BSPP Presidential Meeting 1996

P H Gregory paper-reading competition for young researchers

The genetics and pathogenicity of pigment mutants of the take-all fungus Gaeumannomyces graminis
CP Kelly*, AE Obsourne** and CE Caten*
* School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. 
** The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich NR7 4UH, UK.

Pigmented hyphae of the take-all fungus Gaeumannomyces graminis (Gg) are observed both on infected host tissue and when the fungus is grown in vitro. The pigment has previously been partially purified and has properties characteristic of melanin. Growth of isolates in the presence of inhibitors of melanin biosynthesis such as tricyclazole and pyroquilon gave rise to characteristic changes in hyphal pigmentation, but did not affect growth rate. Melanin has been shown to be essential for pathogenicity in some other fungi (Bell & Wheeler, 1986 Annual Review of Phytopathology 24, 411-451) and in order to determine whether this is also the case in Gg, we set out to isolate pigment mutants and to examine their pathogenicity.

An albino and several pale mutants were isolated following irradiation of protoplasts with ultra-violet light. The pale mutants produced only mild disease symptoms on wheat and oats, when compared to the wild type, and the albino was non-pathogenic. Attempts have been made to cross these mutants back to their wild type parent in order to determine the genetic basis of the mutant phenotype and to test whether reduced pigmentation and reduced pathogenicity co-segregate. However, only two crosses have been achieved because of the normal homothallic breeding system of Gg. The first cross, involving a white waxy mutant, indicated that the mutant phenotype was determined by a single gene. All the mutant progeny showed reduced pathogenicity, confirming that the loss of pathogenicity is associated with the pigment loss. In the second cross, a pale brown phenotype was again the result of a mutation in a single gene, but the precise relationship of this to pathogenicity appeared more complex. These results suggest that melanin production is essential for pathogenicity in Gg.

Identification of species of Pestalotiopsis using artificial neural networks based on spore morphometric data
Alexandra Morgan
School of Pure and Applied Biology, University of Wales, Cardiff, UK.

Numerous species of Pestalotiopsis, a fungal plant pathogen, have been described, the majority of which are separated by small differences in dimensions, pigmentation and appendage characters of conidia. Conflicting descriptions of several taxa are typical of the problems involved in clearly defining species boundaries. Radial basis function (RBF) artificial neural networks (ANNs) were trained and optimized for the identification of 23 species of Pestalotiopsis and the related species Truncella truncata based upon morphometric measurements of spores.

Cloning a gene which encodes a glycoprotein present at the fungal-plant interface formed in the Colletotrichum - bean interaction
Sarah Perfect*, Richard O'Connel** and Jonathan Green*
* School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. 
** IACR Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Bristol BS18 9AF, UK

Colletotrichum is a large genus of plant pathogenic fungi which cause anthracnose diseases on a wide range of temperate and tropical crops. C. lindemuthianum is a hemibiotrophic species causing anthracnose of bean, Phaseolus vulgaris. During the initial biotrophic stage of infection, the fungus differentiates infection vesicles and primary hyphae within host epidermal cells. These specialized intracellular hyphae invaginate the host plasma membrane, from which they are separated by a matrix layer. Monoclonal antibodies (MAbs) raised to isolated infection structures have been used to identify proteins present at the fungal-plant interface.

One of these MAbs, designated UB25, recognizes a protein epitope in a 40kDa N-linked glycoprotein specific to intracellular hyphae. Indirect immunofluorescence and EM-immunogold labelling show that the glycoprotein is present in the infection peg, and the fungal walls and matrix surrounding the intracellular hyphae. However, the glycoprotein is not present in intercellular hyphae or secondary necrotrophic hyphae, which suggests that it is specific to biotrophic structures growing in contact with host protoplasts. The glycoprotein may therefore be involved in the establishment and maintenance of biotrophy.

A cDNA library has been constructed from total RNA isolated from infected bean hypocotyl epidermis. The MAb UB25 hs been used to immunoscreen the library and positive clones have been isolated and sequenced. Analysis of the deduced amino acid sequence revealed the presence of two distinct domains, one of which is rich in serine, threonine and proline. The functional significance of this will be discussed. In addition, Southern analysis indicates that the glycoprotein recognized by UB25 is fungally encoded and is present in several Colletotrichum species.

Stem tip dieback of cassava: identification and virulence of the causal organism
Emmanuel Moses
Department of Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK.

A disease of cassava characterized by prominent dieback symptoms was first described in Zaire in 1989 but the causal agent was not established. A similar disease is widespread in Ghana and isolations from affected plants consistently yielded a fungus with morphological features corresponding to the genus Colletotrichum. When four Ghanaian cultivars of cassava were inoculated with these isolates they developed symptoms which were indistinguishable from those of the plants from which the isolations were made. Koch's postulates were completed by reisolating the fungus from the inoculated plants and showing that its morphology was the same as the one originally isolated.

The identity and taxonomic position of the cassava pathogen was established by sequencing domain 2 of ribosomal DNA and comparing the nucleotide sequences with those of other members of the genus whose identities are well established. Out of 193 nucleotides analysed, the cassava isolate differed from authentic C. gloeosporioides isolates by only one, demonstrating that the cassava pathogen was a form of C. gloeosporioides.

The host range of the casssava pathogen was tested by inoculating other plant species that are widely grown as crops in Ghana. Fruits of tomato, banana and pepper developed lesions within two days of inoculation and lesions were especially severe if the fruit were wounded.

The early occurrence of water-soaking symptoms in inoculated stems of cassava suggested that the fungus was affecting the permeability of the plasmamembranes of host cells. When the fungus was grown on Czapek-Dox nutrients supplemented with cassava extract or citrus pectin, the culture filtrates killed cells of the plant isolated from leaves by enzyme digestion.

On dialysis of culture filtrates, about half of the cell-killing activity was found in the high molecular weight fraction and the same proportion was precipitated by 60% acetone. The acetone precipitate also possessed macerating activity as determined by the reduction of weight of cucumber slices and pectate lyase activity as determined by the increase in absorption of light at 232 nm of a reaction mixture consisting of acetone precipitate, sodium polypectate, calcium chloride and buffer at pH 9.5.

When the acetone was removed by evaporation from the acetone supernatant of culture filtrates, most of the residual cell killing activity partitioned into ethyl acetate. This ethyl acetate fraction was further separated by flash chromatography on silica gel. Three toxic compounds were isolated which, on mass spectrometry, were found to share a prominent peak at m/z 355 as well as several peaks at other m/z values suggesting that all three compounds were similar.

In conclusion, the work has shown that dieback of cassava is caused by C. gloeosporioides and that the fungus is capable of producing enzymes and toxins which may be of importance in its virulence.