BSPP News Spring 1999 - Online Edition

The Newsletter of the British Society for Plant Pathology
Number 34, Spring 1999

BSPP Undergraduate Vacation Bursaries 1998

The possible control of Leptosphaeria maculans on oil-seed rape by Cyanthus striatus, a bird's nest fungus

Disease severity on oilseed rape infected by Leptosphaeria maculans (anamorph Phoma lingam) is less severe when Cyanthus striatus is present in the field. L. maculans is an important pathogen, as the causal agent of black-leg and canker. Competition between the two fungi for cellulose was thought to be the cause of antagonism between them. I measured cellulase production by burying cotton strips with the fungi, then, after inoculation, measuring the tensile strength of the strips. The results were not as expected: C. striatus did not produce more cellulase than L. maculans, although, of the two fungi, growth rates of C. striatus were consistently higher.

However, pairing both fungi on agar plates produced interesting results. At first, C striatus appeared to be inhibited by L. maculans, but after one week, it produced a line of brown pigment facing towards the pathogen. The hyphae of C. striatus proceeded to grow up and onto the L. maculans colony (see illustration), eventually overgrow
ing it completely. Although the two fungi do seem to compete, competition does not appear to be for cellulose. Possible future investigations into the use of calcium may be appropriate, as it is known that calcium oxalate crystals are produced by the bird's nest fungus.

I am extremely grateful for this opportunity, which I found enlightening and inspiring and provided me with relevant work experience. I wish to thank Dr Avice Hall for her patient encouragement and supervision, as a result of which I aspire to a career in plant pathology and microbiological research.

Monica Maksymiak, University of Hertfordshire

Using the polymerase chain reaction to detect fungal spores

The polymerase chain reaction (PCR) has the potential for specific and sensitive detection of fungal plant pathogens. The aim of my project was to investigate the possibility of using the PCR for detect
ing small numbers of spores of plant pathogenic fungi.

Initially, Penicillium roqueforti was used as a model fungus as it sporulates profusely in vitro and specific PCR primers were available. The existing PCR protocol was optimised using purified template DNA extracted from mycelium. A selection of Taq polymerases were tested in conjunction with various denaturation reagents over a range of annealing temperatures and MgCl2 concentrations. Consequently, a protocol was developed that enabled amplification of a 300 bp product from purified DNA of P. roqueforti but did not amplify DNA from other Penicillium spp.

The optimised PCR protocol was then used to investigate the detection limit of P. roqueforti spores. Where spores were added directly to the PCR, the limit of detection was 104 spores. Microscopic studies showed that boiling did not disrupt the spore wall. However, milling with Ballotini beads ruptured spores and resulted in greatly increased sensi
tivity of the PCR assay. Sensitivity was further enhanced by the use of a simple DNA extraction procedure which, in conjunction with milling, reduced the detection limit to less than 100 spores.

Whilst P. roqueforti produces tough spores adapted for dry-dispersal, many fungi produce delicate, thin-walled spores. For example, the plant pathogen Phoma lingam produces thin-walled conidia which are splash-dispersed. As a specific PCR assay for this fungus was available at IACR-Rothamsted, detection of Ph. lingam conidia was investigated. When added directly to the PCR, less than 100 conidia of Ph. lingam
could be detected. Sensitivity was not improved by milling and DNA extraction.

Many thanks to the BSPP for the 10 week placement at IACR-Rothamsted, to Dr Roger Williams for his supervision, and to Dr Alastair McCartney and Dr Elaine Ward for their advice and support. The work I have been involved with has been interesting and challenging, and I have no doubt that my final year of undergraduate study, and any subsequent employment, will benefit from the experience I have gained.

Lesley Lunn, University of Hertfordshire

Detection Techniques for Chrysanthemum Stunt Viroid

The purpose of my research at HRI Wellesbourne was to develop reliable protocols, using nucleic acid hybridization, to detect chrysanthemum stunt viroid (CSVd). CSVd is an important pathogen of chrysanthemum crops, infecting large numbers of plants each year. The early detection of infected plants would allow selection of viroid-free propagating material and reduce the risk of further plant contamination and crop damage. A stock of healthy and infected chrysanthemum plants, whose status had been confirmed by independent testing at HRI, was used to determine the reliability, repeatability and sensitivity of the hybridization techniques developed.

RNA extracts were made from both healthy and infected chrysanthemums using a high-phosphate concentration extraction buffer. Serial dilutions were then made from each extract, 100 ul of each dilution being applied to a membrane using a dot-blot apparatus. These membranes were then run through a hybridization protocol with a DIG labeled probe. After hybridization the membranes were washed with increasing stringency before being incubated with an anti-DIG antibody, which would bind to any hybridized probe. Finally substrate for the antibody was added to the membrane and the chemiluminescent reaction detected by exposure to film.

At first a DNA probe was used in the hybridization. This was produced by labeling, with the template DNA was attached to DIG. The template DNA used was from linearised pMOS Blue plasmids containing the chrysanthemum stunt viroid sequence inserted into the multiple cloning site between the EcoRI and XbaI restriction endonuclease sites. Unfortunately the DNA probe performed poorly, hybridizing non-specifically to healthy chrysanthemums and negative controls. It was thus decided to produce an RNA probe. To produce the RNA probe the insert had to be cut out from the plasmid and re-inserted in the correct orientation in a pT3/T7 plasmid. This plasmid was then used to transform competent E.coli. Bacterial colonies showing transformation and the correct insert orientation were then taken, the plasmid purified and linearised to allow RNA transcripts to be run from it. The RNA probe was still being developed when my placement came to an end but results looked promis-ing. During my time, I also developed a PCR technique to detect CSVd in infected chrysanthemum samples.

