Undergraduate Bursary Reports The following reports are by the first set of five undergraduates to receive BSPP Undergraduate Bursaries, which gave them two months' work experience in a plant pathology laboratory during their summer vacation in 1996. Biotrophic fungal diseases of Senecio and its relatives Two different projects were carried out at Glasgow University. The first project entailed collecting isolates of Erysiphe fischeri (groundsel powdery mildew) and preparing a single spore isolate from each. Work started very slowly in July because there was little infection on the groundsel but eventually 5 isolates were obtained from different plants. Three of these isolates were from infected plants within 100 yards of each other in the Botany Department experimental gardens while the other two were from half a mile away. The isolates were first transferred to plants growing in an isolation plant propagator and single conidial chain isolates were prepared from each on leaves of a known susceptible line of groundsel. When the mildew cultures had multiplied up sufficiently, inoculations were carred out onto plants of a set of 50 differential lines of Senecio vulgaris. Infection was assessed using a scoring system devised by Fraser S Campbell (1990) Genetic Interactions between Erysiphe fischeri (Blumer) and Members of the Genus Senecio. (Ph.D. Thesis, University of Glasgow). Because of the late appearance of mildew infection this summer on S. vulgaris, testing and scoring of all the isolates was not completed. Two of the isolates were tested on the 50 lines of S. vulgaris and two on 10 lines. It is intended that when the isolates have been characterised completely and are bulked up, they will be compared using molecular genetic techniques at the Scottish Crop Research Institute in Invergowrie. The second project involved the investigation of the host ranges of Erysiphe fischeri (powdery mildew), the new Albugo sp. (white blister rust), only recorded on groundsel in Britain since about 1980 and Puccinia lagenophorae (yellow rust), using 12 different lines of Senecio vulgaris together with one cultivar each of 5 other Senecio species (S. madagascariensis, S. viscosus, S. sylvaticus, S. squalidus, S. sylvaticus) and two cultivars of scorzornera and salsify known to be susceptible to Albugo tragopogonis. The objective of this project was to assess how closely related the new Albugo species on groundsel is to the one which commonly occurs on Senecio squalidus, scorzornera and salsify. This comprised mainly of survey work to determine the extent of infection on each line and the date they were infected. Some of the results from this latter project were presented at a meeting of the Scottish Mycology and Plant Pathology Group held in the University of Glasgow in September 1996. Sinead C Collins Verticillium dahliae on linseed My BSPP undergraduate bursary enabled me to gain some experience in a plant pathology laboratory at IACR-Rothamsted during my 1996 summer vacation. At Rothamsted I gained practical experience in both field and laboratory based work, and assisted my supervisor, Dr B D L Fitt, to meet some specific research objectives. Whilst I was at Rothamsted, I worked primarily with the fungal pathogen Verticillium dahliae on linseed. I did two field experiments, one of which investigated the effects of crop rotation on the pathogenicity of V. dahliae on linseed. The other assessed the presence of the fungus in linseed plants prior to the apperance of visual symptoms on the plants, and then assessed the subsequent development of the visual symptoms on the linseed. I also did a pathogenicity experiment which investigated effects of V. dahliae isolated from different previous hosts on growth of linseeed plants in a controlled environment. In conjunction with these experiments I wrote a short literature review entitled "Verticillium dahliae: the risk to UK arable and horticultural crops". In the field experiment which investigated effects of crop rotation on disease development, there appeared to be no difference in the development of the disease on linseed in plots which had different previous crops in 1995. The field experiment which assessed the presence of Verticillium dahliae prior to the appearance of visual symptoms showed that it was possible to isolate the fungus from all parts of the linseed plant stem prior to the appearance of visual symptoms. In the results of the controlled environment experiment which investigated the effects of V. dahliae isolates from different previous hosts on the growth of linseed plants, there was a difference in the pathogenicity of the isolates which had different previous hosts. The isolate which had potato as its previous host was observed to be the most damaging. I would like to thank both Dr B D L Fitt who gave me this opportunity, and the BSPP Council for the bursary which enabled me to take it. Allison Cook Viral diseases of Surfinia petunias In 1994-1995, trailing petunias (Surfinias) suffered a mixed viral infection resulting in huge commercial losses due to the necessary destruction of plants. Two most important viruses in this infection were reported to be a tobamovirus related to tobacco mosaic virus, and potato virus Y (strain n), a potyvirus. The main interest of this project was to carry out some preliminary work towards generating transgenic resistance against both viruses in Surfinia, with two main aims (i) to develop a transformation system for Surfinias and (ii) generate constructs which would confer