Diagnostic protocols for sweetpotato

Contributors to this section: CIP, Lima, Peru (Carols Chuquillanqui, Segundo Fuentes, Ivan Manrique, Giovanna Muller, Willmer Pérez, Reinhard Simon, David Tay, Liliam Gutarra); CIP, Nairobi, Kenya (Ian Barker); FERA, UK (Derek Tomlinson, Julian Smith, David Galsworthy, Rebecca Weekes).

Case studies - CIP potato and sweet potato

Contributors to this section: CIP, Lima, Peru (Carols Chuquillanqui, Segundo Fuentes, Ivan Manrique, Giovanna Muller, Willmer Pérez, Reinhard Simon, David Tay, Liliam Gutarra); CIP, Nairobi, Kenya (Ian Barker); FERA, UK (Derek Tomlinson, Julian Smith, David Galsworthy, James Woodhall).

International Potato Centre (CIP) case studies on germplasm health testing of potato and sweet potato held in tissue culture

In the mid 2000s, CIP set out to ensure all tissue-culture material held at its headquarters at CIP was tested for listed pathogens to an internationally recognised standard ISO 17025. The principles of quality assurance and ISO accreditation are provided in the page on Quality assurance within pest diagnostic laboratory procedures. Accordingly, in partnership with Food and Environment research Agency (FERA) in the UK, the CIP tissue culture germplasm collection held at Lima acquired ISO17025 accreditation in 2007/8 for the pests for which diagnostic protocols are provided in this section. (See potato and sweet potato protocols pages).

Equivalent systems are not in place for other germplasms collections held by the CGIAR.

In the following we provide an overview of the CIP accreditation process and suggest this as an example for institutes to observe in order to develop equivalent ‘best practices’ for any clonal crop.

CIP germplasm health

Contributors to this section: CIP, Lima, Peru (Carols Chuquillanqui, Segundo Fuentes, Ivan Manrique, Giovanna Muller, Willmer Pérez, Reinhard Simon, David Tay, Liliam Gutarra); CIP, Nairobi, Kenya (Ian Barker); FERA, UK (Derek Tomlinson, Julian Smith, David Galsworthy, James Woodhall).

CIP germplasm health

Introduction

Collecting, conserving and utilising plant genetic resources and their global distribution is a core function of the GCIAR centres. At CIP, wild and cultivated genetic resources of potato, sweetpotato and other root and tuber species are collected, securely conserved through integrated ex situ, in situ and on-farm approaches and disseminated to users worldwide.

The safe handling of this germplasm involves minimising the risk of distributing plant pathogenic organisms along with the host plant. In order to manage this risk, effective testing (indexing) procedures are required to ensure that distributed material is free of pests that are of concern to the end users of the germplasm.

A summary of the numbers and countries that have accessed germplasm from CIP can be found at External Distribution Summary - GADC (login required).

The purpose of this document is to record the assessment of the effectiveness of these testing processes for potato and sweetpotato germplasm. The document will outline the approaches adopted, assessment of results recorded for the testing of the genebank accessions during the period 2007 and demonstration of the “fitness for purpose” of the in-vitro diagnostic methodology being used.

Summary of CIP Germplasm Status

Total number of accessions

5046 HS2 status potato accessions
2175 HS2 status sweetpotato accessions

Number health status assessments in 2007

749 potato accessions
358 sweetpotato accessions

Number of infected samples reported by 3rd Party post entry quarantine assessment in the last 5 years – 3 accessions (see Annex 3 - letter from USDA).

Number of symptoms reported from field grown material in 2006-2007 - 2 accessions both of which tested negative to in-vitro diagnostic tests.

Virus incidence in Health Status 0 (HS0) Material (Dec. 2005)

Health status checking of 4,415 native potato accessions maintained in vitro showed that 64% of the accessions were negative to the quarantine pathogens (PSTVd, PVT, PLRV, PVY, PVX, PVS, APMoV, APLV). From the infected accessions, 72% were infected with PVS virus alone or in combination with other viruses (Table 1). No single incidence of PSTVd infection has been recorded in the last 5 years in any of the materials tested.

