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Tegniese Nuus • Technical News potatoes SOUTH AFRICA Can a leaf sample from a field planting be considered as an official sample in the certification process? Artikel: Anel Espach, Plantovita INTRODUCTION Currently in the Potato Certification Scheme the use of tuber samples to test for the presence of virus and bacterial diseases are prescribed as part of the certification process. Properties of the pathogen and disease allow the bacterial tests to commence as soon as the sample has arrived at the laboratory. Virus tests on the other hand can only proceed when the tubers are test-ready. Being test-ready implies that dormancy has been broken, the tubers were allowed to sprout, and sprouts of ALL tubers in a sample are of a minimum length of 3-5mm. Based on research projects, 28 days proved to be the time before which most cultivars are not test ready and the time at which the virus content is sufficient to reliably detect using the ELISA-based method. Leaf samples have been used in potato production as a management tool to allow the grower to predict the virus presence of the progeny tubers in a planting. Decision making by the grower is thereby facilitated after test results from leaf samples are received. The use of leaf samples as a management tool is based on the assumption that high virus content in the leaf sample indicates an increased probability that a high percentage of tubers are also infected with virus. Questions arose from growers regarding the possibility to use Page 30 • CHIPS • Januarie / Februarie • January / February 2013 leaf samples instead of tuber samples for certification of seed potatoes – thus not only as a management tool, but treating the results from leaf samples as official results in certification of the represented seed lot. Aspects that have to be taken into account when considering what kind of sample should be used, include, but are not limited to: 1. 2. 3. 4. Applicable and influencing properties of viruses in question (Potato Virus Y - PVY and Potato Leaf roll virus - PLRV) Historic data collected in the certification and testing scenario in South Africa The current testing scenario employed at Potato Laboratory Services, South Africa Logistical and practical aspects to be considered Advantages and disadvantages of using leaves as sample for virus detection include: • • Tannins and starch can cause false readings in a test assay, with specific reference to the ELISA-based assays currently used. The limited amount of tannins and starches in leaf material is advantageous in the generation of reliable results. It is not necessary to break dormancy in leaf samples – samples can thus be tested when submitted. This should save time in the testing process. Leaf samples have the disadvantage of not being a true representation of what the virus status of the tubers are and that the leaves does not have a shelf life. Any test method has limitations and aspects to consider during the implementation of such a method. Before any method is implemented knowledge about the advantages and disadvantages of and the value to an industry, have to be assessed to enable the adoption Tegniese Nuus • Technical News potatoes SOUTH AFRICA of a method offering the generation of valuable information. The purpose of determining/predicting the fraction of tubers with virus content is to allow the client to predict yield and to produce at optimal potential. To employ a test method that gives a biased indication of what the fraction of tubers is with virus content does not give the client the opportunity to produce at optimal potential. 1 THE VIRUSSES PLRV Potato leaf roll virus is introduced into the vascular tissue of the plant by feeding aphids. Although it is introduced into the phloem, PLRV only infects a small portion of the available phloem cells and does not move effectively in the mesophyll tissue (movement into the mesophyll tissue is considered short distance movement). For complete systemic infection it is required that the virus can move locally over short distances, from cell to cell in the host through plasmodesmata and then further movement over longer distances through the phloem. In resistant plants, the virus mostly occurs in the internal phloem bundles and cannot move from cell-to-cell without the aid of helper proteins from other viruses. This implies that exiting the phloem tissue into mesophyll cells can only happen when the plants are also infected with other viruses (Barker H, 1987). Movement of PLRV through sieve elements (long distance movement) is a passive process and is strongly dependent on metabolite fluxes which is for most viruses the case. Derrick and Barker (1997) showed that movement of virus from point of infection upwards or downwards could be detected from 7 days after graft inoculation. The virus resides and CHIPS • Januarie / Februarie • January / February 2013 • Page 31 Tegniese Nuus • Technical News potatoes SOUTH AFRICA multiplies in the companion cells of the phloem tissue of the plant. In practice, this implicates that current season infection may take a while to reach tubers and that the amount of virus particles does not reach detectable levels quickly. Distribution of the virus in above ground parts (leaves and stem) does not correlate with the distribution in below ground parts (tubers). In mixed infections, synergistic interactions between viruses can exist that can influence the resistance of potato to other pathogens as well. PLRV specific resistance can manifest as further limited movement from the foliage to the tubers – with the consequence that