Phytophthora ramorum Domestic 

Regulatory Program Manual
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Department of Agriculture.

First Edition Issued 2020
08/2022-05 Phytophthora ramorum Manual TOC-1

Phytophthora 

ramorum 

Contents

Figures LOF-1-1

Tables LOT-1-1

Introduction 1-1-1

Phytophthora ramorum Inspection and Sampling Protocol for 

Nurseries 2-1-1

Interstate Confirmed Nursery Protocol 3-1-1

Confirmed Retail Nursery and Retail Nursery Dealer Protocol 4-1-1

Trace Investigations

 Introduction 5-1-1

 Trace Forward Protocol for Nurseries that Received Plant Material Shipped 

from a Confirmed P. ramorum-Infested Nursery 5-2-1

 Trace Back Protocol for Nurseries that Shipped Plant Material to a Confirmed P. ramorum-Infested Nursery 5-3-1

Confirmed Residential and Commercial Landscape Protocol 6-1-1

Biology and Symptoms of Phytophthora ramorum 7-1-1

Sampling and Submission Protocol 8-1-1

Biosecurity Measures for Nurseries 9-1-1

Water Sampling and Processing Protocol 10-1-1

Soil and Container Mix Sampling and Processing Protocol 11-1-1

Treatments and Disinfectants 12-1-1

Resources A-1-1

Glossary Glossary-1-1

Index Index-1-1
9/2023-06 Phytophthora ramorum Manual LOF-1

Phytophthora 

ramorum 

Figures

Figure 3-1-1 Schematic of Destruction and Quarantine Radii of Positive 

Plants 3-1-10

Figure 4-1-1 Schematic of Destruction and Quarantine Radii of Positive 

Plants 4-1-8

Figure 6-1-1 Diagram Showing Destruction Radius, Quarantine Radius, and 

Delimitation Survey 6-1-7

Figure 6-1-2 P. ramorum Nursery Questionnaire 6-1-8

Figure 6-1-3 Follow-up Survey for Residential Locations with Infected Plants 

 6-1-13

Figure 8-1-1 Instructions for Using PPQ Form 391, Specimens for 

Determination 8-1-3

Figure 10-1-1 Bottle of Bait (BoB) Materials and Leaf Pieces Plated into 

PARPH Media 10-1-5

Figure 10-1-2 Example of a Bait Bag Construction 10-1-7

Figure 10-1-3 Pattern of Mesh Screen to Use for Constructing a Bait 

Station 10-1-8

Figure 10-1-4 Completed Bait Station Construction 10-1-9

Figure 10-1-5 Characteristic Structure of P. ramorum 10-1-15

Figure 10-1-6 Vacuum Apparatus for Water Filtration and Filter Plated onto 

PARPH-V8 Plate 10-1-16

Figure 11-1-1 Baiting Soil Samples and Plating Baits 11-1-6

Figure 11-1-2 Characteristic Structure of P. ramorum 11-1-7

Figure 12-1-1 Steam Sock in Place on Soil Surface 12-1-5

Figure 12-1-2 Concrete Blocks Used to Support Tarp from Surface to Allow 

Steam Distribution from Steam Sock 12-1-6

Figure 12-1-3 Tarp with Sand Snakes in Place to Secure Edges 12-1-7

Figure A-1-1 P. ramorum Nursery Questionnaire A-1-3
9/2023-06 Phytophthora ramorum Manual LOT-1

Phytophthora 

ramorum 

Tables

Table 1-1-1 Reporting Issues With or Suggestions for the Manual 1-1-4

Table 2-1-1 Minimum Number of Plant Samples to Take Based on the Number of Regulated Plants Within the Nursery 2-1-8

Table 3-1-1 Interstate Confirmed Nursery Protocol (CNP) Procedures

 3-1-5

Table 3-1-2 Legend of Destruction (D) and Quarantine (Q) Radii of Positive 

Plants Schematic 3-1-11

Table 3-1-3 Disinfest the Nursery 3-1-12

Table 3-1-4 Quarantine Period for Plants in Q-Radii 3-1-13

Table 3-1-5 Minimum Number of Samples to Take Based on the Number of 

Regulated Plants 3-1-16

Table 3-1-6 Examples of CCPs, Remediations, Mitigations, Best Management Practices (BMPs), and Changes to Business/Cultural 

Practices 3-1-23

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol 

Steps 4-1-5

Table 4-1-2 Legend of Destruction (D) and Quarantine (Q) Radii of Positive 

Plants Schematic 4-1-8

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol 

Steps (continued) 4-1-9

Table 5-2-1 Trace Forward Protocol Instruction 5-2-2

Table 5-3-1 Trace Back Protocol Instructions 5-3-2

Table 6-1-1 Confirmed Residential Protocol Instructions 6-1-3

Table 6-1-2 Treatments and Disinfectants for Residential or Commercial 

Landscaped Settings 6-1-15

Table 6-1-3 Summary of Disinfectant Activities 6-1-17

Table 10-1-1 Temperature Versus Days for Floating Baits in Water 10-1-7

Table 10-1-2 PARPH-V8 Selective Medium: for Phytophthora Species

 10-1-17

Table 10-1-3 PAR-V8 Selective Medium: for Phytophthora Species

 10-1-18

Table 11-1-1 Number of Composite Samples to Collect Per Block 11-1-2

Table 11-1-2 PARPH-V8 Selective Medium: for Phytophthora Species

 11-1-10

Table 11-1-3 PAR-V8 Selective Medium: for Phytophthora Species

 11-1-11

Table 12-1-1 Summary of Disinfestant Activities 12-1-3

Table 12-1-2 Labeled Soil Fumigants 12-1-8
3/2024-06 Phytophthora ramorum Manual/Introduction 1-1-1

Chapter

1

Introduction

Contents

APHIS Mission 1-1-1

Phytophthora ramorum Program Objective 1-1-1

PPQ Mission 1-1-1

Manual Objective 1-1-2

Scope 1-1-2

Revisions 1-1-3

Reporting Issues With or Suggestions For the Manual 1-1-4

APHIS Mission

The Animal and Plant Health Inspection Service (APHIS) is an Agency within 

the United States Department of Agriculture (USDA). The mission of APHIS 

is to protect the health and value of American agriculture and natural 

resources.

Phytophthora ramorum Program Objective

The goal of the APHIS P. ramorum program is to limit the spread of P. 

ramorum from regulated nurseries and quarantine areas to nonregulated 

nurseries and nonquarantine areas through regulatory strategies and adopting 

(voluntary and mandatory) best management practices.

PPQ Mission

APHIS Plant Protection and Quarantine (PPQ), and operational program, 

safeguards agriculture and natural resources from the risks associated with the 

entry, establishment, or spread of animal and plant pests and noxious weeds.

Intended Users

Users of this manual include State and Federal regulators who conduct or 

oversee surveys and inspections for P. ramorum in nurseries, on residential 

properties, and at managed landscapes and public gardens. It is publicly 

available for nursery owners, homeowners, managers of large landscapes and 

public gardens, and others interested in the Federal processes involved with the 

P. ramorum program.
Introduction

Manual Objective

1-1-2 Phytophthora ramorum Manual/Introduction 3/2024-06

Manual Objective

The objective of the Phytophthora ramorum Manual is to help users detect the 

presence of P. ramorum in interstate shipping nurseries. This chapter describes 

methods for sampling plants, surface water, drainage water, water for 

irrigation, container mix, and any other articles designated by an inspector as 

possible sources of P. ramorum inoculum at the nursery.

Scope

The chapters in this manual are:

Introduction on page 1-1-1

Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

on page 2-1-1

Interstate Confirmed Nursery Protocol on page 3-1-1

Confirmed Retail Nursery and Retail Nursery Dealer Protocol on page 

4-1-1

Trace Investigations on page 5-1-1

Trace Forward Protocol for Nurseries that Received Plant Material 

Shipped from a Confirmed P. ramorum-Infested Nursery on page 5-2-1

Trace Back Protocol for Nurseries that Shipped Plant Material to a 

Confirmed P. ramorum-Infested Nursery on page 5-3-1

Confirmed Residential and Commercial Landscape Protocol on page 

6-1-1

Biology and Symptoms of Phytophthora ramorum on page 7-1-1

Sampling and Submission Protocol on page 8-1-1

Biosecurity Measures for Nurseries on page 9-1-1

Water Sampling and Processing Protocol on page 10-1-1

Soil and Container Mix Sampling and Processing Protocol on page 11-1-1

Treatments and Disinfectants on page 12-1-1

Example of PPQ Form 519, Compliance Agreement on page A-1-3

Glossary on page Glossary-1-1

Index on page Index-1-1
Introduction

Revisions

3/2024-06 Phytophthora ramorum Manual/Introduction 1-1-3

Revisions

The Manuals Unit of PPQ issues revisions using a USDA Stakeholder Registry 

announcement. Each announcement provides the following information:

Transmittal number used to track revisions

Purpose of the revision

Page number(s) on which the revision(s) is located

Authorities

This protocol incorporates requirements and procedures outlined in the 

regulations declared under the Plant Protection Act of June 20, 2000, as 

amended, Section 412(a), 7 U.S.C. 7712(a), which authorizes the Secretary of 

Agriculture to prohibit or restrict the movement in interstate commerce of any 

plant, plant part or article, or means of conveyance if the Secretary determines 

the prohibition or restriction is necessary to prevent the dissemination of a 

plant pest within the United States. These protocols are also issued in line with 

P. ramorum regulations found at 7 CFR 301.92 et seq.

Since the regulations were first published in 2022, P. ramorum has been 

detected in a significant number of nurseries. These detections prompted a 

standard protocol to be used by State and Federal regulators when responding 

to P. ramorum found in nurseries. To ensure there is consistency in responding 

to P. ramorum infestations, this manual describes the official activities 

performed within and around nurseries by USDA–APHIS staff in cooperation 

with State agriculture regulatory officials.

For States with regulations for quarantine pests, and/or specifically for P. 

ramorum equivalent to the Federal regulations or Federal Orders, State 

personnel may conduct specific actions required by the protocol, within and 

around the nursery, under State authority with Federal support. For States 

without regulations for quarantine pests and/or P. ramorum equivalent to the 

Federal regulations, specific actions required by this protocol within and 

around the nursery will be conducted under Federal authority, in cooperation 

with State and/or Federal personnel.

Consult the latest APHIS Lists of Proven Hosts of and Plants Associated with 

Phytophthora ramorum prior to beginning any survey, inspection, or 

delimitation.
Introduction

Reporting Issues With or Suggestions For the Manual

1-1-4 Phytophthora ramorum Manual/Introduction 3/2024-06

Reporting Issues With or Suggestions For the Manual

Refer to Table 1-1-1 to determine where to report problems or disagreements, 

or improvements that directly affect the contents of the manual.

Table 1-1-1 Reporting Issues With or Suggestions for the Manual

If you: Then:

 Are unable to access the online manual

 Have a suggestion for improving the 

format (layout, spelling, etc.)

CONTACT PPQ Manuals Unit at PPQ.IRM.ISMU.Manuals.Feedback@usda.gov

Disagree with a policy or procedure, or 

have an urgent situation requiring an 

immediate response

CONTACT Phytophthora ramorum National Policy Manager at william.d.wesela@usda.gov or 301-851-2229.

4700 River Road

Riverdale, MD 20737
3/2024-07 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 2-1-1

Chapter

2 Phytophthora ramorum

Inspection and Sampling 

Protocol for Nurseries

Contents

Introduction 2-1-1

Quick Guide for Conducting Nursery Inspection and Sampling 2-1-1

Timing Nursery Inspection and Sampling 2-1-3

Notifying the Laboratory 2-1-4

Preparing for Nursery Inspection and Sampling 2-1-4

Plant Symptoms and Sampling for P. ramorum 2-1-4

Plant Symptom Resources 2-1-4

Sampling by Symptom Type 2-1-5

Inspecting and Sampling the Nursery 2-1-7

Sampling Instructions for Plants 2-1-8

Sampling Instructions for Water 2-1-10

Sampling Instructions for Soil 2-1-10

Sampling Instructions for Pots and Containers 2-1-10

Sampling Instructions for Cull Piles 2-1-11

Sampling Instructions for Other Articles 2-1-11

Introduction

The objective of the Phytophthora ramorum Inspection and Sampling Protocol 

is to detect the presence of P. ramorum in interstate-shipping nurseries. This 

protocol describes methods for sampling plants, surface water, drainage water, 

water for irrigation, container mix, and any other articles designated by an 

inspector as possible sources of P. ramorum inoculum at the nursery.

Quick Guide for Conducting Nursery Inspection and Sampling

When sampling, remember to move from low-risk to high-risk areas to prevent 

potential spread of the pathogen.

1. Determine when to sample each nursery based on the time of year when 

climatic conditions will be most conducive for P. ramorum disease 

expression (refer to Timing Nursery Inspection and Sampling on page 

2-1-3).

2. After determining when to sample, notify the laboratory beforehand to 

ensure supplies are available and the laboratory is prepared to receive the 

samples (refer to Notifying the Laboratory on page 2-1-4).
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Quick Guide for Conducting Nursery Inspection and Sampling

2-1-2 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 3/2024-07

3. Ensure all supplies and equipment are available for the planned survey and 

review symptoms before arriving at the nursery (refer to Preparing for 

Nursery Inspection and Sampling on page 2-1-4).

4. Prior to inspection day, review:

A. APHIS Lists of Proven Hosts of and Plants Associated with 

Phytophthora ramorum

B. Obtain and review the nursery inventory, if available

C. Obtain and review any available maps of the nursery to determine areas 

to inspect and sample (refer to Inspecting and Sampling the Nursery on 

page 2-1-7)

D. Nursery history, if available (e.g., has it been previously positive for P. 

ramorum? Is it new? Has it recently changed management or 

ownership? What are the best management practices and sanitation 

measures?)

E. Fungicides can mask the presence of P. ramorum. Obtain the nursery’s 

fungicide spray schedules to better understand how fungicide 

treatments may impact symptom expression. For inspector safety, 

plants should not be sprayed immediately prior to inspection.

5. Determine the approximate number of plant samples to take from each 

regulated plant genus (refer to Table 2-1-1).

6. On inspection day, begin by conducting a visual of the nursery as a whole. 

Note topography, water sources, drainage patterns, areas of high risk (cull 

piles, low-vigor plants, etc.) to compare your observations to any nursery 

map provided and verify or note any of the following items (refer to 

Inspecting and Sampling the Nursery on page 2-1-7).

7. Take samples of:

8. Plants: sample symptomatic plant tissue. Ensure that at least the minimum 

number of samples are collected (refer to Table 2-1-1) and keep plant 

genera samples separate from one another. Each sample must be bagged 

separately (refer to sample on page Glossary-1-6). Record the collection 

location of each sample on the outside of the sample bag. Each bag should 

have a unique identification number and the date. During implementation 

of this protocol, every plant sampled is on regulatory hold and should not

be subject to scheduled nursery maintenance (refer to Plant Symptoms and 

Sampling for P. ramorum on page 2-1-4). While taking samples, visibly 

and indelibly flag or mark plants and areas sampled. Also mark sampled 

areas on a map of the nursery and take pictures, including areas of surface 

water (refer to Inspecting and Sampling the Nursery on page 2-1-7). 

Properly label and store collected samples for shipping to the laboratory 

(refer to Sampling and Submission Protocol on page 8-1-1).
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Timing Nursery Inspection and Sampling

3/2024-07 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 2-1-3

A. Water: sample water in and around the nursery. Each area is its own 

discrete sample. Collect surface water in and around plant material 

blocks. Collect from holding ponds, drainage ditches, water around cull 

piles, etc. (refer to Water Sampling and Processing Protocol on page 

10-1-1).

B. Pots and containers: if containers are recycled and stored at the 

nursery or if used pots are purchased, sample residual container mix 

from pots or other containers. Scrape container mix from pots filling a 

labeled, self-sealing plastic bag. Use the Soil and Container Mix 

Sampling and Processing Protocol on page 11-1-1.

C. Cull piles: examine any area where plants have recently been disposed. 

If regulated plants are present, sample symptomatic plant material and 

keep plant genera samples separate from one another. If there is any 

surface water, take at least one sample from each cull pile area.

D. Other articles designated by an inspector as possible sources of P. 

ramorum inoculum: at the inspector’s discretion, sample any and all 

other possible sources of P. ramorum inoculum.

E. Container mix: only container mix from used container piles is 

sampled in this protocol (refer to Soil and Container Mix Sampling and 

Processing Protocol on page 11-1-1).

9. Sanitize tools and change or sanitize gloves between samples to prevent 

cross-contamination (refer to Inspecting and Sampling the Nursery on page 

2-1-7).

10. Complete a PPQ Form 391 Specimens for Determination (or State 

equivalent) for each sample. Forward all samples to the appropriate 

laboratory (either NPPLAP-accredited or APHIS diagnostic laboratory; 

refer to Sampling and Submission Protocol on page 8-1-1 and U.S. State 

and Territory Plant Health Directors on page A-1-2 and APHIS Lists of 

Proven Hosts of and Plants Associated with Phytophthora ramorum).

Timing Nursery Inspection and Sampling

Nurseries should be inspected and sampled at a time of year when nursery 

conditions are optimal for P. ramorum disease expression.

Disease expression typically begins between 30 and 90 days after bud 

break. Inspection and sampling should begin after the spring flush is 

underway when some of the leaves have fully expanded. Plants can 

express symptoms throughout the growing season, though isolating the 

pathogen may be more difficult during hot and dry periods.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Notifying the Laboratory

2-1-4 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 3/2024-07

Nursery beds under shade cloth or overhead irrigation, greenhouses, and 

hot houses should be considered micro-climates where optimum 

conditions can occur outside of the typical fall/spring window.

Plan inspections and samplings when nurseries receive shipments in the 

spring and fall when regulated plants will be present and nursery 

conditions will be optimal.

Research suggests the most favorable climate for expressing symptoms 

(i.e., “conducive environmental conditions”) is when ambient 

temperatures are between 3 °C (37.4 °F) and 28 °C (82.4 °F) (optimum 20 

°C (68 °F)) and free moisture is present on regulated plant tissue for at 

least 12 hours over 10 or more days. In most areas, the most favorable 

timing is in the spring and fall.

Notifying the Laboratory

Notify the laboratory of an upcoming sampling date. This will also ensure the 

laboratory is prepared to receive the samples and prepared to process them 

promptly.

Preparing for Nursery Inspection and Sampling

Refer to Sampling Supplies and Equipment Checklist on page 8-1-4 and Water 

Sampling and Processing Protocol on page 10-1-1.

Plant Symptoms and Sampling for P. ramorum

Plant Symptom Resources

Inspectors must be trained to identify symptoms associated with P. ramorum

on regulated plants. At a minimum, they should review photographs of the 

wide range of possible symptoms before starting the inspection and sampling. 

Photographs of typical and atypical symptoms are available in Biology and 

Symptoms of Phytophthora ramorum on page 7-1-1.

Foliar symptoms of P. ramorum infection are highly variable and can range 

from pinpoint discolorations on the petiole and leaf surface to large “V”-

shaped lesions along the leaf mid-vein. Inspect the lower, more shaded 

portions of plants and the interior of the canopy where moisture and high 

humidity may persist. Pay special attention to leaf areas in which water would 

NOTICE

Symptoms of P. ramorum are variable and the greatest chance of detecting P. ramorum infections is through the collection of any unhealthy-looking plant tissue for laboratory analysis. Avoid desiccated or excessively decayed tissue.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Plant Symptoms and Sampling for P. ramorum

3/2024-07 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 2-1-5

linger such as the midrib and leaf tips. Check for leaves inside the pot of 

asymptomatic, regulated plants because infection could cause premature leaf 

drop and symptomatic leaves could be found only in the pot or on the ground. 

Many Phytophthora spp., other pathogens, and environmental stressors can 

cause symptoms that cannot be distinguished from P. ramorum infection by 

visual inspection. Do not presume to know what all P. ramorum symptoms 

look like. Collect samples of leaves with symptoms that could be caused by 

abiotic stressors. If there is not enough symptomatic, regulated plant material 

to fulfill the required number of samples (refer to Table 2-1-1), surveyors shall 

also collect any symptomatic material on nonregulated plant material or any 

symptomatic material on adjacent landscape plants.

Sampling by Symptom Type

Leaf Spots and Lesions

1. Collect symptomatic leaves

A. Some plants, i.e., Camellia or Loropetalum, may have very small 

pinpoint lesions.

B. Some leaves have very subtle symptoms, such as flecking or chlorotic 

spots.

C. For plants with very small leaves or needles, submit samples as twig 

sections with the leaves attached; in these cases, try to ensure the 

sample has a minimum of 2 square inches of symptomatic tissue.

D. If there are not enough symptomatic leaves on the plant, collect 

symptomatic leaves that have dropped into the pot, provided they are 

not exhibiting dessication or extensive decay.

E. If necessary to get the required amount of symptomatic tissue, collect 

composite leaves from up to five adjacent plants to make a composite 

sample.

Twig Dieback

1. Cut the twigs below the cankered region (1 inch into healthy tissue). The 

sample must include canker margins and 1 inch of healthy tissue on either 

side.

2. Sterilize pruning equipment between samples using a diluted (10%) bleach 

solution, a quaternary ammonium solution at labeled rates, or disinfectant 

spray (with ETOH).