Health status checking of 1,216 native sweetpotato accessions showed that 74% of the accessions were originally virus-free. SPFMV (alone or in combination with other virus) has the higher frequency of infection (17%) (Table 2).

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Executive summary

The acquisition, conservation and distribution of potato and sweetpotato germplasm is a core function of CIP and serves to underpin germplasm conservation and breeding programs worldwide. Clonally propagated crops such as potato and sweetpotato are infected by a number of viral, viroid, phytoplasma and bacterial diseases, which can be transmitted through exchange of in vitro material. CIP aims to only distribute material of the highest health status to prevent the possible movement of pathogens around the world and any consequent damage to food security, livelihoods and economies. CIP also complies with all National and International phytosanitary treaties, regulations and requirements relating to both phytosanitation and also the Convention on Biological Diversity (CBD).

CIP wishes to develop and validate a pathogen testing scheme, which is fit for the purpose of eliminating (within acceptable statistical limits) the risk of spread of economically important pathogens, particularly to developing countries, which often have only poor phytosanitary capacity of their own. The scheme also reflects that there is considerable demand for CIP germplasm to support ongoing breeding programs which deliver critical benefits through improved yields, nutritional quality and resistance to diseases, heat and drought to some of the world’s poorest farmers. Thus the scheme needs to carefully balance any potential risks of pathogen spread with acceptable delivery times for germplasm and available resources for testing. The scheme also prioritises pathogens of high economic importance as well as those known to be a problem in Peru. There is also recognition that there are a number of potato pathogens of relatively low economic importance but which are of importance to seed potato exporting Nations. The latter typically have comprehensive post entry quarantine testing programs aimed at more extensive testing for such pathogens. There is lastly also a dichotomy between the relatively well characterized pathogens of potato and the much less well characterized pathogens of sweetpotato. The scheme fundamentally aims to detect and eliminate clonally spread pathogens rather than their identification per se.

The current CIP scheme is based on the two FAO/IPGRI technical guidelines for the Safe Movement of Germplasm. The scheme for potato was modified in 2006 to include the growing out of a potato plant to at least flowering and to provide two independent methods for the testing of priority pathogens in line with current EPPO guidelines. Additional testing is carried out in line with any importing country special requirements. This document provides evidence for the validation and performance of these methods, particularly the new potato scheme.

Evidence is drawn from 4 separate sources of information and reflects our best endeavours and recognising the current lack of any available reference material and systematic proficiency testing in the field of pathogen testing of clonally propagated crops. Firstly, available diagnostic reagents and tests were applied to known infected and healthy controls which were then serially diluted to demonstrate the end points of detection. Secondly, data from the testing of 749 actual potato accessions in 2007 and 737 sweetpotato accessions in 2006/07 was analysed to infer test performance. Thirdly, in a unique structured experiment in 2007, some 80 known infected in vitro plantlets (10 replicates each of 8 accessions representing 7 different potato viruses) were subjected to testing through the entire screening process. Lastly, worldwide anecdotal evidence is available from colleagues and collaborators in the field and post-entry quarantine testing labs receiving CIP material as to its health status as an additional check on the process.

Additional extensive validation data for CIP radioactive and non-radioactive NASH methods for the detection of Potato spindle tuber viroid (PSTVd) are also available and shown in Müller G. et al. CIP also successfully took part in a European led (SASA) ring-test for this pathogen (data available) in 2002.

Data from the serial dilutions of infected and healthy sap for both potato and sweetpotato indicates that there is a wide margin of safety in relation to test sensitivity. The test pathogens were detected down to dilutions of approximately 1000 fold (200x less than working dilution) in the case of potato and down to 400 to 800 fold (80x to 160x less than working dilution) in the case of sweetpotato. Thus there is a considerable margin for the detection of low level infections and even evidence for potential for sample bulking if required. Multiple strains of SPFMV and PYV were also tested and all were detected. ELISA values for a few potato viruses were perhaps low probably reflecting the difficulty of propagating viruses in the summer in a tropical environment although the signal to noise ratios were acceptable (more than 9x background as a minimum).