more virus free tubers are borne from plants with primary (in-season) infection. It has been reported by Barker and Harrison (1985) that PLRV occurs in lower concentrations in leaves of cultivars with resistance. Some cultivars have genetically controlled resistance to the virus infection, but no cultivar is immune to infection and there are no known sources of major gene resistance. The proportion of virus-free progeny tubers is greater in cultivars resistant to infection and accumulation than cultivars that are susceptible. Resistance mechanisms and virus distribution through the plant thus describe the difference in virus incidence in leaves compared to tubers. With respect to visual observations - visual inspections and indexing plants by visual inspections are complicated and inconsistent especially at high temperatures due to masking of disease symptoms in warm conditions. It has been reported by Loebenstein et al (1997) that PLRV is not always detectable in leaves particularly when plants are grown at temperatures around 30°C and higher. Symptoms of PLRV infection may be absent in infection that occurred later in the season making sampling of symptomatic leaves a challenge. PVY As mentioned previously, any test method has limitations and must be considered in implementation of such a method. Disadvantages of using tuber samples can be illustrated in the PVY testing scenario. PVY is underestimated by using tuber material as sample during the ELISA test, whether dormant or actively sprouting. This was shown in a research project conducted at the University of Aberdeen in Idaho by Jonathan Whitworth et al in February 2012 and confirmed results obtained by Singh and Somerville (1986). Siitari and Kurppa (1987) demonstrated that virus detection in tubers have lower sensitivity in comparison with leaf samples due to the interference of tannins and starches. The underestimation was because of distribution of virus through the plant and the effect on sensitivity of the tuber matrix. To limit the effect these disadvantages have on the test result, scientists allow the tubers to sprout or they employ the so-called “growout” method and then after sprouting use as little tuber flesh as needed to complete the test. The use of fresh leaf material is of utmost importance since leaf material showing signs of deterioration do contribute to false positive results in test regimes. A number of potato varieties display latent infection (no or little symptoms) in the foliage. This makes indexing by visual inspection (as also in the case of PLRV) extremely inconsistent and rendering it essential to use tuber samples for virus testing. Singh and Santos-Rojas (1983) compared detection of PVY primary infection using the ELISA and indicator hosts. In this study, they found that using the strains and cultivars specific to the region (Canada), that the ELISA could only detect primary infection in the leaves from 4 weeks post inoculation. This implies that very recent infection in leaves would go undetected when leaf samples are used as sample. Also, PVY symptoms were more obvious in green-house plants than field grown plants, emphasizing the effect that environmental conditions have on pathogenesis. Although it can take no more than a few hours to reach exponential phase of replication in a cell, it can take days to weeks to develop a complete systemic infection. As in the case of PLRV, detection of in season infection remains inconsistent. The distribution of virus into progeny varies significantly – causing leaf samples to be an unreliable sample in predicting the virus content of progeny tubers. 2 HISTORIC DATA COMPARING LEAF AND TUBER SAMPLES Leaf samples have been used more regularly as management tool in the potato industry since 2004. The experience is that growers in some regions use this tool more frequently than those in other regions for reasons only speculated about. The leaf samples are drawn by the growers themselves and submitted to a laboratory participating in the Potato Laboratory Services (accredited by ICCSP) group. Since leaf samples are drawn just before haulm killing, and when the grower sees it fit, Page 32 • CHIPS • Januarie / Februarie • January / February 2013 Tegniese Nuus • Technical News potatoes SOUTH AFRICA arrangements has to be made with the regional laboratory when the sample needs to submitted. It mostly happens that routine, scheduled testing has to be re-scheduled to accommodate leaf samples since the condition of leaf samples is crucial. Decayed leaf samples give rise to false positive results. The growers thus determine time of sampling, growth stage of the plant, sample size and representation from a planting. For some growers, Potato Certification Service submits seed lists containing information about the sample/s. Other growers prefer to communicate with the laboratory directly. Samples are sent to the regional laboratories and stored in a storage facility that allow temperature and humidity control. The samples are allowed to sprout in the storage facility to facilitate sensitive determination of virus presence in tubers. The reasons for sprouting are based on advantages and disadvantages of leaf and tuber samples as matrix and include the generation of sprouts to allow limited tuber flesh into the sample and ensure that virus titres are at detectable levels. Tubers are used as testing matrix because as mentioned, detection of virus in leaves does not compare sufficiently to virus occurrence in tubers. Since 2004, samples have been submitted to the individual laboratories. Generated results have been compared where information allowed it, to results of tests performed on tuber samples. In total, data from 741 leaf samples were collected and compared with tuber samples from different regions. Comparison of 428 samples was possible. A national average of 30% of the samples had results that graded seed lots in the same generation. 