Legislative/Regulatory

Question to assist you in your review can be found at the bottom of the draft protocol.

Retail Confirmed Nursery Protocol, July 26, 2007
Official Regulatory Protocol for Retail Nurseries (rCNP) Containing Plants Infected with Phytophthora ramorum

United States Department of Agriculture (USDA)
Animal Plant Health Inspection Service (APHIS)
Plant Protection and Quarantine (PPQ)
Center for Plant Health Science and Technology (CPHST)
Emergency and Domestic Programs (EDP)
Eastern Region (ER)
Western Region (WR)

INTENDED USE
Retail nurseries represent a different type of risk from Phytophthora ramorum than nurseries which specialize in propagating and growing plants. The nature of the retail business tends to require that plants are moved more often in order to present them to the public for sale. Plants are not intended to remain on site for an extended period of time and plants do not tend to receive cultural controls like pruning or pesticides on the same frequency as they would during the plant production process.

As retail nurseries are at the end of the production and distribution process, they represent a lower risk in distribution of infected plants to other nurseries and facilities in the plant distribution system. As retail nurseries are the final point in the plant distribution system, they are the last point before infected plants would be moved directly to the environment. It is also important that retail nurseries do not become a point where non-infected plants could become infected on the way to the place of final planting.

GOAL
The purpose and goal of this protocol is to ensure that any infestations of this pathogen are consistently and effectively addressed, mitigated, and eradicated when Phytophthora ramorum is detected in a retail setting. The actions described in the protocol are designed to ensure that any infestations of this pathogen are consistently and effectively addressed and the pathogen eradicated, while minimizing the expected downtime for the retail nursery. Cooperation by nursery management personnel is essential. Early detection and reporting of potential P. ramorum plant infections are crucial, to ensure that spread is contained.

Retail Nurseries found with P. ramorum infestations more than once

P. ramorum infestations in nurseries may be re-introduced, or the effort to eradicate the disease may fail. In the event that a nursery has P. ramorum detected on site after the initial release from the Emergency Action Notification (or state equivalent), it is necessary to implement additional measures to ensure that the risks associated with P. ramorum are properly mitigated (see Appendix 11). The goal is to find and eradicate the pathogen if present in nurseries. Any interpretation of this protocol that is contrary to this goal is a misinterpretation of the protocol.

Definitions

Associated plants: Associated plants are those reported found naturally infected and from which P. ramorum has been cultured and/or detected using PCR (Polymerase Chain Reaction). For each of these, traditional Koch’s postulates have not yet been completed or documented and reviewed (see Appendix 1).Destruction block: Block of plants to be destroyed. Within a nursery, for purposes of the retail protocol, the destruction block is defined as all P. ramorum infected HAP and all other HAP within 2 meters of any infected HAP.

HAP: Host and associated host plants listed on the official APHIS List of Regulated Hosts and Plants Associated with Phytophthora ramorum.

Host plants: Naturally infected plants verified with completion, documentation, review and acceptance of traditional Koch’s postulates and listed in the "APHIS List of Regulated Hosts and Plants Associated with Phytophthora ramorum".

Infected plants: Plants officially confirmed as being infected with P. ramorum, based on the use of APHIS approved diagnostics, and following the PASS system.

Nursery/Facility: Any location where nursery stock is grown, propagated, stored, or sold, or any location from which nursery stock is distributed. Locations that grow trees for sale without roots (e.g., as Christmas trees) are considered to be nurseries.

PASS (Potentially Actionable Suspect Sample): A presumptive positive P. ramorum sample diagnosed or identified by a provisionally approved laboratory or diagnostician with identification authority that would require confirmatory testing by an official APHIS Laboratory due to the nature of the plant sampled and the necessity for Federal confirmation. (For more information see: "PASS System Policy" at http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/protocols.shtml

Quarantine block: For purposes of the retail protocol this is an area identified as a 2 meter radius around the destruction block (see Appendix 2) designed to determine if P. ramorum has spread beyond the destruction block. (Use of Quarantine block is an adaptation from the definition: "An area in which a specific pest does not occur, or occurs at a low level and is officially controlled, that either encloses or is adjacent to an infested area, an infested place of production, a pest-free area, a pest-free place of production or a pest-free production site, and in which phytosanitary measures are taken to prevent spread of the pest." [ISPM Pub. No. 10, 1999]).

Quarantine release survey: This is the second quarantine period inspection that occurs near the end of the quarantine period. This survey includes visually inspecting all HAP within the nursery and sampling any unhealthy plant tissue, soil of destruction and quarantine block(s) and drainage or recirculated irrigation water, as per Appendices. When the quarantine period is completed and all plant, soil and water samples taken are negative for P. ramorum the nursery can be released.

Retail Nursery: A nursery whose business is the sale of plants to the end user, typically a home owner.

Suspect infected plants: These are plants with visible symptoms likely of P. ramorum infection; and/or HAP that are a part of an infested block or derived from an infested block or Quarantine block and/or plants that have tested positive using PCR or culturing, but have not been confirmed positive for P. ramorum by APHIS.

