1999 BSPP Undergraduate Vacation Bursary Reports

PCR primers for rapid identification of plant virus families infecting ornamental crops

Plant viruses are a big problem in the UK protected ornamentals industry.  Many viruses with isometric and rod-shaped particles have been detected by serology and electron microscopy techniques.  However identification has been slow and the hope is to develop new tests that will aid the rapid identification of these viruses.  The polymerase chain reaction (PCR) provides a sensitive technique for detecting plant viruses.  PCR amplifies a specific region of DNA if the flanking sequences (primers) are known.  Work concentrated on ‘universal primers’, as these are complementary to a number of related sequences, as in a virus family.

Work began on potyviruses. These contain a genome of one positive-sense single-strand RNA of about 10kb.  Reverse-transcription followed by PCR (RT-PCR) was used to amplify regions of the genome.  Getting this system to work was difficult, and experiments were done with various reaction cycles and reagent concentrations.  However the focus was on cucumoviruses working mainly with cucumber mosaic virus (CMV) and tomato aspermy virus (TAV).  CMV was of particular interest as the virus is found in a wide range of crops, causing yellowing and mosaic symptoms in leaves and TAV is a serious problem in chrysanthemum.  Like potyviruses, CMV also has an RNA genome, but differs in that it is divided into four  or five subunits.  Primers were chosen from the conserved region in the coat protein gene of RNA3: CMV1 (downstream) CMV 2 (upstream) (Aust.J.Agric.Res 44:41-51) were used in the RT-PCR to give a single product of 500bp for CMV and 600bp for TAV.  Standard PCR conditions were used, and the best products were obtained with high MgCl2 concentrations and an annealing temperature of 58°C.  This test distinguished between the two viruses and further endonuclease digestion of the CMV product with EcoRI and MspI classified isolates into two distinct subgroups.  Subgroup II was mainly found in the isolates tested as this group dominates in temperate climates.  TAV was resistant to endonuclease digestion like subgroup I, however the heavier amplification product of 600bp classified it separately.  Commercially these tests could aid in the early diagnosis of infected plants so that healthier crops result, particularly in conjunction with serological tests.  The sensitivity of PCR gives the advantage of being able to detect even very low levels of the virus.  Work will continue in validating the findings against other virus families to check the specificity of the primers for cucumoviruses.

Thanks to BSPP for this opportunity that has provided excellent experience for my final year project, as well as confirming my interest in a career in biological research.  Thank you also to all staff in the Plant Pathology and Microbiology Department at HRI Wellesbourne for their support and encouragement particularly my supervisor Dr Nicola Spence.

Hardip Kaur
University of Birmingham

Systemic induced resistance to Late Blight (Phytophthora infestans) in tomatoes

With ever increasing public concern about the quality, safety and health attributes of food products, commercial organic production is becoming more and more important.  The main principal of organic production is to produce food naturally with little or no artificial chemicals.  However, many traditional organic production systems are not financially viable as they involve high cost labour processes. In commercial tomato production, current protection against late blight relies on copper-based treatment. A number of new treatments have become available on the market which are designed to provide induced resistance to blight in various crops including potatoes and tomatoes.  These products are not widely used in the UK at present, but may  provide  possibilities to prevent  high levels of blight infestation.

As my summer bursary project I conducted a  number of experiments to compare the effectiveness of these organic treatments to conventional copper oxychloride treatment.  Two products were used, these being Mycosin Plant Tonic and Milsana Plant Tonic.  These so-called “plant tonics” are largely based on vegetable matter making them suitable to be used in organic production systems.

Having grown tomato plants from seed, the first of two experiments were carried out.  This experiment involved treating three batches of  plants, one with Milsana, one with Mycosin and one with copper oxychloride.  Application rates were as per manufacturer’s recommendations. Detached leaf tests were then carried out at set intervals with the leaves being infected with a droplet of Phytophthora infestans spores  which was prepared in a solution.  The results from this experiment were obtained by measuring the spread of the fungus on the leaves after set numbers of days.  It was observed that the conventional copper oxychloride treatment was more effective than the new treatments with some leaves showing little or no signs of fungal growth after three days.  Those plants treated with Mycosin showed less infection than those treated with Milsana although the differences were negligible.

The second series of experiments involved treating batches of plants with half-rate copper-oxychloride and half-rate of the two plant tonics.  These experiments were carried out as before with results being measured as level of infection of leaves on detached leaf tests at set intervals.   The results showed that the half-rate copper then half-rate Mycosin provided good resistance, with the batch treated with half-rate Milsana showing a greater level of infection.

I feel that this opportunity to carry out this research within the Crop Health Department of SAC Aberdeen has benefited me by equipping me with a number of new skills particularly laboratory techniques and Good Laboratory Practice.
I wish to thank the BSPP for enabling me to carry out this project and the Crop Health Department at SAC for the use of facilities and equipment.  Particular thanks to Dr R. Clayton for help and advice, Mrs Linda Stevenson and Mrs Valerie Griffin-Walker for practical assistance.

