Commission Implementing Regulation (EU) 2022/1195 of 11 July 2022 establishing measures to eradicate and prevent the spread of Synchytrium endobioticum (Schilbersky) Percival

Article 1

Subject matter

This Regulation sets out measures for the purpose of eradicating Synchytrium endobioticum (Schilbersky) Percival, and preventing its spread within the Union territory.

Article 2

Definitions

For the purposes of this Regulation, the following definitions apply:

(1) ‘specified pest’ means Synchytrium endobioticum (Schilbersky) Percival;

(2) ‘specified plants’ means plants of Solanum tuberosum L. other than seeds.

Article 3

Surveys and laboratory tests of the specified pest

Article 4

Designation of infested production sites and infected specified plants

Plots demarcated by the competent authorities as contaminated, in accordance with Article 2(1) of Directive 69/464/EEC, before 1 January 2022, shall be deemed to be designated as infested production sites.

Article 5

Establishment of demarcated areas

The demarcated area shall consist of:

(a) an infested zone, including at least the production site designated as infested; and

(b) a buffer zone, surrounding the infested zone.

The delimitation of the buffer zone referred to in the first subparagraph, point (b), shall be based on sound scientific principles, the biology of the specified pest, the level of infestation, the distribution and frequency of cultivation of specified plants in the area concerned, the environmental and geographical conditions, as well as the specific risk of spread of resting spores.

Article 6

Eradication measures

In an infested zone, all of the following measures shall apply:

(a) no specified plants shall be planted, grown or stored;

(b) no other plants, intended for replanting outside the infested zone, shall be grown or stored, both in the ground or anywhere else;

(c) soil shall be removed from plants other than those referred to in points (a) and (b), by appropriate methods ensuring that there is no identifiable risk of spreading the specified pest, before these plants are moved from the infested zone into the buffer zone, or out of the demarcated area, or immediately after;

(d) machinery shall be cleaned from soil and plant debris, before or immediately after being moved out of the infested zone and before entering any production site located in the buffer zone or outside of the demarcated area;

(e) any soil or debris originating from an infested zone may only be moved and used or deposited outside that zone under conditions ensuring that there is no identifiable risk of spreading the specified pest.

Plants other than those referred to in paragraph 2, points (a) and (b) from which soil has not been removed may only be moved out of the demarcated area if the following two conditions are fulfilled:

(a) they are transported for the purpose of removing soil from those plants by appropriate methods ensuring that there is no identifiable risk of spreading the specified pest;

(b) the transport and the removal of soil take place under official supervision, and appropriate measures have been put in place to effectively prevent the spread of the specified pest.

The competent authorities shall ensure that:

(a) in the buffer zone, no plants intended for replanting outside the demarcated area are grown;

(b) in the buffer zone, only specified plants are grown of a variety, which is resistant to the pathotypes of the specified pest found in the infested zone or to all pathotypes known to occur in their Member State, as provided for in Article 7, and other than for the production of specified plants for planting; and

(c) any soil or debris originating from the buffer zone is moved and used or deposited outside the demarcated area under conditions, such that there is no identifiable risk of spreading the specified pest.

Article 7

Potato varieties resistant to pathotypes of the specified pest

Article 8

Notification of the confirmed presence of the specified pest on a resistant potato variety

Member States shall notify to the Commission and the other Member States, each year by 31 January, the details of the confirmations made pursuant to paragraph 2 as regards the preceding year.

Article 9

Revocation of the measures

Article 10

Entry into force

This Regulation shall enter into force on the third day following that of its publication in the Official Journal of the European Union.

This Regulation shall be binding in its entirety and directly applicable in all Member States.

ANNEX I

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(Nevșehir), 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).

Cultivar	S. endobioticum pathotypes
1(D1)	2(G1)	6(O1)	18(T1)	38(Nevșehir)
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’).

ANNEX II

Survey template as referred to in Article 3

Template for presenting results of potato wart disease surveys carried out during the calendar year preceding the year of reporting.

Please use this table only for the survey results for potatoes harvested in your country.