TRIGGER EVENTS FOR USE OF PROTOCOL
This protocol is to be implemented by APHIS-PPQ and/or its State Plant Regulatory cooperators when the presence of P. ramorum has been confirmed in a retail nursery. This may be from samples taken as part of a trace forward survey*, trace back survey*, a P. ramorum nursery survey*, annual compliance survey, or found by other means. Confirmed samples must have been diagnosed using a methodology approved by USDA, APHIS, PPQ and consistent with the Potentially Actionable Suspect Sample (PASS) protocol*.

*See http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/ for links with details on trace forward survey, trace back survey, P. ramorum nursery survey, and the PASS protocol.

AUTHORITIES

- For States with equivalent quarantines for P. ramorum, specific actions required by this protocol within and around the nursery are expected to be conducted by the State personnel under State authority with Federal support.

- For States without equivalent quarantines for P. ramorum, specific actions required by this protocol within and around the nursery will be conducted under Federal authority, in cooperation with State personnel.

COMMUNICATE AND NOTIFY
Communicate suspect finds using the bullets below as soon as one of the following has occurred:

1. A positive PCR determination
2. A culture that matches the morphology for P. ramorum (i.e. isolation of P. ramorum)

• Immediately notify the State Plant Health Director (SPHD) and the State Plant Regulatory Official (SPRO) of the State in which the nursery is located. The SPHD will notify the Regional Office and National Headquarters Office.

• SPHD’s and SPRO’s, shall notify facilities within their states that are impacted by the trace backs and provide a list of these facilities to their PPQ Regional offices. See "Conduct Investigations" Section.

• Laboratories need to notify, the SPHD, and the SPRO, the Regional Office, National Program Manager, and the submitter. Ideally the SPRO should notify the owner of the nursery, but either the SPRO (if State authority is used) or the SPHD (if Federal authority is used) may notify the owner of the nursery.

• The SPRO and SPHD will use state channels, including public affairs offices to make any public announcements, as necessary. The SPHD will ensure that the USDA APHIS Office of Legislative and Public Affairs is aware of any pending release, via the Regional Office and National Headquarters Office.

CONDUCT INVESTIGATIONS
Trace Forward Investigation: If the nursery has distributed HAP to another nursery, the Trace Forward Protocol shall be applied.

Implement the current Trace Forward Protocol present on the Phytophthora ramorum website located at http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/Trace Back Investigation:

Implement the current Trace Back Protocol present on the Phytophthora ramorum website located at http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/Nursery Sites:

Determine whether additional locations (nursery sites) are maintained by the same nursery personnel, or if HAP move to other sites or between sites.

• Equipment: Determine if equipment used at the site is shared with other nursery sites or field areas. Document any shared equipment utilization in different nursery sites or field areas. Equipment movement without appropriate biosecurity measures (see Appendix 9) between nursery sites requires that all nursery sites utilizing the equipment be included under this protocol.

• Plants: Determine if HAP move between sites. If so, than all sites receiving HAPs must be included under this protocol.

SECURE THE NURSERY
When the presence of Phytophthora ramorum has been confirmed in the nursery:

• The nursery operator may choose to destroy plants that have been placed under quarantine at any time within the 90 day quarantine period, however inspection and sampling must take place prior to destruction and the conditions specified in the Alternate Release Strategy below are applied. A questionnaire (see Appendix 12) is provided in this protocol as an aid to assist in an evaluation of the nursery.

• All infected HAP and all other HAP within 2 meters of any infected HAP shall be destroyed (see Appendix 2).

• All HAP within a 2 meter perimeter beyond the 2 meters surrounding the infected HAP (i.e. the retail destruction block) shall be held for a 90 day quarantine period.

• All HAP in the nursery that is not infected and not within 4 meters from the infected plants shall not be held under this protocol. However, this hold may also include "any other product or article that an inspector determines to present a risk of spreading Phytophthora ramorum, if an inspector notifies the person in possession of the product or article that it is subject to the restrictions in the regulations" (7CFR part 301.92-2) within the infested nursery site.

• Once inspection and sampling are complete, any held plants may be consolidated and segregated. If the plants are not consolidated and segregated, then the affected portion of the nursery must be closed to the public. With the approval of the regulatory officer, segregated plants may be moved to a site within the nursery or to a location away from the nursery. Any movement of the segregated plants must be done in an approved manner, under the direction of a regulatory official, in order to minimize the spread of the disease at the nursery. Segregation must include storage on a hard impermeable surface (e.g. concrete or asphalt) and not within 2 meters of any other plant.

• If the plants are consolidated and segregated as specified above, the soil beneath the infected plants must be sampled and tested prior to consolidation and determined to be negative for P. ramorum.

Survey the Nursery
The goal of the survey is to locate P. ramorum in the nursery. A detailed and thorough inspection should be conducted at the field level to determine the presence of P. ramorum. Samples should be collected from unhealthy looking plants, including any plants with any minute symptoms such as tiny leaf spots or brown leaf tips.

Delimiting Survey and Establishing Destruction and Quarantine Block(s):

• Examine all plants (nursery stock and decorative) within the nursery and sample any unhealthy plant tissue found.

• Samples must be analyzed using a methodology approved by APHIS (see Appendix 5).

• The final destruction and quarantine block(s) is (are) established when diagnostic results from all delimiting samples have been reported. The 90 day quarantine period begins when the delimiting survey is complete.