Kenneth Ross
SAC, Aberdeen

Screening of Pseudomonas syringae pv. phaseolicola avr mutants

I was fortunate enough to be granted a BSPP undergraduate vacation bursary which enabled me to spend a profitable 8 weeks at Wye College in the plant pathology lab working with Pseudomonas syringae pv. phaseolicola, the cause of bean halo- blight disease. My work involved analysis of genes for virulence cloned from a plasmid in P. syringae.  One genomic clone, pAV522 containing about 25kb of insert DNA, was found to partially restore virulence to plasmid-less derivatives.  I was given a set of mutants of pAV522 which contained Tn3gus transposon insertions at various locations in the cloned DNA.  I screened the mutants to see whether any would fail to restore virulence, which would indicate insertion of the transposon into a possible virulence gene.

I used two types of pathogenicity test to screen the mutants - pod tests and leaf inoculations. These were carried out on three bean cultivars. During my time in the lab I made up various media and re-plated cultures so they were always fresh for pod inoculations. For leaf inoculations fresh broths of the bacteria were always made to a specific concentration of bacterial cells measured using a specrophotometer.

After testing the pAV522 mutants on pods twice I identified possible “hits” in virulence genes and the mutants were further tested on pods and leaves. By the end of the research project it was quite satisfying to see that at least one positive “hit” may have been identified. I also screened mutagenised populations of a plasmid cured strain on both pods and leaves. There was also time for  a population study looking at the numbers of bacteria in the leaves of different cultivars at different times after inoculation.

I enjoyed myself very much and acquired practical skills in microbiology and plant pathology. I also gained a greater knowledge of pathogenicity and defence mechanisms in bacteria and plants as well as techniques used in the research lab such as PCR, gel electrophoresis, HPLC and digestion of DNA with restriction endonucleases. After finishing my degree I look forward to pursuing a career in plant pathology.

Caroline Smith
University of Nottingham

Genetic variability in Tapesia yallundae

Eyespot is a fungal disease of cereals, especially winter wheat and barley, caused by the fungus Tapesia spp. It has been identified in most of the temperate regions of the world, and can cause major yield loss when infection is severe. The genus Tapesia contains two closely related species: T. yallundae and T. acuformis. These species can be distinguished by colony morphology and growth characteristics and also by molecular markers.

The object of my project was to examine the relationship between isolates of T. yallundae collected from Europe and New Zealand using a variation of the polymerase chain reaction (PCR). Random amplification of polymorphic DNA (RAPD) PCR is a technique that uses a single random primer to amplify the parts of the genome to which it anneals. This produces a unique “fingerprint” for a particular isolate. The analysis of the bands produced can be used to generate a genetic tree showing how closely the isolates are related within the species.

The results could either show that the isolates from New Zealand were distinct from those from Europe, or that the isolates are closely related. Both of these outcomes could have interesting implications on the evolution and spread of the disease across the world.

In all 37 isolates were examined, 9 from Europe, 2 from the USA and 26 from New Zealand. The New Zealand isolates were from 2 different collections: 11 from Nottingham, which originate from south New Zealand, and 15 from Paul Bowyer and Rosie Bradshaw at IACR-Long Ashton, which originate from south-east New Zealand.

Isolates of T. yallundae were grown up in YEG medium for approximately 10 days at 19°C, then were harvested and washed in phosphate buffer at pH7. The DNA was then extracted from freeze-dried mycelia and quantified by gel electrophoresis against lambda DNA standards. Working stocks of the DNA solutions were then set up at a concentration of 5ng/microlitre, determined by running trial RAPD PCR using different concentrations of DNA for the optimum results from the DyNAzyme polymerase. The RAPD PCR reactions were run using 20 different primers for each isolate and the products were run out on a 1.4% agarose gel. The presence or absence of strong, reproducible bands was then scored for each isolate.

The scored bands were put into a computer program and  a genetic tree was formed. To reduce the risk of there being an outlying result that would affect the tree, a technique called bootstrapping was applied to the data. This technique mimics repetition of the experiment by altering the results randomly to see if there is any effect on the tree structure. This was done 70 times. An average tree was then produced.

The average tree formed by the bootstrapping procedure was very similar to the initial tree, indicating that the original results were reliable. The branches of the tree showed a percentage, with branches with a value over 80% considered significantly different.

Overall, the isolates from south New Zealand formed a heterogenous group with the isolates from Europe and the USA. This suggested that these isolates were imported fairly recently by settlers coming to New Zealand from Europe. However the isolates from south-east New Zealand formed a distinct group separate from all other isolates. This suggested that T. yallundae may have already been present in New Zealand before European colonisation, and had evolved sufficiently tobecome divergent from its European counterparts.Other significant branches were found within the tree. One branch separated a group of nine isolates from different countries. Interestingly, more divergence was found within isolates from the same field in New Zealand than was present between some isolates from different countries!

Helen assessing her latest RAPD results with fellow eyespot researcher Henry Wood.

I would like to thank the BSPP for giving me this opportunity to experience working in a research environment at Nottingham University. I have no doubt that this will benefit me in my final year of study, and has confirmed that my future lies in a career in microbiological research. Many thanks to Dr Paul Dyer for his advice and support, and to all those in the lab for their encouragement.

Helen Meakin
University of Nottingham

Fungicide-resistant strains of Botrytis cinerea on Ericaceous plant nurseries in the West of Scotland

In recent years, there has been an increase in damaging infections caused by Botrytis cinerea on Calluna vulgaris and Erica spp. on growers’ holdings in the West of Scotland. Cuttings, potted-on plants and stock plants have been subject to some of the most serious damage. Many growers have reported significant losses despite integrating fungicide applications with current cultural control practices. Fungicide programmes are one of the most important strategies for the control of B. cinerea. However, the number of fungicides currently available for control of Botrytis on Ericaceous plants is limited. Many of the fungicides available give inadequate control, and information on safety and efficacy is often limited. In addition, recent evidence has indicated that resistance to specific fungicides may be widespread.

