title / A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE PREDICTION OF TUBER SUSCEPTIBILITY TO BLACKSPOT BRUISING IN THE FIELD
/ DEFRA
project code / HP0217
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE PREDICTION OF TUBER SUSCEPTIBILITY TO BLACKSPOT BRUISING IN THE FIELD
DEFRA project code / HP0217
Contractor organisation and location / School of Biological and Biomedical Sciences
University of Durham
Science Laboratories
South Road
Durham
DH1 3LE
UK
Total DEFRA project costs / £ 54,783.00
Project start date / 01/03/01 / Project end date / 28/02/03
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 5
Projecttitle / A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE PREDICTION OF TUBER SUSCEPTIBILITY TO BLACKSPOT BRUISING IN THE FIELD
/ DEFRA
project code / HP0217
A. Introduction
Blackspot bruising is the blue-black tuber discoloration encountered when susceptible potatoes impact on a hard surface, and is caused by a chemical reaction initiated when tuber cells are physically damaged. This phenomenon greatly affects the quality of the crop and its value.
A 2-year, BPC/DEFRA funded programme at the University of Durham under the direction of Dr Ron Croy has made significant advances in the understanding of this important problem facing the potato industry. This knowledge has led to the development of a detection system for assessing blackspot susceptibility. The detection system will form the basis of a kit for assessing the crop prior to harvest or during storage and will provide an 'early warning' of a high bruise risk. This will facilitate implementation of appropriate, gentle harvesting and transport procedures.
When a tuber impacts on a hard surface the tissues are damaged and they respond rapidly by synthesising oxygen free radicals - a highly active form of oxygen which rapidly reacts with many biological materials including membranes (lipids), proteins and nucleic acids. The results from this research have confirmed that the level of free radicals produced indicates how susceptible the tuber is to bruising - a high level indicates a high susceptibility to bruising while a low level indicates good resistance to bruising. The project has demonstrated the generation of radicals in amounts very closely correlated with the level of bruise susceptibility in 13 different varieties including Cara, King Edward, Russet Burbank, Maris Piper and Desiree; as yet no varieties have failed to demonstrate the correlation. As a consequence of the close relationship between these highly active molecular species and bruise susceptibility it is anticipated that a diagnostic kit
based on a free radical assay will be able to predict bruising status to between 90 and 95% accuracy. This is the first demonstration of such a clear relationship between a measurable factor and bruise susceptibility.
The method of detection of bruise susceptibility is relatively simple and is based on an assay specific for the superoxide radical. The level of radical generation is measured using a dye which changes colour when it reacts with the factor and this colour change is measured to assess the bruise susceptibility. The test is carried out by selecting samples of the potato crop directly from the field or from storage and exposing these to a standard impact using the impactor (figure 2). The tuber tissue responds within two hours and then a standard size of core of tissue is excised using the corer (figure 3), and placed into the dye solution. Following a 20 minute incubation period the solution is mixed with a second solution which brings about a colour change. The depth of colour of the final solution is measured or compared by eye with a colour chart and indicates how prone the crop is to bruise damage. The findings from this work have recently been published in the prestigious journal Plant Physiology (Johnson et al., 2003).
Currently the research is aimed at developing the chemistry and hardware components of the assay to provide a fully workable and ‘user-friendly’ kit of components for field use by the potato industry.
In the future, investigations will employ cutting-edge biochemical and molecular technologies to identify other factors present in growing tubers which govern the underlying mechanisms controlling blackspot. This will involve fundamental studies of the tuber developmental processes which lead to the generation of the factors which pre-determine bruise susceptibility.
B. Project aims
The overall aim of this project was to exploit the novel observations made by Dr Croy’s group, (and recently reported in the paper by Johnson, Doherty and Croy, 2003 – Appendix 3) which showed a direct correlation between bruising susceptibility and free radical generation. These observations were to be adapted into a system for predicting bruise status in field grown potato crops. The project was directed towards the production of a convenient and easy-to-use bruise susceptibility diagnostic kit and to test the performance of the detection system under simulated and real field conditions.