70% of the samples showed a difference between the generated leaf and tuber sample test results in terms of generation graded. The inconsistency in results can be attributed to several factors: Although testing is destructive, the remainder of the tuber samples already submitted to the testing process is stored at the testing facility until the disseminated results are accepted by the authority and / or grower. If not, the interested party can call a dispute and the stored sample can be submitted for the dispute test in the case of PVY. For PLRV, store samples are drawn. • Virus properties • Disease properties • Sampling inconsistency 4. LOGISTICAL AND PRACTICAL ASPECTS IN CONSIDERATION 3 THE CURRENT SCENARIO AT POTATO LABORATORY SERVICES Tuber samples are drawn by Potato Certification Services (PCS) representing seed lots from plantings registered as seed plantings. After breaking of the dormancy of tubers and subsequently sufficient sprouting, the samples are subjected to the DAS-ELISA assay specific for the pathogen in question. Generated results are disseminated from the laboratories to the certification authority as percentage of tubers infected. This testing regime allows authorities, growers and clients of growers to predict the percentage of infected tubers in a seed lot at the time of harvesting. At laboratories nationally, roughly 3400 - 400 tuber equivalent samples are tested each year. From the beginning of a season, when in some regions the samples grown in that particular region are submitted to the laboratory in the first six weeks of the season, it takes up to four months and longer to test all submitted samples at optimum capacity. Although the 28 day waiting period contributes to this time, once dormancy is broken, samples can only be tested as capacity of the laboratory allows. This imply that if 500 samples are submitted to a laboratory in a season, dormancy is broken and samples is test-ready all at once, even with five roller presses 500 samples can only be completed after more than one month. This is however not the situation. Dormancy cannot be broken all at once due to cultivar differences and different planting dates. Issues like the latter stretches a testing season for another two months or more. The average capacity of laboratories is three roller presses to allow sample CHIPS • Januarie / Februarie • January / February 2013 • Page 33 Tegniese Nuus • Technical News potatoes SOUTH AFRICA processing. Facilities and cost implications do not allow further expansion. The shelf-life of tubers allows storage and keeping until testing schedule permits. When tuber samples are used, tubers can be routinely counted to monitor sprouting and start testing. Also, tubers have a shelflife that allows scheduled testing as capacity permits. Since the condition of leaf samples is of utmost importance, testing of fresh leaf material is a pre-requisite to testing leaf samples. No laboratory has access to sufficient cold room space to maintain leaf samples for two days in a good enough condition, nor sufficient roller press facilities to accommodate and allow the testing of leaf samples as they are submitted in peak season activity. After two days of cold-storage the leaves start to go down and risk for false positives increase. If leaf samples are the chosen matrix, then in season the plantings have to be sampled and the samples sent to the laboratory immediately to reach the laboratory within 24 hours (maximum). Packing of sample is important to allow extended keeping of the condition. After receipt, samples need to be tested immediately. In the event of peak season sampling activities, laboratories can receive more than 50 samples per day. This is much more than the capacity of any laboratory allows. CONCLUSION A facility’s capacity is the one thing that can be changed to accommodate more samples, however, the intrinsic properties of the viruses and the crops they infect cannot be changed and remain the motivation why tuber samples are used to predict the virus content of a tuber seed lot and not the leaves. C REFERENCES 1 2 3 4 5 6 Barker H and BD Harrison (1985). Restricted multiplication of potato leaf roll virus in resistant potato genotypes. Annals of Applied Biology, 107:205-212 Derrick PM and H Barker (1997). Short and long distance spread of potato leaf roll luteovirus: effects of host genes and transgenes conferring resistance to virus accumulation in potato. Journal of General Virology, 78:243-251. Barker H (1987). Invasion of non-phloem tissue in Nicotiana clevelandii by potato Leaf roll Luteovirus is enhanced in plants also infected with Potato Y Potyvirus. Journal of General Virology, 68:1223-1227. Siitari H and A Kurppa (1987). Time-resolved Fluoroimmuniassay in the detection of Plant viruses. Journal of General virology, 68:1423-1428. Singh RP and TH Somerville (1987). Factors affecting the detection of Potato virus Y in tubers by Enzyme-linked immunosorbent assay (ELISA). Indian Journal of Plant Pathology 4(1): 91-97. Loebenstein G, Akad F, Filatov V, Sadvakasova G, Manadilova, A, Bakelman H, Teverovsky E, Lachmann O, and David A (1997). Improved detection of Potato LeafrollLuteovirus in Leaves and Tubers with a Digoxigenin-LabeledcRNA probe. Plant Disease 81:489-491. Page 34 • CHIPS • Januarie / Februarie • January / February 2013