Cankers on Boles and Branches of Regulated Plants

1. Some regulated plants do not have foliar symptoms but get cankers on 

boles or branches. Bole or branch cankers consistent with P. ramorum

disease must be sampled.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Plant Symptoms and Sampling for P. ramorum

2-1-6 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 3/2024-07

2. In some States, nursery inspectors may sample trees, while in other States, 

forestry or other officials may be asked to sample trees.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Inspecting and Sampling the Nursery

3/2024-07 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 2-1-7

Inspecting and Sampling the Nursery

Two basic principles governing the inspection and sampling process are:

1. P. ramorum cannot be diagnosed by a visual inspection of symptoms alone 

and only laboratory testing can provide a definitive diagnosis; and

2. If there is any doubt as to whether the symptoms observed could be caused 

by P. ramorum, collect a sample.

3. Before the inspection season begins, review the APHIS Lists of Proven 

Hosts of and Plants Associated with Phytophthora ramorum; if possible, 

time nursery inspection to periods when surface water is likely to be 

present. For example, this could be after a rain event, after irrigation, or 

early in the morning.

4. Prior to inspection day, if available, obtain and review a nursery plant 

inventory, a plant location map, an aerial map, and a topographic map of 

the nursery to determine areas to sample. Create a sampling plan based on 

the number of regulated plants present in the nursery and plan the areas to 

visit within the nursery (refer to Table 2-1-1). Initiate the survey and 

progress through the nursery beginning with the lower-risk areas, if 

possible. Within this protocol, the term “lower-risk” is meant to convey 

that these areas are less conducive to P. ramorum. More than one survey 

team can be deployed to the nursery and should be assigned specified areas 

(i.e., low risk versus high risk).

5. Begin the inspection by conducting a visual overview of the nursery as a 

whole to compare your observations to any nursery map provided and 

verify or note any of the following items. Identify cull piles, “plant 

hospitals” where low-vigor plants are kept for sale, and areas that may 

include plant returns. Determine irrigation water source (well, municipal, 

treated, or recycled). Note topography of the nursery and nursery drainage 

patterns and systems and irrigation method (i.e., overhead, drip, etc.). 

Confirm low-lying areas, surface water, nursery layout, the general 

condition of the plants, and the nursery environment.

6. Decontaminate inspection personnel, tools, and equipment between blocks 

in the nursery, between regulated plant genera within a block (refer to 

block on page Glossary-1-1), and between nursery sites. Wear rubber boots 

or other waterproof boots without sole crevices that can be treated with 

disinfectant. Sanitize or change gloves between samples. Use a spray bottle 

containing a diluted (10%) bleach solution, a quaternary ammonium 

solution at labeled rates, or disinfectant spray to treat all tools between 

samples. Brush loose dirt from boots, then spray boots to point of runoff 

with disinfection solution, or use foot bath, between nursery blocks. 
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Inspecting and Sampling the Nursery

2-1-8 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 3/2024-07

Decontaminate all equipment between each sample and before leaving a 

nursery.

7. Indicate inspected and sampled areas on the nursery map. Note plants 

sampled, surface water areas sampled, cull piles sampled, etc. Photograph 

sampled areas, including areas of surface water.

Sampling Instructions for Plants

Visually inspect all plants within a nursery paying careful attention to plants on 

the official APHIS Lists of Proven Hosts of and Plants Associated with 

Phytophthora ramorum. Collect samples from all symptomatic regulated, 

plants. Additional samples can be collected at the inspector’s discretion, all 

other nonregulated plant tissue with symptoms suggestive of P. ramorum.

Read Biology and Symptoms of Phytophthora ramorum on page 7-1-1 and 

view photos prior to entering the nursery. Each sample should consist of at 

least 2 square inches of symptomatic plant tissue; collect as many leaves as 

necessary to represent 2 square inches. It is strongly encouraged that each 

sample is from 1 plant; however, if there are not enough symptomatic leaves 

on the plant, collect symptomatic leaves that have dropped into the pot, 

provided they are not exhibiting desiccation or deterioration. The leaves still 

need to be mostly green. If the inspector is certain that leaves on the ground 

adjacent to the pot are from that plant, they can be used to complete the 

sample. Otherwise, symptomatic leaf debris (with distinct spots and margins) 

from the ground should be a separate sample and labeled as such.

The amount of leaf samples is necessitated by:

Down-stream confirmatory testing

Genetic analysis

Initial testing

Using Table 2-1-1 and the nursery inventory, determine the minimum number 

of samples to collect within the nursery.

If the number of regulated plants in a nursery is greater than one number (e.g., 

405 is greater than 400), move to the next greater number listed (500). If there 

are more symptomatic plants, always take more samples than the calculated 

minimum. This allows pinpointing the location of P. ramorum in the nursery 

and lessening the potential regulatory impact on the nursery.

NOTICE

The more plant samples composited into one, the larger the resulting destruction 

and quarantine radii will be if the composite sample is confirmed to be positive for P. 

ramorum. This is why, if possible, it is important for the plants in a composite sample 

to be adjacent to one another.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Inspecting and Sampling the Nursery

3/2024-07 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 2-1-9

After the survey results from the laboratory are reported to the inspector, the 

inspector may release all ELISA-, PCR, or culture-negative plants (unless

within the quarantine radii or destruction radii of a confirmed-positive 

detection).

Table 2-1-1 Minimum Number of Plant Samples to Take Based on the Number of Regulated Plants 

Within the Nursery

Regulated 

plants per 

nursery:

Minimum number of samples to collect (95% confidence of detecting a 1.0% disease incidence)1

:

25 25

50 50

100 100

200 173

300 211

400 234

500 250

600 262

700 270

800 277

900 283

1000 287

2000 308

3000 316

4000 320

5000 322

6000 324

7000 325

8000 326

9000 326

10000 327

20000+ 332

1 Numbers are the minimum number of regulated plants that must be sampled in a confirmed-positive nursery to ensure 

detection at a 95% confidence level for a 1.0% incidence of disease.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Inspecting and Sampling the Nursery

2-1-10 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 3/2024-07

Sampling Instructions for Water

Examine all areas within the nursery for surface water, particularly after a rain 

event or after irrigation occurs. With pin flags, demarcate each area in which 

water is collected. If demarcation is not possible, draw a sketch, take photos, 

and flag nearby areas. Label the water container and the flagging with 

corresponding numbers so any confirmed-positive samples can be located 

within the nursery. If helpful, take photos of each area in which water is 

collected. Areas of water sampling are not on hold awaiting diagnostic results, 

but must be visibly and indelibly marked in case of positive confirmation.

Irrigation water—sample all types of irrigation water except from a 

municipal source or well water. Sample at end dispensers (sprinklers, 

nozzles, drip, etc.) instead of source pipe. Sample retention ponds 

regardless of the source, because they are likely to contain runoff from 

production areas.

Standing water—as the first priority, sample standing water in and around 

blocks of regulated plant material and the drainage from regulated plant 

material blocks. Drains in greenhouses and hoop house systems 

containing regulated plant material can be accessed for sample collection 

after a rain event or after irrigation occurs. Collect a minimum of one 50- 

to 800-ml water sample per sample site from each general area in which 

surface water occurs, a minimum of one sample from each drainage ditch 

into which runoff from regulated plant blocks collects. Sample any water 

that is around or drains from cull piles as well.

Nonrecycled retention ponds—collect a minimum of one 800-ml water 

sample from each nonrecycled holding pond.

Refer to the Water Sampling and Processing Protocol on page 10-1-1.

Sampling Instructions for Soil

Standing water will be sampled in place of substrate soil sampling. No 

substrate soil samples are required for this sampling protocol.

Sampling Instructions for Pots and Containers

If containers are recycled and stored at the nursery or if used pots are 

purchased, sample residual container mix from pots or other containers; scrape 

container mix from pots filling a labeled one-liter self-sealing plastic bag. 

During the inspection and sampling protocol, there are no holds on the nursery 

or container pile associated with sampling containers while waiting for 

diagnostic results. Refer to the Soil and Container Mix Sampling and 

Processing Protocol on page 11-1-1.
Phytophthora ramorum Inspection and Sampling Protocol for Nurseries

Inspecting and Sampling the Nursery

3/2024-07 Phytophthora ramorum Manual/P. ramorum Inspection and Sampling Protocol for Nurseries 2-1-11

Sampling Instructions for Cull Piles

Examine any area in which plants have recently been disposed. If regulated 

plants are present, sample symptomatic plant material and keep plant genera 

samples separate from one another. If there is any surface water, take at least

one sample from each cull pile area. Demarcate for avoidance and do not

disturb the cull/compost pile or that area of the cull/compost pile while waiting 

for diagnostic results because the material collected is symptomatic plant 

tissue.

Sampling Instructions for Other Articles

At the inspector’s discretion, sample any and all other possible sources of P. 

ramorum inoculum.
3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-1

Chapter

3 Interstate1

 Confirmed 

Nursery Protocol

Protocol for Interstate Nurseries1 Confirmed 

Positive for Phytophthora ramorum

Contents

Introduction 3-1-2

Goal 3-1-2

Trigger Events for Use of the Interstate Confirmed Nursery Protocol 3-1-2

Disclaimers 3-1-3

Challenges 3-1-3

Field-Grown Stock 3-1-3

Interstate Confirmed Nursery Protocol Steps 3-1-3

Interstate Confirmed Nursery Protocol (CNP) Procedures 3-1-4

Schematic of Destruction (D) and Quarantine (Q) Radii of Positive Plants 

3-1-10

Notification Requirements for Interstate CNP 3-1-10

Disinfest the Nursery 3-1-11

Delimitation Sampling for Confirmed Plant Positives and Sample Handling 

and Submission Protocol 3-1-13

Collecting Samples 3-1-14

Trigger Sequence for Entire Block Destruction 3-1-14

Determining the Number of Samples to Take 3-1-15

Conduct Investigations 3-1-16

Associated Nursery Site Inspections 3-1-16

Trace Forward Investigations 3-1-16

Trace Back Investigations 3-1-17

Alternative Quarantine-Release Strategy 3-1-17

Post-Confirmed Nursery Protocol Monitoring 3-1-18

Critical Control Point Assessment Procedures for P. ramorum-ConfirmedPositive Nursery Sites 3-1-19

Repeat Positive Nurseries 3-1-20

Planning the CCP Assessment 3-1-20

CCP Assessment Day 3-1-20

Critical Control Points 3-1-23

References 3-1-27

1 Interstate shipping retail nurseries, brokers, interstate and certain intrastate wholesale and 

production nurseries.
Interstate Confirmed Nursery Protocol

Introduction

3-1-2 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

Introduction

The intended use of this protocol is for nurseries that have been confirmed 

positive for P. ramorum in plants, water, or other regulated articles. The 

nursery types are:

Interstate commerce brokers with a nursery site or holding lot

Interstate-shipping propagation, wholesale, and re-wholesale nurseries

Intrastate-shipping wholesale nurseries that distribute plants for interstate 

shipping (i.e., using an interstate shipper to broker plants to other States)

Online interstate retail plant sellers

This protocol does not cover retail nursery dealers who only ship intrastate. 

Intrastate retail nurseries are covered in Chapter 4 Protocol for Intrastate Retail 

Nurseries and Retail Nursery Dealers When Phytophthora ramorum Is Present 

(rCNP) on page 4-1-1.

Goal

The goal of this protocol is to limit the spread of P. ramorum, a quarantined 

plant pathogen, and to simplify the movement of P. ramorum-free nursery 

stock. When procedures described in this protocol are implemented, plant-toplant spread and movement of the pathogen through nursery shipments should 

be minimized. Cooperation by nursery management personnel is essential. 

Early detection and reporting of potential P. ramoram plant infections are 

crucial to ensure the spread is contained.

Trigger Events for Use of the Interstate Confirmed Nursery Protocol

This protocol shall be implemented by USDA–APHIS–PPQ, in cooperation 

with State Plant Regulatory Officials (SPROs), when the presence of P. 

ramorum has been confirmed in interstate-shipping nurseries from samples 

collected by regulatory officials. Samples may have been collected during 

surveys or inspections such as Cooperative Agricultural Pest Survey (CAPS), 

State Nursery Cleanliness Survey, national survey, State inspections, trace 

forward investigation, trace back investigation, or found by other means. 

APHIS regulatory authority can be used in all of these cases. The Interstate 

Confirmed Nursery Protocol (CNP) is triggered for any confirmed-positive 

sample, such as plants, water, soil, containers, container mix, or any other 

article.
Interstate Confirmed Nursery Protocol

Disclaimers

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-3

Samples must be diagnosed using a method approved by USDA–APHIS–PPQ 

and consistent with the Potentially Actionable Suspect Samples (PASS)

protocol (refer to the PPQ Phytophthora ramorum website for diagnostic 

information and the PASS protocol).

Disclaimers

Any interpretation of this chapter or its procedures not consistent with the 

goal listed above, is a misinterpretation and misrepresentation.

Challenges

P. ramorum is a micro-organism and difficult to detect. It can infect plants; 

infest container mix, soil, and water; and persist in these substrates despite the 

best eradication efforts. Detection and management of this pathogen is 

informed by continually improving science. These protocols and regulations 

will be adjusted accordingly, based on the understanding of the pathogen 

biology.

Field-Grown Stock

Field-grown stock can present different challenges and field personnel may 

need to adapt this protocol to those situations after discussion with the P. 

ramorum program National Operations Manager (NOM) until other 

appropriate modifications are incorporated.

Interstate Confirmed Nursery Protocol Steps

In chronological order, the steps for the Interstate Confirmed Nursery Protocol 

(CNP) are as follows.

1. Communicate and notify

2. Conduct trace investigations (concurrently with securing the nursery)

3. Secure the nursery (concurrently with conducting trace investigations)

A. Disinfest the nursery

B. Delimit the nursery

C. Delimiting survey results received

NOTICE

When planning to announce or make a public statement about the detection of a federally regulated pest, the State Plant Regulatory Official (SPRO) and/or the public 

information officer for the State department of agriculture must first contact the State 

Plant Health Director (SPHD) and/or USDA–APHIS–Office of Legal and Public 

Affairs (LPA).
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3-1-4 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

4. 90-day (minimum) quarantine activities

5. Release of plants in the nursery

6. Alternate quarantine-release strategy

7. Critical Control Point (CCP) assessment

8. Post-quarantine release monitoring

Interstate Confirmed Nursery Protocol (CNP) Procedures

NOTICE

Prior to an APHIS-confirmed positive determination, the National Plant Protection 

Laboratory Accreditation Program (NPPLAP)-approved laboratory must communicate all suspect positive diagnostic samples to inspectors as soon as one of the following has occurred:

• Culture that matches the morphology for P. ramorum as determined and reported 

by an APHIS-approved laboratory; or

• Positive PCR using APHIS-approved work instructions by an APHIS-approved laboratory

For all Potentially Actionable Suspect Samples (PASS), laboratories must immediately forward the plant material and DNA to the PPQ–S&T Plant Pathogen Confirmatory Diagnostics Laboratory (refer to Contact Information for the Phythopthora 

ramorum Program on page A-1-1) with a domestic ARM routing receipt (DARR) and 

notify the State’s State Plant Health Director (SPHD; who generates DARR for laboratory), the State Plant Regulatory Official (SPRO) for the State of sample origin, and 

the National Operations Manager (NOM).
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-5

Table 3-1-1 Interstate Confirmed Nursery Protocol (CNP) Procedures (page 1 of 5)

If the step number: Subnumber: Then:

1—Communicate and 

notify

When a sample is confirmed positive, laboratories need to notify the SPHD 

and the SPRO, the National Operations Manager (NOM), the National Program Manager (NPM), and the submitter. The SPRO (if State authority is 

used) or the SPHD (if Federal authority is used) may notify the owner of the 

nursery. These steps start immediately after SPHD and SPRO are notified of 

a confirmed-positive article in their State. SPHD and SPRO should designate an official to lead the activity.

1.1 The designated official will notify the confirmed-positive nursery of the confirmed-positive and instruct the nursery to place a hold on all regulated 

plants at the nursery or as many plants as deemed necessary by the inspector. Regulated plants outside the D and Q radii will be released from hold 

once the block is cleared by inspection.

1.2 SPHD and/or SPRO will notify the NOM of the nursery notification and the 

hold on the plants.

1.3 SPHD and/or SPRO will provide a list of the identified facilities found through 

trace back and trace forward investigations to the NOM within five business days of a confirmed P. ramorum-positive sample in a nursery (refer to 

2—Conduct investigations on page 4-1-5). The NOM will notify SPHDs and/

or SPROs of States sending or receiving these shipments.

1.4 SPHDs and/or SPROs will notify affected retail nurseries and retail nursery 

dealers within their States.

2—Conduct trace 

investigations (concurrently with securing the nursery)

2.1—Trace forward and trace 

back investigations

1. Determine from provided information if the nursery has distributed regulated plants to another nursery. If so, implement Trace Forward Protocol 

for Nurseries that Received Plant Material Shipped from a Confirmed P. 

ramorum-Infested Nursery on page 5-2-1. Submit the trace forward list(s) 

to the NOM within five business days. If a nursery does not meet the 

five business day requirement, the SPHD, SPRO, and NOM should work 

together to decide potential consequences, such as holding the compliance agreement.

2. Determine from provided information if the confirmed-positive plants were 

received from another nursery. If so, implement Trace Back Protocol for 

Nurseries that Shipped Plant Material to a Confirmed P. ramorumInfested Nursery on page 5-3-1. Submit the trace back list(s) to the NOM 

within five business days. If a nursery does not meet the five business 

day requirement, the SPHD, SPRO, and NOM should work together to 

decide potential consequences, such as holding the compliance agreement.

2.2—Associated nursery 

sites

1. Determine from provided information if additional locations (i.e., nursery 

sites) are owned and operated by the same nursery company.

2. Determine from provided information if nursery personnel are deployed to 

multiple locations. 

3. Determine from provided information if regulated plants have moved to 

other sites or among nursery sites. If so, all nursery sites receiving regulated plants must be surveyed.

4. Determine from provided information if equipment used at the infested 

site is shared with additional locations (i.e., nursery sites, field areas, 

etc.). Document any shared equipment use in those additional locations. 

Equipment movement among nursery sites must use appropriate biosecurity measures (refer to Biosecurity Measures for Nurseries on page 

9-1-1).
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3-1-6 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

3—Secure the nursery (concurrent with 

conducting trace 

investigations)

3.1 1. All regulated plants in the nursery should be held until they are cleared by 

inspection. To ensure material does not move prior to being cleared, use 

PPQ Form 523, Emergency Action Notification (EAN), or State equivalent.

2. Refer to the Schematic of Destruction (D) and Quarantine (Q) Radii of 

Positive Plants on page 3-1-10 for guidance. 

A. Arrive on site as soon as practicable. Identify locations of confirmedpositive sample sources. Obtain maps of the nursery (with bed layouts, 

if possible) and maps of the surrounding area for assessment.

B. Establish the quarantine (Q) and destruction (D) radii for each confirmed-positive source as described below. 

C. For any type of confirmed positive, hold all plants within each established Q-radii for the entire quarantine period (a minimum of 90 days 

of conducive environment for disease development). Instruct nursery to not use fungicides registered for Phytophthora spp. in the Q 

radii. 

D. During implementation of this protocol, every plant on regulatory hold 

should not be subject to scheduled nursery maintenance.

3. Restrict access to any D-radii until the inspector/nursery is prepared to 

begin disinfestation procedures; refer to Disinfest the Nursery on page 

3-1-11.

4. Put nursery under initial compliance agreement for first-time detection. 

(Contact the PPQ NPM or NOM for a copy of the Compliance Agreement.)

5. Hold may include “any other product or article that an inspector determines to present a risk of spreading P. ramorum, if an inspector notifies 

the person in possession of the product or article that it is subject to the 

restrictions in the regulations” (7 CFR part 301.92-2) within the infested 

nursery site.

3.2—Confirmed positive 

plant(s)

1. Establish and demarcate D-radii by visibly and indelibly flagging 2 meters 

out from the confirmed-positive plant(s) (refer to Schematic of Destruction (D) and Quarantine (Q) Radii of Positive Plants on page 3-1-10). 

Hold all plants within the 2-meter D-radius until all plants, pots, and potting medium are destroyed. A regulatory official must oversee plant 

destruction.

2. Establish an additional 2-meter radius around the D-radii and hold all

plants within that 2-meter radius for the Q-radius. Restrict fungicide use 

on plants in the Q radii and restrict access to this area.

3. Each new confirmed-positive plant requires a new D-radius and Q-radius.

3.3—Confirmed-positive 

surface water 

or soil

1. Establish and demarcate the confirmed-positive area by visibly and indelibly flagging 1 meter out from the margin of standing water. Make sure to 

include algal deposits, Nostoc spp., and aquatic plants in the margin of 

the demarcated area.

2. Restrict access.

3.4—Confirmed-positive 

cull pile

1. Establish and demarcate the area by visibly and indelibly flagging 1 meter 

out from the perimeter of the cull pile.

2. Restrict access.

3.5—Confirmed-positive 

used containers

Flag for hold until sanitation is applied (refer to Treatments and Disinfectants

on page 12-1-1).

Table 3-1-1 Interstate Confirmed Nursery Protocol (CNP) Procedures (page 2 of 5)

If the step number: Subnumber: Then:
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-7

3a—Disinfest the 

nursery

Depending on the complexity of the situation, disinfestation can occur before 

or after the delimiting survey.