My project provided me with the opportunity to investigate the incidence of fungicide-resistant strains of B. cinerea on selected nurseries in the West of Scotland. In addition, a small-scale glasshouse trial was undertaken to compare several novel fungicides for control of B. cinerea during propagation of C. vulgaris cuttings.

Samples of infected plant material were collected from three Ericaceous plant nurseries situated in the West of Scotland. The majority of Botrytis infection was found on stock plants. Infected material was surface sterilised and plated onto potato dextrose agar (PDA) and incubated at 18-20°C to encourage sporulation, for re-isolation onto fresh plates. A total of 21 isolates originally collected from different varieties of C. vulgaris were selected for further investigation. Isolates were tested for resistance to carbendazim (mycelial growth test), iprodione (mycelial growth test) and dichlofluanid (mycelial growth and spore germination tests). Fungicide-amended PDA was inoculated with 5mm agar discs of B. cinerea, and plates were incubated at 20°C. Colony diameters were measured after 3 days and % incubation was calculated. Thirteen isolates showed signs of resistance to carbendazim at 100 micrograms/ml. However, only four isolates showed signs of resistance to iprodione at 10 micrograms/ml. Spore germination tests were undertaken to examine resistance to dichlofluanid as this is the primary mode of action of the fungicide and should, therefore, give a more accurate indication of the sensitivity of isolates. Concentrations of 0, 1, 2, 3 and 5 micrograms/ml were used, and % germination was assessed on each plate. Three of the five isolates tested had mean values for % germination greater than 82% at the highest concentration (5 micrograms/ml) indicating resistance to dichlofluanid.

Separate assessments were also made of growth rates, production of conidia and sclerotia, and also osmotic sensitivity of all 21 isolates. Growth rates did not differ significantly between isolates. Resistant isolates tended to produce more conidia than sensitive isolates and showed a higher degree of osmotic sensitivity.

A small-scale glasshouse trial undertaken to assess the effect of novel fungicides on the control of B. cinerea on C. vulgaris cuttings yielded promising results. Treatments with a novel experimental fungicide were more effective than all other treatments. However, it remains to be seen whether the experimental fungicide will be approved for use on Ericaceous plants.

I am extremely grateful to the BSPP for providing me with the opportunity to undertake this 10-week summer project. The experience has given me an invaluable insight into field and laboratory research. I have developed basic laboratory skills, and an appreciation of planning and time management together with a genuine interest in plant pathology. I would like to thank Dr Mark McQuilken for constructive conversations, support and expert guidance throughout the 10 weeks. Also thanks to Kenneth Weierud, Jacqueline Gemmell and Jim Thomson and other staff and research students in the Department of Plant Biology, SAC, Auchincruive for being very friendly and helpful. I am now looking forward to undertaking my final year dissertation on biological control of Pythium spp., with Dr McQuilken as supervisor.

David Thomson
SAC, Auchincruive, Ayr

Molecular diversity and epidemiology of Raspberry Bushy Dwarf Virus

During my 10 week placement at HRI-Wellesbourne, I was involved in the continuation of a study into the taxonomy and distribution of raspberry bushy dwarf virus (RBDV). The virus has been associated with considerable loss of yield in some varieties. A recent increase in its occurrence means that it can now be found in commercial crops on about 30% of UK farms.

I began my work by verifying the results obtained by previous research into the genomes of different viral isolates. RBDV has a segmented genome with three positive-sense RNA strands. Regions on two of the strands were amplified by nested PCR using two sets of primers designed during previous work. I looked for possible subdivision of the genomes already classified, by digesting both PCR products with five different restriction endonucleases with four-base recognition sites, generating banding patterns specific to each genotype. No further subdivisions were observed within existing genotypes.

Furthermore, leaf samples containing standard viral isolates were collected from HRI-East Malling where they are maintained. The virus was trapped from leaf extract by immunocapture followed by reverse transcription and nested PCR, using the two sets of primers. PCR product was then available from each of the standards. For the purposes of the project, further DNA could be produced by re-amplification, using the internal primers and product from the external primers. An attempt was also made to maintain the isolates by inoculation of Chenopodium quinoa plants in the glasshouse. However, for whatever reason, this was unsuccessful. It is known that best conditions for inoculation with RBDV occur in spring.

Representatives from each genotype and all standards were purified from reaction mixtures and sent for sequencing off-site. Due to some poor PCR reactions in the late stages of my project, as well as inefficient initial purification protocols, too low a yield of some isolates was obtained for sequencing to be carried out. It was necessary in some cases to try to optimise the PCR reaction by varying magnesium and template concentrations, as optimum conditions appeared to vary from isolate to isolate.

The sequences were used for the production of phylogenetic trees relating the viral genomes on the basis of the two PCR products from each representative isolate. These trees, together with data from the original field survey showing the distribution of genotypes across the UK, can now be used to describe the current epidemic in much greater detail.

Thank you to the BSPP for providing me with this opportunity to experience scientific research first-hand. I would also like to thank everyone who helped me at HRI, especially Dr Dez Barbara and his team for allowing me to join them in their laboratory for ten weeks. I have learnt a great deal of practical skills, including specialised plant pathology and molecular biology, as well as more general lab procedures. I worked independently in many endeavours, which gave me a lot of confidence in my ability. It has all been very interesting, and I know it will stand me in good stead for my future career.

