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D- 97-12: Seed Potato Certification Program – Bacterial Ring Rot Testing Program for Field Grown Seed Potatoes

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Effective Date: July 6, 2011
(5th Revision)

Subject

This directive contains the guidelines for required testing of field-grown seed potatoes pursuant to the Seeds Regulations Part II against Clavibacter michiganensis subsp. sepedonicus (Spieckermann & Kotthoff 1914) Davis, Gillaspies, Vidaver & Harris 1984 (C.m. sepedonicus), the causal organism of Bacterial Ring Rot (BRR) in potatoes. In particular, it covers timing of sampling, the sample size, and the extent to which laboratories can combine samples for testing.

Changes made during this revision were administrative in nature as a result of regular periodic review.

Table of Contents

Review

This directive will be reviewed every three years unless otherwise needed. For further information or clarification, please contact the Canadian Food Inspection Agency (CFIA).

Endorsement

Approved by:

space to insert name
Chief Plant Health Officer

Amendment Record

Amendments to this directive will be dated and distributed as outlined in the distribution below.

Distribution

  1. Directive mail list (Regions, PHRA, USDA)
  2. Provincial Government, Industry (as determined by author)
  3. National Industry Organizations (Canadian Horticulture Council)
  4. Internet

Introduction

In 1997, a regulatory change was introduced setting new standards for testing for the presence of Clavibacter michiganensis subsp. sepedonicus, the causal agent of Bacterial Ring Rot (BRR) in potatoes. As a result, a minimum of two seed lots (even if only seed classes of Pre-Elite, Elite I or Certified are grown/produced on the farm unit), including all seed lots shipped as Elite-II, Elite-III, Elite-IV and Foundation classes, must be tested for C. m. sepedonicus on each farm unit. Directive D-97-12 (Original, dated July 31, 1997) was developed to provide details on seed lot selection and sampling procedures, including: determination of sample size, details related to tuber or stem sampling, packaging and shipping, and procedures for the combination of samples by the laboratory during testing.

The directive D-95-18 : "Seed Potato Certification Program - Investigation Procedure after Clavibacter michiganensis subsp. sepedonicus has been detected on a Seed Potato Farming Unit" should also be referenced for the appropriate investigation procedures following the detection of C.m. sepedonicus on a farm unit.

It is important to note that the probability of detection of C.m. sepedonicus depends on several variables including the sample size, sample collection, and disease incidence. For example, the probability of detecting C.m. sepedonicus at an incidence of 0.1% is approximately 33% in a sample of 400 tubers or stems and approximately 70% in a sample of 1,200 tubers or stems.

This directive deals exclusively with testing required for field grown seed potatoes. The Seeds Regulations Part II also requires specific C.m. sepedonicus testing for nuclear stock class material, and its equivalent. The Plant Protection policies regarding these are covered in two other Directives:

Scope

This directive outlines the guidelines for sampling field grown seed potatoes and testing for C.m. sepedonicus required to determine the absence of C. m sepedonicus and is intended for the use of the CFIA inspection and laboratory staff, private laboratories approved by CFIA, and growers.

References

This directive supersedes D-97-12 (4th Revision).

Definitions, abbreviations and acronyms

Definitions for terms used in the present document can be found in the Plant Health Glossary of Terms.

1.0 General Requirements

1.1 Legislative authority

The Plant Protection Act, S.C. 1990, c.22

The Plant Protection Regulations, SOR/95-212

Seeds Act R.S., c.S-8 and amendments 1976-77, c.28 and 1985, c.47.

Seeds Regulations and their amendments, SOR/91-526, SOR/93-331, SOR/95-179, SOR/95-215, SOR/97-118, SOR/97-292, SOR/2000-183, SOR/2000-184 and SOR/2002-198.

1.2 Regulated pests

Clavibacter michiganensis subsp. sepedonicus, the pathogen causing BRR in potatoes.

