FAQ

The ELISA test is considered to be a safe, inexpensive and rapid method for the detection of plant viruses. The inexpensive nature and relative simplicity of the test allows it to be used as a workhorse within the agricultural sector and it is used to screen thousands of samples per annum.

The opinion was that field evaluations of the occurrence of viruses do not correlate well with laboratory test results in respect of tuber samples. This led to greater uncertainty regarding the reliability of the tuber sample test. Between 2005 and 2007 a quality control project was registered with the National Research Committee of Potatoes South Africa. The generated results confirmed the reliability of the ELISA test as a tool in the certification of tubers. See Quality control PVY and Quality control PLRV.

ELISA is a robust, cost – effective and reliable serology test that allows high throughput of samples in the commercial Certification laboratory. The ELISA test requires adequate sprouting of all tubers in a sample to achieve optimal test results due to its lower ability to pick up latent or very low titres of virus infections. Because of the waiting period due to sprouting requirements, time between sampling and testing can become an issue for the seed grower, when choosing the ELISA. The PCR is a highly sensitive molecular test and because tubers can be tested directly after harvest there is no waiting period for the seed grower. Due to its very sensitive nature PCR is able to detect latent and very low titres of disease and seasonal infections. PCR is however, not a robust test, requires more skill, more expensive equipment and the testing material is more costly. The PCR test will therefore be more expensive. Both the ELISA and PCR tests have been evaluated by Plantovita in official projects under the guidance of the Potatoes South Africa Research Committee. Protocols for application of any of the two methods were developed and standardized . Both the ELISA and PCR were approved as reliable testing methods for the Certification of tubers by the Independent Certification Council, The National Seed Growers Committee and the Research Committee of Potatoes South Africa. The protocol development in case of the PCR was also evaluated and approved by a molecular expert at the University of Stellenbosch. It is for the Seed grower, considering the differences in time, costs and sensitivity, to decide, which method is preferred for the testing of samples for Certification purposes.

Samples were selected from previously tested samples and after secondary sprouting occurred, these samples were tested in a quality control ELISA test for the occurrence of PVY and the results compared with those of the original ELISA tuber test. Between 2005 and 2007 a total of one hundred and sixty-five X 200 tuber samples were tested and 93% of the results of these tests correlated with the results of the original tests. For the same period the results of 89% of sixty-five X 400 samples tested correlated with the results of the original tests. Samples in respect of which the results of the original tests could not be confirmed, had a virus infection of ≤2.7%. It was also found that the inclusion of 400 tuber dispute samples (store samples) statistically decreases the ability to acquire the original result by 50% as a store sample is a different sample although from the same seed lot.

Due to the location of the PLRV in the tuber, the PVY quality control method could not be used for the PLRV quality control project. In 2007 it was decided to continue with the PLRV quality control by planting 400 and 200 tuber samples in the greenhouse and harvesting the leaves for PLRV quality control testing. For the report period 2007 twenty-nine X 200 tuber samples and four X 400 tuber samples were successfully tested for PLRV applying this technique. It was again found that the inclusion of 400 tuber dispute samples (store samples) statistically decreases the ability to acquire the original result by 50% as a store sample is a different sample although from the same seed lot.

It was also found that the ELISA test underestimates rather than overestimates the virus content of a sample due to the location of the virus and sampling in the laboratory, as well as the test’s tendency to miss very low titres of virus.

The ELISA test is considered to be a safe, inexpensive and rapid method for the detection of plant viruses. The inexpensive nature and relative simplicity of the test allows it to be used as a workhorse within the agricultural sector and it is used to screen thousands of samples per annum. Unfortunately ELISA tests are not completely fail-safe. Virus levels within potato tubers which are screened by means of the ELISA test for use as seed potatoes are normally low while the tubers are dormant. The detection of viruses in these potatoes by means of the ELISA test is difficult and absorbance values may fall below the set cut-off value. For this reason, seed tuber screening is performed on sprouting rather than dormant tubers. Although this results in more reliable readings than direct tuber testing, it does delay the certification of seed potatoes.

Results obtained in Switzerland by Mr. Jan Rek over several years of studying the influence of different factors affecting the reliability of the ELISA test have confirmed the following:

• • When performing the ELISA test, the physiological state of the tuber (cultivar, artificial breaking of dormancy, seasonal differences, etc.) at the time of testing also affects the reliability of the test.

• • The time of infection (secondary or primary) also plays a roll.

According to Mr. Rek’s studies, a 100% reliable detection of the occurrence of PVY and PLRV in tubers is optimal five and two weeks respectively after an artificial breaking of dormancy with Rindite irrespective of the cultivar and time of infection concerned.