Member State	Category	Cropping area (ha)	Visual inspection of tubers	Laboratory testing	Initial size of the infested area (1) (ha)	Updated size of the infested area (2) (ha)	Notification numbers of the new outbreaks notified, as applicable, in accordance with Implementing Regulation (EU) 2019/1715	Additional information
Number of lots	Number of suspicious lots	Number of tested samples	Number of positive samples
	Potato tubers for planting
Potato tubers other than for planting
(1) Total size of the infested area previous to the year covered by the report. (2) Total size of the infested area in the year covered by the report.

ANNEX III

The protocol for the assessment of the resistance of a variety shall include the following steps.

(i) the Glynne-Lemmerzahl method (Glynne 1925, Lemmerzahl 1930, Noble & Glynne 1970); (ii) the Spieckermann method (Spieckermann & Kothoff 1924); or (iii) the SASA (Science and Advice for Scottish Agriculture) method, consisting of all of the following steps: — tuber preparation: Tubers shall be removed from the cold store around 10 days before intended inoculation, washed gently, dried and stored in the dark at room temperature to induce sprouting. A highly susceptible variety (‘Morene’ or a variety with comparable susceptibility) shall be included in each inoculation to serve as positive control; — germination of resting spores: Conditions to induce germination of resting spores shall be set up 21 days prior to inoculation. Approximately 10 mg of extracted spores shall be sprinkled onto the surface of 10 ml of sterile distilled water in small plastic Petri dishes and incubated in the dark at 20 °C until germination. The content of each Petri dish shall be diluted with another 10 ml of sterile distilled water for the inoculation; — inoculation and incubation of sprouts: When the sprouts reach 1 mm in length, they shall be ringed with melted Vaseline. The Vaseline ring shall be unbroken to hold the spore suspension without leaking and high enough for the suspension to cover the sprout. A single sprout or a single cluster of sprouts shall be ringed on each tuber. The tubers shall be placed in plastic boxes, lined with damp tissue paper with the ringed sprouts facing upwards. The Vaseline rings shall be filled with spore suspension, using a pipette or a squeeze bottle until the sprout is completely submerged. The plastic boxes shall be covered with lids and incubated for 4 days at 10 °C in the dark, after which the Vaseline rings shall be removed and the boxes shall be placed open in a glasshouse at 15–18 °C under periodic misting (3 times per day for 30 min). In cases where the infection failed, for example because the sprout rotted or failed to develop, the tuber may be retested using another sprout; — assessment: Sprouts shall be examined for infection 28 days after the inoculation, using a stereo microscope with 10–15x magnification and a light microscope. Reactions of score 4 or 5, as set out in the table, shall be observed on the positive control on at least 80 % of tubers. At least one tuber shall show a score of 5.

(i) a variety shall be considered ‘highly resistant’, if all tubers in all replicates have a score of 1; (ii) a variety shall be considered ‘resistant’, if all tubers in all replicates have a score between 1 and 3; (iii) a variety shall be considered ‘slightly susceptible’, if one or more tubers score 4 (if only one tuber scores 4, the test may be repeated, in order to exclude impurity in the variety lot); (iv) a variety shall be considered ‘extremely susceptible’, if at least one tuber in one replicate scores 5. Standard scoring notations for potato testing populations Standard score Group of resistance Resistance description Description 1 R1 Extremely resistant Early defence necrosis; no visible sorus formation. 2 R1 Resistant Late defence necrosis; sorus formation partially visible, sori immature or necrotic before maturity. 3 R2 Weakly resistant Very late defence necrosis; single ripe sori or sorus fields developed, but completely surrounded by necrosis; up to five non-necrotic summer sori permitted, clear necrosis in other zones of the same tuber piece. No formation of warts or resting spores. To decide between groups 3 and 4, it may be necessary to prepare thin slides of infected tissue: if there are no resting spores, the score shall be 3. 4 S1 Slightly susceptible Scattered infections; sori or sorus fields non-necrotic, few in number; late necrosis can be present on other infection sites on the sprout; the sprout can be slightly malformed (thickened). Resting (winter) sporangia are present. To decide between group 3 and 4, it may be necessary to prepare thin slides of infected tissue: if resting spores are present, the score shall be 4. 5 S2 Extremely susceptible Dense infection fields, numerous ripe non-necrotic sori and sorus fields, fields with dense non-necrotic infection sites, predominant wart formation.