1.3 Regulated Commodities

Field grown seed potatoes (Solanum tuberosum)

2.0 Specific Requirements

2.1 Regulatory requirements

All seed potato lots, except for Pre-Elite, Elite I, and Certified classes, that are shipped from a farm unit must be tested in an approved CFIA laboratory and found not to be infested with C.m. sepedonicus. Furthermore, a minimum of two lots per farm unit (except where only one lot was produced on the farm unit), intended for planting, must be tested. The testing can be done either on stems sampled before harvest (in a field), or on tubers collected from the lot prior, during or after harvest.

2.1.1 Shipping and issuance of documentation

C.m. sepedonicus testing must be conducted on at least two lots from a farm unit and any seed lot shipped as Elite II, Elite III, Elite IV or Foundation class before an inspector can proceed with the issuance of Seed Potato Tags, Record of Bulk Movement for Seed Potatoes, Certificate of Authorization, or equivalent. The grower is responsible to maintain a copy of all C.m. sepedonicus laboratory testing results for his farm unit.

A grower may be allowed to ship seed potatoes at the Pre-Elite, Elite I and Certified classes without any further testing for C.m. sepedonicus, only if the minimum testing of two seed lots has been completed and they were found not to be infested with C.m. sepedonicus.

2.1.2 CFIA inspector and direct supervision

In some circumstances, sample collection may be required to be conducted by CFIA inspectors or under their direct supervision. However, in most circumstances growers are responsible to properly collect and identify samples submitted to CFIA-approved laboratories.

Direct supervision means that the CFIA inspector must be present on-site and witness that all required tubers and stems are collected randomly, and are representative of the identified seed lot.

2.2 Choice of lot(s) or crop(s) to be tested, when necessary

When no lots on the farm unit (or only one) are shipped as seed, a requirement exists to test a minimum of two lots per farm unit to meet seed grower's eligibility requirements under the Seeds Regulations. Seed lot selection should be discussed between the grower and the inspector, but the final decision is made by the CFIA inspector. The selection is made by taking into consideration the following:

Note: Occasionally, only one seed lot may have been produced by a farm unit, therefore it would be the only lot requiring sampling and testing, at the specified rate to maintain grower's eligibility under the Seeds Regulations.

3.0 Sampling procedure

Except as otherwise specified, samples are to be collected by the grower as instructed by a CFIA inspector. Stems or tubers for sampling must be selected at random to provide an unbiased representative sample of the crop or lot. Samples from each crop or lot must be submitted to the laboratory in separate, closed bags and should be individually labelled as prescribed in section 3.7.

Each sample collected by, or under the direct supervision of, a CFIA inspector to meet a specific country's import requirements, or to meet the specifications of directive D-95-18, must be sealed by a CFIA inspector. Sample integrity must be maintained from the time of collection to the time it is delivered to the laboratory. Representatives from CFIA laboratories and CFIA-approved laboratories will reject, upon delivery to the laboratory, any samples with a broken seal.

All samples taken by a CFIA inspector or under his direct supervision and sent to a CFIA laboratory must be entered into the Laboratory Sample Tracking System (LSTS) prior to submission.

3.1 Sample size

Table 1: Sample sizes for C.m. sepedonicus testing.
Field (crop/lot) sizeSample size
Less Than 0.025 ha 1%; minimum 5, maximum 50 tubers
0.025 ha to less than 1.000 ha 100 stems or tubers
1.000 ha to less than 4.000 ha 200 stems or tubers
4.000 ha to less than 40.00 ha 400 stems or tubers
40.00 ha or more 800 stems or tubers

Note: under the intensified testing regime a minimum of 1000 tubers or stems is required for crops grown in fields of 1 ha or greater.

3.2 Testing regimes

The required testing regimes will differ depending on the disease status and history of the farm unit.

The Normal Testing Regime may not, in all instances, meet the phytosanitary import requirements of a foreign country. Therefore, for export phytosanitary certification purposes, extra sampling and testing for C.m. sepedonicus may be required to fulfill the importing country's specific requirements.

3.2.1 Normal testing regime: farm unit with no history of C.m. sepedonicus

The minimum sample size required is dependent on field size. The size of samples, i.e. number of stems per crop, or number of tubers per lot (field sample size) required is a function of the field(s) size. Field sample sizes are given in Table 1.