Refer to Table 3-1-3 for disinfestation procedures. Refer to Treatments and 

Disinfectants on page 12-1-1.

3b—Delimiting survey 3b.1—All confirmed positivesFor more information on the delimiting survey, refer to the Delimitation Sampling for Confirmed Plant Positives and Sample Handling and Submission 

Protocol on page 3-1-13.

1. Ensure conducive environmental conditions are present, as described in 

Timing Nursery Inspection and Sampling on page 2-1-3.

2. Ensure necessary sanitation measures are applied by regulatory officials 

while in the confirmed nursery.

3. Inspect all regulated and nonregulated plants within the nursery.

4. Sample any symptomatic tissue found and submit samples to the appropriate laboratory. Refer to Table 3-1-5 on page 3-1-15 to determine the 

number of samples to collect.

5. Refer to individual delimiting procedures for specific confirmed-positive 

material.

6. Place all sampled plants on hold.

7. Disinfest tools and equipment associated with any confirmed-positive 

materials.

For each type of confirmed-positive material, follow the specific delimiting 

instructions below. In addition, use the Phytophthora ramorum Inspection 

and Sampling Protocol for Nurseries on page 2-1-1 for guidance.

3b.2—Confirmed-positive 

plant(s)

1. Immediately inspect all plants in the Q-radii and inspect other plants in 

that block. Take the number of samples required in Table 3-1-5 on page 

3-1-15.

2. Sample any unhealthy tissue, provided it is not exhibiting desiccation or 

extensive decay.

3. Inspect all regulated and nonregulated plants within the nursery.

4. Sample surface water from underneath confirmed-positive plant(s), as 

well as throughout the D-radii and adjacent downslope areas (refer to 

Water Sampling and Processing Protocol on page 10-1-1 and Soil and 

Container Mix Sampling and Processing Protocol on page 11-1-1).

5. Mitigate all soil and gravel under plants within the D-radii and adjacent 

downslope areas to be able to allow host plants in the area. If there is no 

mitigation, then no host plants are allowed in the area.

For retail nurseries, once the delimiting survey and sampling are complete, 

any held plants may be consolidated and segregated. 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 a manner that will safeguard and prevent the 

spread of the disease at the nursery, and be conducted under the direction 

and supervision of a regulatory official. If the plants are not consolidated and 

segregated, the affected portion of the nursery must be closed to the public. 

Segregation must include storage on an impermeable surface (e.g., concrete, asphalt, or a 45-mil thick pond liner) and not within 2 meters of any 

other plant. The impermeable surface should be sloped to drain away from 

regulated plants.

Table 3-1-1 Interstate Confirmed Nursery Protocol (CNP) Procedures (page 3 of 5)

If the step number: Subnumber: Then:
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3-1-8 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

3b—Delimiting survey

(cont.)

3b.3—Confirmed-positive 

surface water 

or soil

1. Examine all plants in the Q radius of demarcated sample area as well as 

all plants within a 10-meter perimeter beyond the Q-radius demarcations.

2. Sample any unhealthy tissue, provided it is not exhibiting desiccation or 

extensive decay.

3b.4—Confirmed-positive 

cull pile

If not sampled in initial inspection, sample surface water or symptomatic 

plants adjacent to and/or downslope from cull pile.

3b.5—Confirmed-positive 

used containers 

1. Inspect any plants within 2 meters of container storage area.

2. Sample symptomatic plants.

3. Sample surface water or soil within the 2-meter radius.

3b.6—Perimeter survey1. Survey for symptoms on all plants located within 10 meters of the 

infested nursery.

2. Sample all symptomatic plants.

3c—Delimiting survey results received3c.1—Confirmed-positive 

results from 

the delimiting 

survey

1. Conduct a second delimiting survey of the entire nursery as described 

above in 2a and 2b. Wait until all diagnostic results are final because 

subsequent delimiting surveys may be necessary if further confirmedpositive results are reported.

2. With each new confirmed-positive diagnostic result, restart the 90-day 

quarantine period. Please note that confirmed-positive surface water or 

soil may require longer to process and receive diagnostic results.

3c.2—Confirmed-positive 

results from 

the second 

delimiting survey

At the inspector’s discretion, after two positive delimitation surveys, the 

entire block of plants may be destroyed if the distribution of positive plants 

found outside the initial quarantine radius suggests an extensive and random pattern of infestation (refer to Trigger Sequence for Entire Block 

Destruction on page 3-1-14 for more details).

3c.3—Negative results 

from the delimiting survey

1. Release sampled plants or other articles from hold if they test negative 

for P. ramorum. However, continue to hold all plants or other articles 

within the Q-radius around confirmed-positive plants or other articles for 

the 90-day period.

2. The 90-day quarantine starts when samples are submitted.

4—90-day (minimum) quarantine 

activities

1. The 90-day (minimum) quarantine period begins the day samples are collected if: 

A. The delimiting survey is completed; and 

B. All delimiting sample results are negative; and

C. PPQ Form 523, EAN or sufficient State equivalent is issued.

2. Update hold notice for specific plants on hold (e.g., official communication, PPQ Form 523, or State equivalent).

3. Within the Q-radius, do not allow applications of fungicides registered for 

Phytophthora spp. control during the quarantine period.

4. Visually inspect plants within Q-radii a minimum of two times. Sample 

any symptomatic plants, as above. Conduct the first inspection approximately halfway through the quarantine period. Near the end of the quarantine period, a second visual inspection in the Q-radius should be 

performed while a visual survey of the entire nursery is being completed.

5. During the quarantine period, all sample results must be negative for P. 

ramorum or the quarantine period shall be extended for an additional 90 

days.

Table 3-1-1 Interstate Confirmed Nursery Protocol (CNP) Procedures (page 4 of 5)

If the step number: Subnumber: Then:
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-9

5—Release of plants 

in the nursery

Plants placed under regulatory control may be released from that control by 

PPQ or its designated authority after the quarantine period, if the following 

three conditions are met:

 There are no additional detections of P. ramorum in regulated and nonregulated plants based on PPQ-approved plant inspection, sampling, 

and testing protocols during the preceding 90-day quarantine period; 

and

 If testing water, soil, and growing media is required, those sample results 

are negative for P. ramorum based on PPQ-approved sampling and testing protocols for the preceding quarantine period; and

 Any resulting samples from the second visual survey at the end of the 

90-day quarantine period are negative for P. ramorum.

6—Alternate quarantine-release strategyA nursery may avoid a quarantine period through the voluntary Alternate 

Quarantine Release Strategy if the following conditions are met. The Alternate Quarantine Release strategy is not available to nurseries that meet the 

criteria for Continuously Positive Nurseries. (Refer to Continuously Positive 

Nursery section on page 3-1-20).

1. The nursery must destroy everything (e.g., all plants, containers, growing media, etc.) in each D-radius by approved methods listed in the specific nursery compliance agreement; and

2. Inspect and sample all regulated plants within the Q-radius. Then destroy 

all regulated plants within the Q-radius along with their containers, 

media, debris, etc.; and

3. Mitigate soil of each D- and Q-radius, as per Disinfesting Soil and Container Mix on page 12-1-4. Sample and test drainage or recirculated irrigation water, as per Sampling Instructions for Water on page 2-1-10; and

4. Complete critical control point assessment (refer to 7—Conduct Critical 

Control Point (CCP) assessment on page 3-1-9); and

5. Revisit the nursery a minimum of 90 days after completing the alternate 

quarantine-release strategy and conduct a nursery-level survey inspection. The nursery is subject to “post-quarantine-release monitoring” (refer 

to 8—Post-quarantine release monitoring below).

7—Conduct Critical 

Control Point (CCP) 

assessment

After completing the delimiting survey and implementing or planning disinfestation procedures for each confirmed-positive article, use the CCP 

assessment and reference material (refer to Critical Control Point Assessment Procedures for P. ramorum-Confirmed- Positive Nursery Sites on page 

3-1-19) to identify remediation and mitigation options, business/cultural 

practices, and best management practices (BMP) for the nursery’s site-specific plan to address P. ramorum. Refer to Compliance Agreement (obtained 

from PPQ NPM or NOM) for further instructions.

8—Post-quarantine 

release monitoring

Previously confirmed-positive nurseries shall be surveyed twice per year in 

successive years until there are 3 consecutive years of negative sample 

results. These nurseries are not under any other regulatory action unless 

there are additional P. ramorum detections. If there are further P. ramorum 

detections during the 3-year monitoring, the nursery must enter into a 

revised compliance agreement and restart 3 consecutive years of negative 

sample results.

Table 3-1-1 Interstate Confirmed Nursery Protocol (CNP) Procedures (page 5 of 5)

If the step number: Subnumber: Then:
Interstate Confirmed Nursery Protocol

Interstate Confirmed Nursery Protocol (CNP) Procedures

3-1-10 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

Schematic of Destruction (D) and Quarantine (Q) Radii of 

Positive Plants

Notification Requirements for Interstate CNP

SPHDs and/or SPROs will notify nurseries and the NOM within 24 hours of 

final determination of results from samples collected in their State. 

Figure 3-1-1 Schematic of Destruction and Quarantine Radii of Positive Plants

Table 3-1-2 Legend of Destruction (D) and Quarantine (Q) Radii of Positive Plants Schematic

Area color: Name: Once D-radii and Q-radii are flagged, then:

Gray (*1) Destruction (D) radii Destroy all plants, pots, medium, and leaf debris

Light gray (*2) Quarantine (Q) radii Hold all plants from sale for 90 days or opt for the Alternative QuarantineRelease Strategy on page 3-1-17

Gray (*3) D-radii in block with 

nonregulated plants

Destroy all plants; nonregulated plant nursery stock could still move the 

pathogen

Light gray (*4) Q-radii in block with 

nonregulated plants

Hold all plants from sale for 90 days; nonregulated plant nursery stock could 

still move the pathogen.

Dotted (*5) Rest of the block 

with regulated 

plants

Release all plants for sale only when found to be asymptomatic during the 

delimiting survey

White (*6) Rest of the block of 

nonregulated plants

Release all plant materials for sale if found to be asymptomatic during the 

delimiting survey
Interstate Confirmed Nursery Protocol

Disinfest the Nursery

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-11

SPHD and/or SPROs will provide a list of the identified facilities found 

through trace back and trace forward investigations to the NOM within five 

business days of a confirmed P. ramorum-positive sample in a nursery (refer 

to Conduct Investigations on page 3-1-16). The NOM will notify SPHDs and 

SPROs of States sending or receiving these shipments, and SPHDs and/or 

SPROs will notify affected nurseries within their States.

Disinfest the Nursery

Conduct the Critical Control Point (CCP) Assessment only after completing 

the delimiting survey and implementing or planning disinfestation procedures 

for each confirmed-positive article. Then, use the CCP assessment and 

reference material to identify remediation and mitigation options, business/

cultural practices, and best management practices (BMP) for the nursery’s sitespecific plan to address P. ramorum. Refer to Compliance Agreement 

(obtained from PPQ NPM or NOM) for further instructions.

Table 3-1-3 Disinfest the Nursery (page 1 of 2)

If material is: Then:

Confirmed-positive 

plant(s)

1. Destroy all plants, pots, medium, and leaf debris in the D-radii, per Treatments and Disinfectants on page 12-1-1; refer to Schematic of Destruction (D) and Quarantine (Q) Radii of Positive Plants on page 3-1-10.

2. Remove and destroy all plant debris including container mix and any other plant parts found 

within the D-radii; refer to Treatments and Disinfectants on page 12-1-1 for proper removal 

and destruction.

3. A regulatory official must oversee plant destruction.

4. For field-grown stock, contact the NOM.

5. Sample surface water or soil underneath the D- and Q-radii; refer to Water Sampling and 

Processing Protocol on page 10-1-1 and Soil and Container Mix Sampling and Processing 

Protocol on page 11-1-1.

Confirmed-positive 

surface water

1. Photograph area for the nursery owner and the CCP assessment team; site-specific conditions may apply depending on CCP assessment.

2. Maintain flagging for avoidance until remediation is chosen by the nursery owner with 

approval from the regulatory inspector and written into Exhibit D of the CA. Refer to Compliance Agreement (obtained from PPQ NPM or NOM) for further instructions.

Plants sitting in confirmed-positive surface water

Remove and destroy all plants, container mix, and pots sitting in confirmed-positive surface 

water as well as all plants within a 2-meter buffer, because these items have been exposed to 

water containing a quarantine organism. Site-specific recommendations may apply depending 

on CCP assessment. Confirmed-positive surface water samples initiate the 90-day quarantine 

period whether or not the plants standing in the water are confirmed positive.

Confirmed-positive 

irrigation water

1. Cease using confirmed-positive irrigation source until treated; irrigation water sources must 

be free of P. ramorum as determined by water-testing protocols described in Soil and Container Mix Sampling and Processing Protocol on page 11-1-1.

2. Mitigate the irrigation water if it was sampled and tested positive for P. ramorum during the 

survey and delimitation of the infestation at the nursery; refer to Treatments and Disinfectants on page 12-1-1.

3. Confirmed-positive irrigation water samples initiate the minimum 90-day quarantine period 

for plants receiving positive irrigation water.
Interstate Confirmed Nursery Protocol

Disinfest the Nursery

3-1-12 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

Confirmed-positive 

cull pile

1. Immediately demarcate the cull pile to avoid pathogen dispersal (as a quarantine hold).

2. Dispose of all material (plants, plant material, water, growing media, or soil) from the cull pile 

if any material is confirmed positive for P. ramorum. For disposal, use one of the approved 

methods described in Treatments and Disinfectants on page 12-1-1.

3. Use the CCP assessment to address site-specific conditions and determine appropriate mitigation measures. Refer to Compliance Agreement (obtained from PPQ NPM or NOM) for 

further instructions.

Confirmed effluent 

water (e.g., culvert/

ditch, stream, nonrecycled retention 

pond) is positive

1. Immediately demarcate the area around the confirmed-positive effluent water.

2. Identify remediation, mitigations, or business/cultural practices via the CCP assessment with 

the nursery owner.

3. Use the CCP assessment to address site-specific conditions and determine appropriate mitigation measures. Refer to Compliance Agreement (obtained from PPQ NPM or NOM) for 

further instructions.

Confirmed-positive 

soil (nursery substrate)

1. Locate and reestablish boundary demarcation.

2. Place barrier mitigation and/or adopt appropriate avoidance practices while determining the 

disinfestation/remediation strategy.

3. Use the CCP assessment, Treatments and Disinfectants on page 12-1-1, and mitigation 

options available from nursery associations, county extensions, and State nursery practices 

manuals. Refer to Compliance Agreement (obtained from PPQ NPM or NOM) for further 

instructions.

Confirmed-positive 

tools or equipment

1. Disinfest using options in Biosecurity Measures for Nurseries on page 9-1-1 and Treatments 

and Disinfectants on page 12-1-1.

2. Choose and institute cultural practices to ensure future sanitation (e.g., refer to the Best 

Management Practices Manual to Reduce the Risk of Introducing Soil-Borne Plant Pathogens into Horticultural Nurseries and Managed Wildland Landscapes).

3. Use the CCP assessment, Treatments and Disinfectants on page 12-1-1, and mitigation 

options available from nursery associations, county extension agents, and State nursery 

practices manuals. Refer to Compliance Agreement (obtained from PPQ NPM or NOM) for 

further instructions

Table 3-1-3 Disinfest the Nursery (page 2 of 2)

If material is: Then:

Table 3-1-4 Quarantine Period for Plants in Q-Radii (Figure 3-1-1)

If step number: For: Then:

1 —Quarantine period 

activities

The quarantine period, a minimum of 90 days of conducive weather conditions, 

begins when the nursery delimitation survey(s) are complete and all test results are 

negative. Plants, water, or other articles in Q-radii remain on hold for the full period.

During the quarantine period:

1. Do not use fungicides registered for Phytophthora spp. in the plant’s Q-radii.

2. Regulatory officials will inspect plants in the Q-radii and regulated plants in the 

nursery a minimum of two times; once about halfway through the anticipated 

quarantine period and once near the end so test results coincide with the end of 

the period. All symptomatic regulated plants must be sampled and tested; the 

second inspection can serve as the quarantine release survey.

3. If confirmed-positive samples result from quarantine period surveys, return to 

steps 2 through 4 in Table 3-1-1 on page 3-1-5; quarantine period begins again.
Interstate Confirmed Nursery Protocol

Delimitation Sampling for Confirmed Plant Positives and Sample Handling and Submission Protocol

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-13

Delimitation Sampling for Confirmed Plant Positives and Sample 

Handling and Submission Protocol

The delimitation survey begins once the confirmed-positive results are 

reported to the inspector. Conduct delimitation, inspection, and sampling after 

(on the same day(s)) the D-radii and Q-radii are established. The delimitation 

survey instructions for all other articles other than plants are provided in 3b—

Delimiting survey on page 3-1-7. 

2—Quarantine release 

survey

The quarantine release survey is the second of the two quarantine period inspections. It occurs near the end of the quarantine period. This survey includes Q-radii 

plant inspection and all regulated plants within the nursery. Sample and test any 

unhealthy plant tissue.

Transition 

from EAN to 

new or 

revised CA

Q-radii: after 90 days of conducive weather conditions, if the quarantine release survey reveals no symptomatic plants or further confirmed-positive plants in Q-radii, and 

reveals no further positive surface water or soil in Q-radii, release Q-radii.1

To retain interstate shipping status, or to otherwise distribute plants for interstate 

shipment (brokered), the nursery must enter into a compliance agreement.

1 Surface water, effluent water (e.g., culvert, ditch, and nonrecycled retention pond water), soil, or cull piles may take longer 

than 90 days to disinfest/remediate; ensure avoidance/exclusion mitigations are in place for these confirmed-positive areas. 

This short-term mitigation and the longer-term remediation plan needs to be written into Exhibit D of the CA (refer to Compliance Agreement, obtained from PPQ NPM or NOM, for further instructions) prior to the end of the quarantine period. The 

end of the quarantine period infers the close of the Emergency Action Notification (EAN) and the beginning of the CA or the 

modification of the CA to contain measures to address P. ramorum in the nursery. Close the EAN after the following conditions are met:

Tests of all symptomatic plants from the quarantine release survey are negative

If surface water, effluent water (culvert, ditch, and nonrecycled retention pond water), soil, or cull pile is still confirmed 

positive awaiting remediation, ensure exclusionary/avoidance mitigation is in place and the specific temporary 

mitigation and the permanent remediation plan is written into Exhibit D of the CA prior to the end of the quarantine 

period. Contact the PPQ National Operations Manager (NOM) for a template and conformance instructions

If the irrigation source water was confirmed positive, ensure it has been treated and found negative prior to the 

quarantine release survey, or, an alternate source is in place until the water tests negative. Ensure the alternate source 

and/or sanitation/mitigation plan is written into Exhibit D of the CA

The new CA is signed

Table 3-1-4 Quarantine Period for Plants in Q-Radii (Figure 3-1-1)

If step number: For: Then:

NOTICE

REMINDER: disinfest personnel, tools, and equipment between blocks in the nursery, 

between regulated plant genera within a block, and between nurseries. Wear rubber 

boots or other waterproof boots without crevices. Sanitize or change gloves between 

samples. Use a spray bottle containing a diluted (10%) bleach solution, a quaternary 

ammonium solution at labeled rates, or disinfectant spray (with ETOH) to treat all

tools between samples. Between nursery blocks, brush loose dirt from boots then 

spray boots with disinfectant solution in spray bottle, or use a foot bath. Disinfest all

equipment used between each sample and before leaving the nursery.
Interstate Confirmed Nursery Protocol

Delimitation Sampling for Confirmed Plant Positives and Sample Handling and Submission Protocol

3-1-14 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

When delimiting due to confirmed-positive detection in plants, focus on 

sampling the individual blocks in which the confirmed-positive plants were 

found. Follow the sampling method below for each individual block with a 

confirmed positive(s). Inspectors should sample all symptomatic material 

present. Each sample should consist of a minimum of 2 square inches of 

symptomatic plant tissue. The minimum number of plants to be sampled will 

depend on the total number of plants in the confirmed-positive nursery block.

Collecting Samples

Collect samples from all symptomatic regulated plants and, at the inspector’s 

discretion, all other nonregulated plant tissue with symptoms suggestive of P. 

ramorum (refer to Biology and Symptoms of Phytophthora ramorum on page 

7-1-1). Foliar symptoms of P. ramorum infection are highly variable and can 

range from pinpoint discolorations on the leaf surface to large V-shaped lesions 

along the leaf mid-vein. Include inspection of the lower portions of plants 

where conditions favoring P. ramorum would be present. Moisture will tend to 

be present for longer periods of time on the plant surfaces on the lower 

portions of the plants, which can also result in higher humidity depending on 

plant spacing. Shading on the lower portions of the plants can promote cooler 

temperatures and offer protection from the effects of ultraviolet (UV) rays on 

spores. Pay attention to leaf areas at which water would run off or persist the 

longest such as the midrib and leaf tips. In some regulated plants (Camellia and 

Rhododendron) low levels of infection can cause premature leaf drop, resulting 

in infected plants that appear to be asymptomatic. As a result, leaves found in 

the pot should also be checked for possible symptoms and collected for 

laboratory analysis.

