BSPP News Spring 2001 - Online Edition

The Newsletter of the British Society for Plant Pathology
Number 38, Spring 2001 

2000 BSPP Undergraduate Vacation Bursaries

Cultural and molecular analysis of the UK Leptosphaeria maculans (stem canker) population.

My project at Rothamsted was based on the fungus Leptosphaeria maculans, which infects oilseed rape (Brassica napus). The fungus causes stem canker, one of the most damaging diseases of oilseed rape in Europe, capable of causing substantial yield losses. Over the period of 1993-95, it was estimated to be responsible for economic losses exceeding £30 million per annum in the UK.

The L. maculans fungus can cause many symptoms on the rape plant. These range from initial leaf spots, causing the leaf surface to rupture, and later in the season, damaging stem cankers, which can cause lodging of the whole plant,  to premature ripening and even death. Recent studies have shown that there are two types of the fungus that infect oilseed rape and these have been given many names A- & B-type, Tox+ & Tox0 and also highly virulent (HV) & weakly virulent (WV). DNA polymorphisms, ribosomal DNA sequences, and estimates of chromosome number and size indicate that the two virulence types are very different genetically and are probably separate species.

My project involved the study of the UK population of L. maculans with the aim of determining whether the symptoms on the rape stems were the result of infection by the A- or B-type. Samples, which consisted of oilseed rape stems varying in height from 30-90cm, were sent to me by Dr Peter Gladders (ADAS, Boxworth). Samples came from all areas of the country ranging from Northumberland to Kent and Wales to Suffolk.

My first task was to transfer the fungus from the plant material onto agar. This would allow me to distinguish A- & B- types, as the B-type produces a distinct yellow pigment when grown on PDA (potato dextrose agar).  This was done by cutting out the lesion on the rape stem, cutting it in half, surface sterilising in sodium hypochlorite and then transferring the section to PDA directly. Unfortunately this method resulted in a high level of contamination, as competitive fungal species were able to grow at a faster rate than the L. maculans fungus. As a result of this, subsequent plant material was placed on distilled water agar first and then sub-cultured onto PDA when sufficient growth had taken place. This work was carried out in a laminar flow cabinet to try and minimise further contamination.  The second half of the lesion was sealed in a polythene bag and frozen with its sample number and the height up the stem from which the lesion had been taken, to be analysed later on in the project. Due to the aforementioned isolation problems, some of this material was also used to make "re-isolations".

The cultures of L. maculans grew successfully. The B-type fungus was found to be present in 20 sites across the UK from 98 sample sites that were tested. It is hoped that by using polymerase chain reaction (PCR) analysis, the proportion of B-type isolates may rise as the use of PCR removes some of the problems caused by overgrowth of isolates by faster growing fungi. The B group was not concentrated over a narrow area of the country, but was present in several locations.

The second half of my project involved the use of a PCR-based technique to try to identify which strain of the L. maculans fungus had caused the canker and confirm the results from agar testing. Primers to differentiate between the A- & B-types, based on the internal transcribed spacer (ITS) sequences of the ribosmoal DNA, were received from collaborative partners at INRA, Versailles. However, problems with the PCR work were experienced. The B-type complementary primer did not facilitate amplification of the B-type specific PCR product, even when tested with known B-type DNA. Also, when the A- and B-type specific primers were used together in a multiplex reaction, only the A type specific PCR product was amplified. We tried a number of ways to resolve the problems. One was changing the enzyme used from normal Taq DNA polymerase to Platinum (TM) Taq DNA polymerase. This has no enzyme activity until heat denaturation is performed, due to the presence of a propriety antibody that inhibits polymerase activity.  It can therefore reduce or eliminate non-specific amplification. Unfortunately this was unsuccessful, as was trying to amplify DNA from plant material.

After carrying out many other reactions it was concluded that sequence differences between UK and French isolates may be responsible for the problems experienced. We therefore decided to try and sequence the ITS regions from UK B-type isolates and try and design primers of our own.  DNA was extracted from mycelium of three single spore isolates using a standard extraction procedure. This DNA was used as a template to amplify the ITS regions and these PCR products are currently being sequenced to enable the design of new primers.

I would like to thank my supervisor Dr Neal Evans, for putting me forward for a BSPP Bursary, Drs Jon West and Bruce Fitt for their knowledge on L. maculans and Dr Simon Foster for helping me with my molecular work. I would also like to thank everybody in the Plant Pathology Department for making me feel welcome for the 10 weeks that I spent there this summer. I would also like to thank Dr Peter Gladders and his ADAS "team" for supplying stem samples. Finally, many thanks to the BSPP for granting me the opportunity to work at Rothamsted.  I have to say that it has been an extremely rewarding and fun experience.