• Establish destruction block(s) by flagging a 2 meter circle around all infected plants. (see Appendix 2).

• Limit access to destruction block. Ensure that proper sanitation measures are applied (see Appendix 8).

• The HAP in the destruction block shall be destroyed in an appropriate manner (see Appendix 8).

Soil and Growing Media Sampling:

• Soil from within the destruction and quarantine block(s) must be sampled, and

• Growing media from non-HAP within the destruction block(s) and from all types of plants in the quarantine block(s) must be sampled, and

• Soil and growing medium from nursery blocks down slope from destruction and quarantine block(s) must also be sampled.

• Growing media from the plant potting area shall be sampled.

• Soil is the substrate underneath pots and growing medium is located within pots with the plants in the blocks.

• If reported positive, determine the content, origin, storage and handling of growing media used at the nursery site. See Appendix 6 for detailed soil and media sampling protocol. Keep soil samples separate from growing media samples.

Water Sampling:
Determine the source of water used at the nursery site and where drainage water flows. Note the type of irrigation system(s) in use, areas of standing water and any safeguards against water back flow in the irrigation system, as well as any water treatment practices if recirculated water is used. Water is to be sampled; See Appendix 7 for detailed water sampling protocol. Water sampling is not required for irrigation water from municipal water facilities that treat their water prior to release, but any retention pond or area where water collects at the nursery site must be sampled.

Cull Pile Sampling:
In a retail nursery this is likely not present, however if a cull pile is present, record the location of any cull piles as these may be contaminated with infected plant material or associated soil and/or growing media. Check any cull piles for P. ramorum symptomatic plants and plant material and sample if observed. Determine how the nursery disposes of culled plant material. Sample and test soil at the down slope edge of the cull pile for the presence of P. ramorum.

Compost Pile Sampling:
In a retail nursery this is likely not present, however if a compost pile is present, record the location of any compost piles as these may be contaminated with infected plant material or associated soil and/or growing media. Check any compost piles for P. ramorum symptomatic plants and plant material and sample if observed. Determine how the nursery disposes of composted plant material. Sample and test soil at the down slope edge of the compost pile for the presence of P. ramorum.

DISINFEST THE NURSERY

Plant Destruction:
Where a P. ramorum infected plant(s) is found, all HAP and plant parts within a destruction block will be removed and destroyed using one or more of the techniques detailed in Appendix 8.

Debris Removal:
All plant debris including growth medium, leaves, stems, flowers, roots, and any other plant parts found within the destruction block will be removed and destroyed using one or more of the techniques detailed in Appendix 8.

Cull Pile Treatment:
If any plants, plant material, growing media or soil from a cull pile is positive for P. ramorum, all material in the cull pile shall be properly disposed. See Appendix 8 for recommended destruction/disinfestation options.

Compost Pile Treatment:
If any plants, plant material, growing media or soil from a compost pile is positive for P. ramorum, all material in the compost pile shall be properly disposed. See Appendix 8 for recommended destruction/disinfestation options.

Non-porous Surfaces:
Non-porous surfaces will be disinfested. See Appendix 8 for recommended disinfestation options.

Porous Surfaces:
If the nursery is situated on a porous surface, remedial action must be developed and implemented with the written approval of a regulatory official in order to prevent contact of new HAP with soil or any other surface which cannot be immediately disinfested, and/or tested to determine if free of P. ramorum. A durable, impermeable ground barrier may be used as an inexpensive temporary measure to prevent contact of new HAP with soil. The condition of the barrier must be monitored and mainatined.

Water Treatment:
If water tests positive for P. ramorum, treatment is required (see Appendix 8 for recommended disinfestation options) and an additional delimitation of the nursery must be completed. For nurseries with established quarantine block(s) undergoing a 90 day quarantine period, the 90 day quarantine period re-starts after the second delimiting survey is completed. Also, plants and growing media that may have been irrigated with infested water must also be resampled and retested within the new 90 day quarantine period.

Soil and Growing Media Treatment:
If soil, growing media or plant debris in a destruction or quarantine block test positive, soil treatment is required. The destruction block is the most likely area of soil or growing media infestation (underneath and around the diseased plants, and in containerized stock) and the most likely area where reinfestation of new host material would occur. See Appendix 8 for recommended destruction/ disinfestation options.

Equipment and Personnel:
See Appendix 8 for recommended disinfestation options.

Biosecurity Measures:
Biosecurity measures are designed to minimize the risk of introduction or, spread and survival of the pathogen in a nursery. See Appendix 9 for recommended biosecurity measures.

NINETY (90) DAY QUARANTINE ACTIVITIES
These concurrent activities follow completion of the delimiting survey:

• Any non-HAP that were present in a destruction block will be held in place, or moved under official supervision to a safeguarded area with a non-porous surface, during the quarantine period and be subject to the same conditions as the HAP in the quarantine block(s).

• This quarantine period begins when the delimiting survey is completed (i.e. the last sample is taken and an EAN is issued) and lasts until such time as both plant parts and climatic conditions conducive to disease expression have occurred for at least 90 days. If the quarantine period (90 days) does not include climatic conditions conducive for disease development then the quarantine period shall be extended to an appropriate length to include conducive climatic conditions for a total of 90 days. During the quarantine period, inspection, sampling, and testing must reveal no further detection of P. ramorum.