The purpose of the perimeter survey: 1) to ensure P. ramorum has not spread 

outside the infested nursery to the surrounding environment; and 2) to verify 

the infestation in the nursery did not originate in the surrounding environment.

Trigger Sequence for Entire Block Destruction

At the inspector’s discretion, the below sequence may trigger the destruction of 

an entire block of plants where the original P. ramorum-positive articles were 

detected. Two positive delimitations in the same block may trigger an entire 

block destruction if the distribution of positive plants found outside the initial 

quarantine radius suggests an extensive and random pattern of infestation. The 

trigger sequence is as follows:

1. An initial positive plant is detected in the block.

2. In the same block, positive plants are detected in the first delimitation 

survey, triggering a second delimitation.

3. In the same block, positive plants are detected during the second 

delimitation survey outside any positive plant quarantine radius, and 

suggest an extensive and random pattern of infestation in the block.
Interstate Confirmed Nursery Protocol

Delimitation Sampling for Confirmed Plant Positives and Sample Handling and Submission Protocol

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-15

Determining the Number of Samples to Take

Determine the minimum number of symptomatic plant samples of regulated 

plants to take within a confirmed-positive nursery. Samples should be targeted, 

not random. Inspectors should sample all symptomatic plant material present, 

including leaf tissue from the pots, or, if the inspector is absolutely certain the 

leaves are from a given plant, leaves may be collected from the ground 

immediately adjacent to the plant.

Using a permanent marking method, label the sample bag with the following: 

date; genus species; cultivar; collector’s identification number; location of 

sample site; and sample number. Visibly and indelibly mark the sampled plant 

(with flagging tape, stake, etc.), and label with the corresponding sample 

number, date, and other identifying information as required. This will facilitate 

Table 3-1-5 Minimum Number of Samples to Take Based on the Number of Regulated Plants

Regulated 

plants: Minimum number of samples to collect (95% confidence of detecting a 1.0% disease incidence)1

:

25 25

50 50

100 100

200 173

300 211

400 234

500 250

600 262

700 270

800 277

900 283

1000 287

2000 308

3000 316

4000 320

5000 322

6000 324

7000 325

8000 326

9000 326

10000 327

20000+ 332

1 Numbers are the minimum number of regulated plants that must be sampled in a confirmed-positive nursery to ensure 

detection at a 95% confidence level for a 1.0% incidence of disease.
Interstate Confirmed Nursery Protocol

Conduct Investigations

3-1-16 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

any additional work in the event of a confirmed-positive sample or the need for 

a second sample.

Conduct Investigations

Concurrent to notifying and securing the nursery, require the nursery to 

research shipments of the past 6 months for the trace forward and trace back 

investigations and to provide this information within five business days.

Associated Nursery Site Inspections

Determine whether additional locations (nursery sites) are maintained by the 

same nursery owner or if regulated plants are moved to or among other sites or 

between sites during the 6-month time period preceding the confirmed-positive 

detection.

Equipment or other articles: determine if equipment or other articles used 

at the site is shared with other nursery sites or field areas; document any 

shared personnel, equipment, used containers, tools, etc., in different 

nursery sites or field areas. Equipment movement without appropriate 

biosecurity measures (refer to Biosecurity Measures for Nurseries on 

page 9-1-1) between nursery sites requires all nursery sites to be surveyed

Plants: determine if regulated plants are moved among sites, and if so, all

sites receiving regulated plants must be surveyed

Trace Forward Investigations

At the time P. ramorum is confirmed in a nursery, it is necessary to determine if 

the nursery has shipped plants that could potentially be infected. The first step 

of the trace forward investigation is to determine if the nursery is required to 

notify receiving States, refer to Notification Requirements for Interstate CNP

on page 3-1-10 for further instructions.

Initiate the trace forward investigation by identifying all plants shipped 

(domestic and international) within 6 months of the first (and any subsequent) 

confirmed-positive detection of P. ramorum at a nursery meeting the following 

criteria: 1) plants of the infected species/cultivar; 2) all regulated plants that 

originated in the D- and Q-radii; and 3) any plants of the high-risk genera: 

Camellia spp., Kalmia spp., Pieris spp., Rhododendron spp. (including azalea), 

and Viburnum spp. regardless of their location in the nursery. This combination 

of shipped plants is referred to as the “high-priority target plants.” These 

plants, including their shipment date(s); quantities; and respective destinations, 

make up the trace forward list. Identify these high-priority target plants using 

the best available information and to the lowest taxon possible (e.g., if the 
Interstate Confirmed Nursery Protocol

Conduct Investigations

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-17

plants can be identified to cultivar, trace forward activities may be conducted at 

the cultivar level).

Within 30 calendar days, the NOM will forward domestic trace forward lists to 

the States that have received plants. The NOM will forward international trace 

forward lists to the National policy Manager (NPM) who will work with 

Phytosanitary Issues Management (PIM) to inform internal trading partners of 

shipments to their countries. The plants sent to the receiving States must be 

inspected at the receiving nurseries (trace forward sites). Once the trace 

forward information is collected and communicated to the receiving States (or 

countries), the receiving regulatory officials will implement the trace forward 

protocol, refer to Trace Forward Protocol for Nurseries that Received Plant 

Material Shipped from a Confirmed P. ramorum-Infested Nursery on page 

5-2-1.

Trace Back Investigations

At the time P. ramorum is confirmed in a nursery, determine if confirmedpositive plants were shipped from another nursery. Trace back plants include 

all plants of the same genus of the infected plant regardless of size, location, or 

lot, back to the original propagation source (if it still exists).

The NOM will forward domestic trace back invoices to the States that have 

shipped plants. The NOM will forward trace back invoices from international 

sources to the NPM to inform PIM and the international trading partners of 

potentially positive shipments from their country. To view the trace back 

protocol refer to Trace Back Protocol for Nurseries that Shipped Plant Material 

to a Confirmed P. ramorum-Infested Nursery on page 5-3-1.

Alternative Quarantine-Release Strategy

A nursery may avoid a quarantine period through a voluntary management 

decision if the following conditions are met.

The alternate quarantine release strategy is not available to nurseries who meet 

the criteria of Continuously Positive Nurseries (refer to Continuously Positive 

Nurseries on page 3-1-19).

If this nursery does not meet Continuously Positive Nursery Criteria, 

follow:

1. Steps 1 through 5 in section 6—Alternate quarantine-release strategy on 

page 3-1-9 of Table 3-1-1 and all steps in Table 3-1-3 are complete.

NOTICE

Forward the trace forward list(s) to PPQ’s National Operations Manager (NOM) within 

five business days.
Interstate Confirmed Nursery Protocol

Conduct Investigations

3-1-18 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

2. All plants, pots, media, debris, etc. in the D-radii and all regulated plants 

and their pots, media, debris, etc. in the Q-radii are destroyed.

3. The CCP assessment is complete.

4. Surface water, effluent water (e.g., culvert, ditch, and nonrecycled retention 

pond water), soil, or cull piles may take some time to disinfest/remediate; 

ensure avoidance/exclusion mitigations are in place for these confirmedpositive areas.

5. The compliance agreement shall be updated based on the CCP assessment 

and shall contain measures to address P. ramorum in the nursery and the 

agreement is signed by the nursery representatives and regulatory officials. 

(Refer to Compliance Agreement, obtained from PPQ NPM or NOM.) The 

nursery is under a compliance agreement for a minimum of 3 years from 

this date.

6. The short-term mitigations and the longer-term remediation plan are 

written into Exhibit D, updating PPQ Form 519, compliance agreement 

(CA). (Refer to Compliance Agreement, obtained from PPQ NPM or 

NOM.)

7. Inspect for compliance.

Post-Confirmed Nursery Protocol Monitoring

Under the Compliance Program, if a nursery in the regulated or nonregulated 

area tests negative after 3 years (all samplings (6 or more) during the 

conducive time), it shall be released from compliance. For nurseries in the 

quarantine area, the sampling returns to the 7 CFR § 301.92 regimen.

Nurseries under compliance are required to notify state regulatory officials 

when host plants are shipped. They must notify state regulatory officials in 

both origin and receiving states at the time of shipment. The nursery must also 

notify state regulatory officials about shipments of all infected species/

cultivars found in the nursery, as well as shipments of Camellia spp., Kalmia

spp., Pieris spp., Rhododendron spp. (including azalea), and Viburnum spp. 

The USDA and state departments of agriculture will provide the information 

and requirements about the Notify system and its use.
Interstate Confirmed Nursery Protocol

Critical Control Point Assessment Procedures for P. ramorum-Confirmed- Positive Nursery Sites

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-19

Critical Control Point Assessment Procedures for P. ramorumConfirmed- Positive Nursery Sites

After positively confirming the presence of P. ramorum at a nursery site and 

completing the delimiting survey or completion of the alternative release 

strategy, a Critical Control Point (CCP) assessment of the nursery operation 

will be conducted by a team composed of State, Federal, and other subject 

matter experts (SMEs). CCPs are points in a nursery process or procedure that 

can result in the unintended spread or introduction of P. ramorum. Determining 

CCPs allows applicable best management practices (BMPs) and/or mitigations 

to be provided to the nursery in a systems approach. A systems approach 

consists of a defined set of phytosanitary procedures, at least two of which 

have an independent effect in mitigating pest risk associated with the 

movement of commodities. It is a proactive process to reduce the risk of 

infestation by correcting unsafe nursery practices leading to safe production 

practices that result in healthy plants. By addressing CCPs, nurseries can 

reduce the risk of a potential hazard and take corrective steps leading to P. 

ramorum mitigation and/or avoidance. Mitigation measures will be written in 

Exhibit D, updating PPQ Form 519, compliance agreement. Please contact 

the PPQ S&T Representative for a copy of the fillable CCP template prior 

to the assessment.

Continuously Positive Nurseries

Nurseries that continue to find positive plants over a period of time will be 

assessed and additional mitigations will be required in Appendix D of the 

compliance agreement to ensure P. ramorum is not being moved. Best 

management practices (BMPs) can reduce or eliminate P. ramorum in a 

nursery setting and the required mitigations are part of the requirements for 

shipping plants in a nursery that has been found positive for P. ramorum.

A continuously positive nursery is defined as: a regulated nursery that has been 

under compliance for five consecutive years or more and has had a positive 

detection within the last six months. 

Once a nursery falls under the definition of a continuously positive nursery, 

then the following actions will be required, as identified by the CCP team:

 Upon initial designation as a continuously positive nursery, State, federal, 

and other Subject Matter Experts (SMEs) will conduct a CCP inspection of 

the entire nursery (i.e., a systems approach audit). To bring in new perspectives, USDA recommends including SMEs who were not present at the first 

CCP. Inspectors will review the previous CCP and other historical information about the nursery. State and federal partners should agree in advance 
Interstate Confirmed Nursery Protocol

CCP Assessment Day

3-1-20 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

who will lead the audit and what the process will be to streamline the CCP 

assessment. 

 If possible, the nursery will designate a location where all incoming plants 

will arrive and be held for a minimum of three weeks.

 When inspectors find a positive plant, the continuously positive nursery will 

be required to, at minimum, destroy the D and Q radius. The nursery may be 

required to destroy the entire block if identified by the inspector. 

 Nurseries will place only non-hosts on positive blocks that have mitigated 

soil. 

Planning the CCP Assessment

The CCP assessment team should include at least two members who 

participated in the initiating inspection and sampling event. Schedule the 

assessment with the nursery owner and team well in advance.

To initiate the assessment, the team must consider any available 

information prior to arrival. Once at the site, the assessment team gathers 

additional information through discussions with nursery owners and managers, 

the inspection sampling team, and possibly county, State, and Federal 

personnel with knowledge of the nursery. Note that at the time of the site visit, 

not all nursery processes or situations may be present due to weather or change 

of seasons.

CCP Assessment Day

Initially, the CCP assessment focuses on the areas of the nursery associated 

with confirmed-positive P. ramorum samples such as plants, water, soil, and 

pots. However, the assessment of the nursery operations may identify CCPs 

relating to larger nursery areas beyond the areas associated with confirmedpositive samples.

Information Needed PRIOR to the CCP Assessment

This list is not meant to be inclusive.

1. Maps of the nursery (with bed layouts, if possible) and maps of the 

surrounding area for assessment planning purposes

2. Site assessment

A. Perimeter—look for possible sources of inoculum (water or regulated 

plants)

B. Site history

NOTICE

Under 7 CFR 301.92, it is illegal to knowingly move the pathogen P. ramorum without a permit authorizing movement.
Interstate Confirmed Nursery Protocol

CCP Assessment Day

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-21

a. Prior ownership

b. Prior crops on site

c. Plants previously grown in ground

d. Failed crops or plants (specifically regulated plants)

e. History of prior confirmed-positive detections

f. History of pesticide use

g. History of on-site flooding

h. Weather patterns, rainfall, etc.

i. Location of high-risk areas (e.g., locations of regulated plant 

material and high-traffic areas)

3. Access to property

A. Nursery rules for entering the site

B. Public versus landscapers’ access

C. Any available logs or records of who has been on site

D. Movement of plants and equipment between properties

4. Production practices/standard operating procedures (SOPs)

A. Crop rotation practices

B. Tracking movement of regulated plant material on site

C. Are regulated plant materials treated or handled differently?

D. Are holdovers mixed in with new stock or plants moved to fill in 

blocks?

5. Training

A. Has there been any staff training for scouting P. ramorum symptoms?

B. Are workers equipped with flagging as standard equipment to mark 

plants after scouting?

Please contact the PPQ–S&T representative for a fillable CCP template to use 

during the assessment. This template may also be used prior to the assessment 

team’s arrival for information-gathering purposes and to focus the team’s 

efforts and optimize time.
Interstate Confirmed Nursery Protocol

CCP Assessment Day

3-1-22 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

After the CCP Assessment

After the information gathering and site assessment are completed, the team 

identifies the CCPs associated with the known, confirmed-positive sample 

sites and may include other components of the nursery operation. Send a copy 

of the completed CCP template to the PPQ S&T Representative. The State 

and PPQ regulatory officials/SMEs will provide as much information as 

possible about mitigation measures to assist the nursery owner. Nursery 

owners can request assistance from subject matter experts (county and 

university extension, nursery associations, etc.) to better understand required 

and recommended remediation measures, mitigations, and BMPs 

corresponding to the CCP assessment.

Note to CCP assessment team: surface water, effluent water (culvert, ditch, and 

nonrecycled retention pond water), soil, or cull piles may take longer than 90 

days (the quarantine period) to disinfest/remediate. If the CCP assessment 

team makes specific mitigation recommendations, identify short-term 

mitigation versus longer-term remediation. Both short-term and longer-term 

measures agreed upon by the nursery owner/inspector will be written into 

Exhibit D, updating the compliance agreement with appropriate time periods.

NOTICE

Based on the CCP assessment, remediation, mitigations, and/or specific business/

cultural practices will be determined to address the presence of the pathogen within 

the nursery. Appendix D will contain the findings of the CCP assessment detailing 

the affected areas and will describe the remediation, mitigations, and/ or BMPs discussed with the nursery and agreed upon by the regulatory official.
Interstate Confirmed Nursery Protocol

Critical Control Points

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-23

Critical Control Points

The following table provides examples of CCPs, remediations, mitigations, best management 

practices, and cultural changes that can be applied for P. ramorum. This list is not exclusive: other 

mitigations may be equally effective. For guidance on other potential mitigations, consult the BMP 

guides (refer to the References section on page 3-1-28), your state extension personnel, or the PPQ 

S&T representative.

Table 3-1-6 Examples of CCPs, Remediations, Mitigations, Best Management Practices (BMPs), and 

Changes to Business/Cultural Practices1

 (page 1 of 4)

Identified critical control 

point (CCP):

Mitigations that may have preceded CCP assessment:Examples of remediations, mitigations, BMPs, and 

changes to business/cultural practices:

Regulated 

plant material

 Double-bagged, identified material in (2 mil) plastic bags and 

deep burial (>2 m) burial in a site 

approved by regulatory authorities

 Incinerated at a site approved by 

regulatory authorities

 Heat sterilization; dry heat or 

steam (refer to the USDA Treatment Manual Schedule T415b)

 Avoid accepting returned plant material; destroy or dispose of any returned regulated plant material

 Designate or assign specific personnel to work with regulated plant material for monitoring and management 

purposes

 Do not allow plant foliage to be in contact with the 

ground

 Do not mix incoming crops with existing regulated plant 

material

 Designate an area for unloading and holding regulated 

plant material for 30 days’ monitoring

 Purchase from nurseries licensed or certified under all

phytosanitary laws and applicable Federal and State 

regulations

General operation sanitation Disinfestation of nonporous surfaces, concrete floors, benches, 

plastic sheeting, and tools

 Adequately control weeds on the nursery site as they 

may potentially harbor the pathogen

 After every crop rotation, disinfest propagation mist 

beds, sorting area, cutting benches, machines, and 

tools to minimize the spread or introduction of pathogens

 Develop or review processes of cleaning carts and trailers used in moving plant materials, including tires

 Develop process for ensuring workers’ clothing is clean 

and management tools are routinely cleaned and sanitized

 Do not allow trucks to sweep out debris on site

 Install foot baths in all high-risk areas, including for visitors to the production areas

 Prevent buildup of fallen leaves and plant debris from 

regulated plants in production areas and monitor with 

every crop rotation or quarterly, whichever is more frequent

General operation sanitation

(cont.)

 Disinfestation of nonporous surfaces, concrete floors, benches, 

plastic sheeting, and tools

(cont.)

 Properly dispose of any leaves or plant debris resulting 

from nursery operations or cleanup of areas or beds

 Routinely clean loading and shipping areas following 

shipment arrivals or after loading activities
Interstate Confirmed Nursery Protocol

Critical Control Points

3-1-24 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

Potting media  Double-bagged, identified material in (2 mil) plastic bags and 

deep burial (>2 m) burial in a site 

approved by regulatory authorities

 Incinerated at a site approved by 

regulatory authorities

 Heat sterilization; dry heat or 

steam (refer to the USDA Treatment Manual Schedule T415b)

 Do not reuse container mix from regulated plants

 Ensure area on which the growing media sits drains 

freely

 Ensure cull piles are clearly separated from container 

mix components

 Ensure growing container mix and/or components are 

from an area known to be free of P. ramorum

 Move container mix piles away from potential P. ramorum contamination sources

 Pasteurize potting media

 Place container mixes in containers, bins, or on hard 

surfaces that can be cleaned, and not in contact with 

site soil

 Purchase components from suppliers with the ability to 

supply media free of plant pathogens and pests and 

meets quality requirements

 Raise storage areas above the level of the surrounding 

land to a height of 10 to 12 cm to prevent all runoff water 

from entering the area or surrounding site with surface 

drains or diversion banks

 Sample and test media and media components at delivery or before use

 Steam-sterilize any container mix that is reused or composted according to strict national standards

Potting area  Disinfestation of nonporous surfaces, concrete floors, benches, 

plastic sheeting, and tools

 Clean and disinfect all equipment used to transport 

media, e.g., front-end loader buckets, wheel barrows, 

mobile bins, trolleys, or plastic containers between uses

 Ensure staff members regularly wash their hands and 

maintain good hygiene

 Limit or divert traffic through soil-mixing area

 Regularly clean and disinfest potting facilities

 Regularly clean up and discard split media around potting facilities

 Schedule specific times to pot regulated plants and 

clean equipment and area before or after potting operations

 Use clean equipment to mix or load planting media

The following table provides examples of CCPs, remediations, mitigations, best management 

practices, and cultural changes that can be applied for P. ramorum. This list is not exclusive: other 

mitigations may be equally effective. For guidance on other potential mitigations, consult the BMP 

guides (refer to the References section on page 3-1-28), your state extension personnel, or the PPQ 

S&T representative.

Table 3-1-6 Examples of CCPs, Remediations, Mitigations, Best Management Practices (BMPs), and 

Changes to Business/Cultural Practices1

 (page 2 of 4)

Identified critical control 

point (CCP):

Mitigations that may have preceded CCP assessment:Examples of remediations, mitigations, BMPs, and 

changes to business/cultural practices:
Interstate Confirmed Nursery Protocol

Critical Control Points

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-25

Nursery beds  To avoid contact between infested 

soil/surface water and regulated 

plants, install permanent impermeable, nonporous barriers

 Steam soil

 Soil fumigation (e.g., dazomet, 

methyl bromide)

 Solarize soil

 Maintain substrate, whether this is through additional 

gravel, repairing or replacing landscape cloth or covering, or leveling to improve or increase drainage

 Prevent buildup of fallen leaves and plant debris from 

regulated plants in production areas and monitor with 

every crop rotation or quarterly, whichever occurs most 

often

Irrigation water  Treat recycled water or water 

used for irrigation water with chlorine levels of 2 ppm or 2 mg/liter 

or greater

 Avoid overhead irrigation of regulated plants or irrigate 

in a manner to avoid prolonged leaf wetness and splash

 Eliminate accumulations of surface water

 Irrigate regulated plant material around dawn, when 

possible, in order to prevent extended periods of leaf 

wetness

 Monitor and test (quarterly at a minimum) untreated irrigation water from any source other than a well or a 

municipal water supply to confirm it is free of the pathogen

 Monitor water treatment systems to verify the appropriate treatment measures are being applied

 Prevent surface water by not overwatering. Irrigate regulated plants based on water needs

 Treat water used for irrigation by using one or a combination of the following: bromine; chlorine; sodium hypochlorite; calcium hypochlorite; chlorine dioxide; ozone; 

activated peroxygen; ultraviolet radiation; copper ionization; heat treatment/pasteurization; or filtration

Water drainage  Divert soil and water movement from adjacent properties populated with regulated plants to prevent nursery 

contamination

 Ensure runoff from all cull piles is directed away from 

media components, media mixing areas, growing beds, 

nursery roadways or paths, and irrigation water to prevent contamination

 Insert a well-drained physical barrier (e.g., raised 

benches, effective gravel layer, asphalt, or concrete) 

between native soil and containers to prevent pathogen 

splash dispersal from potentially infested ground

The following table provides examples of CCPs, remediations, mitigations, best management 

practices, and cultural changes that can be applied for P. ramorum. This list is not exclusive: other 

mitigations may be equally effective. For guidance on other potential mitigations, consult the BMP 

guides (refer to the References section on page 3-1-28), your state extension personnel, or the PPQ 

S&T representative.