Anthony Scott-Tucker
The University of Birmingham

PCR detection of fungal spores in real air samples

Conventional methods for identifying airborne fungi requires skill in microscopic and cultural techniques, it is laborious, time consuming and can be unreliable. Novel techniques such as immunoassays and DNA based detection methods may be faster and more reliable. Molecular techniques, such as the polymerase chain reaction (PCR), have the potential to characterise all the microorganisms in the airborne samples by detecting the DNA sequences present in the sample.

This project began by developing a PCR based method for detecting specific fungal spores, using Penicillium roqueforti as a model fungus. The method had been tested with P. roqueforti spores alone or added to background air samples. Therefore, research into the PCR’s sensitivity when faced with a real air sample was required, as non-target material present in real air samples may effect the sensitivity of the PCR assays.

A novel rotating-arm sampler, that collected spores in Eppendorf tubes and a conventional rotating-arm sampler were used to collect air samples in the wind tunnel. The spores in the air samples were disrupted by milling with Ballotini beads. The milled samples were either used in the PCR reaction or the DNA was extracted and added to the PCR. The samples were analysed in a PCR which uses fungal consensus primers (ITS 4 and ITS 5).  The product from this reaction was used in a further PCR (nested PCR) which used primers specific to P. roqueforti. The ITS 4/5 PCR detected DNA in nearly all the spore disrupted (milled) samples from the Eppendorf rotating-arm sampler, including the background, whereas the nested PCR did not detect DNA in any of the background samples or air samples that lacked P. roqueforti spores. Nested PCR when combined with milling and applied to wind tunnel air samples was only slightly less sensitive than the same assay applied to pure P. roqueforti spore samples.

The conventional rotating-arm sampler collected spores on a tape covered in a wax coating. The tape and wax coating or the wax coating alone was processed as above before analysis with ITS 4/5 or nested PCR. PCR products were detected from both types of samples – wax coat and wax coat plus tape – whether the sample had been milled or milled plus DNA extraction. Tests were carried out on the unexposed tape and wax that is used for the trapping surface on the conventional sampler. PCR assays were done on samples of tape alone, wax alone and tape plus wax. The samples were treated exactly as the exposed samples i.e. milled or milled plus DNA extracted before being assayed. The results showed that the trapping surface used in the experiments did not significantly interfere with the PCR reaction and that milling followed by DNA extraction can be used to process air samples taken with conventional sticky samplers.

I would like to thank the BSPP for making my ten week placement at IACR-Rothamsted possible. Also thanks to Dr Alastair McCartney and Dr Roger Williams for their supervision, and Margaret Perry, Simon Foster and Windy Heran for all their advice and support. I have really enjoyed the work I have undertaken, whilst the experience and techniques I have gained will benefit me in my final year at university and also in any future employment.

Elizabeth Sims
University of Hertfordshire

The search for SNPs in Phytophthora infestans

In Europe a displacement of the ‘old’ clonal population of Phytophthora infestans occurred during the late 1970’s when new A1 and A2 mating type strains were introduced from Mexico. Since 1980 greater genetic variation was seen, particularly where both A1 and A2 varieties were found.  A recent survey of 500 isolates in Scotland indicated 79% A1 and 19% A2 with mixtures of both isolates occurring at some sites (mainly private gardens). Molecular analysis of this population using AFLPs suggest the occurrence of sexual recombination which has an impact not only on population structure but also field epidemiology. There are, however, still many unanswered questions such as why A2 isolates are relatively infrequent in the UK and what factors are responsible for the generation and maintenance of pathogen variation.

Better markers are needed to characterize populations of P. infestans. Ideally, markers should be co-dominant and inherited in a simple, independent and stable manner.  Single Nucleotide Polymorphisms (SNPs) are mutations at single base pair positions in genomic DNA.  Such minor allelic differences exist in normal individuals in some populations and to be a useful molecular marker must occur at a detectable frequency. Similarly, single base insertion/deletion variants (indels) are potentially useful bi-allelic polymorphisms.  In the field of human genetics tens of thousands of SNPs are being examined in order to identify associations with heritable disease though these will also revolutionise the study of human population change.

Two thirds of human SNPs involve C to T mutations possibly related to 5-methyl cytosine deamination reactions.  The other three types occur at similar levels to each other. SNP’s typically occur at a frequency of one base pair per thousand. However, genome wide there are regional differences; coding exons have a four-fold lower rate of polymorphisms with about 50% of them resulting in non-synonymous codon changes.  Other peculiar regions show diversities of 5-10%.  Sequence variations are created continuously which are typically eliminated by drift or they may remain for a period as rare variations.

In this project the potential for using SNP markers in P. infestans was assessed. Publications relating to P. infestans gene sequences in the EMBL database were examined and five genes selected. The single copy genes chosen were actin (ActA, ActB), nitrate reductase (2 regions selected), a transcriptional fusion of glyceraldehyde-3-phosphate and triose-phosphate isomerase (TigA), the calmodulin gene, and the RAS gene (one of the few which possess introns).   Primers were designed to amplify c. 900bp fragments (including untranslated and translated regions) of these genes and these regions from 6 isolates were sequenced.  Of the six isolates three were of international origin (Argentina, Equador and the USA) and another three from Scottish locations.

