Microsoft Word - Necrotic Virus Management Plan October 25, 2004.doc

Canada/US-Management Plan for Potato

Viruses that Cause Tuber Necrosis

October 25, 2004

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Plant Protection and Quarantine

4700 River Road, Unit 140

Riverdale, MD 20737

Canadian Food Inspection Agency

Plant Protection Division

59 Camelot Drive

Ottawa, Ontario

K1A 0Y9

TABLE OF CONTENTS

1. Introduction and Goals…………………………………………………….. 3

2. Definitions and Descriptions………………………………………………. 3

3. Virus Management Methods………………………………………………. 6

3.1 PVY Complex……………………………………………………………….. 6

3.2 Potato Mop Top Virus………………………………………………………. 7

3.3 Tobacco Rattle Virus………………………………………………………... 7

3.4 Alfalfa Mosaic Virus……………………………………………………........8

3.5 Shipping Point Inspection …………………………………………………... 8

4. Scoping survey for PVY Complex …………………………………………. 9

5. Research Needs……………………………………………………………… 9

6. Training needs ……………………………………………………………… 10

7. Implementation …………………………………………………………….. 10

8. Revision ……………………………………………………………………... 10

9. Endorsement …………………………………………………………….….. 10

10. References …………………………………………………………...……. 11

Appendices ………………………………………………………………….… 12

Appendix 1 - North American PVY Strain Survey – Crop Year 2004

1. Introduction and Goals

The Canadian Food Inspection Agency (CFIA) and the U. S. Department of Agriculture

Animal and Plant Health Inspection Service (APHIS) announced, on December 23, 2002,

that a joint potato virus management plan will be developed to maintain high quality seed

potato production.

This plan proposes immediate measures to manage the risks associated with several pests

that occur in both countries, including Potato mop top virus (PMTV), Potato virus Y

(PVY) complex and Tobacco rattle virus (TRV), through seed potato certification

measures. Through surveys and research, data will be collected and evaluated to better

understand the biology of these viruses and their vectors, thus enabling the establishment

of further control measures.

CFIA and APHIS are working cooperatively to protect each country's agricultural and

environmental resources and agree to take actions that are least disruptive to trade.

CFIA and APHIS also agree to work collaboratively with respective seed potato

certification agencies and potato organizations to harmonize and make transparent, seed

certification procedures to control viruses causing internal tuber necrosis. Successful

management of viruses that cause internal tuber defects and their respective vectors will

contribute to maintaining high seed potato phytosanitary quality and minimize impacts on

commercial potato production.

This plan replaces the previous PVY

management plan dated March 2, 1994 and

amended October 5, 2001. The objective of this plan remains to facilitate trade of

potatoes within and between Canada and the USA, while minimizing the impact on the

tobacco industry caused by the tobacco-necrotic isolates of PVY infecting the potato

crop. Additionally this plan has the objective to manage the incidence of PMTV and

TRV and minimize the impact of these viruses on US and Canadian potato industries.

2. Definitions and Descriptions

Crop:

A variety and class of seed potatoes, growing in an aseptic environment, a protected

environment or in one or more fields of a farm unit.*

Farm Unit:

a. A single tract of land, operated for the production and marketing of seed potatoes

under the control of one grower, or

b. A number of separate tracts of land, operated as a single unit, with the use of common

equipment, facilities or storage, for the production and marketing of seed potatoes under

the control of one grower.

Field:

Identifiable area of land on which seed potatoes of a particular variety and class are

planted or have been produced.

Flush-Through Production System:

System of seed potato production in which material is allowed to remain in the system for

a limited number of generations.

Inspection:

Official visual examination of plants, plant products or other regulated articles to

determine if pests are present and/or to determine compliance with phytosanitary

regulations.* This includes visual examination as part of a federally recognized quality

assurance program.

Lot:

A population of certified seed potatoes of one variety and class that is identified by one

certificate number.

Nuclear material:

Plant material produced in an aseptic or protected environment.