Data from the actual testing of real samples of sweetpotato (2006/07) and potato (2007) also indicates that the battery of tests applied appears to work well in the detection of viruses. Frequency histogram plots of ELISA and NCM-ELISA data shows the expected distribution of a functioning test i.e. separated populations presumably reflecting healthy and infected plants with a clear separation between the two populations. The relatively few borderline samples was also a good indication of the clear separation. It is clear that the different laboratory tests and bioassays complement each other and demonstrates the necessity of providing both an “a” and a “b” test. It is also evident that the ELISA tests should be carried out on both in vitro and in vivo material as the two tests clearly complement each other (both detecting samples that the other did not) for reasons that remain unclear. In the case of potato only one sample out of 539 accessions tested negative in all tests but showed symptoms on the grown out potato. These symptoms may not of course have been symptoms of a disease but in any case point to the fact that the majority of potato pathogens commonly encountered in the CIP germplasm can be detected by the available laboratory and glasshouse tests.

The unique potato testing validation scheme provides the best and most direct evidence of the efficacy of the detection procedure for a range of commonly encountered viruses. All 80 in vitro plantlets were detected by one or more test method. The in vitro ELISA and NASH test detected 80/80 samples, The ELISA and NASH in vivo test at flowering detected 77/80 samples, the host range test in vivo detected 62/80 samples and symptoms were apparent in some 50/80 grown on potato plants. If for example we substituted a failure rate of 1/80 for the in vitro ELISA tests then the probability of failure of detection by all 4 tests (averaging across all the virus accessions) would be p= 0.000039 or approximately 1 in 25 000. This theoretical failure rate would of course vary from virus to virus with the chance of failure of detection of a virus like PYV, with the lowest detection rates in host range and symptoms on the grown out plant, being higher than the average.

Additional data from the validation experiment was also used to examine the validity of current threshold levels and also the use of single wells in ELISA. Examination of possible threshold levels revealed that 0% of known infected samples fell below 1.5 times the healthy background and only 1.5% of samples fell between 1.5 and 2 times the healthy background values. All the remaining positives (98.5%) fell above 2 times the healthy background limit. This data supports the proposal to designate samples falling under 1.5 times as healthy, between 1.5 and 2 as pending (re-test) and above 2 as infected (reject). Analysis of well to well co-variance between replicate wells was less than 25%. An analogous situation might be the trace detection pesticides or other contaminants where a co-variance of less than 40% would be acceptable.

Lastly there is no anecdotal evidence from feedback from recipients of CIP material of systematic pathogen escapes from detection. The single detection incidents by USDA APHIS remain the only extant examples of pathogen escape from post-entry quarantine labs. CIP collaborators examining CIP material in the field also send back reports of any problems. For example in 2006 single accessions of potato in Bhutan and Kazakhstan were seen to have suspicious symptoms. Good quality digital photos were examined by experts who were of the opinion that the symptoms were not as the result of pathogen infection. Samples were also sent to the Plant Clinic at the UK Central Science laboratory for extensive testing and were reported negative.

The data presented indicates that CIP testing procedures for viruses and viroids of potato and sweetpotato are performing well and the addition of additional tests to the potato procedure was justified and necessary. The sweetpotato program appears to be functioning well with no recent evidence of test failure. There remains a discussion as to whether to add an additional grafting test to detect PLRV and phytoplasmas and also the option to add an additional PCR step to improve detection of the latter. Additional validation data will be collected in 2008 to assess the incidence of phytoplasmas in the collection and the most efficient means of detection. A validation experiment similar to that performed for potato will be also be carried out for sweetpotato in 2008.

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References and further reading

Borkhardt B, Vongsasitorn D, Albrechtsen SE. 1994. Chemiluminescent detection of potato spindle tuber viroid in true potato seed using a digoxigenin labeled DNA probe. Potato Research 37: 249–255.

Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19: 11–15.

Feldstein PA, Hu Y, Owens RA. 1998. Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid. Proceedings of the National Academy of Science. USA. 95, 6560–6565.

International Potato Center (CIP). 1997. Preparation of 32 P-labeled probes by RNA transcription. In: Salazar LF,  Jayasinghe U, editors. Techniques in Plant Virology. Training Manual. Sections 3, 4, 5. International Potato Center, Lima, Peru.