The extent of variation in these regions varied.  No clear candidates were seen in ActB and TigA but more than ten polymorphisms were observed in the nitrate reductase untranslated region and short intron. Restriction digest assays were designed to two of these SNPs and the extent of the polymorphisms assessed in a wider range of Scottish isolates.  Both looked strong candidates as useful markers, showing variation within international and Scottish isolates.  Unfortunately there was insufficient time to study this further.

I would like to thank the BSPP for financial support and David Shaw (U.W. Bangor), David Cooke, Jim Duncan, Naomi Williams and David Guy (SCRI) for their help and guidance over the 10 weeks in ‘Bonny Dundee’.

Russel Palmer
University of Wales, Bangor

Resistance of aerial tubers to potato blackleg

Blackleg of potatoes, caused by E. carotovora subsp. atroseptica (Eca), is a major economic problem to the potato industry.  It is a bacterial soft rot which can cause a rapid reduction in the quality and market value of the potato crop.  With no effective chemical control and only limited cultivar resistance, alternative biological control methods are being investigated.

The intended aim of the project, supervised jointly by Drs Ruairidh Bain and Glynn Percival of SAC, was to compare the resistance to E. carotovora subsp. atroseptica of aerial tubers (tubers produced above ground in leaf axils) and conventionally produced subterranean tubers.  Previous work has shown that the exposure of conventional tubers to light for 10 to 30 days can reduce susceptibility to a number of diseases.  Aerial tubers are exposed to light throughout the growing season.  Light treatment is known to increase the concentration of toxic glycoalkaloids within tubers, but the treatment is suitable for seed potatoes because glycoalkaloid concentrations in progeny grown from light-treated seed are within acceptable limits.  Due to the aerial tubers needed in the study not being ready early enough, only a small amount of time was spent on this aspect of the project. Instead, experiments that examined the effect of potato metabolites on Eca survival in vitro and the effect of light treatment on Eca survival on conventional tubers were carried out.

Firstly, in vitro studies of the bacteriostatic effects of potato metabolites on Eca were undertaken.  A range of sugars, a, b and g glycoalkaloids, solanidine and chlorogenic acid were tested at various concentrations. Bacterial cultures were incubated with potato metabolites singly and in combination for 24 hours and then plated out onto the semi-selective medium, crystal violet pectate (CVP). Fructose was the only sugar that markedly reduced the number of Eca colonies.  In contrast, glycoalkaloids, solanidine and chlorogenic acid all markedly reduced colony numbers.

Another experiment involved exposing conventionally produced tubers, previously inoculated with Eca, to light for 0, 10 and 20 days.  After the exposure period sap was extracted from the tuber peel and plated onto CVP.  Significantly fewer Eca colonies were recorded from tubers stored under light, indicating that light exposure can reduce the number of Eca following artificial inoculation into tuber lenticels.  It was also found that tubers exposed to light had less percentage rot than dark controls when placed into anaerobic conditions to induce soft rot.  These results provide evidence that light treatment of seed tubers could be a suitable method of controlling blackleg of potatoes.

The susceptibility to Erwinia soft rot of aerial tubers and conventionally produced tubers of four cultivars, i.e. Cara, Desiree, Nadine and King Edward, was compared.  The tubers were uniformly wounded by removing a shallow plug of tissue with a cork borer and inoculated with a suspension of Eca.  The aerial tubers were consistently less susceptible than the subterranean tubers.  For some varieties the difference was statistically significant.

I am extremely gratefully to the BSPP for giving me the opportunity to carry out this highly interesting and enlightening summer laboratory work at SAC, Auchincruive.  The range of techniques and experience that I have gained will undoubtedly be of immense benefit to me not only in my future studies but my subsequent career in biological research.

Lorna Hall
University of Strathclyde

Biocontrol of Cocoa Disease

The monies kindly donated by the BSPP enabled me to spend thirteen weeks of my summer vacation at CATIE, Turrialba, Costa Rica, under the able supervision of Dr Ulrike Krauss and her two affable assistants Adolfo Martinez and Eduardo Hidalgo.  Dr Krauss is involved in a five-year research project investigating the efficacy of indigenous mycoparasites at controlling two diseases of cacao, Moniliasis (Frosty pod) being endemic to all but the cacao growing regions of Brazil in Latin America, and Phytophthora (Black pod) being pandemic to cacao plantations globally.

Based at CATIE (Centro Agrónomico Tropical de Investigación y Enseñanza) on the outskirts of Turrialba in the Valle Central, the work undertaken involved: attendance of a workshop, the installation and sampling of a field survival trial (FST) of biocontrol agents, and a laboratory-based investigation of mycoparasite efficacy.  I was also given the opportunity to visit various trial sites throughout the cacao growing regions of Costa Rica and Panama, learning and assisting in the preparation and application of biocontrol inoculum and familiarising myself with the methodology employed by accompanying extensionists.

The workshop “Research Methodology in Biocontrol of Plant Diseases, with Special Reference to Fungal Diseases of Cocoa” organised by Dr Krauss at CATIE was most informative.  This workshop covered all aspects of the methodology employed in the identification and biocontrol of fungal diseases related to cocoa production and was attended by researchers and facilitators from the UK, the USA, Latin America, the Caribbean and Africa.  With laboratory practicals and site visits supplementing the presentations and discussion, this was a most invaluable experience, and an excellent introduction to the problems encountered in the implementation of disease control methodology in cacao production was obtained.

