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Development of a robust assay to estimate the viability of potato cyst nematodes Globodera spp. - HP0143

This proposal seeks to develop an improved laboratory based method for determining the viability of potato cyst nematodes (PCN); techniques developed during this project will be evaluated against the current method. · The current technique utilises Meldola blue stain and has an element of subjectivity in the assessment which that makes it unreliable for routine usage. As a result, the test is little used in practice with PCN extracted by commercial laboratories and tends only to be used under the carefully controlled conditions of research laboratories. Meldola Blue (and other stains) is used only in a limited number of samples for assessing the effects of fumigant nematicides such as 1,3-dichloropropene. · When commercial assessments of PCN population densities are made it is assumed, in the several thousand tests made each year, that all the eggs are viable; this may not be the case and population densities may be being overestimated and in some situations nematicides used where they are not required. · A robust alternative to Meldola blue would then be used routinely in fumigation assessments and could also be used as an additional test when population density assessments are made for decision making in commercial situations and for research work.· RT-PCR assay techniques will be developed and validated against viability staining, hatching bioassays and plant infectivity assays.· On completion of the project we envisage that an improved method for the laboratory assessment of PCN viability will have been developed. The method could then be used worldwide in commercial and research applications where an accurate assessment of PCN viability is needed. An `in-field` assay is unnecessary, as the nematodes need to be physically extracted from the soil before their viability can be assessed.· The proposed research builds on the current collaboration between Harper Adams, IACR-Rothamsted, SCRI and industry on the DEFRA/LINK project CSA 5701A `Integrated management strategies for potato cyst nematodes` .The research will utilise nematode populations obtained from the existing research programme and would give added value to both CSA 5701A and the current proposal.· DEFRA support is appropriate as the work should yield results that allow the recommendation of a standard national procedure for testing the viability for PCN which will have commercial, research and statutory plant health work applications.
· The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are the most problematic pests of the UK potato crop and cause estimated annual losses of 9% of potato production. The corresponding loss at market value has been estimated at £43M, based on the mean value of the crop from 1990-1995 1 . A recent survey showed that approximately 64% of potato growing land is infested with PCN2. Approximately 28,000 ha of potatoes are treated annually with the nematicides at a cost of £9 million to potato producers. · Cysts containing up to 600 eggs can survive in soil for over 15 years in the absence of a host crop. The viability of the nematode within each egg can be influenced by the application of nematicides and parasitic microorganisms such as fungi and bacteria. · Current management strategies rely on the estimation of PCN population densities from soil samples. Currently estimations are undertaken by a range of commercial laboratories on behalf of the crop protection industry and individual growers. The methodologies used are subject to inherent counting errors and are labour intensive3. Part of the method involves a subjective assessment of whether an individual nematode is alive or dead based on the physical appearance of eggs and juveniles. It is also assumed that if nematodes appear healthy that they are viable and potentially capable of hatching and invading a potato root: this may not be the case and could therefore result in an overestimate of viable nematode numbers. Additionally, certain chemical control treatments (such as fumigation with 1,3-dichloropropene) work by directly reducing nematode viability. · In these situations viability can be subjectively assessed using a stain such as Meldola blue or Acetic orcein4, 5. Several fluorescent stains have been used to distinguish live and dead nematodes 6,7 although results were variable. · The Polymerase Chain Reaction (PCR) is a rapid and sensitive method for detecting and identifying different species of nematodes 2,8,9,10 and other microorganisms 11,12. The use of conventional PCR can amplify target DNA from both viable and non-viable cells 13. The detection only of viable microorganisms in biological samples is usually required to ensure positive test results are associated with active agents and not from dead cells. Most messenger RNA (mRNA) molecules have a short half-life in living cells and their presence may therefore indicate viability. Consequently, this part of the proposed work will be directed towards the development of a reverse-transcription polymerase chain reaction (RT-PCR) based amplification of mRNA method that specifically identifies only living cells14. This technique has potential for application to PCN viability.· The proposed work seeks to develop a reliable technique to determine the viability of eggs and juveniles from field samples. The accuracy and the effectiveness of the developed method in detecting the viability of field populations and the efficacy of fumigant nematicides will be compared with conventional methods. A quantitative diagnostic test will be an invaluable tool for all situations where information on the viability of PCN is needed.
1. Identify the most effective currently available staining assay for PCN viability.
2. Evaluate the comparative effectiveness of the optimum staining technique (from Objective 1) with a hatching bioassay and plant infectivity assay.
3. Develop a PCN viability assay based on messenger RNA.
4. Evaluate the messenger RNA techniques against the optimum technique from Objective 2.
5. Validate the chosen optimum technique from Objective 4
Project Documents
• Final Report : Development of a robust assay to estimate the viability of potato cyst nematodes Globodera spp.   (1334k)
Time-Scale and Cost
From: 2003

To: 2004

Cost: £35,686
Contractor / Funded Organisations
University - Harper Adams Agricultural College
Sustainable Production              
Fields of Study