3.2.2 Normal testing regime: new seed potato growers

During the first two years that a new grower enters into the seed potato certification program, sample collection following the Normal Testing Regime (section 3.2.1) must be done by CFIA inspectors or under their direct supervision. This allows inspectors to instruct the grower on proper collection of samples and to monitor that sample collection follows proper procedures. Following the first two years, periodic audits may be conducted by the CFIA to ensure that appropriate procedures continue to be followed.

3.2.3 Intensified testing regime: farm unit where C.m. sepedonicus has been detected within the past 6 years

Due to the increased probability of recurrence of the disease on farm units where C.m. sepedonicus has been recently detected, intensified monitoring of sample collection, as well as increased sample size, is required to gain confidence that C.m. sepedonicus has been eradicated.

Sample collection must be done by CFIA inspectors or under their direct supervision, for at least 6 years following the detection of C.m. sepedonicus on a farm unit.

This includes the first three years under the Intensified Testing Regime, followed by three years under the Normal Testing Regime.

Under the Intensified Testing Regime, a minimum of 1,000 stems or tubers are required from each crop(s) or lot(s), irrespective of the class, produced in a field of 1 hectare or more, by the identified farm unit. For fields smaller than 1 hectare, the sample sizes specified in Table 1, Normal Testing Regime, should be followed.

Samples taken outside the normal testing regime will be submitted to the CFIA Charlottetown Laboratory at no cost to the grower. See additional explanations under section 3.7.

3.2.4 Voluntary sampling and testing by growers to meet directive D-95-18 requirements

Growers have the option of testing more stems or tubers than specified in this directive. Those wishing to conduct sampling and testing at a higher rate and at their own expense are encouraged to proceed accordingly. It should be noted that if a situation occurs whereby a grower is included in an investigation as specified in directive D-95-18, any previously conducted voluntary sampling and testing will only be officially recognized and considered valid if:

Results from any additional testing carried out at a grower's own initiative will be reviewed at the time an investigation under directive D-95-18 is launched, and assessed for their acceptability. Seed lots with results considered as meeting or exceeding the requirements specified in D-95-18 should not have to undergo any additional sampling and testing and be withheld from shipment due to confirmation of C.m. sepedonicus on another farm unit.

3.3 Stem sampling

Stem samples can be collected only after the potatoes have been grown for a minimum number of days. This minimum is equal to at least 75% of the number of days to maturity (maturity varies depending on the variety; consult Table 2 and Appendix 1 for more information) or 90 days of growth.

For example, a field planted June 1 (day 152 in 2009) with a variety for which the expected maturity is 100 days (i.e. maturity on September 8, day 252) would be eligible for sampling 75 days after planting ((252 - 152 = 100) X 0.75 = 75 days), which occurs on August 14 (day 227; 152 + 75). Knowing that many factors can affect the number of growing days of a particular crop, this rule is given as an indication to help in determining a reasonable starting date for sampling. It is the responsibility of the inspector to approve the earliest date a field would be eligible for stem sampling.

Table 2 may also be used to estimate an approximate number of growing days required for the variety to attain maturity before stem sampling can be initiated. The specific number of days after planting but before sampling for some varieties grown in Canada are given in Appendix 1. If a variety is not listed in Appendix 1 and the maturity is not easily found in the scientific literature, then the number of days after planting should be based on the longest maturity period.

Table 2: Sampling time for Stems
Variety maturity Table Note *Days to maturity Table Note *Days after planting before sampling
very early - first early 65 - 70 55 Table Note **
early - second early 70 - 80 60
medium early 80 - 90 65
early mid-season 90 - 100 75
mid-season 100 -110 80
medium late 110 - 120 85
late 120 - 130 90
very late 130 - 140+ 90

Table Notes

Table note *

Extracted from: Potato Varieties in Canada, NB Dept. of Agriculture, ISBN 0-888-38-834-9, 1997.

Return to table note *  referrer

Table Note **

Varieties harvested before 65 days must be sampled after the field has been grown for at least 75% of the intended number of growing days.