ANNEX IV

1. Conditions for revocation of the measures

1.1.After a minimum of 50 years since the last detection of the specified pest, if there is a gapless record of crops in the infested zone showing that the provisions of Article 6(2) and (3) have been complied with during the whole time and that the infested zone has not been used as permanent grassland.

Where the infested zone has been used as permanent grassland, the measures may only be revoked where no signs of infection with the specified pest have been discovered in soil samples which were taken by applying the scheme to be used to obtain the soil for the testing set out in point 1.2;

or

1.2.After a minimum of 20 years, since the last detection of the specified pest, if there is a gapless record of crops showing that the provisions of Article 6(2) and (3) have been complied with during the whole time and that the infested zone was not used as permanent grassland; and

— no signs of infection with the specified pest have been discovered in two bioassays (as described in point (3) with susceptible potato cultivars; or

— no signs of infection with the specified pest have been discovered in 1 bioassay (as described in point (3) with susceptible potato cultivars and no viable resting spores have been found during a direct examination of the soil from the infested zone by microscope following an extraction of spores with one of the methods provided for in point 2 of Annex I.

The scheme to be used to obtain the soil for the testing shall include all of the following steps:

— the infested zone shall be divided into units of 0,33 ha each;

— 60 subsamples shall be taken from each unit to a depth of 20 cm and evenly distributed throughout the area or pooled according to known infested foci;

— the subsamples shall be thoroughly mixed, so as to obtain 3 samples per ha.

2. Partial revocation of the measures

After a minimum of 10 years since the last detection of the specified pest in areas of the infested zone, the partial revocation of the measures provided for in Article 6 may be considered for these areas, where there is a gapless record of crops showing that the provisions of Article 6(2) and (3), have been complied with during the whole time and that the infested zone was not used as permanent grassland, and:

(a) no signs of infection with the specified pest shall be discovered in two bioassays, as described in point 3, with susceptible potato cultivars; or

(b) no signs of infection with the specified pest have been discovered in one bioassay, as described in point 3, with susceptible potato cultivars and less than 5 viable resting spores per gram of soil have been found during a direct examination of the soil from the infested zone by microscope following an extraction of spores with one of the methods provided for in point 2 of Annex I.

The scheme to be used to obtain the soil for the testing shall include all of the following steps:

— the infested zone shall be divided into units of 0,33 ha each;

— 60 subsamples shall be taken from each unit to a depth of 20 cm and evenly distributed throughout the area or pooled according to known infested foci;

— the subsamples shall be thoroughly mixed, so as to obtain 3 samples per ha.

Where these conditions are not met, the partial revocation of the measures may be considered again following a waiting period of minimum 2 years. In determining the length of that waiting period, Member States shall take into account the level of infection and/or the number of viable spores detected.

3. Bioassays for the purpose of revocation of the measures

Several tubers of the specified plants shall be incubated in pots together with at least 5 l of soil under temperature, moisture and light conditions, which are favourable to potato growth. A cultivar which is highly susceptible to all pathotypes shall be used (such as Deodara, Evora, Morene, Tomensa, Maritiema, Arran Chief).

The growing potato plants shall be cut back when reaching a height of about 60 cm. After approximately 100 days, the newly formed tubers shall be examined for warts.

Negative controls of soil free from the specified pest and positive controls of infested soil must always be included in the test. The test is considered valid, if warts are produced in tubers of the positive control and no warts are produced in tubers of the negative control. Temperature and humidity conditions in the glasshouse shall be recorded. Warts produced in test samples shall be examined microscopically for the presence of summer sporangia and/or resting spores.

The entire test shall be carried out under conditions preventing any further spread of the specified pest.