I hope to continue this project and expand it with the aim of submitting it for my final year research project at The University of Nottingham.

Helen Margaret Wicks, University of Nottingham

Information required for quarantine pest risk analysis

"And now for something completely different …"  Instead of squinting down a microscope at small but yet potentially damaging organisms I was to spend my vacation looking at these phytopathogenic creatures and other plant pests from a global viewpoint, specifically that of international plant quarantine, which is designed to prevent the introduction and/or spread of quarantine pests or to ensure their official control. The term 'pest' refers here to any organism that causes plant damage.

Naturally, each country has quarantine procedures in force but problems arise when phytosanitary regulations between countries differ and terminology varies, and by difficulties in sourcing the requisite information for official documents. The first two issues are being addressed by organisations such as FAO and WTO that seek to standardise international plant quarantine. CABI is concerned with the third point: information sourcing, and in particular the problems presented by filling in pest risk analysis (PRA) forms.

PRAs are lengthy documents requiring referenced scientific information and expert judgements to enable an evaluation of the risk that a potential pest could cause. This then aids decision-makers in plant quarantine (PQ) to ascertain whether the pest or its commodity host is of quarantine importance and if so then how best to control its spread. PRAs can be requested for example whenever a new commodity is traded, a new pest is discovered, any pest is intercepted during trade, new information on a pest is learned or an associated policy is changed.

 Accurate and up-to-date information is required on a wide range of topics including pest taxonomy, biology, abiotic factors, host range, and geographical distribution but these data are rarely found together in one convenient location or media form, especially when the pest is a new or emerging species where information is often scarce or not widely published. CAB International has already developed a comprehensive global database – the Crop Protection Compendium (CPC) in CD ROM format containing soft-linked detailed datasheets, distribution data and more, for pests, hosts and countries. Their next aim is to enhance this database to include as much information (text, website links, references) that would be useful to those who are involved in PQ work. My part in all this was to research the locations and availability of data required for PRA using literature, database and internet searches and to assess its usefulness for the CPC.

To summarise my results: there is a wealth of information to be sourced, albeit often fragmented. Much can be incorporated into new or current datasheets. Other data will be useful in providing a background to PRA and to guide people new to the subject through the basic procedures. For some databases and websites found on the Internet it would be extremely useful to provide a link to them (e.g. taxonomic databases or 'alert' sites like the New Disease Reports perhaps).

It was a pleasure to work for an organisation with such philanthropic values, and I enjoyed the friendly atmosphere at the headquarters in Wallingford. With special thanks to my supervisors Peter Scott and Lucinda Charles for their guidance and support, and of course to BSPP without whose help I would not have had this marvellous opportunity to work here and examine phytopathogens from this new perspective.

Charlotte Hogg
SAC, Auchincruive

Combining induced systemic resistance and systemic acquired resistance to control tomato pathogens

The fungi, Botrytis cinerea and Fusarium oxysporum are responsible for large crop losses in commercial glasshouses and field grown tomatoes. The purpose of my project at HortResearch, Ruakura was to determine if two different types of host defence stimulation, systemic acquired resistance (SAR) and induced systemic resistance (ISR), could induce disease resistance in tomato plants, and also to determine if SAR and ISR were effective at working together to control these fungal pathogens.

The SAR elicitors I used were salicylic acid (SA), Chitosan or a combination. The bacterium Pseudomonas fluorescens and a species of Trichoderma fungus were applied as potential ISR elicitors.
    Initially, the SAR and ISR treatments were applied individually to investigate the best combination of both types of elicitor. Then in subsequent experiments the Trichoderma was applied with a SA/chitosan elicitor combination to determine if SAR and ISR could work in synergy. To determine the effects of the different treatments on plant defence mechanisms, I carried out enzyme assays to measure peroxidase (POX) activity levels per mg of protein. Peroxidase is an important indicator enzyme involved in the resistance response within the tomato plant. Throughout my experiments, high POX activities were recorded in plants treated with a SA/chitosan and a Trichoderma combination or where SA only was applied on a daily basis.

Figure 1. Detached leaf bioassay.

In order to relate the POX activity with disease suppression I compared the disease incidence in the different treatments using detached leaf and stem  bioassays. I found that the elicitor combination of  SA/chitosan with Trichoderma resulted in the least amount of Botrytis growth on the tomato leaf. The lowest level of disease was recorded using the stem assay where plants were treated with SA on a daily basis.

