BSPP Presidential Meeting 1997

Session III - Recognising the cause

Diagnosis - the means to an end
Dr David Stead
Central Science Laboratory, Ministry of Agriculture, Fisheries & Food, Sand Hutton, York YO4 1LZ

I will define the end in this case as production of plants and their products with minimal loss due to pests and diseases. For much of the work we do at CSL it could have been prevention of alien pests and diseases becoming established in the UK. The use of appropriate diagnostic methods is an essential means of achieving these ends.For example, correct diagnosis facilitates selection of the best means of control.Diagnostic methods are of equal value to the extension pathologist giving advice on control based on a field diagnosis and to the pathologist screening propagating material for a pest /pathogen listed in a certification scheme.

Diagnostic methods can be placed in 3 broad categories:

• methods which allow diagnosis in symptomatic plants or products
• methods which allow detection of pests and pathogens in symptomless infections
• methods which allow indexing for pest/pathogen free material.

There is some obvious overlap between the last two.

The last decade has seen a marked reduction within the UK in diagnoses in the first category, especially through regular crop inspections for visual symptoms. However, there has been a large increase in diagnoses in the last two categories, ie. those that test for the presence or absence of the pest or pathogen.The increased emphasis on detection has been accompanied by advances in technology especially in the development of nucleic acid -based methods and in the development of diagnostic kits.

This talk reviews some of the key elements of modern diagnostic methods, including - specificity, sensitivity, sampling, speed, cost, reliability and how the results should be used to achieve the end.The talk is illustrated with pests and diseases currently of concern to UK agriculture and in particular to CSL. In particular it discusses the problems that may occur if the trend towards indexing continues.

Central diagnostic facilities in support of local problems
Dr Ghita Cordsen Nielsen
The Danish Agricultural Advisory Centre, The National Department of Plant Production, Udkaersvej 15, Skejby, DK-8200 Aarhus N., Denmark.

The Danish agricultural advisory system is organised at two levels - a national and a local level. The local level involves about 85 local advisory centres, organised and run by the local farmers' unions and associations. The advisors working at these centres provide the individual farmers with guidance and other services.

At the national level the Danish Agricultural Advisory Centre (DAAC) provides the local centres with the latest information from both Danish and foreign research. The Centre also undertakes its own programme of investigations on practical issues. The plant clinic is located at the DAAC and operating at the national level.

The Danish Agricultural Advisory Centre has a staff of about 370 whereas a local centre typically has 20-70 employees and serves between 500 and 2,000 members. The entire advisory service serves about 70,000 farmers which corresponds to 95% of the Danish farmers.

The DAAC is responsible for the main services that are most appropriately organized at the national level and has 5 main tasks:

• Specialized advisory services
• Communication of knowledge and information
• Development activities
• Trials and investigations
• Education , in-service training and courses
• Service activities

The DAAC performs these duties in many ways. Today I will mention only one service activity and that is the central diagnostic facility.

The diagnostic facilities

The central plant clinic of The National Department of Plant Production employs a senior adviser and a laboratory technician who treat the about 600 samples which the local advisers send in annually.

The advantages of a central diagnostic organisation are:

• We build up expert knowledge which benefits all the local advisers (and farmers). In this way we can hopefully give more qualified answers.
• We only need laboratory facilities in one place
• The total amount of time used to solve a specific problem is less as not all the local advisers have to deal with this problem.
• The clinic knows about the special problems of a specific growing season. This knowledge can be used for example in newsletters to the local advisers who can use the information in locally adapted newsletters to the farmers.
• In order to be able to follow the general problems during the growing season we operate a monitoring system for pests and diseases in the main crops in co-operation with local advisers.
• The DAAC also develops new field experiments and investigations - for instance in case the local advisers present us with problems we cannot solve or prevent. We organise and carry out about 2,000 field trials every year in collaboration with the local advisers. About 40% of them are related to plant protection.
• New problems in plant production are more easily found when you get many plant samples. In the late 1980s for example we received an increasing number of samples showing symptoms of sulphur deficiency. Over the past few years we have seen black scurf and stem canker (Rhizoctonia solani) in organic potatoes.