Positive Lot, Crop or Field:

A lot, crop or field, where the presence of a pathogen has been confirmed, based on

recognized tests.

PVY Complex:

Potato virus Y (PVY) is the type-species of the Potyvirus genus. It is a positive strand

monopartite RNA virus that infects primarily solanaceous plants including potato,

tomato, tobacco, and pepper. PVY occurs as a complex of virus strains that can be

distinguished by reactions on tobacco and potato cultivars. Recognized strains covered

by this plan include, but are not limited to:

Strain Group Symptoms Other Distinguishing

Characteristics

PVY

Mosaic, leaf and stem-

necrosis, leaf drop or veinal

necrosis in potato

Mosaic in tobacco, mild to

severe mosaic in potato;

hyper sensitive response in

potato with Ny and Nc

genes

PVY

Mosaic or mild mosaic in

potato, veinal necrosis in

tobacco

Serologically

distinguishable from

PVY

O/C

; coat protein

sequence phylogenetically

distinct from PVY

O/C

PVY

NTN

note: may include several

distinct isolates/groups

Mosaic or veinal necrosis in

tobacco, necrotic arcs and

ring spots in tubers of some

potato cultivars

Serologically

distinguishable from

PVY

O/C

; coat protein

sequence phylogenetically

distinct from PVY

O/C

PVY

N:O

Symptoms may vary. Some strains are not

serologically or

phylogenetically distinct

from PVY

O/C

Quality Assurance System: A planned and regularly monitored framework of controls,

based on documented procedures, which is applied to critical operational activities in the

production of potatoes or the performance of a service (e.g. diagnostics) or activity (e.g.

conducting a test) to ensure operational efficiency and reliable compliance with specified

quality standards. (NAPPO RSPM 3, 2002)

Shipping Point Inspection:

Inspection of seed potatoes shortly before or at time of shipment to determine the product

complies with seed potato grade standards recognized by the US and Canada seed potato

regulatory agencies.

Test:

Official examination, other than visual, to determine if pests are present or to identify

pests*.

Tuber Necrosis:

Necrotic dark brown arcs, rings and/or flecks in the tuber tissue often associated with

necrotic rings on the tuber surface. Tuber necrosis is also referred to as spraing

depending on the virus.

* FAO Glossary of Phytosanitary Terms, 2002

3. Virus Management Methods

3.1 Potato Virus Y (PVY) Complex

3.1.1 Nuclear Material

In-vitro material

 All plants in a tissue culture bank must be tested and found free of PVY.

 Any material introduced into tissue culture must be tested and found free of PVY.

 Any tissue culture material testing positive for PVY cannot be used to produce

certified seed potatoes, unless the virus is eliminated.

Protected Environment (greenhouse / screenhouse/growth chamber)

Certification rules concerning production in protected environment must include the

following

 Seed potato production in a protected environment will be tested at least annually

for PVY at a target rate of 1% of the plants, with a minimum of 5 plants per

crop/lot. It is recommended that testing takes place every crop cycle.

 Any crop/lot grown in a protected environment that tests positive for PVY must

be destroyed, unless it is found to be PVY

alone, in which case it can be

downgraded.

 If aphid vectors are detected in the protected environment, when the crop is

growing, testing must be conducted to determine freedom from PVY.

3.1.2 Field Grown Seed

 Inspection for PVY complex symptoms will be included in seed certification

agencies’ inspection procedures.

 A sample of suspect plants observed during field inspection will be tested to

determine if the symptoms are caused by PVY. :

If PVY presence is confirmed

the tolerances for mosaic must be enforced.

3.1.3 Field Generation 2

All Field Generation 2 (FG2) material will be sampled and tested for the presence of

PVY and PVY

. A total of 400 leaves will be collected and tested from up to 10 seed

lots/crops per farm unit. The collection of the 400 leaves will be proportional and

random across the crops or lots to be tested.

 Tolerances of the state and federal certification programs apply.

 If a farm unit has less than 10 FG2 crops/lots the sample size for the farm will still

be 400 leaves.