I then set up trials to determine the effect of different growing conditions on induction of disease resistance in tomatoes against B. cinerea. The Trichoderma enhanced disease resistance within the plant more effectively in a soil based media compared to a hydroponic growing system. I believe that the SAR and ISR elicitor could enhance disease resistance if used in combination with tomato plants grown in a soil based media.

To determine induced resistance against F. oxysporum, I set up split root assays to prevent the elicitor treatments being in direct contact with F. oxysporum. However, F. oxysporum did not infect any of the plants in this experiment and measurement of disease control could not be gathered. This could be due to the isolate I used being stored for too long, resulting in some loss of pathogenicity. In inoculum concentration experiments I found that when high concentration doses of F. oxysporum were applied to the tomato plants they became infected. I can recommend that higher doses of inoculum be used in future studies. Other F. oxysporum experiments which had treatments in contact with the pathogen showed signs of Fusarium wilt within two weeks of inoculation, but the treatments seemed to have no effect on disease levels with severe wilt in all treatments.

I am very grateful to the BSPP for the grant which enabled me to travel to New Zealand and work with the Sustainable Disease Management group at Ruakura. I was very fortunate to work with an extremely helpful and friendly group of people who never tired of my questions. I would like to thank Dr Tony Reglinski and everyone at Hort Research who made my time enjoyable and taught me the laboratory skills which will be so valuable for future research that I hope to undertake.

Marianne White
University of Glasgow

Differences in resistance to MBC fungicides in isolates of Mycosphaerella linicola (pasmo) from linseed

Winter-sown linseed was introduced commercially to the UK in 1995 as an alternative to spring-sown linseed. Spring linseed poses problems for harvesting because of its late maturity and therefore can be difficult to include in arable rotations. However, there has currently been little research into diseases of winter linseed; surveys have shown widespread, severe epidemics of pasmo (caused by Mycosphaerella linicola) in many commercial crops, causing considerable yield losses. Experiments have shown that carbendazim (MBC) sprays can control pasmo, and thus produce good yield responses. However, development of resistance to MBC fungicides is widespread in other plant pathogens. This project investigated the effects of MBC fungicides on growth of M.linicola isolates obtained from winter linseed crops, which had or had not been sprayed with MBC fungicides.

Winter linseed plants with pasmo symptoms were obtained from 28 commercial crops in the West Midlands and the North of England (via ADAS, Boxworth) and from 12 experimental plots at Rothamsted; details of whether or not these crops had been treated with MBC fungicides were obtained. Different techniques to isolate the fungus were tested and direct isolation onto PDA+ plates was the most successful. The cyrrhi (of conidiospores) were picked off from the tops of pycnidia (under low-power microscopy) and spread directly on to agar plates. The M.linicola colonies were grown under near UV light at 20oC. Because M.linicola is a very slow-growing fungus, it was necessary to sub-culture to avoid the cultures becoming overgrown with saprophytic fungi. In total, 21 different isolates of M.linicola were obtained; six from ADAS samples and the remainder from Rothamsted. The spores of these isolates were suspended in sterile distilled water and spore counts of each were adjusted to achieve a concentration of 100,000 spores per ml in each suspension. The suspension of each isolate was then sprayed onto PDA plates (incorporating antibodies).

The fungicide carbendazim was serially diluted to produce concentrations of 100ppm, 20ppm, 4ppm and 0.8ppm. Filter paper discs (5mm diameter) were then soaked in each concentration for 1 min. and placed on the inoculated plates (four replicates per plate). The zones of inhibition of growth of M. linicola were measured after 4 days. There was no inhibition expressed at the two lowest fungicide concentrations. Nine of the 21 isolates were inhibited strongly by the fungicide at the two highest concentrations. Of these isolates, four were from samples sprayed once with an MBC compound and five were from unsprayed plots. However, 12 of the 21 samples showed reduced inhibition (and therefore possible resistance) at the two highest concentrations. Of these, four were from samples that had been sprayed once with an MBC compound, three were from samples sprayed twice with an MBC compound and five were from samples that had been spayed with non-MBC compounds. The results show some evidence to suggest that resistance to MBC fungicides may be developing in M. linicola populations; this merits further investigation.

I would like to thank the BSPP for granting my bursary and giving me the opportunity to gain invaluable experience in this interesting field, Peter Gladders (ADAS) for supplying linseed samples and the Plant Pathology department at IACR-Rothamsted, particularly Sarah Perryman, Dimitra Gkilpathi and Bruce Fitt, from whom I learnt so much.