Quantitative diagnostics - have we arrived?
Dr Paul Nicholson
Cereals Research Department, John Innes Centre, Colney Lane, Norwich NR7 7UH, UK

In an attempt to answer the question posed (by Nigel Hardwick) in the title I will draw on examples and experience from our work with cereal diseases. Two economically important disease complexes affect cereals in the UK - 'stem-base disease' (SBD) and 'Fusarium ear blight' (FEB). Visual diagnosis of these disease complexes is difficult, with several species often occurring together in the same tissue. Even attempts to evaluate the relative proportion of each species in plant samples by isolation into axenic culture only reveals what can be grown out of the plant rather than what is within the plant.

The inability to detect, identify and quantify individual species within plant tissues has seriously hindered the study of these diseases. In addition, the inability to diagnose correctly may result in the adoption of inappropriate of poorly timed control measures.

Molecular techniques are being developed to overcome many of the problems associated with the study of SBD and FEB. Among the most sensitive techniques available in the polymerase chain reaction (PCR). We have developed PCR-based assays for detection of the SBD and FEB fungi directly in extracts from plant tissue. These assays have been designed to enable simultaneous detection of several pathogens in each reaction and so provide and integrated system. These assays have been refined to enable quantification of each species, allowing the relative contribution of each component to the disease of the plant to be estimated. This paper reports aspects of this work and some preliminary results achieved using these systems.

Quantifying Fusarium diseases of cereals
Dr Philip Jennings, J.A. Turner, J.N. Banks and R.H. Rizvi
Central Science Laboratory, Sand Hutton, York YO4 1LZ

The major pathogens causing Fusarium diseases of cereals in the UK include Fusarium avenaceum (Gibberella avenacea), Fusarium culmorum, Fusarium graminearum (Gibberella zeae), Fusarium poae and Microdochium nivale (Monographella nivalis formerly Fusarium nivale). Symptoms caused by different species are often indistinguishable and pathogen isolation using traditional methods is time consuming. Subsequent species identification requires expertise and results are not quantitative. Control of these pathogens is often difficult due to differences in fungicide sensitivity and in determining the correct timing of fungicide applications. Development of a rapid, simple to use, diagnostic method for the identification of these species would allow more accurate epidemiological investigation, leading to improved disease control. Enzyme-linked immunosorbent assay (ELISA) is a specific, rapid and quantitative method, which has been used in the identification and detection of a range of viral and fungal diseases. Monoclonal antibodies (MAbs) were raised against F. avenaceum, F. culmorum, F. graminearum, F. poae and M. nivale, with a view to developing an ELISA for their detection in plant material. Cross-reactivity studies carried out against fourteen Fusarium species plus M. nivale, ten other field fungi and eight storage fungi indicated that cell lines had been produced that secreted MAbs specific to F. avenaceum, F. culmorum, F. poae or M. nivale, but not F. graminearum. The MAbs detected antigen from plate washings; this both simplified and reduced the time required for their identification. The limit of detection was estimated to be between 0.4 and 2 ìg antigen/ml. Screening against twenty different isolates for each species showed that selected cell lines produced species-specific MAbs. However, other cell lines produced MAbs which recognised only the isolate to which it was raised. Initially, no MAb detected antigen from infected plant material. Further investigation indicated that the type of plant material and its treatment affected antigen detection. The presence of crushed grain, stem-base or root material totally inhibited antigen detection. However, inhibition was reduced when plant material was soaked, but increasing the soaking time increased the inhibition. The form of the antigen also affected antigen detection. MAbs did not detect spores or non-active mycelia in plant material. However antigen was detected when actively growing mycelia was produced from diseased stem-base and root material. The use of bio-amplification techniques was necessary for successful detection in infected grain. Further refinement of the protocol, possibly by the use of more complex ELISA, is being undertaken to minimise inhibition and produce a more sensitive assay.