Between 2005 and 2007 a project was registered with the National Research Committee of Potatoes South Africa with the aim to determine whether time has an effect on the virus percentage of a specific sample. A total of 5 samples from the potato regions in KwaZulu-Natal, the Western Free State, Mpumalanga, the Sandveld and the Northern Cape were tested for this project. The total number of tubers per sample were 3750. The samples were tested for the presence of PVY and PLRV at 21, 28, 35 and 42 days after a PROGIP treatment. Four repetitions per test were done (4 x 200 tubers 21 days after treatment, 4 x 200 tubers 28 days after treatment, etc.). Tubers were tested individually. It was found that the detection of viruses in any specific sample was optimal 28 days after treatment with a sprout stimulant.

A field sample is drawn after two field inspections and sent to a regional laboratory for testing for the presence of PVY and PLRV. The result of this test determines the certification status of the seed lot concerned. After the second field inspection and the drawing of the field sample the grower should kill the overhead foliage in order to limit an increased risk of virus infection by means of vectors. It is, furthermore, the duty of the grower to ensure that no regrowth takes place. Regrowth is a possible breeding-ground for vectors of viruses. Post-control samples are drawn from the bags after the harvest. A certain period of time has therefore elapsed between the drawing of the field and the post-control samples. Should the foliage not be killed timeously after the field inspection and the drawing of the field sample and regrowth is not controlled adequately, the lapse of time presents an opportunity for virus infection mainly by means of aphids. This gives rise to the difference between the results in respect of the field and post-control samples originating from the same unit.

At present a seed lot is certified after completion of a laboratory test to determine the presence of PVY and PLRV in a representative tuber sample. The use of leaf samples as management tool is based on the assumption that a high virus content in leaves could be an indication of a high percentage virus content in the daughter tubers. Predicting the fraction of tubers containing virus, enables the client to calculate the potential yield if produced optimally. Properties of PLRV and PVY give occasion to an uneven distribution of the diseases through the plant. Uneven distribution necessitates the use of sprouted tuber material. Using a method which misinterprets the virus content prevents the opportunity to produce optimally.

In view of the fact that the ELISA test can under exceptional circumstances also trace the presence of other organisms (including saprophytes present in the soil and on the plant) and that the testing process can generate readings on account of non-specific reactions, it is necessary that the presence or absence of Ralstonia solanacearum be confirmed in cases where readings are higher than the cut-off values.

On account of possible non-specific reactions during the ELISA test, the quarantine status of the disease, the zero tolerance applicable to the disease in the South African Seed Potato Certification Scheme and the tremendous economic impact for growers when the disease occurs, positive ELISA readings have to first be confirmed alternatively before a final result is sent to the grower.

The confirmation methods used at present entail conventional isolation and identification by means of biochemical tests as well as PCR-based methods. This implies that the sap and the remaining tubers of possibly infested sub-samples have to be processed and plated out on to semi-selective media after the ELISA test has been carried out.

Sap which has been received is plated out immediately on semi-selective media. The suspect tubers are first incubated for 14 days to afford the bacteria an opportunity to multiply especially if the infection is latent.  After incubation the tubers are processed for plating onto the semi-selective media while also checked for typical symptoms of bacterial wilt.

If suspect colonies are present on the growth medium at any stage, the colonies are isolated and purified after which Ralstonia species complex specific PCR analysis is done and the phylotype of the isolates are determined.  To complete the process,  a tomato bio-test is done and the pathogen re-isolated from the inoculated plants.

If no suspect colonies were observed on the media during the analysis of sap AND tubers, a negative result can be released.  A positive result can be obtained after plating the sap OR tubers.

The main reason is to afford the bacteria – if present – an opportunity to multiply to traceable levels.

The warming of samples entails the incubation of the seed potatoes at 25ºC for 14 days.

Experience has shown that loose skins do have an effect on the ELISA test for the tracing of bacterial wilt. It has also been found that Fusarium and Verticillium in tubers can influence the test. The growers can be assured that ALL readings higher than the cut-off value have to be confirmed irrespective of the knowledge that the afore-mentioned can influence the test. Therefore no results are finalised before the confirmation procedure has been followed.

Apart from the quarantine status and difficult control of the disease, bacterial wilt is a highly contagious disease offering no benefits to any grower of potatoes. Latent (low levels of) infection is not necessarily visible and therefore all measures have to be taken in order to prevent the spreading of the disease from seed potato to seed potato, from sample to sample of different units and to other categories of samples (for example, G0 material)

During transportation the samples must –

• be packed in new bags/containers;
• be packed and transported separately;
• if two or more samples are transported together, be separated by means of suitable partitions (for example thick tarpaulins); and
• if samples are rotten and/or tubers are suspected of being infected, be packed separately in clearly marked bags/containers.

Samples destined for testing must always be accompanied by a seed list or consignment note so that the samples can be clearly identified and verified upon receipt.

