Undergraduate Bursary Reports
The following reports are by the first set of five undergraduates to receiveBSPP Undergraduate Bursaries, which gave them two months’ work experience in aplant 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 firstproject entailed collecting isolates of Erysiphe fischeri (groundselpowdery mildew) and preparing a single spore isolate from each. Work startedvery slowly in July because there was little infection on the groundsel buteventually 5 isolates were obtained from different plants. Three of theseisolates were from infected plants within 100 yards of each other in the BotanyDepartment experimental gardens while the other two were from half a mile away.
The isolates were first transferred to plants growing in an isolation plantpropagator and single conidial chain isolates were prepared from each on leavesof a known susceptible line of groundsel. When the mildew cultures hadmultiplied up sufficiently, inoculations were carred out onto plants of a set of50 differential lines of Senecio vulgaris. Infection was assessed usinga scoring system devised by Fraser S Campbell (1990) Genetic Interactionsbetween 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 ofthe isolates were tested on the 50 lines of S. vulgaris and two on 10lines. It is intended that when the isolates have been characterised completelyand are bulked up, they will be compared using molecular genetic techniques atthe Scottish Crop Research Institute in Invergowrie.
The second project involved the investigation of the host ranges of Erysiphefischeri (powdery mildew), the new Albugo sp. (white blister rust),only recorded on groundsel in Britain since about 1980 and Puccinialagenophorae (yellow rust), using 12 different lines of Senecio vulgaristogether 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 Albugotragopogonis. The objective of this project was to assess how closelyrelated the new Albugo species on groundsel is to the one which commonlyoccurs on Senecio squalidus, scorzornera and salsify. This comprisedmainly of survey work to determine the extent of infection on each line and thedate they were infected.
Some of the results from this latter project were presented at a meeting ofthe Scottish Mycology and Plant Pathology Group held in the University ofGlasgow in September 1996.
Sinead C Collins
Verticillium dahliae on linseed
My BSPP undergraduate bursary enabled me to gain some experience in a plantpathology laboratory at IACR-Rothamsted during my 1996 summer vacation. AtRothamsted I gained practical experience in both field and laboratory basedwork, and assisted my supervisor, Dr B D L Fitt, to meet some specific researchobjectives.
Whilst I was at Rothamsted, I worked primarily with the fungal pathogen Verticilliumdahliae on linseed. I did two field experiments, one of which investigatedthe effects of crop rotation on the pathogenicity of V. dahliae onlinseed. The other assessed the presence of the fungus in linseed plants priorto the apperance of visual symptoms on the plants, and then assessed thesubsequent development of the visual symptoms on the linseed. I also did apathogenicity experiment which investigated effects of V. dahliaeisolated from different previous hosts on growth of linseeed plants in acontrolled environment. In conjunction with these experiments I wrote a shortliterature review entitled “Verticillium dahliae: the risk to UKarable and horticultural crops”.
In the field experiment which investigated effects of crop rotation ondisease development, there appeared to be no difference in the development ofthe disease on linseed in plots which had different previous crops in 1995. Thefield experiment which assessed the presence of Verticillium dahliaeprior to the appearance of visual symptoms showed that it was possible toisolate the fungus from all parts of the linseed plant stem prior to theappearance of visual symptoms. In the results of the controlled environmentexperiment which investigated the effects of V. dahliae isolates fromdifferent previous hosts on the growth of linseed plants, there was a differencein the pathogenicity of the isolates which had different previous hosts. Theisolate which had potato as its previous host was observed to be the mostdamaging.
I would like to thank both Dr B D L Fitt who gave me this opportunity, andthe BSPP Council for the bursary which enabled me to take it.
Viral diseases of Surfinia petunias
In 1994-1995, trailing petunias (Surfinias) suffered a mixed viral infectionresulting in huge commercial losses due to the necessary destruction of plants.Two most important viruses in this infection were reported to be a tobamovirusrelated to tobacco mosaic virus, and potato virus Y (strain n), a potyvirus.