 If the farm unit has more than 10 FG2 crops/lots an additional sample of 400

leaves will be submitted for testing from every group of 10 lots or less.

 If crops/lots are found positive for PVY

, all FG2 crops/lots on that farm, and

lots/crops that are progeny of the same seed lot as the positive crops/lots, must be

sampled and tested at a rate of 400 leaves per lot.

 Regulatory action will only be taken on FG2 crops/lots. PVY

positive crops/lots

of FG2 can only be used for the production of commercial crops, not for

recertification.

The testing of FG2 crops/lots may be done either during the growing season or post

harvest. If done post-harvest, leaves from grown-out plants or sprouts from tubers must

be used.

 Leaf testing will be done by ELISA using an appropriate protocol. An ELISA

positive for PVY

will be considered a suspect positive. Confirmatory tests must

be conducted with PCR and/or bioassays.

3.2 Potato Mop Top Virus (PMTV)

3.2.1 Nuclear Material

 All plants in a tissue culture bank must be

tested and found free of PMTV.

 Any material introduced into tissue culture must be tested and found free of

PMTV.

 Any tissue culture material testing positive for PMTV cannot be used to produce

certified seed potatoes, unless the virus is eliminated.

3.2.2 Field Grown Seed

 Inspection for PMTV related symptoms will be included in seed certification

agencies’ inspection procedures. Training material on PMTV symptoms in plants

will be provided to all field inspectors.

 Plants symptomatic for PMTV must be eliminated before final inspection.

 PMTV will also be regulated through the inspection of seed shipments.

3.3 Tobacco Rattle Virus (TRV)

3.3.1 Field Grown Seed

 Inspection for TRV related symptoms will be included in seed certification

agencies’ inspection procedures. Training material on TRV symptoms in plants

will be provided to all field inspectors.

 Plants symptomatic for TRV must be eliminated before final inspection.

 TRV will also be regulated through the inspection of seed shipments.

3.4 Alfalfa Mosaic Virus (AMV)

 Tubers exhibiting necrosis caused by AMV will be scored as an internal defect

and be allowed for recertification providing the tolerance of 2.0% for internal

necrosis is not exceeded.

3.5 Shipping Point Inspection

3.5.1 Seed for Commercial Production

 All seed shipments moving from a production State or Canada for commercial

production must be inspected by federally recognized inspectors or a federally

authorized cooperator from a facility under a quality assurance system and must

meet the tolerances for internal necrosis of Table 1 below. If a lot exceeds this

tolerance, the grower will have the option to re-grade the lot or remove it from the

seed certification system.

3.5.2 Seed for Recertification

 All seed shipments moving from a production State or Canada for recertification

must be inspected by a federal, state or provincial inspector for tuber necrosis. If a

lot exceeds the tolerance of 0.5% necrosis caused by PVY complex, PMTV or

TRV (see Table 1 below), it is not eligible for recertification but may be shipped

for commercial purposes.

Table 1: Eli

ibilit

of Seed potatoes related to the Percenta

e of Internal Tuber

Necrosis, apparently caused by PVY complex, PMTV or TRV

Percentage Internal Tuber

Necrosis Scored*

Seed intended for

Recertification

Seed intended for

Commercial Production

Less than or equal to 0.5%

Eligible Eligible

Between 0.5 and 2.0% Eligible if negative lab test

for PVY

N/NTN

, PMTV,

TRV

Eligible

More than 2.0%

Not eligible Not eligible

Notes:

* Internal tuber necrosis will be calculated as percent incidence {(number of tubers

with internal necrosis/number of tubers sampled) x100}.

4. Scoping Survey for PVY Complex

A multi year scoping survey, starting with the 2004 crop will be conducted, coordinated

by APHIS and CFIA, in each state and province, where seed potatoes are certified. The

seed lots of the oldest classes in production will be surveyed to determine the incidence

and makeup of any PVY complex at that point in the seed potato production system. The

data will be kept confidential and be reviewed annually on a country-wide basis. The

data will be used confidentially to help determine if the FG2 management protocol is

effectively managing virus levels of PVY complex. No regulatory action will be taken

on lots found to be positive in this survey. PVY certification requirements will not be

changed without proper notification. See Appendix 1 for details of the survey and testing

protocols.

