ANNEX I

Methods of testing for detection and identification of the specified pest referred to in Article 3(2)

1. Testing by means of spores

For detection and identification, summer sporangia and resting spores are used, which are obtained from soil after sieving, or directly from the plant material.

2. Methods for detection

For the extraction of spores of the specified pest from soil, one of the following methods shall be used:

(a) soil sieving method, as described by Pratt (1976) (1);

(b) soil sieving method, as described by van Leeuwen et al. (2005) (2);

(c) zonal centrifuge technique for high throughput sample processing, as described by Wander et al. (2007) (3).

3. Methods for identification

After extraction, the spores of the specified pest shall be identified by one of the following methods:

(a) morphological identification under a light microscope at 100x – 400x magnification;

(b) conventional PCR using primers based on Lévesque et al. (2001) (4) and van den Boogert et al. (2005) (5);

(c) real-time PCR using primers and probes following van Gent-Pelzer et al. (2010) (6);

(d) real-time PCR using primers and probes following Smith et al. (2014) (7).

4. Viability of resting spores

Determination of viability of the resting spores may be achieved by microscopic examination or bioassay. Viability of sporangia may be determined by microscopic examination of sporangia mounted in lactophenol or in water (Przetakiewicz 2015) (8). Sporangia with granular contents or with slightly rounded-off protoplasm may be considered viable. Those permanently plasmolysed or with no apparent content shall be considered dead.

As an alternative, or in case of doubt, a bioassay, as described in point 3 of Annex IV, may be carried out.

5. Determination of pathotypes

The determination of pathotypes shall require fresh warts.

The inoculum for the test shall be produced by one of the following methods:

(a) method of SASA (Science and Advice for Scottish Agriculture), consisting of the two following steps: (i) production of inoculum Old (brown) wart tissue shall be broken into smaller pieces and air dried at room temperature until it becomes hard. The hard tissue shall be ground, either manually or mechanically. The ground material shall be dry-sieved, collecting the fraction from 25 to 75 μm, and then extracted using the chloroform method of Pratt (1976)1; (ii) production of fresh warts Approximately 10 mg of extracted resting spores shall be sprinkled onto the surface of 10 ml sterile distilled water in a small plastic Petri dish and incubated in the dark at 20 °C until germination. Potato tubers with small sprouts about 1 to 2 mm long shall be placed in transparent plastic boxes, lined with damp tissue paper with the marked sprouts facing up. The sprouts shall be ringed with melted Vaseline using a syringe. The ring shall be unbroken and high enough to hold the spore suspension without leaking. The 10 ml of germinating resting spores shall be diluted further to 20 ml with sterile water and placed within the rings using a pipette or a squeeze bottle until the sprout is completely submerged in spore suspension. The plastic boxes shall be covered with lids and incubated for 4 days at 10 °C, after which the boxes shall be opened, the inoculum and Vaseline rings shall be removed and the boxes shall be moved to a misted glasshouse at 15 to 18 °C (16 h light);

(b) method of Spiekermann & Kothoff (1924) (9);

(c) method of Potoček et al. (1991) (10);

(d) method of Glynne-Lemmerzahl (Glynne 1925 (11); Lemmerzahl 1930 (12); Noble and Glynne 1970 (13)).

For determination of all pathotypes known to be relevant for the Union (1(D1), 2(G1), 6(O1), 18(T1) and 38(Nevşehir), a differential infection test with various varieties of the specified plant shall be used as indicated in the table. The infection test shall be carried out following the protocol mentioned under point (d) (Glynne-Lemmerzahl method).

Selective sensitivity of potato cultivars for the determination of S. endobioticum pathotypes

Cultivar S. endobioticum pathotypes 1(D1) 2(G1) 6(O1) 18(T1) 38(Nevşehir) Tomensa/Evora/Deodara S S S S S Irga/Producent R S S S S Talent R R* R* S S Saphir R S R R S Ikar/Gawin/Karolin/Belita R R R R R ‘S’: Susceptible ‘R’: Resistant *: indicates a weak susceptibility of the variety to S. endobioticum (‘presence of non-necrotic sori fields without the formation of warts’).

(1) Pratt MA. 1976. A wet-sieving and flotation technique for the detection of resting sporangia of Synchytrium endobioticum in soil. Annals of Applied Biology 82: 21 – 29.

(2) van Leeuwen GCM, Wander JGN, Lamers J, Meffert JP, van den Boogert PHJF, Baayen RP. 2005. Direct examination of soil for sporangia of Synchytrium endobioticum using chloroform, calcium chloride and zinc sulphate as extraction reagents. EPPO Bulletin 35: 25 – 31.

(3) Wander JGN, van den Berg W, van den Boogert PHJF, Lamers JG, van Leeuwen GCM, Hendrickx G, Bonants P. 2007. A novel technique using the Hendrickx centrifuge for extracting winter sporangia of Synchytrium endobioticum from soil. European Journal of Plant Pathology 119: 165 – 174.

(4) Lévesque CA, de Jong SN, Ward LJ & de Boer SH (2001) Molecular phylogeny and detection of Synchytrium endobioticum, the causal agent of potato wart. Canadian Journal of Plant Pathology 23: 200–201.

(5) van den Boogert PHJF, van Gent-Pelzer MPE, Bonants PJM, de Boer SH, Wander JGN, Lévesque CA, van Leeuwen GCM, Baayen RP. 2005. Development of PCR-based detection methods for the quarantine phytopathogen Synchytrium endobioticum, causal agent of potato wart disease. European Journal of Plant Pathology 113: 47 – 57.

(6) van Gent-Pelzer MPE, Krijger M, Bonants PJM. 2010. Improved real-time PCR assay for the detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants. European Journal of Plant Pathology 126: 129 – 133.

(7) Smith DS, Rocheleau H, Chapados JT, Abbott C, Ribero S, Redhead SA, Lévesque CA, De Boer SH. 2014. Phylogeny of the genus Synchytrium and the development of TaqMan PCR assay for sensitive detection of Synchytrium endobioticum in soil. Phytopathology 104: 422 – 432.

(8) Przetakiewicz, J. 2015. The Viability of Winter Sporangia of Synchytrium endobioticum (Schilb.) Perc. From Poland. American Journal of Potato Research 92:704-708.

(9) Spieckermann A, Kothoff P. 1924. Testing potatoes for wart resistance. Deutsche Landwirtschaftliche Presse 51: 114 – 115.

(10) Potoček J, Krajíčková K, Klabzubová S, Krejcar Z, Hnízdil M, Novák F, Perlová V. 1991. Identification of new Synchytrium endobioticum (Schilb.) Perc. pathotypes in Czech Republic. Ochrana Rostlin 27: 191 – 205.

(11) Glynne MD. 1925. Infection experiments with wart disease of potatoes. Synchytrium endobioticum. Annals of Applied Biology 12: 34 – 60.

(12) Lemmerzahl J. 1930. A new simplified method for inoculation of potato cultivars to test for wart resistance. Züchter 2: 288 – 297.

(13) Noble M, Glynne MD. 1970. Wart disease of potatoes. FAO Plant Protection Bulletin 18: 125 – 135.