The main interest of this project was to carry out some preliminary worktowards generating transgenic resistance against both viruses in Surfinia, withtwo 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 Agrobacteriumtumefaciens mediated methods to introduce a 35S-GUS construct into twovarieties of Surfinia. Leaf discs were then incubated on selective shootregeneration media. Results have shown that both varieties show good shootregneneration and histochemical GUS assays have shown that leaf discus haveebeen transformed. It has also been determined that untransformed shootsgenerated from callus can produce roots when transferred onto rooting media.
To generate constructs for transformation, it was decided to use coatprotein-mediated resistance against PVYn. The coat protein gene of PVYn wastherefore cloned using RT-PCR with coat protein specific primers from tobaccoplants infected with PVYn and cloned into a plant transformation vector underthe control of a CaMV35S promoter. Constructs were generated in which the coatprotein gene was positioned in both sense and anti-sense orientations, ready fortransformation and evaluation of levels of resistance.
This project was kindly supported by BSPP and carried out under thesupervision of Tim Clifford and Dr Gary Foster (University of Leicester) andNicola Spence (HRI).
Studies on pre-formed cellulolytic enzymes in pycnidiospores ofStaganospora nodorum
Staganospora (Septoria) nodorum is an important pathogen of wheat inmany areas of the world. During this ten week studentship, investigations werecarried out into the activity of various enzymes constitutively expressed on orwithin the spore and their possible role in pathogenicity.
Spores of S. nodorum are produced in a mucilaginous matrix. Previouswork in the Plant Science Department has at Oxford University showed thismucilage to have little enzymatic activity, although low levels of cutinaseactivity were detected. However, little is known about enzymes present withinthe 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 thesurface, we were able to investigate the activities of enzymes within the sporesusing a number of techniques. The washed spores showed considerable esteraseactivity using a p-nitrophenol butyrate assay, which can most probably beattributed to cutinase activity within the spore. Use of the esterase-specificstain indoxyl acetate confirmed this by showing “hot-spots” ofactivity on the spore surface.
Tests for other enzymes showed low glucosidase activity together with someindication of very low arabinosidase and galactosidase activity within thespores. However, tests for mannosidase activity and for exo- and endoxylosidaseactivity were negative. Protease activity with a pH optimum of 4 was alsodetected, but inhibition studies using four protease inhibitors provedinconclusive.
Recombinant cutinase (supplied by Dr Mike Morgan, Institute of FoodResearch, Norwich) was then used to screen by plate-trapped antigen ELISA, 35antibodies previously raised against S. nodorum in Oxford. Of these, 8were found to recognise the recombinant enzyme. Five of these were tested fortheir ability to lable spores by immunofluorescence microscopy, of which twowere successful. However, none of the antibodies inhibited the activity ofcutinase in the spores.
Protein extracts from the washed spores were then run on an SDS/DTTpolyacrylamide gel. This revealed a band within the molecular weight rangepreviously described for cutinase, which was detected by Western blotting usingthe Oxford-raised anti-cutinase antibody CU3 BH4.
Changes in esterase levels were measured in spores from two isolates duringthe 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 bothisolates. Esterase activity was also demonstrated in washed spores of Septoriatritici, although at a much lower level than in S. nodorum.
In the wild, S. nodorum is often found growing in close associationwith different bacteria, some of which have previously been shown to increasethe pathogenicity of the fungus. Assays for esterase activity found considerableactivity in several of these bacterial isolates. Furthermore, two bacterialisolates showed some evidence of increasing esterase activity when grown withthe fungus.
This work will form the basis of a publication to be submitted to PlantPathology.
Pectate lyase from a Colletotricum gloeosporioides isolate obtainedfrom cassava
Previous attempts at UCL to polymerize a pectate lyase gene from an isolateof Colletotricum gloeosporioides that is responsible for a seriousdie-back disease of cassava in Ghana yielded a product of about 800bp. As astarting point, attempts were made to repeat this result using the same primers.
DNA was extracted by the CTAB method and, more conveniently, using acommercial kit (Nucleon Phytopure Plant DNA Extraction Kit, Scotlab, Glasgow).The optimum annealing temperature was 62oC and the optimum magnesiumconcentration 2 mM. However, two products were repeatedly obtained ofapproximately 500 and 300 bp. Attempts to sequence these using the ABI PRISM 310DNA 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 increasein absorbance at 232 nm and by the maceration of cucumber mesocarp tissue.