Return to table note **  referrer

Even if a crop in the field identified for stem sampling has reached the minimum number of growing days, the crop must still be in good growing condition to undergo stem sampling. When crops are in advanced senescence or have been exposed to herbicides, or adverse weather conditions, a number of bacteria and fungi are likely to be invading the stems which can interfere with the detection of C.m. sepedonicus.

Therefore, a crop sprayed with a desiccant (top-kill), exposed to frost, or in an advanced stage of maturity (more than 25% of the stems are dead or dying), is no longer appropriate for stem sampling to be conducted and must undergo tuber sampling.

Only one stem section is to be sampled per plant (each stem section coming from a different plant). Stem samples must be selected at random, in an attempt to obtain a sample which is representative of the entire field.

Several fields that are intended to be part of the same lot can be combined into one sample. The total area of all the fields is then used to determine the sample size (three individual one hectare fields = a three hectare field). However, if fields are down graded in class in the second field inspection leading to a change in total sample area. Then the sample size will have to be adjusted to represent the new total area.

The stem section submitted for testing must be approximately 1.0 cm long and sampled directly above the soil level (Diagram 1). Each stem section must weigh between 0.5 - 1.0 g. The process can be described as follows: one main stem of the plant is pulled up, taking care to leave the tubers in the soil; the excess soil is then removed from the base; with pruning-scissors, the base of the stem is then cut off at the original soil level (where the green pigment starts); the last step is to cut a segment of approximately 1 cm in length off the end of the stem (which should then be green) and place it into an appropriate bag (the length of the segment should be adjusted, depending on the diameter of the stem, to get approximately 0.5 - 1.0 g of stem tissue). It is important that the stem section is green. Non-pigmented segments from below the soil level are very difficult to prepare for testing and must be removed. The samples must be within the 0.5-1.0 gram size otherwise they will be trimmed at the lab adding additional costs to the testing.

Diagram 1: How to collect a stem section.

Click on image for larger view
How to collect a stem section - description above

3.4 Tuber sampling

Tubers are best collected at the time they are harvested or brought into storage. Acceptable tuber samples are those collected when all of the tubers have an equal chance of being sampled and are representative of the entire lot. Samples should include a specific number of tubers collected from every load going into storage. Samples from every row, or a set number of rows in the field, or collection of the required number of tubers from those left on the ground once a field is harvested, are all acceptable sampling options. Once the tubers are in a storage bin, it is very difficult to collect a representative sample of the lot, therefore samples should be collected prior to storage.

For tuber samples, whole tubers or tuber cores may be submitted directly to the laboratory. If cores are shipped to the laboratory, original tubers must be kept by the grower in separate, labelled (so it can be linked to a lot) and sealed containers until test results are available.

Cores must be taken at the stolon attachment site and must be conical or semi-spherical in shape, approximately 1 cm in diameter at the top and 1 cm deep (see diagram 2). Each core should weigh approximately 0.5 - 1.0 g, and include as much of the vascular ring radiating from the stolon attachment as possible.

Diagram 2: How to take a core from a tuber.

Click on image for larger view
How to take a core from a tuber - description above

3.5 Combination of samples

Because follow-up measures on a C.m. sepedonicus positive sample are taken on a crop or lot basis, samples from each crop or lot must be submitted to the lab in a closed, separate bag, and individually labelled (so it can be linked to a lot or crop).

Each sample received by the laboratory is logged separately according to the certification number. However, according to official protocols followed by the laboratories, samples may be combined for testing purposes.

The maximum number of cores or stems that may be combined by the laboratory for testing purposes is 200 (bags containing more than 200 cores will be subdivided).

Tubers/stems samples from different farm units may not be combined into one laboratory sample.

Since options are available for sample submissions to the lab, it is advisable to contact the lab manager to make appropriate arrangements.

3.6 Positive samples

Backup material (tubers already cored and stored by the laboratory or by the grower) may be used to investigate positive samples to determine disease source.

In the case of stems, as no backup material exists, if further investigation is required, new samples must be taken if available.

3.7 Sending samples to a CFIA-approved laboratory and to the CFIA Charlottetown Laboratory

The number of stems or tubers required under the Normal Testing Regime should be sent by the grower to a CFIA-approved laboratory for testing.