• During the 90 day quarantine period within the quarantine block(s):

• No fungicides registered for Phytophthora control shall be applied.

• Regulatory officials will visually inspect plants a minimum of two times, once about half-way through the anticipated quarantine period and once near enough to the end to have test results coincide with the end of the quarantine period, according to the protocol detailed in Appendix 4. This second visual inspection in the quarantine block(s) can be done at the same time as the quarantine release survey as described below.

• Regulatory officials will collect water, soil, and media samples and test during the quarantine period according to the protocols detailed in Appendices 6 and 7.

• If a plant sample tests positive for P. ramorum, the destruction block(s) and quarantine block(s) shall be redefined via sampling and the quarantine period reset.

• If water, soil, and/or media samples tested positive for P. ramorum during the delimiting survey, it must be treated per Appendix 8. Once successfully treated, samples of the infested water, soil, and/or media material will be taken and tested during each of the two quarantine period nursery inspections per the protocols detailed in Appendices 6 and 7.
• If irrigation water is found to be positive, then any portion of the nursery that has been irrigated with the P. ramorum infested water shall be placed on hold and the irrigated area re-delimited.

• If a soil sample is found to be positive, the soil shall be treated, then any plants in the block with the infested soil are placed on hold and the area re-delimited.

• The growing media in the potting shed must be tested. Any positives for P. ramorum from the media in the shed confer with the Regional Program Manager.

• A quarantine release survey of the entire nursery must be completed near the end of the 90 day quarantine period. This survey includes visually inspecting all HAP within the nursery and sampling any unhealthy plant tissue, soil of destruction and quarantine block(s) and drainage or recirculated irrigation water. When the quarantine period is completed and all plant, soil and water samples taken are negative for P. ramorum the nursery can be released.

RELEASE THE NURSERY
Nurseries and their plants that have been placed under regulatory control may be released from regulatory control by USDA, APHIS or its designated authority after the quarantine period if the following three conditions are met:

• There are no additional detection of P. ramorum in nursery stock based on USDA, APHIS approved plant inspection, sampling and testing protocols for the preceding quarantine period; and

• Water, soil, and growing media have also tested negative for P. ramorum based on USDA, APHIS approved sampling and testing protocols for the preceding quarantine period if testing of soil, water and media is required: and

• The quarantine release survey is negative for P. ramorum.

POST ERADICATION MONITORING
Nurseries that have been infested will continue to be monitored when disease expression is anticipated for the following two years at the national survey protocol levels. These nurseries are not under any other quarantine or regulatory action, unless there are additional detections.

APPENDIX 1
APHIS List of Regulated Hosts and Plants Associated with Phytophthora ramorum
A current list may be found at the USDA APHIS PPQ website at http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/

Appendix 2
Schematic of Retail Nursery with Infected Host Plant(s)July 9, 2007

APPENDIX 3
Reserved

APPENDIX 4
Delimiting Survey Protocol
Delimiting Survey Protocol to Detect Phytophthora ramorum In Plants at Confirmed Nurseries
Revised: July 19, 2007

Objective:
The objective of this document is to provide guidelines for the delimiting survey in nurseries where the regulated pathogen, Phytophthora ramorum has been confirmed. This survey method is designed using the best available scientific principles to determine apparent freedom from P. ramorum in nursery plants. In order to achieve this freedom from P. ramorum, accurate and successful inspection of HAP (genera for wholesale/production) must be accomplished at an appropriate confidence level to ensure detection of disease.

Sampling method:
The goal is targeted sampling of plant tissue to determine the presence of P. ramorum with a 95% confidence of finding the disease at a very low level (0.5% of plants are infected with P. ramorum) by inspecting a minimum of 850 HAP plants in each block (or all the plants if there are less than 850). A physical sample of the inspected plant is only to be taken if unhealthy plant tissue is present. Do not sample asymptomatic plants.

• Inspector should contact the nursery manager to set up the inspection and find out approximately how many HAP are present in each nursery block (i.e. a nursery map).

• These visually inspected plants should be chosen at random, but if certain areas of the block contain plants exhibiting unhealthy tissue or are more prone to disease development (such as low areas where water might puddle or places where mist or fog persists) these areas should be included in the sampling process.

• Disposable rubber gloves and tyvek booties should be worn and should be changed or disinfested using 10% bleach solution or a quaternary ammonium solution (at the labeled rate) between each block. Additionally, waterproof raingear and rubber boots may be used and disinfested between each block. Washtubs with ~ 1/2 inch of disinfectant to step in for booties and 3 inches in buckets to dip gloved hands should be sufficient.

• To visually inspect a plant, carefully lift the plant from surrounding plants, if possible, and carefully examine all plant leaves and stems for unhealthy tissue particularly for the presence of water-soaked or necrotic lesions consistent with P. ramorum infection, however all unhealthy tissue should be considered suspect. Take care to examine the leaves on the interior as they may exist in a microclimate more conducive to disease development and may be more likely to have disease symptoms. Be sure to properly disinfest booties and gloves between all nursery blocks. Because this is a confirmed nursery, proper use of sanitation is imperative to reduce the potential for pathogen transport from an infested part of the nursery to an un-infested nursery block.