Table 3-1-6 Examples of CCPs, Remediations, Mitigations, Best Management Practices (BMPs), and 

Changes to Business/Cultural Practices1

 (page 3 of 4)

Identified critical control 

point (CCP):

Mitigations that may have preceded CCP assessment:Examples of remediations, mitigations, BMPs, and 

changes to business/cultural practices:
Interstate Confirmed Nursery Protocol

Critical Control Points

3-1-26 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3/2024-07

Pots/containers  Sterilization by steam or disinfestation by alcohols, chlorine, quaternary ammonium, or phenolics Do not store used pots in areas in which water drainage 

could flow or splash into regulated plant beds

 Establish a procedure for cleaning and sanitizing pots 

with clear separation of clean pots from dirty pots

 Regularly control weeds in and around container storage facilities

 Store new and clean disinfested containers above 

ground level

 Store pots on a barrier that effectively separates them 

from underlying substrates

 Use pots that are: 1) new; 2) clean and properly disinfested; or 3) sanitized by steam sterilization or hot water 

dip

Roads/pathways Cover pathways and roads adjacent to beds and 

benches with materials to reduce contamination with soil 

and water

 Maintain roads to avoid surface water; fill potholes and 

maintain drainage such that water will flow away

 Maintain substrate, leveling to improve or increase 

drainage

 Prevent buildup of fallen leaves and plant debris

Conveyance  Develop or review processes of cleaning tires and carts 

or trailers used in moving plant material

 Do not allow trucks to sweep out debris into nursery

 If a known P. ramorum-infested area has been visited, 

wash and sanitize shoes, tools, and vehicles that may 

have contacted contaminated soils before traveling to 

disease-free areas

 Regularly clean and disinfest transport equipment

 Require pick-up and delivery trucks to properly clean 

and sanitize truck bed, undercarriage, and tires prior to 

entering nursery operations

 Unload incoming deliveries onto a hard, impermeable 

surface area that is clean and free of any debris; collect 

all debris from plants, surface area, and delivery trucks

1 Although this table tries to cover the most common potential hazards and the appropriate rectifications, it is unlikely to cover 

all of them. Hazards, CCPs, and potential responses will differ between nurseries.

The following table provides examples of CCPs, remediations, mitigations, best management 

practices, and cultural changes that can be applied for P. ramorum. This list is not exclusive: other 

mitigations may be equally effective. For guidance on other potential mitigations, consult the BMP 

guides (refer to the References section on page 3-1-28), your state extension personnel, or the PPQ 

S&T representative.

Table 3-1-6 Examples of CCPs, Remediations, Mitigations, Best Management Practices (BMPs), and 

Changes to Business/Cultural Practices1

 (page 4 of 4)

Identified critical control 

point (CCP):

Mitigations that may have preceded CCP assessment:Examples of remediations, mitigations, BMPs, and 

changes to business/cultural practices:
Interstate Confirmed Nursery Protocol

References

3/2024-07 Phytophthora ramorum Manual/Interstate Confirmed Nursery Protocol 3-1-27

References

The following are examples of programs based on assessments to identify 

CCPs leading to BMPs and mitigations addressing associated risks.

Best Management Practices Manual to reduce the risk of introducing Soilborne Plant Pathogens into Horticultural Nurseries and Managed 

Wildland Landscapes 

The Systems Approach to Nursery Certification (SANC) Program

Phytophthora ramorum: Best Management Practices

Presidio Phytophthora Management Recommendations

United States Nursery Certification Programs (USNCP)
3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-1

Chapter

4 Confirmed Retail Nursery 

and Retail Nursery Dealer 

Protocol

Protocol for Intrastate Retail Nurseries and 

Retail Nursery Dealers When Phytophthora 

ramorum Is Present (rCNP)

Contents

Introduction 4-1-1

Goal 4-1-2

Trigger Events For Use of the Confirmed Retail Nursery and Retail Nursery 

Dealer Protocol 4-1-3

Challenges 4-1-3

Confirmed Retail Nursery and Retail Nursery Dealer Steps 4-1-4

Schematic of Destruction (D) and Quarantine (Q) Radii 4-1-9

APHIS List of Regulated Hosts and Plants Proven or Associated with 

Phytophthora ramorum 4-1-13

Introduction

This protocol covers retail nurseries and retail nursery dealers who are 

intrastate shippers only. Any nursery that ships interstate is covered in Chapter 

3 Interstate Confirmed Nursery Protocol on page 3-1-1.

Retail nurseries and retail nursery dealers represent a different type of risk 

from Phytophthora ramorum (P. ramorum) than nurseries that specialize in 

propagating and growing plants. The nature of the retail business tends to 

require that plants be 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 at the same frequency as they would during the plant production 

process.

As retail nurseries and retail nursery dealers are at the end of the production 

and distribution process, they normally represent a lower risk of distributing 

infected plants to other nurseries and facilities in the plant distribution system. 

Retail nurseries are the final location in the plant distribution system as well as 

the final location before infected plants are moved directly to the environment. 
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Goal

4-1-2 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 3/2024-07

It is important that retail nurseries and retail nursery dealers do not become a 

location where noninfected plants could become infected en route to the point 

of final planting.

If the facility of concern is an intrastate commerce propagation, wholesale and 

rewholesale nursery, intrastate commerce grower, or an intrastate commerce 

broker with a nursery site or hold lot, please note the following:

If a State is interested in quarantining less than the entire State for P. 

ramorum, it must enforce restrictions on the intrastate movement of 

regulated, restricted, and associated articles that are substantially the same 

restrictions as those imposed by Federal regulation on the interstate 

movement of regulated, restricted, and associated articles.

Intrastate movement for nurseries that have been confirmed positive for P. 

ramorum in plants, water, or other regulated articles is not covered by 

Federal regulations. State inspectors have authority to limit movement 

intrastate. Creation and implementation of an intrastate protocol should 

closely resemble the Federal protocol for the movement of interstate 

material. Please refer to Interstate Confirmed Nursery Protocol on page 

3-1-1 for guidance.

Goal

The goal of this protocol is to limit the spread of Phytophthora ramorum, a 

quarantine plant pathogen, and to simplify the movement of P. ramorum-free

nursery stock. When procedures described in this protocol are implemented, 

plant-to-plant spread and movement of the pathogen through nursery 

shipments should be minimized. Cooperation by nursery management 

personnel is essential. Early detection and reporting of potential P. ramorum

plant infections are crucial to ensure spread is contained.

NOTICE

Online retail plant sellers would not be considered a retail nursery and should refer 

to Chapter 3 Interstate Confirmed Nursery Protocol on page 3-1-1 when positive 

plants are found.

! WARNING

Any interpretation of this protocol or its procedures NOT consistent with the goal 

listed above, is a misinterpretation of the protocol.
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Trigger Events For Use of the Confirmed Retail Nursery and Retail Nursery Dealer Protocol

3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-3

Trigger Events For Use of the Confirmed Retail Nursery and Retail 

Nursery Dealer Protocol

This protocol is to be implemented by USDA–APHIS–PPQ, in cooperation 

with State Plant Regulatory Officials (SPROs) when the presence of P. 

ramorum has been confirmed in a retail nursery or retail nursery dealer. Any 

detection of P. ramorum, a quarantine plant pathogen, requires a regulatory 

response. Samples may have been collected during surveys or inspections such 

as a Cooperative Agricultural Pest Survey (CAPS), State Nursery Cleanliness 

Survey, Plant Protection Act (PPA) Section 7721, or other surveys; State 

inspections, trace forward investigation, trace back investigation, or found by 

other means. The Retail Nursery and Retail Nursery Dealer protocol can be 

used in all of these cases.

Samples must be diagnosed using a method approved by USDA–APHIS–PPQ 

and consistent with the Potentially Actionable Suspect Sample (PASS)

protocol (for diagnostic information and the PASS protocol, refer to the PPQ 

Phytophthora ramorum website).

Challenges

P. ramorum is a micro-organism and difficult to detect. It can infect plants; 

infest container mix, soil, and water; and persist in these substrates despite the 

best eradication efforts. Based on the understanding of the pathogen biology, 

these protocols and regulations will be adjusted accordingly. Detection and 

management of this pathogen is informed by continually improving science.

Additional challenges related to retail nurseries and/or retail nursery dealers 

include the following:

Disposition of plants when not confirmed positive is a challenge when 

ownership is not clear

If found positive, held plants, which are moved and commingled, lose 

their identity to surrounding plants that may be implicated for destruction 

and quarantine

Lack of interest in ensuring plants are free of pathogens when plants are 

under consignment at a retail location

NOTICE

When planning to announce or make a public statement about the detection of a federally regulated pest, the State Plant Regulatory Official (SPRO) and/or the public 

information officer for the State department of agriculture must first contact the State 

Plant Health Director (SPHD) and/or USDA–APHIS–Office of Legal and Public 

Affairs (LPA).
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

4-1-4 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 3/2024-07

Large retail chains create a unique pathway that is further complicated by 

the increasing use of consignment sales in ornamental plants

Movement of nursery plants to plant brokers who bring material from 

larger nurseries and disseminate to multiple States increases the 

likelihood of accidental exposure to contamination of P. ramorum to 

other nursery products

Plant traceability back to origin is difficult when multiple brokers/

nurseries are involved

Plants under consignment complicate orders for plant destruction of 

confirmed positive plants because ownership is not local

Potential reduction of effectiveness of the notification system is likely to 

occur when a nursery with a compliance agreement sells plants to a 

broker (receiving State is notified) and then the broker sells plants to a 

retail nursery dealer in multiple States and the receiving States are not

notified

Securing plant material at retail nursery dealers is a challenge, examples 

include space limitations and loss of communication between inspectors 

and managerial shifts at the facilities

Confirmed Retail Nursery and Retail Nursery Dealer Steps

Any confirmed-positive article, such as a plant or water sample, triggers the 

confirmed nursery protocol. In chronological order, the steps for the 

Confirmed Retail Nursery and Retail Nursery Dealer protocol (rCNP) are as 

follows:

1. Communicate and notify.

2. Conduct trace investigations (concurrently with securing the nursery).

3. Secure the nursery (concurrently with conducting trace investigations).

4. Survey the nursery (delimitation inspection).

5. Disinfest the nursery.

6. Conduct 90-day (minimum) quarantine activities.

7. Release plants in the retail nursery or retail nursery dealer.

8. Conduct post-quarantine release monitoring.
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-5

NOTICE

Prior to an APHIS-confirmed positive determination, the National Plant Protection 

Laboratory Accreditation Program- (NPPLAP) approved laboratory must communicate all suspect positive diagnostic samples to inspectors as soon as one of the following has occurred:

• Culture that matches the morphology for P. ramorum as determined and reported 

by an APHIS-approved laboratory; or

• Positive PCR using APHIS-approved work instructions by an APHIS-approved laboratory

For all Potentially Actionable Suspect Samples (PASS), laboratories must immediately forward the plant material and DNA to the PPQ–S&T Plant Pathogen Confirmatory Diagnostics Laboratory (refer to Contact Information for the Phythopthora 

ramorum Program on page A-1-1) with a domestic ARM routing receipt (DARR) and 

notify the State’s State Plant Health Director (SPHD; generates DARR for laboratory) 

and State Plant Regulatory Official (SPRO) for the State of sample origin, and the 

National Operations Manager (NOM).

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps 

Step number: Subnumber: Then:

1—Communicate and 

notify

Laboratories need to notify the SPHD and the SPRO, the NOM, and the submitter. The SPRO (if State authority is used) or the SPHD (if Federal authority is used) will notify the owner of the nursery.

1.1 The designated official will notify the confirmed-positive nursery of the confirmed-positive and instruct the nursery to place a hold on all regulated 

plants at the nursery and any other plants deemed necessary by the inspector.

1.2 SPHD and SPRO will notify the NOM of the nursery notification and the hold 

on the plants.

1.3 SPHD and/or SPRO will provide a list of the identified facilities found through 

trace back and trace forward investigations to the NOM within five business 

days of a confirmed P. ramorum-positive sample in a nursery (refer to 2—

Conduct investigations on page 4-1-5). The NOM will notify SPHDs and/or 

SPROs of States sending or receiving these shipments.

1.4 SPHDs and/or SPROs will notify affected retail nurseries and retail nursery 

dealers within their States.

2—Conduct investigationsConduct trace back and trace forward investigations concurrently with securing the nursery, as shipping lists must be provided to the NOM within five 

business days. If a nursery does not meet the five business day requirement, 

the SPHD, SPRO, and NOM should work together to decide potential consequences, such as holding the compliance agreement.
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

4-1-6 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 3/2024-07

2—Conduct investigations

(cont.)

2.1—Trace 

back investigation

At the time P. ramorum is confirmed in a retail nursery or retail nursery dealer 

facility, determine the source of the plant(s) to initiate the trace back investigation. Trace back plants include all plants from the source nursery of the 

same genus as the infected plant regardless of size, location, or lot, back to 

the original propagation source (if it still exists). Collect invoices for those 

purchases and provide to the NOM within 10 days. For more detailed 

instructions, please refer to Trace Back Protocol for Nurseries that Shipped 

Plant Material to a Confirmed P. ramorum-Infested Nursery on page 5-3-1.

Provide the trace back list(s) (including plant taxonomic details) to 

PPQ’s NOM within 5 business days.

2.2—Trace 

forward 

investigation

Most retail establishments may not keep records of plant sales, however, it 

is necessary to inquire in order to perform a trace forward investigation. Any 

trace forward records must be sent to the NOM within five business days. If 

a nursery does not meet the five business day requirement, the SPHD, 

SPRO, and NOM should work together to decide potential consequences, 

such as holding the compliance agreement. Retail plant sales are likely to be 

intrastate and will be subject to the appropriate State regulations. If the retail 

nursery or retail nursery dealer has sales records, determine if the establishment has sold plants that could potentially be infected to another facility such 

as a landscaper. Initiate the trace forward investigation by identifying all

plants (meeting the following criteria) sold within 6 months of the first confirmed-positive detection of P. ramorum at a nursery: 1) plants of the infected 

species/cultivar; 2) all regulated plants that originated in the destruction 

radius; and 3) any plants of the high-risk genera: Camellia spp., Kalmia spp., 

Pieris spp., Rhododendron spp. (including azalea), and Viburnum spp. 

regardless of their location in the nursery. This combination of shipped plants 

is referred to as the high-priority target plants. These plants, including their 

shipment date(s); quantities; and respective destinations, make up the trace 

forward list. Identify these high-priority plants using the best available information and to the lowest taxon possible (e.g., if the plants can be identified to 

cultivar, trace forward activities may be conducted at the cultivar level).

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:

NOTICE
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-7

3—Secure the nursery When the presence of Phytophthora ramorum has been confirmed in the 

nursery or retail nursery dealer, safeguard infected high-risk plants and highrisk plants nearby. The Schematic of Retail Nursery with Positive Plants 

(Figure 4-1-1 and Table 4-1-2) is included, but may not be appropriate for 

use in retail nursery dealer settings.

3.1—Establishing 

destruction 

and quarantine radii for 

confirmedpositive 

plant(s)

3.1.1—All plants within 2 meters from the edge of any infected plants shall 

be held for destruction (destruction radius)

3.1.2—All plants within a 2-meter perimeter (quarantine radius) beyond the 

2 meters surrounding the infected plants (i.e., the retail destruction radius) 

shall be held for a minimum 90-day quarantine period OR plants can be voluntarily relinquished under official supervision at any time (refer to 7—

Release the nursery on page 4-1-13).

3.1.3—Inspect all regulated plants in the nursery that are not within the 

destruction and quarantine radii and hold any plants sampled. If an inspector 

determines any other product or article presented is at risk of spreading P. 

ramorum and the inspector notifies the person in possession of the product 

or article that it is subject to the restrictions in the regulations, the product or 

article can be held (7 CFR Part 301.92-2).

3.1.4—Refer to 5—Disinfest the nursery on page 4-1-11 for destruction and 

treatment protocols.

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

4-1-8 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 3/2024-07

4—Survey the nursery The goal of the survey is to locate P. ramorum in the nursery. A detailed and 

thorough inspection should be conducted in the nursery to determine the 

presence of P. ramorum. Samples should be collected from symptomatic 

plants (refer to Biology and Symptoms of Phytophthora ramorum on page 

7-1-1).

4.1—Delimit 

the nursery

4.1.1—Examine all plants within the retail nursery or retail nursery dealer 

and sample any symptomatic plant tissue found (refer to Plant Symptoms 

and Sampling for P. ramorum on page 2-1-4). Plants currently regulated in 

nurseries are included in the APHIS Lists of Proven Hosts of and Plants 

Associated with Phytophthora ramorum.

4.1.2—Hold all plants of that taxon (taxa) and all plants that are within 2 

meters of the confirmed-positive plant. Or plants can be voluntarily relinquished for destruction under official supervision at any time (refer to 7—

Release the nursery on page 4-1-13).

4.1.3—Submit samples to the appropriate lab for analysis using a methodology approved by APHIS (refer to Sampling and Submission Protocol on 

page 8-1-1).

4.1.4—Release all plants held if sample results are negative.

4.1.5—Establish destruction and quarantine radius (radii) around plant(s) 

with positive diagnostic results (refer to 4.2—Establish destruction and quarantine radii for plants confirmed positive during delimiting survey on page 

4-1-8) and delimit the nursery again.

4.1.6—The 90-day plus quarantine period begins when the delimiting survey 

is complete.

4.2—Establish destruction and 

quarantine 

radii for 

plants confirmed positive during 

delimiting 

survey

4.2.1—Plants at retail nursery dealers can be very transient, making it difficult to implement some of the following measures. Adapt as much of this 

protocol to the situation as practical.

4.2.2—Establish destruction radius (radii) by flagging a 2-meter radius (a 4-

meter diameter circle) around all infected plants (refer to Figure 4-1-1) OR

inspector-witnessed relocation/segregation of plants within the nursery with 

safeguarding OR plants can be relinquished under official supervision at any 

time (refer to 7—Release the nursery on page 4-1-13); include plants on 

carts or other movable shelving situations.

4.2.3—Establish quarantine radius (radii) by flagging a 4-meter radius (an 8-

meter diameter circle) around all infected plants (refer to Figure 4-1-1) OR

inspector-witnessed relocation/segregation of plants within the nursery with 

safeguarding OR voluntary destruction.

4.2.4—Limit access to destruction and quarantine radius (radii) or other designated hold areas. Ensure proper sanitation measures are applied (refer to 

5—Disinfest the nursery on page 4-1-11 and Biosecurity Measures for Nurseries on page 9-1-1).

4.2.5—Inspectors should document all actions taken on EAN or State equivalent.

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-9

Schematic of Destruction (D) and Quarantine (Q) Radii 

Figure 4-1-1 Schematic of Destruction and Quarantine Radii of Positive Plants

Table 4-1-2 Legend of Destruction (D) and Quarantine (Q) Radii of Positive Plants Schematic

Area color: Name: Once D-radii and Q-radii are flagged, then:

Gray (*1) Destruction (D) radii Destroy all plants, pots, medium, and leaf debris

Light gray (*2) Quarantine (Q) radii Hold all plants from sale for 90 days

Gray (*3) D-radii in block with 

nonregulated plants

Destroy all plants, containers, media, and leaf debris; nonregulated plant 

nursery stock could still move the pathogen

Light gray (*4) Q-radii in block with 

nonregulated plants

Hold all plants for sale for 90 days; nonregulated plant nursery stock could still 

move the pathogen

Dotted (*5) Rest of the block 

with regulated 

plants

Release all plants for sale only when found to be asymptomatic during the 

delimiting survey

White (*6) Rest of the block of 

nonregulated plants

Release all plant materials for sale if found to be asymptomatic during the 

delimiting survey
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

4-1-10 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 3/2024-07

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:

4—Survey the 

nursery

(cont.)

4.3—Sample 

other articles of 

concern at the 

nursery

4.3.1—Not all of the following articles of concern will be found at the majority of 

retail nurseries and retail nursery dealers. Sample other articles of concern if 

they occur at the facility.

4.3.2—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, any safeguards against water back flow in the irrigation system, as 

well as any water treatment practices if recirculated water is used.

 Water sampling is not required for irrigation water from municipal water facilities that treat their water prior to release.

 Sample any retention pond or surface water at the nursery (refer to Water 

Sampling and Processing Protocol on page 10-1-1). Bottle of bait is the preferred sampling method for surface water.