Any additional stem or tuber samples required beyond the Normal Testing Regime (for farm units where C.m. sepedonicus has been detected within the past 3 years, as per the Intensified Testing Regime, are to be forwarded by a CFIA inspector, at no cost to the grower, to the

CFIA Charlottetown Laboratory
93 Mount Edward Road
Charlottetown, PEI C1A 5T1

Note: Testing done in anticipation of being part of a C.m. sepedonicus investigation carried out at a grower's own initiative must be done in a CFIA-approved laboratory and at the grower's own expense.

When samples are taken under the Intensified Testing Regime, efforts should be made to avoid dividing samples between a CFIA-approved laboratory and the CFIA Charlottetown Laboratory. However, it is recognized that often at least one sample of a given farm unit will need to be divided. The following procedure is recommended in determining where the samples should be sent:

  1. Determine the total number of stems or tubers that would be collected as per the Normal Testing Regime.
  2. Determine the additional number of stems or tubers to be collected from each lot as per the Intensified Testing Regime. In consultation with the grower, select a number of lots for which the total number of samples is equal to or greater than the Normal Testing Regime total. These samples (possibly including a part of one lot) are submitted to a CFIA-approved laboratory by the grower at their own expense. These samples must previously have been sealed by the CFIA inspector.
  3. The CFIA inspector sends the remaining samples to the CFIA Charlottetown Laboratory at no cost to the grower.

For clarity, an example is presented in Table 3.

Table 3: Comparison of sample size and lab destinations between the Normal

Testing Regime and the Intensified Testing Regime

Sending samples to a CFIA-Approved laboratory and to the CFIA Charlottetown Laboratory
Area (Ha)Normal Testing Regime
Sample Size
Normal Testing Regime
Lab Destination
Intensified Testing Regime
Sample size
Intensified Testing Regime
Lab Destination
2.3 200 CFIA-Approved laboratory 1000 CFIA-Approved laboratory
1.7 200 CFIA-Approved laboratory 1000 800 to CFIA-Approved laboratory
200 to the CFIA Charlottetown Laboratory
4.8 400 CFIA-Approved laboratory 1000 CFIA Charlottetown Laboratory
0.7 100 CFIA-Approved laboratory 100 CFIA Charlottetown Laboratory
0.3 100 CFIA-Approved laboratory 100 CFIA Charlottetown Laboratory
42.2 800 CFIA-Approved laboratory 1000 CFIA Charlottetown Laboratory
Totals 52 ha 1,800 1,800 to CFIA-Approved laboratory 4200 1,800 to CFIA-Approved laboratory

2,400 to the CFIA Charlottetown Laboratory

Note: Unless specified otherwise, additional samples required for the purpose of meeting the import requirements of a foreign country must be sent to a CFIA-approved laboratory for testing and associated testing costs are the grower's responsibility.

3.8 Packaging and shipping

To ensure sample continuity from a specific crop or lot to the laboratory it is crucial to follow proper packaging, shipping, and identification procedures. If sample integrity is in question, the sample will be discarded, and a new sample must be submitted.

When more than one tuber, core or stem sample are shipped in the same container, a complete content list of samples submitted must be placed on the top of each shipping container or with the bill of lading. This list must be signed by the collector.

To ensure sample continuity, packages must be properly sealed so that they cannot be tampered with or opened during transit without the laboratory being aware of this upon arrival of the samples. If sample integrity is in question, the sample will be discarded and a new sample must be submitted.

When outside temperatures are anticipated to drop below freezing (0°C), freezing of the samples must be prevented. Laboratory staff will reject any samples showing signs of freezing.

3.8.1 Tubers

Tubers must be as dry as possible before packing. If tubers are sent in bags, tags bearing proper identification of the sample must be present both inside, and attached to the outside of the bag (on top of the tubers). This is in order to identify the sample in case the outside tag is lost or damaged during transportation.

3.8.2 Cores and stems

Cores and stems, dried and wrapped in paper towels, may be kept in cold storage (4°C) for a maximum of 14 days before processing by the laboratory; refrigeration at all times is important. Cores and stems that become decayed during storage will be discarded by the laboratory and a new sample requested.