Invited into Ulrike’s office one day, I was presented with an A4 sheet of paper covered in sketches and doodles, presented to me as Ulrike’s thoughts on the field survival trial that she asked me to undertake.  After a brief brainstorming session, involving Ulrike, Adolfo, Eduardo and myself, the plans were laid for the experiment to be installed by myself, with the assistance of Adolfo and Eduardo when and if required.

Five treatments were to be applied in this trial; two ultra-violet protectants, one adherent, a positive (unprotected mycoparasite inoculum) control and a (negative) water control.  Laboratory preparations involved the inoculation of PDA agar petri dishes with a Phytophthora host (employing the pre-colonised plate method) the inoculation of growth medium (using the rice bag method of location of Hebbar and Lumsden, 1999) with a strain of Gliocladium.

Three locations (all within close walking distance) in Cabiria, CATIE’s onsite germplasm bank, were chosen for the experiment site, each containing one cacao clone-type (UF29).  Cacao pods (85) of an appropriate size and age for the experiment were randomly selected and tagged, the severity of the disease highlighted here by the number of pods succumbing to the disease before the experiment could be installed.  Finally, the mixing of the inoculum and inoculation with each of the prescribed treatments were undertaken and applied to the respective pods at each location.  The methodology used for this exercise was exactly the same as of that used to apply inoculum at all of the project’s associated trial and experimental sites.

This experimental site was then sampled for 56 days, to determine the field survival of the mycoparasite and the efficacy of the adjuncts.  The data collected are to be analysed and form part of a larger report, and a possible publication, to be prepared in the near future.

The laboratory-based experiments were designed to investigate the host range, comparative growth and compatibility of promising mycoparastic fungi, already in use on experimental and field trial sites associated with the project.  Preparation involved the pre-colonisation of PDA agar petri dishes with each of the mycoparasites under investigation (Gliocladium and Trichoderma spp.), the two mycopathogens (Moniliophthora rorei and Phytophthora palmivora) and two entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae).  These pre-colonised petri dishes were then challenged with a representative of each of the mycoparasites under investigation, incubated and then observed, using the method as described by Krauss et al. (1998).  Again these data are being analysed and will be encompassed in a larger report or publication.

Visits to each of the experimental and trial sites were also undertaken, mostly in the company of Adolfo Martinez.  The experimental sites at La Lola, a CATIE experimental research station situated on the lowland plains, comprised of two relatively abandoned cacao germplasm plots, one plot having a high incidence of “frosty” pod, the other having a high incidence of “black” pod.

The trial sites, six in the Talamanca region of Costa Rica and five in the Bocas del Toro region of Panama, are all situated in cacao-producing small-holdings. Treatment application and evaluation were undertaken with assistance from the farmers, local co-operatives and extension workers.

For each of these visits mycoparasitic fungi were cultured, using the rice-bag method and transported to the sites where the inoculum was prepared using low-tech methodology, the inoculum being applied using hand sprayers.  This was rather hot and sweaty work, but the experience and skills gained from this introduction to extension methodologies and the role played by extension workers will no doubt stand me in good stead for my future ambitions.

I would like to extend my thanks to the BSPP, for ample funding through their undergraduate vacation bursary, without which this experience may never have occurred.  I would also like to extend my thanks to Dr Ulrike Krauss, a most capable and approachable supervisor and to Ulrike’s two assistants, Adolfo and Eduardo, along with all those working in Fitoproteccion.  The memories, experiences and skills gained during my stay at CATIE, a most pleasant working environment, surrounded by the most pleasant of people, will always remain with me.
Pura Vida!

References
Hebbar. K. P. and Lumsden  R. D.  Formulation and Fermentation of Biocontrol Agents of Cacao Fungal Pathogens: Example of Trichoderma Species.  In “Research Methodology in Biocontrol of Plant Diseases, with Special Reference to Fungal Diseases of Cocoa” edited by Krauss. U and Hebbar P. (1999), CATIE, Costa Rica, pp 63-68.

Krauss. U., Bidwell. R. and Ince. J. (1998) Isolation and preliminary evaluation of mycoparasites as biocontrol agents of crown rot of banana.  Biological Control 13: 111-119.

Stephen Piper
University of Edinburgh

External signals  and the role of cAMP-mediated signal transduction  in the early development of Oidium lycopersici

The aim of this study was to investigate the external signals and signal transduction involved in the differentiation of the infection structure by Oidium lycopersici conidia and, through comparison wth Blumeria graminis f.sp. hordei, to draw inferences about the nature of host recognition and specificity in these two species.

The obligate biotroph O. lycopersici, which causes tomato powdery mildew, was first identified as a pathogen of glasshouse-grown tomatoes in 1987, and has since become an economically important disease. O. lycopersici is highly polyphagous and also differs in the morphology of its early development from its more extensively-studied relative B. graminis, the causal agent of barley powdery mildew.

The experimental approach used in this study was determined by the obligately biotrophic nature of the pathogens. Chemicals were exogenously applied to spores that had been inoculated onto either host epidermis, cellulose membrane or water agar, and the effects of these chemicals observed using phase contrast microscopy.

