.

Special emphasis is given on the Zygomycetes which encompass microscopic fungi forming zygospores in sexual interactions. The most prominent and largest order are the Mucorales comprising saprotrophic and facultatively parasitic species, among those, the soil fungi Chaetocladium brefeldii and Parasitella parasitica which parasitise on other mucoralean fungi during the establishment of plasma bridges and the unidirectional transfer of genes to the host. This parasexual interaction utilises the pheromone trisporic acid for the identification of compatible hosts, similarily to sexual interactions. One of the last steps of trisporic acid biosynthesis is catalysed by the 4-dihydromethyltrisporate dehydrogenase. The gene encoding this aldo-keto reductase were found in all families of the Mucorales, Mortierellales, Entomophthorales and Kickxellales. The sequences have been analysed and used for the reconstruction of phylogenetic trees. Phylogenetic analysis in the context of available sequence data (approx. 6,300 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level.

Current literature related to these topics

Voigt K & Wöstemeyer J (1995) The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species-specific PCR-primers based on diagnostic RAPD-bands. Microbiol. Res. 150: 373-377.

Schleier S, Voigt K & Wöstemeyer J (1997) RAPD-based molecular diagnosis of mixed fungal infections on oilseed rape (Brassica napus): evidence for genus- and species-specific sequences in the fungal genomes. J. Phytopathology 145: 81-87.

Wöstemeyer J, Wöstemeyer A & Voigt K (1997) Horizontal gene transfer in the rhizosphere: a curiosity or a driving force in evolution? In: Andrews JH, Tommerup IC & Callow JA (eds.) Advances in Botanical Research incorporating Advances in Plant Pathology 24: 399-429, Academic Press San Diego.

Voigt K, Schleier S & Wöstemeyer J (1998) RAPD-based molecular probes for the blackleg fungus Leptosphaeria maculans (Phoma lingam): evidence for pathogenicity group-specific sequences in the genomes. J. Phytopathology 146: 567-576.

Voigt K, Cigelnik E & O'Donnell K (1999) Phylogeny and PCR identification of clinically important zygomycetes based on nuclear ribosomal-DNA sequence data. J. Clin. Microbiol. 37: 3957-3964.

Voigt K & Wöstemeyer J (2000) Reliable amplification of actin genes facilitates deep-level phylogeny. Microbiol. Res. 155: 179-195.

Voigt K & Wöstemeyer J (2001) Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1a genes. Gene 270: 113-120.

Voigt K, Jedryczka M & Wöstemeyer J (2001) Strain typing of Polish Leptosphaeria maculans supports at the genomic level the multi-species concept of aggressive and non-aggressive strains. Microbiol. Res. 156: 169-177.

Hijri M, Redecker D, MacDonald-Comber Petetot JA, Voigt K, Wöstemeyer J & Sanders IR (2002) Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea. Appl. Environ. Microbiol. 68: 4567-4573.

Voigt K & Wöstemeyer J (2002) Disease management of Phoma infections. In: Kempken F (ed.) The Mycota Vol. XI: Agricultural Applications. Springer Verlag, Berlin, Heidelberg, New York, pp. 193-216.

Voigt K (2002) Management of Fusarium diseases. In: Kempken F (ed.) The Mycota Vol. XI: Agricultural Applications. Springer Verlag, Berlin, Heidelberg, New York, pp. 217-242.

Wöstemeyer J, Burmester A, Wöstemeyer A, Schultze K, Voigt K (2002) Gene transfer in the fungal host-parasite system Absidia glauca-Parasitella parasitica depends on infection. In: Syvanen M, Kado C.I. (eds.) Horizontal gene transfer, 2nd edition, Academic Press, San Diego, CA, pp. 237-247.

Einax E & Voigt K (2003) Oligonucleotide primers for the universal amplification of beta-tubulin genes facilitate phylogenetic analyses in the regnum Fungi. Organisms, Diversity & Evolution 3: 185-194.

Schimek C, Kleppe K, Saleem AR, Voigt K, Burmester A & Wöstemeyer J (2003) Sexual reactions in Mortierellales are mediated by the trisporic acid system. Mycological Research 107: 736-747.

Fliegerová K, Hodrová B & Voigt K (2004) Classical and molecular approaches as a powerful tool for the identification of polycentric rumen fungi. Folia Microbiologica 49: 157-164.

Contact

Kerstin Voigt
Tel.: +49 (0)3641 9493-21 /-10
Fax: +49 (0)3641 949322
e-mail: kerstin.voigt@uni-jena.de