• Sample plant tissue from any and all visually inspected plants that appear unhealthy. Each sample should consist of a minimum of five leaves; for Vaccinium and other small leaf hosts collect the terminal last 3 inches of branch tips, if present, from each unhealthy plant. If, however, only one leaf is unhealthy include only the one leaf with lesions. Examine any other leaves on the plant for the presence of lesions, because chances are much smaller lesions may be present on other leaves of the same plant.

• Samples should be placed in a re-sealable leak proof plastic bag labeled with the appropriate nursery designation and sample number. Samples should be double-bagged in an additional re-sealable leak proof plastic bag with a completed PPQ391 form for each sample submitted.

• Keep the samples cool by placing them in a cooler (around 3^o - 6^o C or 37 - 43 F).

• Overnight mail or deliver the sample to the laboratory as soon as possible to preserve freshness.

• All samples must be analyzed following the APHIS diagnostic protocols.

• Continue inspecting 850 plants in each block that contains HAP (genera for wholesale/production).

• Examine all HAP (genera for wholesale/production) in cull piles for the presence of tissue symptomatic for P. ramorum and take symptomatic tissue from any and all plants with symptoms.

Appendix 5
Diagnostics
Revised: April 2007

Samples must be analyzed using a methodology approved by APHIS. See techniques posted at: http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/

See http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/ for latest approved protocol.

Soil and Growing Media Sampling:

• Infested soil or growing media will look exactly the same as un-infested soil or growing media. Therefore all soil and media must be handled carefully. All tools used to collect soil or media samples must be disinfected with 10% bleach solution, quaternary ammonium solution or flame-sterilized with a propane torch between blocks. All soil and organic material should be removed from the tools prior to disinfection. Care should also be taken not to transfer soil or growing media from one block to the next on shoes or clothing. All sampling equipment should be cleaned and disinfected prior to entering a new nursery block. Care must be taken to ensure that un-infested soil or growing media is not contaminated by infested soil or growing media. If the areas of soil/media infestation are known or suspected sample these quarantine block and work toward the destruction block(s).

Preparing for sampling:

• Soil and growing media samples should be collected as composite samples. Composite samples of growing media should be kept separate from soil samples. A composite sample consists of a mixture of sub-samples. Sub-samples (See Figure 1) are small amounts of soil (or media) removed from the ground (or pot) and added together to form a composite sample. The use of sub-sampling increases the chances of finding P. ramorum if it is present. Samples should contain a maximum of 500-ml (volume) of soil and/or growing media (1/2 of a quart-size Ziploc bag). The number of composite samples collected will depend upon the size of the nursery block being sampled (see Table 1). There should be at least two samples, one for growing media and one for soil, unless all plants and associated growing media were destroyed or the plants are not on soil (e.g. on concrete or asphalt). If the surface of soil is covered with gravel take sub-samples from the soil beneath the gravel. If water permeable weed block is present, either covered with gravel or under gravel, the weed block should be removed prior to soil sampling.

Table 1: Number of composite samples collected based on nursery block size.

• Each composite sample will consist of at least five sub-samples collected from soil or growing media within the targeted area. While five is a minimum, it is preferable to take 24 sub-samples of soil or growing media for each sample, provided the area is large enough (for soil samples) and enough plants are present (for growing media samples). Sub-samples should be collected according the pattern in the diagram below (Figure 1). Alternatively, if fallen leaves or other debris from the infected plants are present; sub-sampling may be targeted towards those areas. The location of each composite sample should be maintained (preferably by GPS but at least by flagging) in case follow-up treatment of the soil or growing media for P. ramorum is required. Composite samples may also be collected from neighboring blocks of un-infested plants using the same steps. If you are collecting from blocks of un-infested plants, collect the composite soil/growing media samples from these blocks first to minimize the risk of contaminating un-infested soil/growing media. If all potentially-infested growing media has been destroyed with the infected plants, collect composite samples from the remaining host plants within 2- to 10-m of the originally infected plants that have been placed on hold. Preferentially target the growing media of those plants that are downslope (e.g., based on watering patterns) of the originally infected plants.

Soil Baiting
It is possible to follow the below procedure and not successfully bait and culture Pr. This may be due to Pr not being present, but may be due to dormancy of Pr. To address this dormancy potential and to better enable the diagnostician to detect Pr when present, mix the soil well and split the soil samples when they arrive in the laboratory. Once the samples are well mixed and split, place one of the split sample halves into cold storage at approximately 4 degrees C for 2 months. Bring samples out from cold room after two months have passed, leave samples at room temperature for two days and repeat soil baiting process. This baiting can be done in conjunction with the final baiting required for the quarantine release survey. The samples should be processed as shown below.To prepare soil bait, briefly soak the pears (select unripe green pears) or Rhododendron leaves in a mild detergent solution to remove any pesticide residues. Rinse the baits well and drain.

Leaving the soil in the Ziploc bag, add enough sterile deionized water to saturate and cover soil with about 2.5 cm (1") of water. Do not mix the soil and water.

Use two pears or leaves per soil sample. With a black sharpie pen, label one side of the pears or leaves with the soil sample number and date processed. The USDA Forest Service recommends the following bait selection criteria in Stream Baiting Protocol: 2007 National Phytophthora ramorum Early Detection Survey of Forests, issued March 20, 2007. See http://fhm.fs.fed.us/sp/sod/sod.shtm for latest approved protocol.