4.3.3—Sampling instructions for soil—

 Standing water will be sampled in place of substrate soil sampling. No substrate soil samples are required for this sampling protocol.

4.3.4—Cull pile and compost pile sampling—determine how the nursery disposes of culled and other waste plant material. Cull and compost piles will be 

uncommon in a retail nursery or retail nursery dealer, however, if cull or compost 

piles are present, record the location of any piles as these may be contaminated 

with infected plant material or associated soil and/or growing media.

 Check cull and compost piles for P. ramorum symptomatic plants and plant 

material and sample if observed.

 If a cull or compost pile is found to be positive, establish and demarcate the 

area by visibly and indelibly flagging 1 meter out from the perimeter of the 

cull pile.

 Sample and test soil for the presence of P. ramorum at the down slope edge 

of the cull pile.

 Determine how the nursery disposes of cull and compost piles.

 Restrict access.

 Determine the appropriate treatment or destruction method (refer to Treatments and Disinfectants on page 12-1-1).

4.3.5—Segregation of plants on hold—

 Once inspection and sampling are complete, any held plants may be consolidated and segregated. If the plants are not consolidated and segregated, 

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 if properly safeguarded. Any movement of the segregated plants must be done in a manner that will safeguard and prevent the spread of the disease at the nursery, 

and be conducted under the direction of an inspector. Segregation must

include storage on an impermeable surface (e.g., a 45-mil thick pond liner or 

concrete or asphalt) and not within 2 meters of any other plants. The impermeable surface should ideally be situated to drain away from regulated 

plants.
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-11

5—Disinfest the 

nursery

Inspectors should use appropriate disinfection measures for conditions found at 

the establishment. For information on disinfesting and sanitizing any nursery, 

refer to Treatments and Disinfectants on page 12-1-1 and Biosecurity Measures 

for Nurseries on page 9-1-1.

5.1—Plant 

destruction

5.1.1—When a P. ramorum-infected plant is found, all other plants, including 

pots and growing media within 2 meters (destruction radius) of the infected 

plant, will be removed and destroyed using one or more of the techniques 

detailed in Treatments and Disinfectants on page 12-1-1. This includes pots and 

potting media. A regulatory official must oversee plant destruction.

5.1.2—Plants can be voluntarily relinquished under official supervision at any 

time (refer to 7—Release the nursery on page 4-1-13).

5.2—Debris 

removal

All plant debris, including growing media, leaves, stems, flowers, roots, and any 

other plant parts found within the destruction radius, will be removed and 

destroyed using one or more of the techniques detailed in Treatments and Disinfectants on page 12-1-1.

5.3—Nonporous surfacesNonporous surfaces will be disinfested beneath plants. For instance, wipe off 

shelves with Lysol or other approved disinfectant (refer to Treatments and Disinfectants on page 12-1-1 for recommended disinfestation options).

5.4—Porous 

surfaces

If the nursery is found to have an infested porous surface, remedial action must

be developed and implemented with the written approval of an inspector. This is 

done in order to prevent contact of plants with soil or any other surface that cannot be immediately disinfested. A durable, impermeable ground barrier (e.g., a 

45-mil pond liner) may be used as an inexpensive temporary measure. The condition of the barrier must be monitored and maintained and foot traffic minimized. Refer to Treatments and Disinfectants on page 12-1-1 for other 

recommended options.

5.5—Equipment and personnelUse recommended disinfestation options and biosecurity measures (refer to 

Treatments and Disinfectants on page 12-1-1 and Biosecurity Measures for 

Nurseries on page 9-1-1). Use appropriate sanitation practices, such as disinfesting clippers used to cut plants or using disposable gloves to prevent crosscontamination.

5.6—Biosecurity measuresBiosecurity measures, such as tool sanitation, equipment disinfestation, and 

appropriate sanitation measures for employees, are designed to minimize the 

risk of introduction to or spread and survival of the pathogen in a nursery (refer 

to Biosecurity Measures for Nurseries on page 9-1-1for recommended biosecurity measures).

5.7—Treatments for cull 

piles, compost 

piles, water, 

soil, or growing 

media

It is unlikely cull piles, compost piles, water, soil, or growing media will need 

remediation at a retail nursery dealer, but they may need remediation at a retail 

nursery. If they are found to be positive, they will need to be treated (refer to 

Treatments and Disinfectants on page 12-1-1).

5.8—Considerations for 

inspectors

5.8.1—Determine if plants will be placed on hold, destroyed on site, destroyed 

off site, or relinquished to an inspector.

5.8.2—Determine sanitation protocols established for the facility including management oversight (e.g., how is information on holds communicated among 

employees and management staff?).

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

Confirmed Retail Nursery and Retail Nursery Dealer Steps

4-1-12 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 3/2024-07

5—Disinfest the 

nursery

(cont.)

5.8—Considerations for 

inspectors

(cont.)

5.8.3—Determine management oversight and responsibility for plants for sale. 

How is information on holds communicated among employees and management staff? How will plants on hold be safeguarded? Who owns the plants and is 

responsible for destruction costs?

5.8.4—Determine which disinfestation methods are appropriate for the site 

(refer to Treatments and Disinfectants on page 12-1-1). For instance, disinfesting concrete or asphalt may not be practical at a retail nursery dealer’s based on 

the movement of the plants at the store.

5.8.5—Determine the number of plants that must be destroyed and the number 

the nursery may voluntarily destroy or relinquish to an inspector. Determine the 

number of positive plants, the number of potentially positive plants, and the location of those plants in the nursery. The inspector can use the information collected to determine the State authority for holds, plant destruction, and other 

components of the nursery disinfestation.

6—90-day (minimum) quarantine activitiesPlants can be relinquished under official supervision at any time (refer to 7—

Release the nursery on page 4-1-13). These activities follow the completion of 

the delimiting survey and may run concurrent with some of the disinfestation 

activities taking place at the nursery. If a retail nursery dealer that has been 

found to be infested has completed all of the following, it shall not undergo posteradication monitoring unless additional plants or articles are found to be positive:

 Plants are only present seasonally; and

 Plants are placed only on nonporous surfaces that have been mitigated

If additional plants or articles are found to be positive, the nursery shall be 

placed in a compliance agreement and have post-eradication monitoring.

6.1—The quarantine periodThe quarantine period begins when the delimiting survey is completed (i.e., the 

last sample is taken and an EAN or State equivalent is issued) and lasts a minimum of 90 days. During the quarantine period, inspection, sampling, and testing 

must reveal no further detection of P. ramorum, or the quarantine period will be 

extended. If a positive sample occurs, the 90-day (minimum) quarantine period 

restarts and the appropriate measures will be taken (refer to 3—Secure the 

nursery on page 4-1-7).

6.2—During 

the 90-day 

(minimum) 

quarantine 

period within 

the quarantine 

radius (radii)

6.2.1—No fungicides registered for Phytophthora control shall be applied.

6.2.2—Inspectors will visually inspect plants a minimum of two times—once 

about halfway through the 90-day (minimum) quarantine period, and once near 

the end of the 90-day (minimum) quarantine period in order to have test results 

coincide with the end of the quarantine period—according to the protocol (refer 

to Phytophthora ramorum Inspection and Sampling Protocol for Nurseries on 

page 2-1-1). Samples will only be taken if symptomatic tissue is observed. The 

second visual inspection in the quarantine radius (radii) can be done at the 

same time as the quarantine release survey as described below.

6.2.3—If positive samples were collected during the delimiting survey, inspectors will collect soil and media samples and test them during the quarantine 

period according to the protocols (refer to Soil and Container Mix Sampling and 

Processing Protocol on page 11-1-1).

6.2.4—If positive water samples were collected during the delimiting survey, 

inspectors will collect water samples and test them during the quarantine period 

according to the protocols (refer to Water Sampling and Processing Protocol on 

page 10-1-1).

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:
Confirmed Retail Nursery and Retail Nursery Dealer Protocol

APHIS List of Regulated Hosts and Plants Proven or Associated with Phytophthora ramorum

3/2024-07 Phytophthora ramorum Manual/Intrastate Confirmed Nursery Protocol 4-1-13

APHIS List of Regulated Hosts and Plants Proven or Associated with 

Phytophthora ramorum

Refer to a current list of APHIS Lists of Proven Hosts of and Plants Associated 

with Phytophthora ramorum.

6—90-day (minimum) quarantine activities

(cont.)

6.3—Quarantine release 

survey

A quarantine release survey of the entire nursery must be completed near the 

end of the minimum 90-day quarantine period. This survey includes visually 

inspecting all regulated plants within the nursery and sampling any symptomatic 

plant tissue, soil of destruction and quarantine radius (radii), 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.

7—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

 If water, soil, and growing media were tested, they must also have 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.

OR

 The nursery operator may also choose to destroy plants that have been 

placed under quarantine at any time within the 90-day quarantine period, 

however, destruction must be under local authority supervision; and

 If not previously tested and determined to be negative, inspectors must

sample and test drainage or recirculated irrigation water as per Water Sampling and Processing Protocol on page 10-1-1 or inspectors must disinfest 

porous and nonporous surfaces (refer to 5.3—Nonporous surfaces on page 

4-1-11). If soil and water samples taken are negative for P. ramorum, the 

nursery can be released; and

 Inspectors must revisit the nursery after approximately 90 days and conduct 

at least a nursery-level survey inspection (refer to Phytophthora ramorum 

Inspection and Sampling Protocol for Nurseries on page 2-1-1).

Table 4-1-1 Confirmed Retail Nursery and Retail Nursery Dealer Protocol Steps (continued)

Step number: Subnumber: Then:
3/2024-07 Phytophthora ramorum Manual/Trace Investigations 5-1-1

Chapter

5-1 Trace Investigations

Introduction

Contents

Trace Forward Protocol for Nurseries that Received Plant Material Shipped 

from a Confirmed P. ramorum-Infested Nursery 5-2-1

Trace Back Protocol for Nurseries that Shipped Plant Material to a Confirmed 

P. ramorum-Infested Nursery 5-3-1

Introduction

Trace investigations are required at nurseries confirmed positive for 

Phytophthora ramorum. The following sections include protocols for trace 

forward (Trace Forward Protocol for Nurseries that Received Plant Material 

Shipped from a Confirmed P. ramorum-Infested Nursery on page 5-2-1) and 

trace back (Trace Back Protocol for Nurseries that Shipped Plant Material to a 

Confirmed P. ramorum-Infested Nursery on page 5-3-1) investigations. These 

two protocols are needed to complete trace investigations related to confirmed 

positive detections of P. ramorum. The Phytophthora ramorum Nursery 

Questionnaire (refer to Figure on page A-1-2) is an available tool to collect 

required and other useful information on nurseries. Select the correct trace 

investigation in the Activity Action section. Contact the NOM for a fillable 

form.

Intended Use

The intended use of these protocols is to identify where suspect Phytophthora 

ramorum-infected plants have been shipped. These protocols include plants 

shipped from the nursery confirmed positive for P. ramorum (Trace Forward) 

and plants shipped to that nursery (Trace Back). Any interpretation of these 

protocols that is contrary to this goal is a misinterpretation of the protocols. A 

detailed and thorough inspection shall take place at the field level to identify 

the presence of P. ramorum. Areas of consideration are to include, but are not 

limited to, plants, plant material and debris, soil, and water.
3/2024-07 Phytophthora ramorum Manual/Trace Forward Protocol 5-2-1

Chapter

5-2 Trace Investigations

Trace Forward Protocol for Nurseries that 

Received Plant Material Shipped from a 

Confirmed P. ramorum-Infested Nursery

Contents

Intended Use 5-2-1

Goal 5-2-1

Trace Forward Protocol Instructions 5-2-2

Intended Use

The intended use of this protocol is to identify where Phytophthora ramoruminfected plants have been shipped. These include:

Interstate commerce brokers with a nursery site or holding lot

Interstate commerce propagation, wholesale, and re-wholesale nurseries

Interstate commerce retail nurseries or other retail outlets

Intrastate commerce nurseries

Residential, commercial sites, and managed landscapes/public gardens

Goal

The goal of this protocol is to determine if P. ramorum-infected plants have 

been shipped from a confirmed-positive nursery. Any interpretation of this 

protocol that is contrary to this goal is a misinterpretation of the protocol. A 

detailed and thorough inspection shall take place at the field level to identify 

the presence of P. ramorum. Areas of consideration are to include, but are not 

limited to, plants, plant material and debris, soil, and water.

NOTICE

If regulatory officials are conducting a trace forward investigation at a non-nursery 

locale (residence, commercial site, managed landscape), they should apply this protocol making appropriate adjustments to the instructions. Refer to Information 

Needed PRIOR to the CCP Assessment on page 3-1-20.
Trace Investigations

Trace Forward Protocol Instructions

5-2-2 Phytophthora ramorum Manual/Trace Forward Protocol 3/2024-07

Trace Forward Protocol Instructions

Table 5-2-1 Trace Forward Protocol Instruction (page 1 of 2)

Chronological or 

concurrent steps: Actions:

1) Communicate 

and notify the trace 

forward nursery

Before inspection day

1. The PPQ National Operations Manager (NOM) will send trace forward information generated 

from the Interstate Confirmed Nursery Protocol Steps on page 3-1-3 to the SPRO and SPHD 

who will determine who responds to the trace forward information.

2. The designated regulatory official will plan an inspection of the receiving facilities (hereinafter 

referred to as trace forward facilities) without delay. If favorable climatic conditions (refer to 

Timing Nursery Inspection and Sampling on page 2-1-3) are not present for disease development/expression when the initial inspection is conducted, an additional inspection must be 

conducted when conditions are conducive.

3. For Federal inspectors, notify the SPRO, or relevant State official, of your plans to inspect.

4. Coordinate the inspection day with the State inspector.

5. If required, Federal and State or county inspectors shall contact the property owners/managers prior to the visit to determine how many trace forward plants are still in stock and to 

arrange for the inspection.

6. If you are unable to visit the nursery within 24 hours of your contact with the nursery owner/

manager, send a PPQ Form 523, Emergency Action Notification (EAN), or State equivalent, 

by email or FAX and request that they sign and immediately return it to you. The EAN will indicate what plants are on hold.

7. Review and bring with you the P. ramorum Biology and Symptoms of Phytophthora ramorum

on page 7-1-1 and Biosecurity Measures for Nurseries on page 9-1-1.

8. Obtain sampling supplies, refer to Sampling and Submission Protocol on page 8-1-1 for 

checklist and review and bring with you.

2) Investigation, 

inspection, and 

quarantine hold 

procedures

Inspection day

1. Identify yourself and agency to the nursery/facility owner/manager and explain the purpose of 

your visit.

2. Obtain copies of the shipping documents relating to the regulated plants shipped from the 

confirmed-positive nursery. Also, determine if the trace forward nursery shipped regulated 

plants to other wholesale or retail nurseries or facilities. If so, obtain those documents from 

the owner/manager.

3. Provide the owner/manager with a copy of the Phytophthora ramorum Nursery Questionnaire 

(refer to Figure A-1-1). Interview, review records, and observe the facility to fill out the questionnaire with the nursery owner/manager. Remember to ask the owner/manager the locations of the cull piles, compost piles, and waste bins or piles.

4. Verify the present or absence of any of the trace forward plants.

A. If trace forward plants are not in the nursery, verify whether additional locations (e.g., nursery sites) are maintained by the same nursery owner, of if any regulated plants moved to 

other sites or between sites during the 6-month period preceding the confirmed-positive 

detection.

B. Through interview or records, if possible, determine if other regulated plants may have 

come in contact with identified trace forward plants, duration of the contact, and where in 

the nursery the contact occurred.

5. Use the Sampling and Submission Protocol on page 8-1-1 and collect any symptomatic plant 

tissue found. While taking samples, visibly and indelibly flag or mark plants and areas sampled with sample ID and date. Also, mark areas on a map where plant samples were collected 

and/or take a GPS point or pictures.
Trace Investigations

Trace Forward Protocol Instructions

3/2024-07 Phytophthora ramorum Manual/Trace Forward Protocol 5-2-3

2) Investigation, 

inspection, and 

quarantine hold 

procedures

(cont.)

6. If trace forward regulated plants are in multiple locations within the nursery, the inspector 

must disinfect boots, tools, and hands between areas (refer to Treatments and Disinfectants

on page 12-1-1).

7. Use PPQ Form 523, Emergency Action Notification (EAN) for the official Federal authorization 

of hold. In Section 16 of the EAN, state that those specific plants are prohibited in movement 

pending further notification by PPQ.

8. Visibly and indelibly flag 2-meter area around the sampled plant to hold for quarantine until all 

diagnostic results are final.

9. Hold the sampled plants and all other regulated plants within the 2-meter radius around the 

sampled plants.

10. Once the delimiting survey and sampling are complete, any held plants may be consolidated 

and segregated. If the plants are not consolidated and segregated, 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 a manner that will safeguard and prevent the spread of the disease at the nursery, and be conducted under the direction of a regulatory official. Segregation must include storage on an impermeable surface (e.g., a 45-mil 

thick pond liner, concrete, or asphalt) and not within 2 meters of any other plant. The impermeable surface should be sloped to drain away from regulated plants.

11. Per Federal and State authorities, inspectors may, at any time, place on hold other plants, 

plant products, or articles (e.g., pots) that present a risk of spreading P. ramorum.

12. Check any cull, waste, or debris piles for P. ramorum-symptomatic plants or plant material. 

Collect samples of symptomatic material.

3) Sample collection and submissionUse Sampling and Submission Protocol on page 8-1-1.

4a) If the trace forward nursery is 

confirmed positive, 

and is an interstate

shipping nursery

Use the Interstate Confirmed Nursery Protocol on page 3-1-1 and contact the PPQ National 

Operations Manager (NOM).

4b) If the trace forward nursery is 

confirmed positive 

and is an intrastate

commerce only 

nursery

Use the Confirmed Retail Nursery and Retail Nursery Dealer Protocol on page 4-1-1 and contact 

the PPQ National Operations Manager (NOM).

5) Questions For PPQ P. ramorum program contacts, refer to Contact Information for the Phythopthora ramorum Program on page A-1-1.

Table 5-2-1 Trace Forward Protocol Instruction (page 2 of 2)

Chronological or 

concurrent steps: Actions:
3/2024-07 Phytophthora ramorum Manual/Trace Back Protocol 5-3-1

Chapter

5-3 Trace Investigations

Trace Back Protocol for Nurseries that Shipped 

Plant Material to a Confirmed P. ramorumInfested Nursery

Contents

Intended Use 5-3-1

Goal 5-3-1

Trace Back Protocol Instructions 5-3-2

Intended Use

The intended use of this protocol is to identify the shipping/infection source(s) 

of confirmed-positive Phytophthora ramorum plants. These sources may 

include:

Interstate commerce brokers with a nursery site or holding lot

Interstate commerce propagation, wholesale, and re-wholesale nurseries

Interstate commerce retail nurseries or other retail outlets

The intended use of this protocol is for nurseries that sell plants for interstate

distribution. For nurseries that only sell plants for intrastate commerce, refer 

to Confirmed Retail Nursery and Retail Nursery Dealer Protocol on page 

4-1-1.

Goal

The goal of this protocol is to determine if suspect Phytophthora ramoruminfected plants have been shipped to a confirmed-positive nursery. The goal is 

to determine if P. ramorum is present at the originating nursery. Any 

interpretation of this protocol that is contrary to this goal is a misinterpretation 

of the protocol. A detailed and thorough inspection shall take place at the field 

level to identify the presence of P. ramorum. Areas of consideration are to 

include, but are not limited to, plants, plant material and debris, soil, and 

water used on plants.

NOTICE

If regulatory officials are conducting a trace forward investigation at a non-nursery 

locale (residence, commercial site, managed landscape), they should apply this protocol making appropriate adjustments to the instructions. Refer to Information 

Needed PRIOR to the CCP Assessment on page 3-1-20.
Trace Investigations

Trace Back Protocol Instructions

5-3-2 Phytophthora ramorum Manual/Trace Back Protocol 3/2024-07

Trace Back Protocol Instructions

Table 5-3-1 Trace Back Protocol Instructions (page 1 of 2)

Chronological or 

concurrent steps: Actions:

1) Communicate and 

notify the trace back 

nursery

Before inspection day

1. The PPQ National Operations Manager (NOM) will send trace back information generated 

from the Interstate Confirmed Nursery Protocol Steps on page 3-1-3 to the SPRO and 

SPHD who will determine who conducts the protocol.

2. The designated regulatory official/inspector will plan an inspection of the receiving facilities 

(hereinafter referred to as trace back facilities) without delay. If favorable climatic conditions 

are not present for disease development/expression when the initial inspection is conducted, an additional inspection must be conducted when conditions are conducive.

3. SPHD and SPRO should communicate on all trace back activities.

4. Coordinate the inspection day with the State inspector.

5. Federal and State or county inspectors should contact the property owners/managers prior 

to the visit to determine how many trace back plants are still in stock and to arrange for the 

inspection.

6. If you are unable to visit the nursery within 24 hours of your contact with the nursery owner/

manager, send a PPQ Form 523, Emergency Action Notification (EAN), or State equivalent, by email or FAX and request that they sign and immediately return it to you. The EAN 

will indicate what plants are on hold.