Cores or stems must be as dry as possible before packing. If using plastic sealable bags, ventilation holes must be present in the surface. Some manufacturers of plastic bags for retail sale (for the purpose of refrigerating vegetables) are now offering finely perforated sealable plastic bags, which can be used very successfully for potato cores or stems. Cores and stems may also be wrapped in paper towels and shipped in plastic bags. Each bag must be properly identified. Bags must be closed and refrigerated as soon as possible, and no more than two hours after coring or sampling (stems). The bags should be kept in cold storage (4°C) overnight and in such a way (i.e., well spread out) that the complete sample is brought down to 4°C before shipping. The sample size indicated on the bags must be correct: the lab will only accept a 2% deviation from this number.

Bags of samples should be packed loosely in insulated cardboard boxes. Ice-packs should be included on top of the samples (as cold air moves downward), but should be sufficiently insulated from the samples so that freezing is avoided.

Ice-packs are effective only for a short period: 24-48 hours depending on insulation. Therefore, shipment of samples must be postponed if the package is likely to be held in transit over a holiday or weekend, except if refrigerated storage is available.

3.9 Identification of samples

Samples from each crop or lot must be submitted in a closed, separate bag, and individually labelled. The following information is required on each label:

Note: Samples not properly identified will not be processed by laboratory staff until correct identification is received. Additionally, any samples presented to a CFIA-approved laboratory with a broken CFIA seal will be rejected due to the possible loss of sample integrity.

Note: The inspector's signature is required if the sample collection is done by a CFIA inspector or under an inspector's direct supervision.

This signature is essential for:

4.0 Laboratory testing

Testing for C.m. sepedonicus under the Normal Testing Regime in Canada is now being done by private laboratories under an Accreditation and Quality Assurance program which approaches ISO 17025 standards, and is administered and audited by the CFIA Charlottetown Laboratory.

All laboratories follow the same official protocols, and their ability to perform these protocols is evaluated on a regular basis. All C.m. sepedonicus positive test results must be confirmed by the CFIA Charlottetown Laboratory.

A list of CFIA-approved laboratories for C.m. sepedonicus testing can be found at the following internet address, under Approved Laboratories in Canada.

5.0 Testing results

For samples with no detection of C.m. sepedonicus, test results are provided to the grower and to the CFIA Regional Officer by the laboratory on a regular basis by fax or e-mail. The information provided to the laboratory by the grower (see section 3.7 for details) should be included.

A list of CFIA Regional Officers, Seed Potato Certification, may be obtained from;

Canadian Food Inspection Agency
Potato Section
Horticulture Division
Plant Health and Biosecurity Directorate
59 Camelot drive
Ottawa, Ontario K1A 0Y9
National Manager's office: Tel: 613-773-7162 / Fax: 613-773-7163

When C.m. sepedonicus is detected by a CFIA-approved laboratory, it is sent to the CFIA Charlottetown Laboratory for confirmation. When this happens, the CFIA Regional Officer must be informed as soon as possible by the CFIA-approved laboratory. The Regional Officer evaluates the situation and decides if it is appropriate to inform the grower immediately. This could be important for example when testing is done on stems, as the grower may decide to delay the harvest of his potatoes, knowing that the seed status of all his lots may be lost.

6.0 Follow-up on laboratory detections

When the CFIA Charlottetown Laboratory has confirmed the presence of C.m. sepedonicus in a sample provided by a CFIA-approved laboratory, the CFIA Regional Officer is informed as soon as possible. It is the responsibility of this CFIA Regional Officer to notify the affected grower. As per the Seeds Regulations, all lots of potatoes on the affected farm unit will be ineligible for certification in the crop year in which C.m. sepedonicus was detected.

An investigation to determine the source of infection is carried out under the supervision of the CFIA Regional Officer. The policy governing this investigation is described in directive D-95-18 "Seed Potato Certification Program - Investigation Procedures after C.m. sepedonicus has been detected on a Seed Potato Farming Unit"

Appendix 1

(see section 3.2 for details)

Sampling time for stems: days after planting (DAP) before sampling, for some potato varieties grown in Canada

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