The techniques that I have learned and the experience I have gained over the last ten weeks has prove invaluable in conrirming that I would like to pursue a career in biological research. I wish to thank BSPP for providing me with this opportunity, and I would also like to thank my supervisors, Dr Dez Barbara and Dr Nicola Spence, and all those in the Plant Pathology and Microbiology Department at HRI Wellesbourne for providing support and making me feel welcome.

Stephen Kissane, University of Birmingham

CHIP: Changing climate and potential impacts on yield and quality in potato

The objective of the CHIP project is to investigate the impact of elevated levels of carbon dioxide or ozone on the growth, development, yield and quality of potatoes. CHIP is an EU-funded project, running from 1998 to 2002, involving eight research groups from countries ranging from Finland and Italy and Ireland to Germany.

Open top chambers are being used to ambient and elevated levels of these gases to the potato crop, which is otherwise grown in standard conditions. Working alongside experience of techniques for measuring plant growth, photosynthetic response curves,
dark-adapted and modulated chlorophyll fluorescence and in situ chlorophyll content.

Although the growing season was completed without pathogen, severe blackening and curling of the youngest leaves began to occur in the middle of the season. This was initially suspected to be the result of infection by potato leaf roll virus, but tests by ADAS established that the symptoms were a variety-specific, hypersensitive response to damage caused by aphids feeding, des-pite the routine application of aphicides.

The final harvest took place during September 1998. Preliminary
analysis of the results indicates a significant effect of CO2 on potato yield but no effect of O3. Analysis of the growth and photosynthetic data obtained and further analytical work on tuber quality have continued through the winter. The data obtained will form the basis of my BSc honours research project.

The summer studentship awarded by BSPP has enabled me to gain an invaluable insight into experience of experimental research under both field and laboratory conditions.

Ann-Marie Tulloch, University of Nottingham

Light-induced biocontrol of Erwinia carotovora subsp. atroseptica on potato tubers

MY GRANT from the BSPP enabled me to take part in the ongoing research into light-induced biocontrol of potato diseases in the Plant Biology Department of the Scottish Agricultural College at Auchincruive. Over a period of 10 weeks, I worked under the guidance of Dr Ruaridh Bain and Dr Glynn Percival on two lines of study.

In the first study, I investigated the effect of exposure to light on the survival of Erwinia caroto-vora subsp. atroseptica, the potato blackleg pathogen, in the lenticels of recently-harvested tubers. The aim of this research was to identify any influence of light-induced photosynthates of potatoes on the survival of the blackleg pathogen.

The tubers were inoculated with
a streptomycin-resistant isolate of the pathogen on the day of harvest and were stored at ambient temperature either in the dark or under light. Bacterial counts from the tubers were done after 10 and 20 days, using the Perombelon peeling method and culturing the peel extracts on streptomycin-enriched crystal violet pectate (CVP) medium. Markedly fewer E. caro-tovora subsp. atroseptica were obtained from the tubers given the light treatment than from those kept in the dark.

My second study was an in vitro investigation of the effect of specific potato photosynthates, the glycoalkaloids alpha-chaco-nine and alpha-solanine and chlorogenic acid on the survival of the blackleg bacterium. Each chemical was examined individu
ally at a selection of concentrations and the combined effect of alpha-chaco-nine and alpha-solanine was also studied. The bacteria were cultured with each chemical, and dilution series were used to obtain bacterial concentrations suitable for counting after plating out.

My bursary allowed me to gain experience of practical laboratory work which I found of great benefit. It is anticipated that this work will contribute to a forthcoming paper. The guidance of Dr Ruaridh Bain and Dr Glynn Percival was greatly appreciated and the helpfulness of the Plant Biology staff was most encouraging. I would like to thank the BSPP for giving me this opportunity.

James Crichton, Scottish Agricultural College 

Black dot of potato tubers: a histological and morphological study of infection and symptom development

Black dot disease of potatoes, caused by the fungus Colleto-trichum coccodes, affects the quality of washed, pre-packed potatoes. In the past it has been a relatively minor disease in the UK, but due to consumer demand for blemish-free tubers, it has become more prominent and is causing serious losses to the potato industry. Symptoms on tubers include minute black bodies (micro-sclerotia) which are barely visible to the naked eye. The surrounding skin becomes dry and discoloured and small cracks develop in the skin which eventually leads to the skin lifting off. Histological and morphological analyses were carried out on the surface layers of infected tubers to determine the relationship between the distribution and development of the fungus and the symptoms.

An area was selected on each tuber which spanned the boundary of a black dot lesion. This formed a transect from infected to non-infected tissue which was divided into six zones of equal size. Using image analysis, measurements were made of surface characteristics such as cracking, skin lifting, sclerotial size and density in each of the zones. After the surface features were measured, the zones were dissected from the tubers, embedded and sectioned for examination by light microscopy.

In the most infected areas sclerotial size varied widely, in the range 0.1 to 1.0 mm diameter. Their distribution was random as indicated by nearest neighbour analysis. The skin in these zones was rough with a well defined mosaic appearance. Cracks of varying length were found which led to skin lifting. In some cases these occurred in the absence of sclerotia.

Histological examination showed that underlying periderm cells in infected areas were difficult to distinguish as they had become flattened or compressed. The degree of compression was reduced with distance from the infected area. The collapse of the periderm cells was not associated specifically with sclerotia, although where there were large sclerotia, cells were stained darker and more cell layers were affected.

I am doing further work towards my Honours thesis to find out how disease development is affected by humidity in store. I would like to thank the BSPP for providing this opportunity and Jacqueline Blackwood for her help throughout the ten week study period.

Catherine McDonald, Scottish Agicultural College, Aberdeen