Ipomoea (Sweetpotato/Kumara) Post-Entry Quarantine Testing Manual

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• N. benthamiana – not susceptible. • N. clevelandii – local lesions, systemic chlorotic spots, rings and lines. • N. tabacum – local lesions, systemic chlorotic spots, rings and lines. The virus symptoms produced on herbaceous indicators are described below. Sweetpotato yellow dwarf virus: • C. quinoa – susceptible, but no information is available on symptoms. Tobacco streak virus: • N. tabacum – systemic vein clearing, then downward curling of the leaf and its margins. 7.1.3 Serological and molecular assays ELISA OR PCR MUST be carried out for the following viruses: • Sweetpotato vein mosaic virus • Tobacco streak virus ELISA OR PCR is OPTIONAL for the following virus: • Sweetpotato mild speckling virus PCR MUST be carried out for the following organisms: • Sweetpotato chlorotic stunt virus • Sweetpotato leaf curl virus • Sweetpotato little leaf phytoplasma PCR OR selective media MUST be carried out for the following bacterium: • Dickeya chrysanthemi 7.1.3.1 Enzyme-linked immunosorbent assay (ELISA) Recommended method 1. Perform the ELISA according to the manufacturer’s instructions. The following controls must be included on each ELISA plate: (a) positive control: infected leaf tissue or equivalent (Table 2); and (b) negative control: sweetpotato tissue that is known to be healthy; and (c) buffer control: extraction buffer only. 2. Add each of the samples and controls to the ELISA plate as duplicate wells. It is not recommended to perform ELISA with plant samples or sap that has been frozen. 3. Measure the optical density 60 minutes after addition of the substrate (or as per manufacturer’s instructions). Ipomoea Post-Entry Quarantine Testing Manual · November 2012 11
Table 2: Source of antisera and positive controls for ELISA Pathogen Antisera Positive/negative Sweetpotato vein mosaic virus and Sweetpotato mild speckling virus Ipomoea Post-Entry Quarantine Testing Manual · November 2012 Agdia Cat No. PSA27200 (Potyvirus group: Pathoscreen kit) 1,2 Tobacco streak virus Agdia Cat No. PSA25500 (Pathoscreen kit) 1,2 control 2 Agdia Cat No. LNC 27200 Agdia Cat No. LNP 27200 Agdia Cat No. LPC25500 1 Catalogue numbers for the complete reagent sets are given, the antisera and reagents can also be purchased separately. 2 The positive control is included if the Pathoscreen set is purchased. Further information about the kits and the supplier listed in Table 2 can be found at the following website: • Agdia Incorporated, USA (http://www.agdia.com). Interpretation of results A result is considered positive if the mean absorbance of the two replicate wells is greater than 2 times the mean absorbance of the negative control. The test will only be considered valid if: (a) the absorbance for the positive and negative controls are within the acceptable range specified by the manufacturer; and (b) the coefficient of variation (standard deviation / mean × 100), between the duplicate wells is less than 20%. If the test is invalid, it must be repeated with freshly-extracted sample. Samples that are close to the cut-off must be retested or tested using an alternative method recommended in the import health standard (see Table 1). 7.1.3.2 Polymerase chain reaction (PCR) The following section describes the molecular tests required for regulated pests listed on the import health standard for I. batatas and I. setosa. The recommended published PCR primers for these tests are listed in Table 3 along with plant internal control primers for RNA and DNA. The inclusion of an internal control assay is recommended to eliminate the possibility of PCR false negatives due to extraction failure, nucleic acid degradation or the presence of PCR inhibitors. It is strongly recommended to extract nucleic acid from indicator plants (I. setosa or I. nil), 3 to 5 weeks after grafting rather than extracting directly from I. batatas test plants. Viruses present in I. batatas can be unevenly distributed in the plant and virus titre can fluctuate over time. Virus levels in grafted indicator plants have been found to be higher in comparison with I. batatas (Kokkinos & Clark, 2006). The PCR reagents listed for the methods described in this section have been tested by the Plant Health & Environment Laboratory, MPI. Alternative reagents may give similar results but will require validation. 12
Page 1 and 2: Ipomoea (Sweetpotato/Kumara) Post-E
Page 3 and 4: 1.7 Dickeya chrysanthemi ..........
Page 5 and 6: The per capita income provided by s
Page 7 and 8: Viruses: Sweetpotato chlorotic flec
Page 9 and 10: 7. TESTING Each of the specific tes
Page 11 and 12: 7.1.1 Graft inoculation Each I. bat
Page 13: 7.1.2 Herbaceous indexing Each I. b
Page 17 and 18: 7.1.3.2.1 Virus reverse transcripti
Page 19 and 20: Recommended method for RNA viruses:
Page 21 and 22: Positive control material, in the f
Page 23 and 24: The reaction and cycling conditions
Page 25 and 26: (a) Primers recAF/recAR (Waleron et
Page 27 and 28: 8. CONTACT POINT This manual was de
Page 29 and 30: Nassar, A; Darrasse, A; Lemattre, M
Page 31 and 32: . 1.5 Streptomyces ipomoea (a) (b)
Page 33 and 34: Appendix 2. Virus symptoms on graft
Page 35 and 36: Appendix 3. Protocols referenced in

Ipomoea (Sweetpotato/Kumara) Post-Entry Quarantine Testing Manual

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