5. Research Needs

Both parties acknowledge the need for and value of multiple virus testing protocols.

Such protocols should be encouraged as a research goal and new protocols incorporated

into the plan when available.

PVY complex

 Research on the susceptibility of US and Canadian potato varieties to PVY

N/NTN.

 Research on recombinants between PVY

and PVY

to develop appropriate tests

for these and any other PVY

isolates.

 Research on the population dynamics and prevalence of recombinant strains to

determine if changes in the population of strains are occurring.

 Research on environmental factors that influence symptom expression to develop

a better understanding of PVY strain symptoms.

PMTV

 Research on susceptibility to PMTV to determine if the pathogen causes an

economic impact on US and Canadian potato varieties. If any varieties are shown

to be extremely sensitive to PMTV, a specific management plan for those

varieties should be developed.

 Research on the control of powdery scab since control of the vector will lead to

control of PMTV.

 Research on improved testing methodologies for PMTV to improve the detection

of the pathogen.

TRV

 Research on improved testing methodologies for TRV to improve the detection of

the pathogen.

 Research on the control of the TRV vector since control of the vector will lead to

control of TRV.

 Research on the efficiency of the nematode vector to acquire the virus from

infected potato plants to determine a better understanding of the dynamics of the

vector/virus interaction.

6. Training needs

 Inspectors involved in shipping point and field inspection of seed potatoes will be

trained annually by state, federal, provincial and/or university officials on

symptoms of PVY

N/NTN

, PMTV and TRV in tubers and plants to assist them in

their inspection efforts.

 APHIS and CFIA will develop a manual to differentiate spraing symptoms from

other causes of internal necrosis.

 An appendix of pest management practices for controlling viruses addressed in

this plan and their vectors will be added upon completion.

7. Implementation

 It is essential that APHIS and CFIA participate in the implementation of this plan,

and oversee the application of all of its elements. Seed potatoes produced and

certified by a federal or state certification agency must comply fully with the

content of the management plan.

 It is understood that some elements of the management plan will require

amendments to regulations, rules, policies and inspection manuals. Therefore,

every certification agency, including CFIA and APHIS, must make the necessary

amendments to achieve full implementation of the plan within 12 months from the

date of signature.

8. Revision

 This plan will be reviewed after three years by APHIS and CFIA or as needed at

the request of either party. Results of all reviews and any recommended

modifications to this plan will be shared with federal, state and provincial

regulatory officials, the potato industry and the tobacco industry for comment.

9. Endorsement

The Canadian Food Inspection Agency and the United States Department of

Agriculture Animal and Plant Health Inspection Service agree to full

implementation of this management plan with an ultimate goal of using existing

seed certification programs to control viruses that cause tuber necrosis.

CFIA USDA APHIS

Date: Date:

10. References

NAPPO PVY

Testing Protocols

Superficial Necrosis, caused by Virus (PTNRD), UN/ECE paper,

TRADE/WP.7GE.6/2004/12,

Appendices

- Scoping Survey for PVY Complex

Appendix 1

Scoping Survey for PVY Complex

1. Purpose: To conduct a scoping survey to assess the presence of different strains of

PVY and their incidence in seed potatoes in Canada and the United States. The

specific objectives of the survey are:

a. to estimate the relative incidence of each strain or novel isolate of PVY (PVY

PVY

, PVY

N:O

, PVY

EU-NTN

, PVY

NA-NTN

, and others),

b. to assess the correlation between strain type and symptom expression in potato