David Chilton
Brunel University

Fungicide Trials on the Study of Rose Black Spot

 The BSPP bursary has given me the opportunity to work at the University of Hertfordshire by contributing to an ongoing research project funded by MAFF, on the biology of the Rose Black Spot fungus, Diplocarpon rosae. The disease is very prominent in the UK and worldwide causing damage both in gardens and commercially. Both amateurs and commercial growers spray heavily to attempt to control this disease (commercial growers spray up to 40 times a year).

In the first part of my 10 weeks I studied the symptoms and learnt the characteristics of black spot on the rose leaf. The main evidence for disease presence are spots 2-12mm in diameter that are black with edges like a burst. These are found mostly on leaves but can be seen on both the stems and thorns of the rose. A closer look reveals acervuli, little black, pin sized dots, with white clumps on top, oozing conidia (spores). My own examination of the diseased roses took place at two local field trial sites for the rose black spot project.  The first hand observations I made were at the Garden of the Rose, St. Albans and the University of Hertfordshire, Bayfordbury. Along with developing my own understanding of the disease, I also had the opportunity to help set up experiments for the study of the epidemiology of the fungus.

The second part of my BSPP experience involved working on fungicide testing to improve control of the disease. Anecdotal evidence has suggested that some isolates of the fungus may be becoming resistant to the commonly used fungicides. The part of the project that I was involved with aims to develop a method to screen for evidence of fungicide resistance. The method used was to make spore suspensions and lawns of the fungus on agar plates. The amount of spores collected and spread depended on the age and overall growth of the isolates. For an effective lawn at least 105 spores per ml were required to ensure a good amount of growth.  However, some isolates were found to be much slower in growth than others, taking longer than the average 6-7 days to produce a lawn.

Once the isolates had developed good germinating lawns, the fungicide trials were started. The active chemicals of the fungicide that I used were buprimiate and myclobutanil and I helped to develop the following method of testing. 35 lawns of each isolate were prepared 24 hours prior to the placement of chemicals and other solutions. The next day sterile filter paper discs were placed at the centre of each plate and a known amount of the fungicide under study was pipetted on, to saturate the disc.  Five plates were used for each treatment. Treatments were field rate of the fungicide, and dilutions of the field rate of 75%, 50% and 25% and three controls. A control of 1% DSMO was used because it was the dilutant for the concentrated form of the chemical. A dilution of 12.5% CuSO4 was also used for a control because it has a known zone of inhibition and the final control was sterile tap water. Zones of inhibition around the paper discs were measured after 8 days. 

Poisoned disc method: A zone of inhibition seen around the disc
treated with the active ingredient Dichlofluanid on isolate DR212.

My overall learning experience of the BSPP 10-week program proved to be very valuable and I enjoyed both the lab and field work. I now understand it takes a lot of time and thought to carry out scientific experiments successfully. Working under the direction of Alefyah Ali and Dr Avice Hall has helped me further my understanding of plant pathology and I hope to use my knowledge to further my education and study for a Masters degree in this field.

Elizabeth Byron
University of Wisconsin, Eau Claire

Colletotrichum lindemuthianum biotrophy-related gene, C1H1

With financial support provided by the BSPP, I have been able to assist studies on the Colletrotrichum lindemuthianum biotrophy-related gene C1H1 in Dr Jon Green's laboratory in the University of Birmingham. Colletotrichum spp. are agronomically important pathogens causing anthracnose on a wide range of crops and plants. C1H1, previously identified in studies by Drs Jon Green, Richard O'Connell and Sarah Perfect, is a proline-rich glycoprotein expressed in intracellular hyphae during in vivo infection. Dr Sarah Perfect prepared several C1H1::GFP constructs to allow further study of C1H1 expression. My project was to analyse these constructs further and study their expression in planta and in vitro. Current results with a C1H1 promoter and terminator attached to green fluorescent protein (GFP) indicate transcription occurs only during the biotrophic phase in planta when the pathogen has successfully infected.

Expression also occurs in "out of control" germ tubes that do not go on to form appressoria. In vitro, both long germ tubes, and appressoria fluoresce, although it appears appressoria fluoresce to a lesser extent. Other members of the group are studying the nutritional and environmental triggers for this expression.

The techniques I have acquired over 10 weeks this summer will prove invaluable as I start my PhD where I will again be working with Colletotrichum and utilising GFP constructs. I would like to thank the BSPP for the 10 week bursary, and all members of Dr Jon Green's laboratory for their support and guidance.

Joanne Perkins
University of Birmingham