C. Project objectives
The objectives of the proposed research were as follows -
i. Develop the existing assays and investigate new chemical detection systems for free radicals. The objectives will be to identify assays with the highest sensitivity and reproducibility with a view to adapting these into a kit format
ii. Use the selected assay systems to test a range of potatoes grown under controlled field conditions to simulate bruise susceptible and resistant tuber materials, to confirm the sensitivity, reproducibility and validity of the assays
iii. Adapt selected free radical assays into simple prototype test kits
iv. Design a tuber impactor that will allow a rapid, reproducible impact force on large numbers of test tubers.
v. Test prototype kits with a wide range of field-grown tuber materials
vi. Investigate the possibilities for the future development of an electronic, free radical biosensor system for diagnosing bruise susceptibility
D. Achievement of project milestones and objectives
Year 1no. / milestone / due date / on schedule / date completed
yes / no
1. / Growth season 1. Production, sampling and storage of defined tuber material for development and testing (ADAS Gleadthorpe). / 10/01 / ü / 10/01
2. / Design, build and test prototype impactor device / 11/01 / ü / 10/01
3. / Supply of other field grown material from ADAS sites, SBEU and other sources.
/ 02/02 / ü / 11/01
4. / Comparison of a range of new detection chemistries with existing ones. Optimise existing and develop new improved assays for free radicals. / 02/02 / ü / 01/02
5. / Test a wider range of tuber materials with existing assays – to provide further proof of concept, detect any deviations from established correlation.
/ 02/02 / ü / 04/02
6. / Submit annual report.
/ 02/02 / ü / 04/02
7. / Submit grower friendly project summary / 02/02 / ü / 04/02
8. / Review of project progress. / 02/02 / ü / 04/02
9. / Modify and refine impactor device as necessary. / 08/02 / ü / 06/02
Year 2
no. / milestone / due date / on schedule / date completed
yes / no
10. / Production, sampling and storage of defined replicated tuber material for kit testing (ADAS Gleadthorpe).
/ 10/02 / ü / 10/02
11. / Supply of field grown material with documented history from 4 other ADAS sites; and further material from storage at ADAS sites, SBEU and other sources.
/ 01/03 / ü / 12/02
12. / Select most appropriate assay systems and adapt into simple kits. / 01/03 / ü / 02/03
13. / Test prototype kits with test tuber materials; produce data on reproducibility of tests and establish scale of bruise susceptibility. / 01/03 / ü / 01/03
14. / Investigate future possibilities for biosensor systems to detect free radicals in tuber tissues. (Report attached – Appendix 2). / 01/03 / ü / 02/03
15. / Produce draft instruction manual for test kit. (Document attached – Appendix 1). / 01/03 / ü / 02/03
16. / Submit final report. (This document) / 01/03 / ü / 08/03 *
17. / Submit grower friendly project summary - section A of this report. / 01/03 / ü / 02/03
18. / Undertake a minimum of 2 presentations at BPC levy payer meetings if required. (See Summary of technology transfer and project deliverables) / 02/03 / ü / 06/03
items attached to this report
* Delay due to requested changes in format and scope of original BPC report. Details subsequently incorporated into the final DEFRA report
CSG 15 (Rev. 6/02) 5
Projecttitle / A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE PREDICTION OF TUBER SUSCEPTIBILITY TO BLACKSPOT BRUISING IN THE FIELD
/ DEFRA
project code / HP0217
Scientific report (maximum 20 sides A4)
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Press the DOWN arrow once to move to the next question.
CSG 15 (Rev. 6/02) 5
Projecttitle / A NOVEL AND RAPID DIAGNOSTIC SYSTEM FOR THE PREDICTION OF TUBER SUSCEPTIBILITY TO BLACKSPOT BRUISING IN THE FIELD
/ DEFRA
project code / HP0217
Scientific Report
A. Introduction
The following section contains the experimental details and conclusions from the work undertaken during this project together with relevant results from other associated fundamental investigations contributed by other personnel/project students in Dr Croy’s group.