7. Review and bring with you the P. ramorum Biology and Symptoms of Phytophthora ramorum on page 7-1-1 and Biosecurity Measures for Nurseries on page 9-1-1.

8. Obtain sampling supplies, refer to Sampling Supplies and Equipment Checklist on page 

8-1-4 for checklist and review and bring with you Sampling and Submission Protocol on 

page 8-1-1 for checklist and review and bring with you.

2) Investigation, 

inspection, and quarantine hold procedures

Inspection day

1. Identify yourself and agency to the nursery/facility owner/manager and explain the purpose 

of your visit.

2. Obtain copies of the shipping documents related to the regulated plants shipped from the 

confirmed-positive nursery. Also, determine if the nursery shipped regulated plants to other 

wholesale or retail nurseries or facilities. If so, obtain those documents from the owner/

manager.

3. Provide the owner/manager with a copy of the Phytophthora ramorum Nursery Questionnaire (refer to Figure A-1-1). Interview, review records, and observe the facility to fill out the 

questionnaire with the nursery owner/manager. Remember to ask the owner/manager the 

locations of the cull piles, compost piles, and waste bins or piles.

4. Verify the presence or absence of any of the trace back plants.

A. If trace back plants are not in the nursery, verify whether additional locations (e.g., nursery sites) are maintained by the same nursery owner, of if any regulated plants were 

moved to other sites or between sites during the 6-month period preceding the confirmed-positive detection.

B. If trace back plants have had any contact, direct or indirect, with other plants or resources 

at the nursery, the entire nursery should/must be inspected.

5. Use the Sampling and Submission Protocol on page 8-1-1 and collect any symptomatic 

plant tissue found. While taking samples, visibly and indelibly flag or mark plants and areas 

sampled with sample ID and date. Also, mark areas on a map where plant samples were 

taken.
Trace Investigations

Trace Back Protocol Instructions

3/2024-07 Phytophthora ramorum Manual/Trace Back Protocol 5-3-3

2) Investigation, 

inspection, and quarantine hold procedures

(cont.)

6. If trace back regulated plants are in multiple locations within the nursery, the inspector 

must disinfect boots, tools, and hands between areas (refer to Treatments and Disinfectants on page 12-1-1).

7. Use PPQ Form 523, Emergency Action Notification (EAN) for the official Federal authorization of hold. In Section 16 of the EAN, state that those specific plants are prohibited in 

movement pending further notification by PPQ.

8. Visibly and indelibly flag a 2-meter area around the sampled plant to hold for quarantine 

until all diagnostic results are final.

9. Hold the sampled plants and all other regulated plants within the 2-meter radius around the 

sampled plants.

10. Once the delimiting survey and sampling are complete, any held plants may be consolidated and segregated. If the plants are not consolidated and segregated, 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 a manner that will safeguard and prevent the spread of the disease at the nursery, and be conducted under the 

direction of a regulatory official. Segregation must include storage on an impermeable surface (e.g., a 45-mil thick pond liner, concrete, or asphalt) and not within 2 meters of any 

other plant. The impermeable surface should be sloped to drain away from regulated 

plants.

11. Per Federal and State authorities, inspectors may, at any time, place on hold other plants, 

plant products, or articles (e.g., pots) that present a risk of spreading P. ramorum.

12. Check any cull, waste, or debris piles for P. ramorum-symptomatic plants or plant material. 

Collect samples of symptomatic material.

3) Sample collections 

and submission

Use Sampling and Submission Protocol on page 8-1-1.

4a) If the trace back 

nursery is confirmed 

positive, and is an 

interstate shipping 

nursery

Use the Interstate Confirmed Nursery Protocol on page 3-1-1 and contact the PPQ National 

Operations Manager (NOM).

4b) If the trace back 

nursery is confirmed 

positive and is an 

intrastate commerce 

only nursery

Use the Confirmed Retail Nursery and Retail Nursery Dealer Protocol on page 4-1-1 and contact the PPQ National Operations Manager (NOM).

5) Questions For PPQ P. ramorum program contacts, refer to Contact Information for the Phythopthora 

ramorum Program on page A-1-1.

Table 5-3-1 Trace Back Protocol Instructions (page 2 of 2)

Chronological or 

concurrent steps: Actions:
3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-1

Chapter

6

Confirmed Residential 

and Commercial 

Landscape Protocol

Contents

Intended Use 6-1-1

Goal 6-1-1

Trigger Events for Use of the Confirmed Residential and Commercial 

Landscape Protocol 6-1-2

Disclaimers 6-1-2

Challenges 6-1-2

Communication and Notification 6-1-2

List of Treatments and Disinfectants for Residential or Commercial 

Landscaped Settings 6-1-15

Intended Use

This protocol specifies actions that should be taken when a confirmed-positive 

Phytophthora ramorum infection is detected in plantings in residential or 

commercial landscape settings. If there are large areas of contiguous regulated 

plant material or large-caliper, infected plants encountered, consult with the 

PPQ National Operations Manager (NOM) for guidance. Guidance should 

include analysis of the environmental risks associated with treatments in 

residential and landscaped areas.

Goal

The goal of this protocol is to ensure any infestations of this serious pathogen 

are consistently and effectively addressed, mitigated, and eradicated when 

possible. Cooperation by the homeowner is essential. Early detection and 

reporting of potential P. ramorum infestations is critical to ensure spread is 

contained. The strategies employed in the protocol are intended to ensure a 

rapid and appropriate response to prevent the spread of the pathogen.
Confirmed Residential and Commercial Landscape Protocol

Trigger Events for Use of the Confirmed Residential and Commercial Landscape Protocol

6-1-2 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

Trigger Events for Use of the Confirmed Residential and Commercial 

Landscape Protocol

This protocol outlines procedures that should be followed when the presence 

of P. ramorum has been confirmed positive in a residential or commercial 

landscape setting. Confirmed samples must have been diagnosed using a 

methodology approved by PPQ and consistent with the Potentially Actionable 

Suspect Sample (PASS) protocol (refer to the PPQ Phytophthora ramorum

website for additional information regarding the PASS protocol).

Disclaimers

Any interpretation of this protocol or its procedures not consistent with 

the goal listed above is a misinterpretation and misrepresentation of the 

protocol.

Challenges

Phytophthora ramorum is a micro-organism and difficult to find and detect. It 

can infect plants; infest container mix, soil, and water; and persist in these 

substrates despite the best eradication efforts. These protocols and regulations 

will be adjusted accordingly, based on the understanding of the pathogen’s 

biology. Detection and management of this pathogen is informed by 

continually improving science.

Communication and Notification

NOTICE

Prior to an APHIS-confirmed positive determination, the National Plant Protection 

Laboratory Accreditation Program- (NPPLAP) approved laboratory must communicate all suspect positive diagnostic samples to inspectors as soon as one of the following has occurred:

• Culture that matches the morphology for P. ramorum as determined and reported 

by an APHIS-approved laboratory; or

• Positive PCR using APHIS-approved work instructions by an APHIS-approved laboratory

For all Potentially Actionable Suspect Samples (PASS), laboratories must immediately forward the plant material and DNA to the PPQ–S&T Plant Pathogen Confirmatory Diagnostics Laboratory (refer to Contact Information for the Phythopthora 

ramorum Program on page A-1-1) with a domestic ARM routing receipt (DARR) and 

notify the State’s State Plant Health Director (SPHD; generates DARR for laboratory) 

and State Plant Regulatory Official (SPRO) for the State of sample origin, and the 

National Operations Manager (NOM).
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-3

Confirmed Residential Protocol Steps

In chronological order, the steps for the Confirmed Residential Protocol are as 

follows:

1. Communicate and notify

2. Secure the site

3. Survey the site and perimeter

4. Delimiting survey

5. Disinfest the site

6. 90-day quarantine activities

7. Release the site

8. Post-disinfestation monitoring

Table 6-1-1 Confirmed Residential Protocol Instructions (page 1 of 4)

If step number: For: Then:

1—Communicate and notify1. Immediately notify the SPHD and the SPRO of the State where the site is 

located. The SPHD will notify the PPQ National Operations Manager (NOM). 

(refer to Resources on page A-1-1). Laboratories need to notify the SPHD, the 

SPRO, the PPQ National Operations Manager, the National Policy Manager, 

and the submitter of the confirmed-positive samples.

2. In the event of a confirmed positive at a residential or commercial landscape 

setting, the appropriate regulatory official in the State (SPRO or SPHD) immediately informs the homeowner or commercial landscape owner of the confirmed positive.

3. Complete the questionnaire Phytophthora ramorum Nursery Questionnaire on 

page A-1-2. Complete the questionnaire as thoroughly as possible during the 

initial contact with the property owner or manager and when the site is 

secured and the confirmed-positive plant(s) and all associated plants are 

safeguarded. Complete the remainder of the questionnaire and Follow-up 

Survey for Residential Locations with Infected Plants (page 1 of 2) on page 

6-1-13 at the time of the delimiting survey.

4. Document any proof of purchase the consumer may have, such as receipts, 

pot labels, etc.

2—Secure the 

site

When the 

presence of P. 

ramorum has 

been confirmed in a 

residential or 

commercial 

setting

1. Place on hold all regulated plant genera within a minimum of a 30-meter 

radius of the infected plant(s) under regulatory control as per the PPQ Form 

523 Emergency Action Notification (EAN) or State equivalent. Safeguard 

these plants and keep them undisturbed until the delimitation survey and confirmation results are complete. Any regulatory control (hold) may also include 

“any other product or article that an inspector determines to present a risk of 

spreading P. ramorum...” (7 CFR part 30.92-2) within the infested site.
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

6-1-4 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

2—Secure the 

site

(cont.)

When the 

presence of P. 

ramorum has 

been confirmed in a 

residential or 

commercial 

setting

(cont.)

2. Complete the questionnaire Follow-up Survey for Residential Locations with 

Infected Plants (page 1 of 2) on page 6-1-13 during the delimitation survey. 

Do not move any equipment used on the residential or landscaped commercial sites without proper treatment and disinfection (refer to List of Treatments 

and Disinfectants for Residential or Commercial Landscaped Settings on 

page 6-1-15).

3. If necessary, detail any additional treatments and/or basic sanitary and precautionary measures on the EAN.

A. The EAN will be used as the official Federal authorization of hold. Detail the 

required treatments and/or basic sanitary and precautionary measures 

(e.g., biocontainment of suspected infected material, etc.) in the EAN. If the 

State initiated action, use the appropriate State notification.

4. If any other plants in the area are showing symptoms consistent with P. ramorum, immediately sample and test those plants for the presence of P. ramorum.

5. If necessary, when the infected plant is located on the boundary between 

properties, regulatory controls may be placed on multiple properties. In the 

event the infected plant is located in a public common area, such as a boulevard or roadside, the regulatory official determines the appropriate area to be 

placed under regulatory control.

3—Survey the 

site and perimeter

The goal of the survey is to locate all P. ramorum-infected plants at the site, 

including the perimeter. A detailed and thorough inspection shall take place at the 

field level to identify the presence of P. ramorum. Collect samples from symptomatic plants, including any plants with minute symptoms such as tiny leaf spots, 

dropped leaves, or brown leaf tips.

Establish the 

destruction 

and quarantine radii (refer 

to Diagram 

Showing 

Destruction 

Radius, Quarantine Radius, 

and Delimitation Survey on 

page 6-1-7)

Determine the destruction radius on a case-by-case basis, but it shall not have 

less than a 2-meter radius in total area (refer to rare exceptions in 2-meter Dradius below). If multiple plants are confirmed positive within 2 meters of each 

other, demarcate a destruction radius around all of them.

The destruction radius is established when diagnostic results from all delimiting 

samples have been reported. The 90-day quarantine period begins when the 

delimiting survey is complete.

1. Observe the slope of the ground on which the confirmed-positive plant(s) are 

located and note the moisture conditions and likely movement of water on the 

site. In sloping areas, the destruction radius may be an elliptical shape that 

angles downslope of the confirmed-positive plant. Determine if the natural and 

irrigation water movement and moisture conditions support increasing the 

destruction radius on the downslope side of the confirmed-positive plant.

2. Determine the plant debris area.

A. If slope is a factor, the destruction radius will be the combined area of the 

elliptical shape and the plant debris area.

B. If slope is not a factor, the plant debris area may increase the destruction 

radius greater than 2 meters.

3. The quarantine radius is a minimum of 10 meters beyond the destruction 

radius and follows the same general shape.

4. Limit access to destruction radius/radii. Ensure proper sanitation measures 

are applied (refer to List of Treatments and Disinfectants for Residential or 

Commercial Landscaped Settings on page 6-1-15).

5. Destroy the P. ramorum-infected plants in an appropriate manner as soon as 

possible (refer to List of Treatments and Disinfectants for Residential or Commercial Landscaped Settings on page 6-1-15).

Table 6-1-1 Confirmed Residential Protocol Instructions (page 2 of 4)

If step number: For: Then:
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-5

4—Delimiting 

survey

1. Inspect all regulated plants within a minimum of a 30-meter radius of the confirmed-positive P. ramorum plant(s) and sample any symptomatic plants. Subsequent detections of P. ramorum as a result of the delimitation survey will 

require all regulated plants within a minimum of a 30-meter radius of the 

newly detected, confirmed-positive plants to be surveyed and all symptomatic 

plants to be sampled.

2. If the infestation is widespread, consult with the PPQ National Operations 

Manager (NOM) to design and implement an appropriate delimiting survey.

3. Document the inspection and map all regulated plant locations.

4. All symptomatic plants shall be sampled, mapped, marked or tagged, and 

tested.

5. Samples must be analyzed using the APHIS-approved methodology.

Soil sampling 1. Take soil samples in the destruction radius at the time of plant removal.

2. When selecting sampling locations, take water drainage patterns into consideration and include soil downslope from the plant removal area. Soil within the 

destruction radius (radii) and the quarantine radius/radii must be sampled 

(refer to Soil and Container Mix Sampling and Processing Protocol on page 

11-1-1).

Water sampling1. If the source of the infected plant is not known, it may be caused by infested 

water.

2. Determine the source of water used at the residential or commercial site. 

Water sampling is not required for chlorinated irrigation water from municipal 

water facilities. If not chlorinated irrigation water from municipal water facilities, bait the water to determine if it is infested.

3. The infected plant(s) might spread disease via water runoff. Evaluate the 

drainage pattern in the area of the infected plant(s). From the point of infection, bait any runoff water as resources permit (refer to Water Sampling and 

Processing Protocol on page 10-1-1).

5—Disinfest the 

site

Plant destruction1. Plants infected with P. ramorum must be removed and destroyed (refer to List 

of Treatments and Disinfectants for Residential or Commercial Landscaped 

Settings on page 6-1-15). A regulatory official must oversee plant destruction.

2. Remove the infected plants and root systems as much as possible. Doublebag with appropriate-sized plastic bags to at least 4-mil thickness. Larger 

plants must be removed at least to the root collar, and the stumps must be 

treated in an APHIS-approved manner to prevent sprouting. Contact the PPQ 

National Operations Manager (NOM) for guidance.

3. Remove and destroy all parts of regulated plants (e.g., branches of larger 

shrubs or trees) within the D-radius (a 2-meter radius (radii) of a confirmedpositive plant). Exception: bole hosts are less prone to disease; therefore, 

unless these plants show symptoms, they may be monitored for infection rather than being destroyed at the inspector’s discretion.

4. Approved methods of destruction include: incineration; deep burial; and steam 

sterilization (refer to List of Treatments and Disinfectants for Residential or 

Commercial Landscaped Settings on page 6-1-15).

5. Using the survey follow-up questionnaire (Follow-up Survey for Residential 

Locations with Infected Plants (page 1 of 2) on page 6-1-13), maintain a 

record of the taxon and number of plants destroyed at each location. Record 

the owner’s name, contact information, address, and the physical location of 

any infected plants. Draw a map, record landmarks, or enter the GPS coordinates for follow-up surveys.

Table 6-1-1 Confirmed Residential Protocol Instructions (page 3 of 4)

If step number: For: Then:
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

6-1-6 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

5—Disinfest the 

site

(cont.)

Debris 

removal

Rake a sufficiently sized area to collect all plant debris associated with the 

destruction radius and at least 2 meters beyond. Double-bag debris, as 

described above. Rake from the outer edge of the area toward the infected 

plant(s). All debris must be destroyed by APHIS-approved methods (refer to List 

of Treatments and Disinfectants for Residential or Commercial Landscaped Settings on page 6-1-15).

6—90-day quarantine activities1. Place all excepted regulated plants not destroyed, but located within the 

destruction radius under a 90-day quarantine.

2. The quarantined plants must be inspected and tested twice during the 90-day 

period. If the plants remain free of P. ramorum during this 90-day period, the 

site will be released from quarantine. The 90-day quarantine must occur 

during a time conducive to the expression of P. ramorum symptoms.

7—Release the 

site

Plants from residential or commercial landscape sites placed under regulatory 

control may be released from that control by PPQ or its designated authority after 

the quarantine period, if the following conditions are met:

1. Regulated plants will not be replanted within a minimum of 2 meters of the 

destruction radius for a period of at least 2 years; and

2. There are no additional detections of P. ramorum on any plants at the site 

based on APHIS-approved plant inspection, sampling, and testing protocols 

during the preceding 90-day quarantine period; and

3. Water and soil have been tested, if necessary, and found free of P. ramorum

based on APHIS-approved sampling and testing protocols for the preceding 

90-day quarantine period.

Alternate 

release strategy

A residential or commercial landscape site may avoid a quarantine period, 

through a voluntary management decision, if the following actions are taken:

1. Performed a delimiting survey and regulated plant genera found free of P. 

ramorum; and

2. Destroyed everything (all plants, pots, media, etc.) in the 2-meter destruction 

radius according to List of Treatments and Disinfectants for Residential or 

Commercial Landscaped Settings on page 6-1-15; and

3. Destroyed the regulated plants in the 10-meter quarantine radii; and

4. Sampled soil and water from destruction and quarantine radii with negative 

results.

Revisit the residential or commercial landscape site after approximately 90 

days of conducive conditions and conduct a survey inspection of the residential or commercial landscape site to include sampling of the soil in the 

destruction radius (2-meter area).

8—Post-disinfestation monitoringSites that have been confirmed positive will continue to have the regulated plants 

monitored (inspected, sampled, and tested) when disease expression is anticipated for the 2 years after the site(s) has been released. These sites are not

under any other regulatory action unless there are additional P. ramorum detections.

Table 6-1-1 Confirmed Residential Protocol Instructions (page 4 of 4)

If step number: For: Then:
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-7

Figure 6-1-1 Diagram Showing Destruction Radius, Quarantine Radius, and Delimitation Survey

Destruction radius: 

1) Inspect all regulated plant 

genera and sample any unhealthy 

plants.

2) Destroy regulated plants ASAP; 

destruction is required when 

sample results from delimitating 

survey are complete. 

3) Sample soil and water where it 

drains.

4)Hold any regulated plants until 

destroyed.

5) Limit access.

Quarantine radius: 

1) Inspect all regulated plant 

genera and sample any unhealthy 

plants.

2) Sample soil and water where it 

drains.

3) All regulated plants are placed 

on a 90-day hold. 

4) Limit access. 

Delimiting survey radius: 

1) Inspect all regulated plant 

genera and sample any unhealthy 

plants.

2) Sample water if necessary.

3) All regulated plants held until 

delimiting survey and results from 

any samples taken are complete.

4) Limit access.
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

6-1-8 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

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Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-9

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Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

6-1-10 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

Figure 6-1-2 P. ramorum Nursery Questionnaire (page 3 of 5)


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Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-11

Figure 6-1-2 P. ramorum Nursery Questionnaire (page 4 of 5)

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Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

6-1-12 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

Figure 6-1-2 P. ramorum Nursery Questionnaire (page 5 of 5)

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Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-13

Figure 6-1-3 Follow-up Survey for Residential Locations with Infected Plants (page 1 of 2)

Follow-up Survey for Locations with Infected Plants 

Locations found to have infected plants will be monitored for 2 years. The affected area will not be under 

any quarantine or regulatory control unless additional outbreaks are detected. 