resistance against the viruses involved. Preliminary leaf disc transformations were carried out using Agrobacterium tumefaciens mediated methods to introduce a 35S-GUS construct into two varieties of Surfinia. Leaf discs were then incubated on selective shoot regeneration media. Results have shown that both varieties show good shoot regneneration and histochemical GUS assays have shown that leaf discus havee been transformed. It has also been determined that untransformed shoots generated from callus can produce roots when transferred onto rooting media. To generate constructs for transformation, it was decided to use coat protein-mediated resistance against PVYn. The coat protein gene of PVYn was therefore cloned using RT-PCR with coat protein specific primers from tobacco plants infected with PVYn and cloned into a plant transformation vector under the control of a CaMV35S promoter. Constructs were generated in which the coat protein gene was positioned in both sense and anti-sense orientations, ready for transformation and evaluation of levels of resistance. This project was kindly supported by BSPP and carried out under the supervision of Tim Clifford and Dr Gary Foster (University of Leicester) and Nicola Spence (HRI). James Hughes Studies on pre-formed cellulolytic enzymes in pycnidiospores of Staganospora nodorum Staganospora (Septoria) nodorum is an important pathogen of wheat in many areas of the world. During this ten week studentship, investigations were carried out into the activity of various enzymes constitutively expressed on or within the spore and their possible role in pathogenicity. Spores of S. nodorum are produced in a mucilaginous matrix. Previous work in the Plant Science Department has at Oxford University showed this mucilage to have little enzymatic activity, although low levels of cutinase activity were detected. However, little is known about enzymes present within the spore itself, which may be important during the early stages of infection. By using spores that had been washed to remove any enzymes adhering to the surface, we were able to investigate the activities of enzymes within the spores using a number of techniques. The washed spores showed considerable esterase activity using a p-nitrophenol butyrate assay, which can most probably be attributed to cutinase activity within the spore. Use of the esterase-specific stain indoxyl acetate confirmed this by showing "hot-spots" of activity on the spore surface. Tests for other enzymes showed low glucosidase activity together with some indication of very low arabinosidase and galactosidase activity within the spores. However, tests for mannosidase activity and for exo- and endoxylosidase activity were negative. Protease activity with a pH optimum of 4 was also detected, but inhibition studies using four protease inhibitors proved inconclusive. Recombinant cutinase (supplied by Dr Mike Morgan, Institute of Food Research, Norwich) was then used to screen by plate-trapped antigen ELISA, 35 antibodies previously raised against S. nodorum in Oxford. Of these, 8 were found to recognise the recombinant enzyme. Five of these were tested for their ability to lable spores by immunofluorescence microscopy, of which two were successful. However, none of the antibodies inhibited the activity of cutinase in the spores. Protein extracts from the washed spores were then run on an SDS/DTT polyacrylamide gel. This revealed a band within the molecular weight range previously described for cutinase, which was detected by Western blotting using the Oxford-raised anti-cutinase antibody CU3 BH4. Changes in esterase levels were measured in spores from two isolates during the growth of the plate cultures by harvesting spores between 7 and 17 days old, with esterase activity showing a steady increase during this period in both isolates. Esterase activity was also demonstrated in washed spores of Septoria tritici, although at a much lower level than in S. nodorum. In the wild, S. nodorum is often found growing in close association with different bacteria, some of which have previously been shown to increase the pathogenicity of the fungus. Assays for esterase activity found considerable activity in several of these bacterial isolates. Furthermore, two bacterial isolates showed some evidence of increasing esterase activity when grown with the fungus. This work will form the basis of a publication to be submitted to Plant Pathology. Robin May Pectate lyase from a Colletotricum gloeosporioides isolate obtained from cassava Previous attempts at UCL to polymerize a pectate lyase gene from an isolate of Colletotricum gloeosporioides that is responsible for a serious die-back disease of cassava in Ghana yielded a product of about 800bp. As a starting point, attempts were made to repeat this result using the same primers. DNA was extracted by the CTAB method and, more conveniently, using a commercial kit (Nucleon Phytopure Plant DNA Extraction Kit, Scotlab, Glasgow). The optimum annealing temperature was 62oC and the optimum magnesium concentration 2 mM. However, two products were repeatedly obtained of approximately 500 and 300 bp. Attempts to sequence these using the ABI PRISM 310 DNA Analyser were unsuccessful. Pectate lyase activity was recorded for culture filtrates of the fungus, grown on Czapek-Dox nutrients supplemented with 1% citrus pectin, as an increase in absorbance at 232 nm and by the maceration of cucumber mesocarp tissue. Jemma Taggart
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