Multiple host-derived signals are known to be involved in the recognition of the barley leaf surface in B. graminis.  Development is abnormal on non-host leaf surfaces and artificial surfaces that supply only a subset of the required signals.  Investigation of the development of O. lycopersici on tomato epidermal peels, cellulose membrane and water agar revealed that, unlike B. graminis, conidial germination is not significantly different on these surfaces. O. lycopersici differentiation was found to be surface-influenced, although the results suggested that the signalling and controls involved are likely to be less complex than those in B. graminis. In particular, there was no significant difference between cellulose membrane and tomato epidermis 24 hours post-inoculation.

The cyclic AMP (cAMP) signal transduction pathway is known to influence a variety of phenomena in phytopathic fungi, including spore morphogenesis and the establishment of infection within a host plant. Recently, investigation of the role of cAMP in B. graminis has identified a unique role for cAMP signal transduction during appressorial germ tube differentiation.

cAMP, the cAMP analogue 8Br-cAMP and AMP were tested on host and artificial surfaces and found to have no effect on either  the germination or differentiation of O. lycopersici conidia. A range of protein kinase and more general inhibitors (H89, staurosporine and cycloheximide) were also found to have no significant effect on the germination or differentiation  of O. lycopersici on tomato epidermal peels. The permeability of the tomato epidermal peels to the exogenous chemicals was tested and found to be adequate. Thus the results strongly implied that the conidia themselves were impermeable to the applied chemicals. This conclusion was further supported by multiphoton microscopic analysis of spores inoculated onto cellulose membrane using a fluorescent cAMP derivative. Interestingly, quinoxyfen, a fungicide specific to powdery mildews but with an unknown mode of action, very effectively inhibited germination of O. lycopersici.

The use of chemical agents to permeabilise the spore to other exogenously applied chemicals was considered, with  low toxicity and minimal effects on spore development the primary selection criteria. The non-ionic surfactant pluronic F-127 was identified as a potential permeabilisation agent, although further investigation is required.

I would like to thank BSPP for providing financial support for this project, and Dr. Sarah Gurr, Alison Hall, Hannah Jones and other members of the Plant Pathology group at the Department of Plant Sciences, Oxford University for their invaluable help and advice.

Annabel Whibley
Magdalen College, Oxford.

Possible biological control of Leptosphaeria maculans  using Cyathus striatus, a Bird’s Nest Fungus.

Oilseed rape is an economically significant crop, which is hampered by the fungal pathogen Leptosphaeria maculans, causal agent of blackleg (canker) on brassicas. Two major groups of L. maculans are known; a pathogenic A-type, and a non-pathogenic B-type. Control with fungicides is unpredictable, but using cultivars with increased levels of erucic acid can be effective. However, if levels are too high, the rape is not suitable for use as cattle feed or cooking oil, but at low levels, yield is substantially reduced by disease incidence.

It has been observed that where Cyathus striatus is present, less of the causal agent of this disease overwinters, and therefore the epidemic builds up more slowly the following spring. Therefore this project was to investigate the possibility of using Cyathus striatus as a biological control agent for Leptosphaeria maculans.

Initially, growth rates of both fungi were measured on various media. C. striatus consistently grew at a faster rate than L. maculans.

Cellulase production by both fungi was assayed by inoculating moistened cotton strips with equal amounts of the fungi and placing them in sealed boxes for two weeks. The tensile strengths of the strips were then measured using a tensiometer. Despite low-level contamination on the majority of strips, C. striatus consistently caused strips to break at significantly lower tension values, indicating that more cellulase had been produced by the fungus. Future investigations could include assaying for lignase activity.

Resource capture investigations involved pairing both fungi on different media; potato dextrose agar, V8 juice agar and sterile rape straw on tap water agar. Again C. striatus consistently grew more rapidly than L. maculans, indicating more efficient primary resource capture and in some cases, when moisture and nutrients were not limiting factors, C. striatus overgrew L. maculans, utilising it as a secondary nutrient source.

When the two fungi were in close proximity on agar, several observations were noted. Firstly, the B-type L. maculans produced a brown pigment, and C. striatus overgrew its leading edge. However the A-type strain (pathogenic), altered the direction of hyphal growth, away from the Cyathus. A clear divide could often be observed, possibly caused by extracellular secretions. A pH difference was also noted between the fungi, the area immediately around the L. maculans being more alkaline.

From the work I have carried out and previous research, there does appear to be potential for the biological control of Leptosphaeria maculans using Cyathus striatus. I have thoroughly enjoyed my contribution to the world of plant pathology and am glad to have learnt many investigative techniques. I would particularly like to thank Dr Avice Hall at the University of Hertfordshire and the BSPP for providing me with this opportunity.

Alison Carmichael
University of Hertfordshire

Identification of Arabidopsis mutants compromised in the oxidative burst associated redox signalling network

Currently about 13% of total world crop production is lost through disease by microbial plant pathogens per annum. Therefore, studies of plant:pathogen interactions are of central importance for agriculture. Plants possess several different lines of defence against potential pathogens, which can be classified as passive or active. Passive defences consist of anatomical features such as the waxy cuticle and the cell wall, in addition to chemical defences, for example, preformed inhibitors such as phenols, alkaloids and antimicrobial proteins such as thionins and defensins. When a pathogen breaches the plant’s first line of passive defences, a battery of active defence mechanisms are induced; these include the rapid production of reactive oxygen species (ROS) and programmed cell death at the site of pathogen attack. These defence mechanisms are part of the hypersensitive response.