B. Materials and methods
Plant materials
Test tubers were grown by ADAS at Gleadthorpe, under conditions designed to produce bruise-susceptible and bruise-resistant crops of the same variety. Initially for the 1st field trial (2001-2) 5 potato varieties exhibiting different degrees of susceptibility to mechanical damage were studied - Cara, King Edward, Maris Piper, Russet Burbank and Saturna (See table 1). This selection was later expanded in the 2nd field trial (2002-3) to 8 varieties as indicated in table 1. The figures refer to the bruise indices for these varieties estimated by our own testing (See later). Tubers were specifically grown and carefully harvested manually by ADAS to avoid any mechanical stress. Harvested tubers were transported to the ADAS storage facility at Low Mowthorpe and stored in the dark at 5oC, to inhibit greening and sprouting, until required.
Table 1 Cultivar bruise susceptibilities
Cultivar / Bruise Index / Cultivar / Bruise Index1st trial / 2nd trial / 1st trial / 2nd trial
Cara / 5.5 / 6.2 / Navan / - / 6.7
King Edward / 4.0 / - / Cultra / - / 6.5
Maris Piper / 3.1 / 6.7 / Ambo / - / 6.3
Saturna / 7.9 / - / Desiree / - / 7.7
Russet Burbank / 9.2 / 7.8 / Pentland Crown / - / 6.2
Bruise susceptibility is on a scale of 0 (highly resistant) to 10 (highly susceptible), calculated through in-house bruise assessment, comparing bruise to a theoretical maximum sized bruise. Data shown are the results from materials harvested from 1st field trials (2001-2) and 2nd field trials (2002-3).
Field Trials - Parameters Used
Three variables were used to generate tuber varieties with a range of susceptibilities. The variables used were – i) sprouted or unsprouted seed, ii) harvested early or late after ‘burn off’ / ‘die back’ and iii) application of normal levels of potassium fertilizer (250kg/ha) or no added potassium. In addition to selecting cultivars showing a range of genetic predispositions to bruising these parameters have been reported to contribute towards bruising. The plots were set up at ADAS Gleadthorpe which has a sandy soil suitable for ‘minimal’ potassium trials (See example plot layout in figure 1).
Blind Testing
In year 1, samples were assayed for radical assays prior to bruise susceptibility assays being performed. However in year 2, a full ‘blind’ testing regime was adopted, with ADAS allocating a code number to each cultivar prior to collection - to permit a full analysis to be performed prior to the cultivar identity being revealed.
figure 1: Diagram of ADAS field plots at Gleadthorpe, 1st field trials
Example of field plot layout used in the first growing season 2001 – 2002. Plot layout was similar in the second growing season 2002-2003 but involved fewer variables and a wider range of varieties (See section B).
Hardware development
Initial work on bruising employed a simple coach bolt with added weights, dropped from a measured height (usually 30cm) onto the surface of the tuber. The need for a more ‘user-friendly’ device which would deliver a defined and measurable impact to the surface of a large number of test tubers was recognised early in the potato bruising work. Preliminary designs were produced as part of the UK and international patents (PCT/GB02/01631, see publication list). Initial designs described a standardised spring loaded device which drives an impacting head along a barrel aligned with the area to be impacted and to exert a precise energy of 0.7 joules. Subsequently, an improved design and the first prototype device were produced in collaboration with the School of Engineering at the University of Durham (figure 2). In addition a simple device for the rapid sampling of small standard samples (cores) of tuber tissue was required. This was also produced by the School of Engineering (figure 3).
Tuber mechanical stress (bruise assay)
Tubers were incubated for 48h at 4oC in the dark and then impacted at the stolon end using either i) a falling weight of 240g and a 300mm drop height imparting a standard energy of 0.7J (Croy et al. 1998) OR ii) the prototype impactor set to deliver the same energy of impact (figure 2). Impacted and control tubers were then incubated at 26oC to promote maximal synthesis of bruise pigments. For bruise index calculations tubers were incubated for 48h then cut in quarters centred at the impact site. The volume of affected tissue was measured and the intensity of pigmentation estimated on a scale of 0 (no discoloration) to 3 (deep blue-black coloration). 30 tubers for each of the test samples were used and the mean bruise index calculated based on bruise extent and intensity. Values were expressed as percentage of a theoretical maximum bruise volume and intensity.