G0 material is the basic material of a healthy seed potato industry. The growers of G0 material produce their material under strictly controlled conditions and at a great cost. In terms of the South African Seed Potato Certification Scheme G0 material must be free from PVY and PLRV, as well as Soft rot causing Enterobacteria and the Ralstonia species complex.

On account of the risk of infection by one of the above-mentioned diseases in the presence of other seed potato material, economic losses for the grower and the zero tolerance applicable to the above-mentioned diseases, it is unwise to transport the seed potatoes without suspicion.

The virus levels within potato tubers screened by means of the ELISA test for use as seed potatoes are normally low while the tubers are dormant. The detection of viruses in these seed potatoes by means of the ELISA test is difficult and absorbance values may fall below the set cut-off value. For this reason, seed tuber screening is performed on sprouting rather than dormant tubers. Although this results in more reliable readings than direct tuber testing, it does delay the certification of seed potatoes. It is important that the grower understands that ALL the tubers representative of a sample must have sprouted to give reliable results. The cultivar as well as the physiological status (age) of G0 material, definitely influence the time needed for all the seed potatoes in a sample to sprout adequately for optimal testing.

Bacterial wilt, caused by the bacteria Ralstonia solanacearum, is one of the most destructive diseases of potato crops and has a very wide host series. The bacteria are soil-borne, can survive in the soil for very long periods and can be distributed, inter alia, by means of irrigation water, storm-water, infected soil and infected seed potatoes. The affected plants wilt and die off whereas the tubers may show a very serious or mild slimy leaching of the bacteria in the eyes or vascular bundles depending on the intensity of the infection. The presence of this disease can lead to great financial losses. In the South African Seed Potato Certification Scheme a zero tolerance exists in respect of the occurrence of bacterial wilt on account of the danger which it constitutes for a healthy potato industry.

Laboratories participating nationally in the Potato Laboratory Service Group include the regional laboratories located in the main potato producing regions in South-Africa. The aim of all the laboratories is to provide an affordable and reliable service to all clients – mainly potato seed growers. A reliable service is a service comparable to services which are nationally and internationally available in terms of quality and technical performance. In the Potato Laboratory Service Group, Plantovita has the function to ensure standardisation between laboratories so that results are comparable. Standardisation is achieved on a technical level and this is obvious if results of laboratories are evaluated. A grower can submit samples to any laboratory and be sure that the generated results will be the same as those generated at all the laboratories.

The comparison is carried out by means of inter-laboratory testing of standardised infected material. The results generated are compared and feedback is provided at the annual Potato Laboratory Service workshop. Should intermediate arrangements and corrective action be necessary based on comparisons undertaken, such actions are implemented immediately. To date the results of inter-laboratory tests done, have proved that standardisation has accomplished comparability.

Within the Potato Laboratory Service Group Plantovita has achieved standardisation of laboratory tests by establishing, implementing and maintaining protocols, by auditing all laboratories annually and by means of inter-laboratory tests. The protocols prescribe all the steps applicable to procedures and systems, and within the technical scope of actions, compared to international standards. The protocols and procedures are maintained and changes are communicated to participants. The implementation of changes is followed up during annual audits.

Not only implemented changes, but also all technical and managerial aspects are monitored during the annual audit. Recommendations are made allowing laboratories to improve over time within each budget. Major non-conformances are dealt with immediately. An audit report is submitted to the Manager: Potato Laboratory Service for final approval of a laboratory. On the basis of the recommendations of the Manager: Potato Laboratory Service, the Independent Certification Council for Seed Potatoes approves or disapproves a laboratory for the testing of seed potatoes. The audit outcome is also forwarded to the Registrar of the Plant Improvement Act, 1976who facilitates the registration of laboratories at the Department of Agriculture, Forestry and Fisheries (DAFF).

Standardisation is, furthermore, monitored by means of inter-laboratory tests which all the laboratories have to perform. Generated results are used to further standardise and correct.

Quality control is a process by means of which entities review the quality of all factors involved in production. Quality control is the more traditional way which businesses have used to manage quality. Quality control is concerned with checking and reviewing work which has been done.

Quality control emphasizes an evaluation of tests to uncover defects and reporting defects to the Quality Manager who decides whether to allow or disallow the release of test results. Quality assurance attempts to improve and stabilize production (and associated processes) to avoid, or at least minimize, issues which may lead to deviations. In the Potato Laboratory Service Group a few control measures are routinely employed to enable quality control in respect of the services rendered. Control measures include an evaluation of test results, where every test performed is examined visually and the data generated is evaluated in terms of the criteria set by the manufacturers of the kits, chemicals, consumables and reagents. This is done for each and every test executed. Plantovita plays a crucial role in approving the results of selected tests.

Furthermore, quality control is achieved by using standardised commercial positive and negative control material, comparing these values and evaluating the performance of tests based on these values. Control charts are completed to monitor trends indicating the overall performance of tests.