Bait Selection

• Use leaves from a population of native or naturalized rhododendrons, if possible. The population should be sufficiently large to supply needed leaves for the survey duration.

• Variation in Pr susceptibility among rhododendron species/cultivars in laboratory inoculation has been published, but field and lab studies have shown that leaves of common native and naturalized species perform acceptably as Pr bait.

• Leaf size can vary considerably among species and cultivars. If bait leaves are quite small (8 cm x 3 cm at the widest point or smaller), use 2 leaves in each pocket of the bait bag.

• If the source of leaves is nursery-grown or naturalized landscape plants, ensure that they have been free of fungicides and other pesticides for a minimum of 6 weeks before using as bait.

• Source plants should be mostly free of dieback and leaf symptoms. Use 1 year-old leaves as free as possible from leaf symptoms (spots, blight, chlorosis), insect damage, and mechanical damage. Do not use newly formed, succulent leaves. Leaves formed in the present year may be used after full leaf expansion and a period of hardening in summer.

• Bait leaves wrapped in paper towels moistened with chlorinated tap or sterile water and sealed in a plastic bag may be stored refrigerated for up to 1 week before use. Do not use well water or stream water for stored leaves.

Carefully push each pear or leaf into the wet soil and water until the bait is immersed halfway. Leave the labeled side of the bait out of the water. Seal the Ziploc bag and leave bait in the soil/water mixture for at least 48- hr at room temperature.

After 48-hr, remove the baits and wash off any clinging soil into Ziploc bag. Set the bait on a moistened paper towel in a sealed container at room temperature for 7-d to let any potential disease symptoms develop. The soil/water mixture must be autoclaved before disposal.

Examine the bait daily for developing symptoms. Pears infected with P. ramorum will display lesions that are round, brown, somewhat leathery in texture, with undefined edges. Colorless, watery, and/or soft lesions are generally caused by other pathogens (especially Pythium spp.).

Rhododendron leaves that have become infected with P. ramorum will exhibit 'diffuse' leaf spots usually with the midvein most affected.

Under the laminar flow hood, cut eight to 10 pieces of pear or leaf from the edge of the developing lesion or leaf spot and insert into the PARP medium. Write the sample number and date processed on the underside of the Petri dish. Seal the dish with parafilm and incubate and treat as described in the USDA approved Guidelines for Isolation by Culture and Morphological Identification of Phytophthora ramorum at http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/protocols.shtml

Appendix 7
Water Sampling Protocol
Revised: April 2007

See http://www.aphis.usda.gov/plant_health/plant_pest_info/pram/ for latest approved protocol.

Phytophthora ramorum is an oomycete, belonging to the group that includes Pythium species.  Collectively these organisms are called "water molds" and are taxonomically related closer to algae than to fungi.  For this reason, water collected from potentially infested nursery blocks must be tested for the presence of P. ramorum. 

There are two potential methods provided here to detect Phytophthora species in water.  The first uses rhododendron leaf baits in mesh bags followed by moist chamber incubation of the leaf baits. As of April 2007, research supports using leaves at least one year old, so that is recommended. Any suspect lesions that develop on the rhododendron leaves would be plated on PARP at 18-20°C (64-68°F).  Any Phytophthora species growing on the PARP would need to be transferred to Corn meal agar or V8 agar for identification to species.

The second method uses water filtration.  Water is removed from the pond, filtered with sterile filters and the filters placed on PARP.  Once the filter is removed from PARP, any resultant Phytophthora colonies are transferred to Corn Meal Agar or V8 agar and identified to species.

In situ Water Sampling with Rhododendron Leaf Baits:
A control sample using a leaf bait in distilled water should be run simultaneously with the leaf bait sample in the nursery site water. The USDA Forest Service recommends the following bait selection criteria in Stream Baiting Protocol: 2007 National Phytophthora ramorum Early Detection Survey of Forests, issued March 20, 2007. See http://fhm.fs.fed.us/sp/sod/sod.shtm for latest approved protocol.

Bait Selection

• Use leaves from a population of native or naturalized rhododendrons, if possible. The population should be sufficiently large to supply needed leaves for the survey duration.

• Variation in Pr susceptibility among rhododendron species/cultivars in laboratory inoculation has been published, but field and lab studies have shown that leaves of common native and naturalized species perform acceptably as Pr bait.

• Leaf size can vary considerably among species and cultivars. If bait leaves are quite small (8 cm x 3 cm at the widest point or smaller), use 2 leaves in each pocket of the bait bag.

• If the source of leaves is nursery-grown or naturalized landscape plants, ensure that they have been free of fungicides and other pesticides for a minimum of 6 weeks before using as bait.

• Source plants should be mostly free of dieback and leaf symptoms. Use 1 year-old leaves as free as possible from leaf symptoms (spots, blight, chlorosis), insect damage, and mechanical damage. Do not use newly formed, succulent leaves. Leaves formed in the present year may be used after full leaf expansion and a period of hardening in summer.

• Bait leaves wrapped in paper towels moistened with chlorinated tap or sterile water and sealed in a plastic bag may be stored refrigerated for up to 1 week before use. Do not use well water or stream water for stored leaves.