2. Principles: To ensure uniformity and comparability of survey results, the survey will

be conducted in both Canada and the United States to conform to the following

principles:

a. every seed producing state/province will be sampled,

b. the survey will include as many commercial potato cultivars as possible,

c. every seed farm in all seed producing states/provinces will be sampled,

d. older field generations of seed potatoes on each farm will be sampled,

e. the sampling rate in each state/province will be between 100 to 400 tubers per lot.,

f. the number of samples taken in Canada and the US will be proportional to the size

of their respective seed potato industries, estimated to require about 80,000 tubers

for Canada and 100,000 tubers for the US.

g. the survey will utilize an ELISA test (see below) for initial detection of PVY in

tuber sprouts,

h. samples will be preserved to allow subsequent strain characterization,

i. testing will be done on samples representing the 2004 crop and subsequent crops,

j. samples will be collected randomly within the seed potato lots selected for testing,

k. results of the survey will be reviewed by September 1, 2005 and subsequent

years,

l. confidentiality of individual grower results will be maintained.

3. Procedure: The survey will be conducted in three stages of testing.

a. PVY-positive tubers or plants will be selected on the basis of an initial ELISA test

using monoclonal 4C3. every seed producing state/province will be sampled,

b. All 4C3-positive tubers/plants will be retested by ELISA using monoclonal 1F5 to

determine whether the serotype is PVY

or PVY

c. The P1 gene of PVY will be amplified by RT-PCR from all 4C3-positive

tubers/plants and sequenced to determine subtype. Selected PVY isolates

representing each of the recombinant and strain types will be observed or assayed

for symptomology on potato foliage and tubers. The following criteria for the

testing procedures have been adopted to ensure uniformity and comparability of

results.

4. Sample preparation (although the survey can be conducted using either tuber

sprouts or leaves of grow-out plants, testing of tuber sprouts appears to be most

feasible and economical).

a. any method (natural or chemically induced) of breaking tuber dormancy is

acceptable,

b. sprouts must be taken from the bud end of the tuber,

c. one to three sprouts will be tested from each tuber for each test,

d. sprouts must be at least 0.5 cm but not more than 6.0 cm long,

e. sprouts from up to three tubers may be combined into a composite sample for the

initial ELISA test,

f. a fresh sprout extract will be used for the second ELISA test,

g. either a third sprout or sap from the second sprout will be used for RT-PCR

amplification of the PVY P1 gene,

h. sprout sap to be used for RT-PCR amplification must be kept on ice and used

within 24 hr.

5. ELISA.

a. the initial ELISA test to select PVY infected tubers will utilize monoclonal

antibody 4C3,

b. the second ELISA test to determine PVY serotype will utilize monoclonal

antibody 1F5,

c. the ELISA tests are to be a triple antibody sandwich (TAS) format,

d. coating antibody for both ELISA tests will be a polyclonal antibody that reacts

with known PVY strains,

e. duplication of test wells is NOT required,

f. each ELISA plate requires a minimum of one control well containing a PVY

isolate, one control well containing a PVY

isolate, and four negative control

wells containing sap from PVY-free sprouts,

g. all negative control wells must have absorbance values of <0.100 for the results of

the ELISA plate to be considered valid (the ELISA plate should be blanked on a

well or wells containing buffer only),

h. the positive/negative threshold will be 0.100 unless the mean of the negative

controls is >0.033 in which case the threshold will be 3X the mean of the negative

controls,

6. RT-PCR amplification and sequencing

a. sprout sap will be combined with buffer and spotted in quadruplicate on

nitrocellulose membranes as specified in the dot blot procedure,

b. RNA will be extracted from dot blots and cDNA generated by reverse

transcription using Primer A (Nie and Singh, 2002),

c. cDNA will be amplified by PCR using Primers S1 and A (Nie and Singh 2002)

for 30-35 cycles,

d. sequencing of amplicons will be contracted to a sequencing facility to obtain

uniform and consistent results,

e. P1 gene sequences will be aligned and analyzed using standard bioinformatics

protocols will be determined.

Reference: Nie, X. and Singh, R.P. 2002. A new approach for the simultaneous

differentiation of biological and geographical strains of potato virus Y by uniplex and

multiplex RT-PCR. J. Virol. Methods 104:41-54.