Jeffries C, James C. 2005. Development of an EU protocol for the detection and diagnosis of Potato spindle tuber pospiviroid*. OEPP/EPPO Bulletin 35: 125–132.

Kanematsu S, Hibi T, Hashimoto J, Tsuchizaki T. 1991. Comparison of nonradioactive cDNA probes for detection of potato spindle tuber viroid by dot-blot hybridization assay. Journal of Virological Methods 35: 189–197.

Kazunori G, Akira K, Makoto K, Chikara M. 2003. A simple and rapid method to detect plant siRNAs using nonradioactive probes. Plant Molecular Biology Reporter 21: 51–58.

Martinez-Soriano JP, Galindo-Alonso J, Maroon CJM, Yucel I, Smith DR, Diener TO. 1996. Mexican papita viroid: Putative ancestor of crop viroids. Proceeding of the National Academy of Science. USA. 93: 9397–9401.

Müller G, Flores B, Meza M, Salazar LF, Barker I. Chemiluminescent detection of potato spindle tuber viroid (PSTVd) using a digoxigenin-labeled RNA probe. International Potato Center, Lima, Peru. Available here.

Palukaitis P, Cotts S, Zaitlin M. 1985. Detection and identification of viroids and viral nucleic acids by ‘dot blot’ hybridization. Acta Horticulturae 164: 109-118.

Puchta H, Herold T, Verhoeven K, Roenhorst A, Ramm K, Schmidt-Puchta W, Sanger HL. 1990. A new strain of potato spindle tuber viroid (PSTVd-N) exhibits major sequence differences as compared to all other PSTVd strains sequenced so far. Plant Molecular Biology 15: 509–511.

Querci M, Owens RA, Bartolini I, Lazarte V, Salazar LF. 1997. Evidence of heterologous encapsidation of potato spindle tuber viroid in particles of potato leaf roll virus. Journal of General Virology 78: 1207–1211.

Querci M, Owens RA, Vargas C, Salazar LF. 1995. Detection of potato spindle tuber viroid in avocado growing in Peru. New diseases and epidemics. Plant Disease Vol. 79 (2): 196–202.

Salazar LF, Balbo I, Owens RA. 1988. Comparison of four radioactive probes for the diagnosis of potato spindle tuber viroid by nucleic acid spot hybridization. Potato Research 31: 431–442.

Salazar LF, Owens RA, Smith DR, Diener TO. 1983. Detection of potato spindle tuber viroid by nucleic acid spot hybridization: Evaluation with tuber sprouts and true potato seed. American Potato Journal Vol. 60: 587–597.

Singh RP, Nie X, Singh M. 1999. Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids. Journal of General Virology 80, 2823–2828.

van Wezenbeek P, Vos P, van Boom J, van Kammen A. 1982. Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA. Nucleic Acids Research. 10: 7947–7957.

Welnicki M, Hiruki C. 1992. Highly sensitive digoxigenin-labeled DNA probe for the detection of spindle tuber viroid. Journal of Virological Methods 39: 91–99.

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Germplasm health testing validation data for potato

Contributors to this section: CIP, Lima, Peru (Carols Chuquillanqui, Segundo Fuentes, Ivan Manrique, Giovanna Muller, Willmer Pérez, Reinhard Simon, David Tay, Liliam Gutarra); CIP, Nairobi, Kenya (Ian Barker); FERA, UK (Derek Tomlinson, Julian Smith, David Galsworthy, James Woodhall).

Potato germplasm health testing

The protocol for health testing of potato material has been modified to increase the range of viruses tested and to reduce the risk of any disease escape. The main improvement is the testing in two different growing conditions (in vivo and in vitro) along with the addition of a “b” test for some viruses (either additional ELISA or additional host range species) and improvements in the host range inoculation buffer. The growing out of a potato plant to beyond flowering also permits the observation of any symptoms on the original host. During 2007, a total of 542 potato accessions were re-classified as HS2 following the new testing procedure. A validation process was also undertaken of the methodology being applied to determine the health status using material known to be infected with various pathogens.

Click here to download a publication on potato germplasm health testing.