Prepare the rhododendron leaves as bait by trimming off the petiole end of each leaf.  Place 3-4 cut leaves into a mesh bag. Label the bag with a plastic tag listing the date, water source (location), and nursery (i.e., nursery license number).  Place the mesh bag into the water source for a minimum of 48-hours to 1-week (preferable).  Do not leave the bait in the water source for longer than 1-week as the bait will begin to decompose.  Place the bags such that the leaves will remain submerged the entire time (i.e., even if water levels fluctuate within the water source).  If possible, place the bait near the influent coming from the area closest to or containing the infested plants.

Remove the bait from the water source and transfer to a sealable bag for transport to the laboratory.  Label the bag with the information on the plastic tag, including the date collected.  Log the leaf samples into the appropriate database.  Assign a unique sample number to the bait(s) from each nursery. 

Water Sampling for Filtration:
Water samples should be collected in a sterile wide-mouth bottle and kept at 5 – 10 C.  Water samples should be taken from the surface to increase the likelihood of obtaining zoospores of Phytophthora. 

Sample size should be approximately 1000 ml.  Samples should be processed within 48 hours of collection or the samples should be discarded and new samples obtained and processed within 48 hours. Number of samples is determined by the size of the nursery pond to be sampled (Table 1)

Table 1: Number of composite samples collected based on pond size.

Steven Jeffers
Associate Professor and Extension Specialist
Clemson University
Department of Entomology, Soils and Plant Sciences
203 Long Hall, Box 340315
Clemson, SC 29634
Tel: 864-656-7157, Fax: 864-656-0274
Email: sjffrs@clemson.edu

APPENDIX 8
Treatment and Disinfection
Revised: April 2007

The following techniques are approved by USDA APHIS PPQ for control of P. ramorum in nurseries found to contain plants infected with P. ramorum.

Infected Plants:
Note: HAP material, including leaf litter, must not be placed in compost piles or be removed from the nursery site as trash or in debris removal. HAP material should be collected and incinerated or double bagged and deep buried in a site approved by USDA, APHIS or delegated regulatory authority.

• Incineration (burning to ash): Infected plants, associated growth media, associated containers (i.e. pots and trays), all leaf debris in and around the area where plants were stored may be disposed of by incineration at a facility or other location (e.g. on site) approved by USDA and permitted within state and municipal statutes or regulations. Off nursery movement must be properly safeguarded and every effort to prevent plant debris or soil from being dislodged from the plants prior to incineration should be taken. Burning may be through open burning or in an incinerator.

• Deep burial: Infected plants, associated growth media, associated containers (i.e. pots and trays), all leaf debris in and around the area where plants were stored must be double bagged using plastic bags of 2 mil thickness or greater and buried to a depth of no less than two meters. The material must be buried at a USDA approved site, onsite, or municipal landfill, which is expected to remain undisturbed. Every effort to prevent plant debris or soil from being dislodged from the plants should be taken.

• Steam sterilization: Dry heat or steam commonly heated to internal temperatures of 212^o F (100^o C) for 30 minutes followed by burial in a landfill, or as otherwise detailed in the USDA Treatment Manual for "insect pests and pathogens in garbage", Schedule T415b. http://www.aphis.usda.gov/ppq/manuals/port/Treatment_Chapters.htm

Non-Porous Surfaces: Most disinfectants are not labeled for use in soil and are only useful for nonporous materials such as concrete floors, nursery pots, and plastic sheeting. A number of disinfectants are registered for use on nonporous surfaces that may effectively reduce populations of Phytophthora species. If it is practical, tools such as knives, pruners, water breakers, water wands and other implements used in the quarantine area should only be used in the quarantine area. If tools and other implements must be moved from the quarantine area, then regular disinfection using an appropriate disinfectant for the control of P. ramorum is recommended prior to removal from the quarantine block. The following table modified from http://cpmcnet.columbia.edu/dept/ehs/decon.html examines the effects of different classes of disinfectants on microbial populations. This list is for explanation and information only. Few disinfectants are specifically labeled for Phytophthora species and are shown in Bold.

All labels for the disinfectants listed below must be strictly adhered to for maximum efficacy and environmental and worker safety.

Summary of Disinfectant Activities

Water:

• For dust abatement, fire suppression, and equipment cleaning: Clorox (sodium hypochlorite) is labeled (EPA Reg. No 5813-50) for treatment of water ( ~50 ppm available chlorine) for controlling the spread of Phytophthora lateralis via water used for dust abatement, fire suppression and equipment cleaning. The active ingredient level must be measured from water collected at the sprinkler head.

• For irrigation: Chlorine levels of 2ppm or 2mg/liter or greater has been correlated with the control of Phytophthora spp. in re-circulated irrigation systems. For irrigation purposes, recirculated, non-municipal water, must be chlorinated at an active chlorine concentration equal to or greater than 2 mg/liter of water; for facilities that recycle water, this chlorine level must be monitored.

Soil and Potting Media:

• Potting media: Potting media must be heated such that the temperature in the center of the load reaches at least 180 degrees F for 30 minutes. Treatment must be conducted in the presence of an inspector or treated with an approved fumigant as detailed below.