Following the HR, levels of salicylic acid (SA) increase locally and systemically. SA is known to activate pathogenesis-related (PR) genes and boost H₂O₂ production in a positive feedback loop. The expression of PR genes is thought to lead to the development of systemic acquired resistance (SAR), providing resistance against a wide range of pathogens. In addition, the accumulation of ethylene and jasmonic acid (JA) leads to the expression of ethylene and JA-dependent genes. Current research suggests that the SA and JA signalling pathways are interconnected.

 There is now a great deal of interest in the generation and perception of ROS in the context of plant disease resistance. Recently, H₂O₂ has been implicated as a signalling molecule (PNAS 95:5818), activating defence and antioxidant genes such as glutathione-S-transferases (GSTs) in local and systemic tissues (Cell 92:773). In this study, gst1 has been used as a molecular marker to screen for mutants in the oxidative burst associated redox signalling network, with the aim of identifying the molecular machinery integral to this signalling pathway. The careful characterisation of such mutants will help shed light on the function(s) of ROS and cognate redox signalling in the establishment of gene-for-gene resistance and SAR.

Previously, gst1 was cloned and its promoter identified and isolated (Grant and Loake, in press). The gst1 promoter was fused to the real time reporter gene, luciferase, and the transcriptional termination sequence of the octopine sythase gene. Arabidopsis thaliana ecotype Columbia was transformed with Agrobacterium, using vacuum infiltration. Several transgenic lines were obtained, and one line selected for further study based on its low background and high activation of LUC activity in response to ROS accumulation. Approximately 20,000 homozygous F2 seeds of this transgenic line were mutagenised with ethyl methanesulphonate (EMS) and the F2 seeds collected in 100 pools.

In order to screen for loss-of-function mutants, 1000 seeds from pools 1 to 35 were germinated on MS agar medium containing 1.5mM H₂O₂, which is known to activate gst1 gene expression. In addition, 20 seeds from each pool were germinated on media lacking H₂O₂, to screen for gain-of-function mutants, which showed gst1::luc gene expression in the absence of H₂O₂. At ten days old, the seedlings were monitored for gst1::luc gene expression using an ultra low light imaging camera system. Seedlings which had lost the ability to switch on the gst1 promoter in response to H₂O₂ were identified by their failure to express LUC activity. In contrast, gain-of-function mutations would express LUC activity even in the absence of H₂O₂. Using the first screen 2% of the total 42,000 seedlings screened, were identified as mutant candidates.

The second stage of screening involved the infiltration of a H₂O₂ generating system, glucose/glucose oxidase (G/GO) into the mutant candidates from the first screen, to further confirm the mutant phenotype. Non mutagenised gst1::luc plants were used as a control to assess LUC activity in potential mutants. Following G/GO infiltration, the percentage of potential mutants was reduced to 0.06% of the 7200 seedlings screened to the second stage. Two loss-of-function mutants, non-inducer of gst1 (nig1 and nig2), but no gain-of-function candidates were identified. In addition, two seedlings, super inducer of gst1 (sig1 and sig2) were uncovered that have a very high level of luciferase expression in the presence of H₂O₂.

Inoculation of these mutant candidates with Pseudomonas syringae pv. tomato (Pst) expressing avrB resulted in the following expression patterns: super induction candidates sig1 and sig2 continued to show high expression of luciferase following inoculation with Pst (avrB), similar to that obtained following G/GO infiltration. One loss-of-function candidate nig1 continued to exhibit very low levels of luciferase expression following Pst(avrB) inoculation. however, the nig2 candidate, which had previously shown low levels of luciferase activity in the presence of H₂O₂, now exhibited high levels of expression following Pst(avrB) inoculation and was therefore not investigated further.

Inoculation of super-induction candidates, sig1 and sig2 with Pst(avrB) activated very high luciferase activity, suggesting the super-induction of the gst1 promoter. In contrast, inoculation of the loss-of-function candidate nig1 with Pst(avrB) showed very low luciferase activity. However, while these results are encouraging, it is possible these mutations may be within the gst1 promoter or luc coding sequence. Inoculation of nig2 with Pst(avrB) exhibited high levels of expression, in comparison to the low expression level of luciferase activity in the presence of H₂O₂. This may suggest that nig2 has a mutation in the ROS signalling pathway, which blocks the activation of gst1 by H₂O₂, but can activate the gst1 promoter via an alternative pathway following inoculation with Pst(avrB).

Recent evidence demonstrates that ROS are essential participants in the establishment of SAR. In particular, H₂O₂ is thought to be an important signalling molecule functioning upstream of SA and ethylene in the establishment of SAR. It has also been proposed that H₂O₂ can act as a diffusable intercellular signal for transcriptional activation of gst and phenylalanine ammonia lyase (PAL). PAL is probably the first enzyme in SA biosynthesis, suggesting H₂O₂ and SAR may be linked in this way. Therefore oncemutant status of the candidates has been confirmed, the mutants can be used to investigate the central role of the ROS-signalling pathway in both gene-for-gene resistance and the establishment of SAR.

Finally I would like to say that the experience of working in a laboratory at Edinburgh University over the summer has given me much more confidence and taught me new skills which I would not have learned on my course. It has also encouraged me to do a  Ph.D and to pursue a career in research. I would like to thank Gary Loake, Byung-Wook Yun and John Grant for their help throughout this project.

Angela Feechan
University of Edinburgh

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