Sampling and testing as referred to in Articles 4 and 7
ANNEX IIISupplementary provisions
ANNEX III Sampling and testing as referred to in Articles 4 and 7 1. Sampling and testing for the official detection surveys, as referred to in Article 4(1) and (2), is carried out as follows: Sampling involving a soil sample with a standard rate of at least 1 500 ml soil/ha collected from at least 100 cores/ha, preferably in a rectangular grid of not less than 5 m in width and not more than 20 m in length between sampling points covering the entire production site. The whole sample is used for further examination, i.e. extraction of cysts, species identification and, if applicable, pathotype/virulence group determination. Testing applying the following methods for the extraction of the specified pest, which are described in the relevant validated and internationally recognised diagnostic protocols: (a) with regard to extraction, methods based on the Fenwick can, Schuiling centrifuge, Seinhorst elutriator or Kort elutriator; (b) with regard to detection and identification, one of the following: (i) isolation of Globodera cysts from the debris based on cyst morphology, followed by species identification based on morphology of individual cysts and juveniles, combined with the conventional PCR test based on Bulman & Marshall, 1997 ( 1 ) (in case of doubt about the presence of G. tabacum , the conventional PCR test based on Skantar et al. , 2007 ( 2 ) may be performed additionally); (ii) isolation of Globodera cysts from the debris based on cyst morphology, followed by species identification based on morphology of individual cysts and juveniles, combined with real-time PCR based on Gamel et al. , 2017 ( 3 ) ; (iii) isolation of nematode cysts from the debris, followed by species detection with real-time PCR based on Gamel et al ., 2017 ( 3 ) . 2. Sampling for the official monitoring survey, as referred to in Article 7, is carried out in accordance with one of the following: (a) the sampling referred to in point 1, with a minimum sampling rate of soil of at least 400 ml/ha; (b) targeted sampling of at least 400 ml of soil after visual examination of roots where there are visual symptoms; or (c) sampling of at least 400 ml of soil associated with the potatoes after harvesting provided that the production site where the potatoes were grown is traceable. Testing for the official monitoring survey, as referred to in Article 7, is carried out in accordance with point 1. 3. By way of derogation from point 1, the standard sampling rate may be reduced to a minimum of 400 ml of soil/ha in each of the following cases: (a) there is documentary evidence that no potatoes or plants listed in point 1 of Annex I have been grown and were present in the production site in the six years prior to the official detection survey; (b) no specimen of the specified pest has been found during the last two successive official detection surveys in samples of 1 500 ml soil/ha and no potatoes or plants listed in point 1 of Annex I, other than those for which an official detection survey is required in accordance with Article 4(1), have been grown after the first official detection survey had been carried out; (c) no specimen of the specified pest or cysts of the specified pest without live content have been found in the last official detection survey, which must have consisted of a sample size of at least 1 500 ml soil/ha and no potatoes or plants listed in point 1 of Annex I, other than those for which an official detection survey is required in accordance with Article 4(1), have been grown in the production site since the last official detection survey had been carried out. 4. Sampling rate may be reduced for fields larger than 8 ha and 4 ha, respectively, in each of the following cases: (a) in the case of the standard rate referred to in point 1, the first 8 ha shall be sampled at the rate specified therein, but may be reduced for each additional hectare to a minimum of 400 ml of soil/ha; (b) in the case of the reduced rate referred to in point 3, the first 4 ha shall be sampled at the rate specified therein, but may be further reduced for each additional hectare to a minimum of 200 ml of soil/ha. 5. The use of the reduced sample size, as referred to in points 3 and 4, may be continued in the subsequent official detection survey referred to in Article 3(1), as long as the specified pests have not been found in the production site concerned. 6. The standard size of the soil sample may be reduced to a minimum of 200 ml of soil/ha, provided that the production site is situated in an area declared free from the specified pest, and designated, maintained and surveyed in accordance with the relevant International Standards for Phytosanitary Measures (ISPM No 4) ( 4 ) . 7. The minimum size of the soil sample in all cases is 200 ml of soil per production site. ( 1 ) Bulman, S.R. & Marshall, J.W. (1997). Differentiation of Australasian potato cyst nematode (PCN) populations using the Polymerase Chain Reaction (PCR). New Zealand Journal of Crop and Horticultural Science 25, pp. 123–129. ( 2 ) Skantar, A.M., Handoo, Z.A., Carta, L.K., Chitwood, D.J. (2007). Morphological and molecular identification of Globodera pallida associated with Potato in Idaho. Journal of Nematology 39(2), pp. 133–144. ( 3 ) Gamel, S., Letort A., Fouville D., Folcher L., Grenier E. (2017). Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida , G. rostochiensis and Heterodera schachtii . Nematology 19 (7):789-804. ( 4 ) ISPM 4. 1995.Requirements for the establishment of pest free areas. Rome, IPPC, FAO. https://www.ippc.int/en/publications/614/