• Soil: Soil must be heated such that the temperature in the center of the load reaches at least 180 degrees F for 30 minutes. Treatment must be conducted in the presence of an inspector or treated with an approved fumigant as detailed below. Methyl bromide has been used for fumigating wood products, but the data on fungi and related organisms in wood are limited. However, methyl bromide has a long history of fumigation of soil in the field and greenhouse. It has commonly been used in combination with chloropicrin for control of Phytophthora spp. and other pests in strawberry beds. Methyl bromide has been used for soil treatment for the mitigation of P. cinnamoni in citrus groves. However, many of the compounds currently in use have been implicated in human and environmental risks. Solarization is not a consideration as a viable option for soil treatment.

All fumigants are restricted use and must be applied according to labels by a licensed applicator. Any use of pesticides in any manner not listed on the label is unlawful.

Summary of Labeled Soil Fumigants

Physical Treatment of Soil:

• Mitigation of infested soil can also be achieved by installing permanent impermeable, non-porous barriers that consist of cement, concrete or asphalt. These barriers must be constructed so that no native soil within the destruction block is visible. The barriers should be graded such that no standing water can be observed.

Equipment and Personnel (Inspectors and employees):

• Access to infested areas and hold areas should be limited, as much as possible, to officials and necessary employees. Everyone entering and leaving the nursery site must scrape off loose pieces of soil into the destruction block. Those working with, or in contact with suspected infested material (including plants), must wash hands using soap or approved disinfectant immediately after completion of task. There are no products currently labeled for use on porous materials for Phytophthora control.
  
• Personnel should not have access to other production areas of the nursery after entering the destruction block on the same day.

• A disinfectant foot bath should be placed near the exit to the destruction blocks and quarantine blocks and used by all personnel entering and exiting the quarantine block and entering and exiting the destruction block at the infested nursery site, where the contact with potentially infested soil or plant debris by footwear is likely. The foot bath must be filled with fresh disinfectant at least on a daily basis or more frequently if contaminated with soil or organic debris, in accordance with label directions. Use of disposable shoe covers may be used in lieu of a footbath, if disposed of immediately upon exiting from the quarantine block or destruction block. The disposable shoe covers must be placed in bags and incinerated, deep-buried or properly disposed in a sanitary landfill.

• The tires (or other parts in contact with the soil or plants, such as the bed of trucks) of vehicles must be cleaned of loose soil and plant debris and disinfested with the appropriate labeled products before leaving the infested site. If at all possible, vehicles should not be allowed in the destruction blocks at all. Any efficacious product labeled for use on non-porous surfaces may be used on tires or vehicle undercarriages.

• Do not visit other nursery sites in potentially contaminated work clothing and footwear. Where it is necessary that visitors enter the nursery, the nursery should ensure that every precaution is taken to prevent the movement of infected plants, contaminated soil or debris by the visitor.

• Wood surfaces suspected of contamination with P. ramorum should be disposed of as stated above under "Infected Plants."

APPENDIX 9
Biosecurity Measures for Nurseries
April 2007

The following measures are designed for production nurseries. Adapt these measures for use in the specific retail nursery that is undergoing eradication of Phytophthora ramorum from the nursery.

In the course of daily work, nursery personnel are frequently required to visit a number of different nurseries sites, greenhouses, fields, and facilities. These actions could potentially provide a pathway for transferring quarantine organisms from one work site to another during the work day. It should also be recognized that even if a single work site is visited per day, precautions must be taken to avoid contaminated clothing and equipment from being used at a new site the following day. Further, visitors to these same facilities present the same risks and additionally could vector disease-causing-organisms from other sites.

Biosecurity measures must be taken by nurseries and be required of nursery personnel and visitors to avoid and mitigate the spread of P. ramorum. The biosecurity measures described here are the minimum measures to be taken by the nursery.

Communications
All nursery personnel should be trained and visitors informed of these biosecurity requirements that have been put in place by the facility. As new scientific data and technology is learned, the facility needs to update their biosecurity requirements and retrain their personnel.

Vehicles: Vehicles can become contaminated with soil; a primary vector for quarantine pests. The following guidelines seek to reduce the likelihood of this pathway.

Avoidance: Once at the inspection site, if possible, the vehicle should only be driven and parked on paved, concrete or gravel areas to avoid contact with soil and organic matter. Visitors should consider requesting a facility employee to drive them to their designated location with one of the nursery’s vehicles. Loading of nursery stock onto other than the nursery’s vehicles should be done in an area with concrete or asphalt pad located near the gate and not in the interior of the nursery.

Cleaning:
Interior of nursery vehicles should be cleaned to ensure no build-up of soil, debris or other items.

Where it is not possible to avoid the vehicle going onto the fields, the vehicle must be driven to the edge of the facility where the tires, wheel wells and accessible areas of the undercarriage of the vehicle must be cleaned of soil and organic matter with a brush or a water hose followed by a spray down with a suitable disinfectant. In situations where the undercarriage has been coated with soil it is recommended that after cleaning and disinfecting at the work site an effort be made to go through a car wash that has the ability to clean the undercarriage before proceeding to another work site. If a car wash is not available, avoid driving onto the next work site. To ensure the entire surface of the tires are cleaned it will also be necessary to move the vehicle forward a foot or so to permit cleaning of the portion of the tire in contact with the ground.

The tires (or other parts in contact w