Property Owner or Manager Survey: 

1. Date of plant collection: __________________________________________________________ 

2. Property owner or manager name: __________________________________________________ 

3. Location: _____________________________________________________________________ 

4. Name(s) and numbers of plants collected for destruction: 

 a. ________________________________________________ # __________________ 

 b. ________________________________________________ # __________________ 

 c. ________________________________________________ # __________________ 

 d. ________________________________________________ # __________________ 

 e. ________________________________________________ # __________________ 

 f. ________________________________________________ # __________________ 

 g. ________________________________________________ # __________________ 

 h. ________________________________________________ # __________________ 

 i. ________________________________________________ # __________________ 

5. Where were the plants purchased? 

 a. ________________________________________________ # __________________ 

 b. ________________________________________________ # __________________ 

 c. ________________________________________________ # __________________ 

 d. ________________________________________________ # __________________ 

 e. ________________________________________________ # __________________ 

 f. ________________________________________________ # __________________ 

 g. ________________________________________________ # __________________ 

 h. ________________________________________________ # __________________ 

 i. ________________________________________________ # __________________ 

 
Confirmed Residential and Commercial Landscape Protocol

Confirmed Residential Protocol Steps

6-1-14 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

Figure 6-1-3 Follow-up Survey for Residential Locations with Infected Plants (page 2 of 2)

6. When were the plants purchased? 

a. ________________________________________________ # __________________ 

 b. ________________________________________________ # __________________ 

 c. ________________________________________________ # __________________ 

 d. ________________________________________________ # __________________ 

 e. ________________________________________________ # __________________ 

 f. ________________________________________________ # __________________ 

 g. ________________________________________________ # __________________ 

 h. ________________________________________________ # __________________ 

 i. ________________________________________________ # __________________ 

7. Any proof of purchase or documentation such as receipts, pot labels, etc.? 

List here: ______________________________________________________________________ 

8. Survey types of regulated plants in the landscape and list named and numbers: 

 a. ________________________________________________ # __________________ 

 b. ________________________________________________ # __________________ 

 c. ________________________________________________ # __________________ 

 d. ________________________________________________ # __________________ 

 e. ________________________________________________ # __________________ 

 f. ________________________________________________ # __________________ 

 g. ________________________________________________ # __________________ 

 h. ________________________________________________ # __________________ 

 i. ________________________________________________ # __________________ 

9. Samples of symptomatic regulated landscape plants taken? No Yes 

10. Soil samples taken? Yes No (plants still in pots) 

11. Comments: 
Confirmed Residential and Commercial Landscape Protocol

List of Treatments and Disinfectants for Residential or Commercial Landscaped Settings

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-15

List of Treatments and Disinfectants for Residential or Commercial 

Landscaped Settings

The following techniques may be used to control P. ramorum in residential and 

commercial landscape sites found to contain infected plants. Prior to use, 

please confirm the chosen method is approved for your State. Always follow 

label directions.

Table 6-1-2 Treatments and Disinfectants for Residential or Commercial Landscaped Settings (page 1 

of 2)

For: Then:

1—Confirmed-positive 

plants

1. Incineration (burning to ash): infected plants, associated container mix, associated 

containers (i.e., pots and trays), and all leaf debris in and around the area where plants 

were stored may be incinerated at a facility or other location (e.g., on site) approved by 

APHIS and permitted within State and municipal statutes or regulations. Properly safeguard off-site movement and take every effort to prevent plant debris or soil from being 

dislodged from the plants prior to incineration. Burning may be through open burning or 

in an incinerator.

2. Deep burial: infected plants, associated container mix, associated containers (i.e., pots 

and trays), and all leaf debris in and around the area where plants were stored must be 

double-bagged using plastic bags to at least a 4-mil thickness or greater and buried to 

a depth of no less than 2 meters to the top of the debris. Bury the material at an 

APHIS-approved site, on site, or in a municipal landfill, which will be undisturbed. Take 

every effort to prevent plant debris or soil from being dislodged from the plants.

3. Steam sterilization: dry heat or steam commonly heated to internal temperatures of 

212 °F (100 °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 

T415-b.

4. Nonporous 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 registered for use on nonporous surfaces 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. The Summary of Disinfect Activities (Table 

6-1-3 on page 6-1-17 examines the effects of different classes of disinfectants on pathogenic micro-organisms. 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.

2—Confirmed-positive 

water

1. 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 spp. 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.

2. For irrigation: chlorine levels of 2 ppm or 2 mg/liter or greater has been correlated with 

the control of Phytophthora spp. in recirculated irrigation systems. Recirculated, nonmunicipal water must be chlorinated at an active chlorine concentration equal to or 

greater than 2 mg/liter of water and monitored to maintain the proper chlorine levels.
Confirmed Residential and Commercial Landscape Protocol

List of Treatments and Disinfectants for Residential or Commercial Landscaped Settings

6-1-16 Phytophthora ramorum Manual/Confirmed Residential Protocol 3/2024-07

3—Confirmed-positive 

soil and container mix

1. Container mix: heat container mix such that the internal temperature in the center of 

the load reaches at least 180 °F for 30 minutes or treat with an approved fumigant. 

Treatment must be conducted in the presence of an inspector.

2. Soil treatment:

A. Heat a load of soil being treated such that the temperature in the center of the load 

reaches at least 180 °F for 30 minutes.

B. Conduct field soil treatments in the presence of an inspector and treat with an 

approved fumigant as per the label. 

If considering the use of solarization for soil treatments, contact the PPQ NOM and 

USDA–PPQ Science and Technology (S&T) representative (refer to Contact Information for the Phythopthora ramorum Program on page A-1-1).

4—Confirmed-positive 

containment soil

Mitigation of infested soil in the destruction radius can also be achieved by installing permanent, impermeable, and impervious barriers that consists of cement, concrete, or 

asphalt 3 inches in depth and extending 6 feet beyond the infested area. Construct these 

barriers such that no native soil is exposed. Grade the barriers such that no standing water 

collects.

5—Equipment and personnel1. Limit or minimize access to infested areas and quarantine areas. Everyone entering and 

leaving the residential or commercial landscape site must scrape off loose pieces of 

soil from their person into the destruction radius. Those working with or in contact with 

suspected infested material (including plants), must wash hands using soap or 

approved disinfectant immediately after completing the task. There are no products currently labeled for use on porous materials for Phytophthora control.

2. Conduct activity in the destruction zone wearing disposable shoe covers and dispose of 

the covers immediately upon exiting the area. Properly dispose of the shoe covers. If 

shoe covers are not used, clean and disinfect shoes with a disinfectant listed in 

Table 6-1-3 upon exiting the area.

3. Tires (or other parts in contact with the soil or plants, such as truck beds) 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, do not allow vehicles 

in the destruction radii. Any efficacious product labeled for use on nonporous surfaces 

may be used on tires or vehicle undercarriages.

4. Do not visit other sites or areas in potentially contaminated work clothing.

Table 6-1-2 Treatments and Disinfectants for Residential or Commercial Landscaped Settings (page 2 

of 2)

For: Then:
Confirmed Residential and Commercial Landscape Protocol

List of Treatments and Disinfectants for Residential or Commercial Landscaped Settings

3/2024-07 Phytophthora ramorum Manual/Confirmed Residential Protocol 6-1-17

Table 6-1-3 Summary of Disinfectant Activities1

Disinfectant: Trade names: Comments: Contact time:

Alcohols (ethyl and 

isopropyl)

(60% – 85%)

Lysol spray® Evaporates quickly so that adequate contact time 

may not be achieved, high concentrations of 

organic matter diminish effectiveness; flammable

10 – 15 minutes

Phenolics

(0.4% – 5%)

Phenocen® Phenol penetrates latex gloves; eye/skin irritant; 

remains active upon contact with organic soil; may 

leave residue

10 – 15 minutes

Quaternary ammonium

(0.5% – 1.5%)

 Consan triple 

action 20

 Physan 20®

 Green-Shield®

 Formula 409®

Effective for nonporous surface sanitation (floors, 

walls, benches, pots). Low odor, irritation. Use 

according to labels

10 – 15 minutes

Chlorine

(100ppm – 1,000ppm)

 10% Clorox®

 10% bleach

Inactivated by organic matter; fresh solutions of 

hypochlorite (Clorox®) should be prepared every 8 

hours or more frequently if exposed to sunlight; corrosive; irritating to eyes and skin. Exposure to sunlight further reduces hypocholorite efficacy. Keep 

solution in opaque container.

10 – 15 minutes

1 Modified table from Columbia Research Environmental Health and Safety (EH&S).
3/2024-07 Phytophthora ramorum Manual/Biology and Symptoms 7-1-1

Chapter

7 Biology and Symptoms of 

Phytophthora ramorum

Contents

Biology and Symptoms of Phytophthora ramorum 7-1-1

Biology 7-1-1

Symptoms 7-1-2

Biology and Symptoms of Phytophthora ramorum

Biology

Hosts of P. ramorum usually fall into one of three disease categories based on 

visual symptoms: canker hosts; leaf hosts; and twig hosts. Infections in leaf 

and twig hosts are rarely fatal, but act as a reservoir for the pathogen. P. 

ramorum has also been shown to infect roots of host plants without resulting in 

above-ground visual symptoms. Host plants with latent root infections can 

remain symptomless for months.

Infected plant debris and P. ramorum spores from infected plants, on or under 

the soil surface, can result in areas remaining persistently confirmed positive 

for months or years following regulated plant material removal and aboveground litter removal. Recent research in Europe has shown this pathogen can 

persist in substrate and be detected in water runoff from P. ramorumconfirmed-positive sites for up to 5 years.

Pathways for long-distance dispersal of the pathogen include movement of 

infected plant material (wood, green material products, and nursery stock), 

soil, water (rain, runoff, streams, rivers, and irrigation water), animals, and 

aerial dissemination during major weather events.

For more detailed information regarding P. ramorum biology, refer to the 

USDA National Invasive Species Information Center website and the 

American Phytopathological Society (APS) website.
Biology and Symptoms of Phytophthora ramorum

Biology and Symptoms of Phytophthora ramorum

7-1-2 Phytophthora ramorum Manual/Biology and Symptoms 3/2024-07

Symptoms

Three different syndromes are attributed to P. ramorum: stem or bole canker; 

leaf blight; and twig blight or dieback. Regulated plant material prominent in 

the nursery trade include Rhododendron, Camellia, Viburnum, Pieris, Kalmia, 

and Syringa. Symptoms on Rhododendron closely resemble those caused by 

other Phytophthora species or those caused by environmental stress (drought, 

etc.), making inspection for the disease more complicated and detection 

challenging. With Lithocarpus species, drooping or wilting of new growth 

occurs before other symptoms appear. Cankers typically occur in the lower 3 

meters and are restricted to above the soil line. Occasionally cankers have been 

found 20 meters above ground. Cankers can eventually kill the tree by 

attacking the phloem and girdling the tree. Bleeding symptoms of the canker 

are easier to detect during dry weather.

For more detailed information regarding P. ramorum symptoms, refer to the 

APHIS–PPQ P. ramorum website.
3/2024-07 Phytophthora ramorum Manual/Sampling and Submission Protocol 8-1-1

Chapter

8 Sampling and Submission 

Protocol

Contents

Sample Handling and Submission Protocol 8-1-1

Sample Handling Information 8-1-1

Sample Submission Information 8-1-2

Sample Forwarding and Reporting 8-1-2

Sampling Supplies and Equipment Checklist 8-1-4

Sample Handling and Submission Protocol

Sample Handling Information

Perform the following tasks in order to correctly and accurately handle and 

submit samples.

1. Always write out the identifying label remarks on the outside of the bag 

with a permanent marking method.

A. Attach labels to the outside of bags because labels inside the bag may 

deteriorate due to moisture and may become illegible.

B. Include on all labels (with a permanent marking method) the following: 

date; collector’s identification number; location of sample site; sample 

number; and other required information.

2. Do not add extra moisture to the sample to keep it fresh. Do not wrap 

leaves in paper towels when shipping. Extra moisture and paper towel use 

can speed deterioration of the sample.

3. Sanitize or remove gloves and place sample bag in a second protective 

bag. To provide extra insurance against accidental release during shipping, 

double bag the labeled specimen bag(s), i.e., first place the specimen in a 

self-locking labeled plastic bag, then place that labeled specimen bag 

inside a second self-sealing plastic bag. Place PPQ Form 391 Specimens 

for Determination (or State equivalent) inside the outer bag.

4. Place samples in a cooler out of the sun as soon as possible. When 

sampling large areas, bring coolers out to the sampling areas. In sunlight, 

samples can heat up quickly when placed in self-sealing plastic bags, even 

for short periods of time. If it is not possible to have coolers in the 
Sampling and Submission Protocol

Sample Handling and Submission Protocol

8-1-2 Phytophthora ramorum Manual/Sampling and Submission Protocol 3/2024-07

sampling area, place the samples in a shaded area until they can be 

collected and placed in a cooler.

5. Refrigerate samples while awaiting shipment. Place double-bagged 

samples in a sturdy cardboard box or heavy styrofoam container so the 

samples are not damaged during shipping. Ship with an ice pack with 

buffer space between the ice and leaves. Thoroughly seal all seams on the 

container with shipping tape. To preserve freshness, mail or deliver the 

sample(s) to the laboratory as soon as possible (if mailing, use overnight 

delivery). Do not ship on Fridays. It is better to hold the sample(s) in the 

refrigerator over a weekend than to have them sit in unknown 

environmental conditions.

Sample Submission Information

Follow the laboratory’s standard operating procedure (SOP). Typically, have 

ready the following required information: 1) tracking number; 2) number of 

samples being shipped; and 3) the disease for which the sample is being tested. 

All samples must have either a completed PPQ Form 391Specimens for 

Determination or an equivalent State documentation. The laboratory may be a 

NPPLAP-accredited State laboratory, a cooperating National Plant Diagnostic 

Network (NPDN) laboratory, or a USDA–APHIS–PPQ laboratory. If 

submitters are not sure to which laboratory they should send samples, contact 

the State Plant Health Director (SPHD).

If PPQ Form 391 is electronic, it can be emailed when notifying the laboratory 

about the pending shipment. Remember to also attach a hard copy to the 

sample. On PPQ Form 391 complete blocks 1 through 5, 7, 10, 11, 16, 22, and 

23 (refer to Figure 8-1-1).

Sample Forwarding and Reporting

Nursery plant samples that are ELISA or ImmunoStrip positive for the genus 

Phytophthora must be forwarded to the cooperating NPPLAP-accredited 

laboratory and/or to an APHIS diagnostic laboratory to determine if the species 

is P. ramorum. Every initial sample from nurseries in nonregulated areas must

be forwarded to APHIS for confirmatory testing. If APHIS confirms a sample 

is positive, all subsequent samples may be diagnosed by any NPPLAPaccredited laboratory.

For laboratories with Federal confirmation authority, the laboratory must

report confirmed positives to the SPRO/SPHD, then to the P. ramorum Field 

Operations Manager within 24 hours of the diagnostic result. All subsequent 

confirmed-positive samples taken at a confirmed-positive nursery must also be 

reported in the same way.
Sampling and Submission Protocol

Sample Handling and Submission Protocol

3/2024-07 Phytophthora ramorum Manual/Sampling and Submission Protocol 8-1-3

Figure 8-1-1 Instructions for Using PPQ Form 391, Specimens for Determination

INSTRUCTIONS: Use PPQ Form 391, Specimens for Determination, for domestic collections (warehouse inspections, 

local and individual collecting, special survey programs, export certification).

DISTRIBUTION of PPQ Form 391

1. Send original along with the sample to your area identifier.

2. Retain and file a copy for your records.

BLOCK INSTRUCTIONS

1 1. Assign a number for each collection beginning with the year, followed by the collector’s initials and collector’s number.

EXAMPLE: In 2001, Brian K. Long collected his first specimen for determination of the year. His 

first collection number is 01-BLK-001.

2. Enter the collection number.

2 Enter date

3 Check block to indicate Agency submitting specimens for identification

4 Enter name of sender

5 Enter type of property specimen obtained from (farm, nursery, feed mill, etc.)

6 Enter address

7 Enter name and address of property owner

8A–8L Check all appropriate blocks

9 Leave blank

10 Enter scientific name of host, if possible

11 Enter quantity of host and plants affected

12 Check block to indicate distribution of plant

13 Check appropriate blocks to indicate plant parts affected

14 Check block to indicate pest distribution

15 • Check appropriate block to indicate type of specimen

• Enter number of specimens submitted under appropriate column

16 Enter sampling method

17 Enter type of trap and lure

18 Enter trap number

19 Enter X in block to indicate isolated or general plant symptoms

20 Enter X in appropriate block for weed density

21 Enter X in appropriate block for weed growth stage

22 Provide a brief explanation if prompt or URGENT identification is requested

23 Enter a tentative determination if you made one

24 Leave blank

Genus species, particularly cultivar name
Sampling and Submission Protocol

Sampling Supplies and Equipment Checklist

8-1-4 Phytophthora ramorum Manual/Sampling and Submission Protocol 3/2024-07

Sampling Supplies and Equipment Checklist

APHIS Lists of Proven Hosts of and Plants Associated with P. 

ramorum

Bigger collection bags in which to carry samples while inspecting and 

sampling

Box for mailing samples

Camera

Clipboard or PDA, PPQ Form 391 Specimens for Determination (or 

State equivalent) nursery inspection and sampling forms, paper, etc.

Cooler, coolant, and newspapers to keep samples cool until mailed

Disposable gloves

Flagging, pin flag, or label sticks to mark sampled plants/blocks

Foot bath bin

GPS (optional)

Hand sanitizer to sanitize gloves between samples

Larger bags for mailing samples (must arrive in the laboratory double 

bagged)

Nursery maps and nursery inventory

Permanent marking method

Pruners to sample twigs and branches

Quaternary ammonium solution or other approved disinfectant, at 

labeled rates 1” deep in bath

Rubber boots

Self-sealing plastic sample bags

Spray bottle of an approved disinfectant for P. ramorum

Toilet brush or other stiff brush for scrubbing dirt off shoes

Writing pen
3/2024-07 Phytophthora ramorum Manual/Biosecurity Measures for Nurseries 9-1-1

Chapter

9 Biosecurity Measures for 

Nurseries

Contents

Introduction 9-1-1

Communications 9-1-2

Vehicles 9-1-2

Avoidance 9-1-2

Cleaning 9-1-3

Nursery Personnel 9-1-3

Access 9-1-3

Boots 9-1-4

Hands 9-1-4

Equipment 9-1-4

Sampling Supplies and Equipment Checklist 9-1-5

Introduction

Decontaminate personnel, tools, and equipment between blocks in the nursery, 

between regulated genera within a nursery, and between nurseries. Sanitize 

gloves with antiseptic rubs/gels/rinses (containing a minimum of 60% ethyl 

alcohol and rub hands vigorously to decrease drying time). Wear rubber boots 

or other waterproof boots without crevices. Sanitize or change gloves between 

samples. Use spray bottle containing a dilute (10%) bleach solution, a 

quaternary ammonium solution at labeled rates, or spray disinfectant (with 

ETOH) to treat all tools between samples. Brush loose dirt from boots then 

spray boots with disinfection solution in spray bottle, or use foot bath, between 

nursery blocks/areas. Decontaminate all equipment used between each sample 

and before leaving a nursery.
Biosecurity Measures for Nurseries

Biosecurity Measure for Nurseries

9-1-2 Phytophthora ramorum Manual/Biosecurity Measures for Nurseries 3/2024-07

Biosecurity Measure for Nurseries

In the course of daily work, nursery personnel are frequently required to visit a 

number of different nursery sites, greenhouses, fields, and facilities. These 

actions could potentially provide a pathway for transferring quarantine 

organisms from one work site to another. It should be recognized that even if a 

single work site is visited per day, precautions must be taken to avoid using 

contaminated clothing and equipment at a new site the following day. Further, 

visitors to these same facilities present the same risks and could vector diseasecausing-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 must be trained and visitors informed of the biosecurity 

requirements enacted by the facility. As new scientific data and technology 

becomes available, the facility must update its biosecurity requirements and 

retrain its 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 in one of the nursery’s vehicles. Load nursery 

stock onto any vehicle, other than the nursery’s vehicles, in an area with a 

concrete or asphalt pad located near the gate and not inside the nursery.
Biosecurity Measures for Nurseries

Nursery Personnel

3/2024-07 Phytophthora ramorum Manual/Biosecurity Measures for Nurseries 9-1-3

Cleaning

To ensure there is no buildup of soil, debris, or other items, clean nursery 

vehicle interiors. When the vehicle must go into a field, the vehicle must be 

driven to the edge of the facility where the tires, wheel wells, and accessible 

areas of the vehicle’s undercarriage must be cleaned with a brush or a water 

hose, followed by a spray down with suitable disinfectant. When the 

undercarriage has been coated with soil, it is recommended that after cleaning 

and disinfecting a vehicle at the work site, a vehicle should go through a car 

wash in order to clean the vehicle’s undercarriage before proceeding to another 

work site. If a car wash is not available, avoid driving the vehicle to the next 

work site. To ensure the entire surface of the tires is cleaned it will also be 

necessary to move the vehicle forward by approximately a foot to clean the 

portion of the tire in contact with the ground.

Vehicle tires (or other vehicle parts in contact with soil or plants, e.g., truck 

beds) must be cleaned of loose soil and plant debris and disinfested with the 

appropriate labeled products before leaving the infested site. Any product 

labeled for use on nonporous surfaces may be used on tires or vehicle 

undercarriages.

A portion of the vehicle must be designated as a “clean area” in which clean 

work supplies and equipment can be kept. A designated “dirty area” of the 

vehicle, such as the trunk of the car or a specified enclosed area of a truck bed, 

must also be identified for use to hold double-bagged clothes or dirty 

equipment requiring cleaning. For situations in which pool vehicles are used, 

adopt a set procedure for all personnel.

Nursery Personnel

Nursery personnel routinely come into contact with potentially contaminated 

soil, plants, and organic matter requiring the personnel to address a number of 

biosecurity measures. If the inspection site has distinct levels of biosecurity for 

different areas in the nursery, it is necessary to work from the areas of lowest to 

highest risk.

Access

Access to infested areas and hold areas should be limited, as much as possible, 

to personnel and employees. Everyone entering and leaving the nursery site 

must scrape off loose pieces of soil into the destruction radius and use a 

disinfection spray on foot wear or use a foot bath. Those working with, or in 

contact with suspected infested material (including plants), must wear gloves 

and rub them with an approved disinfectant between samples and between 

blocks. There are no products currently labeled for use on porous materials for 

Phytophthora control.
Biosecurity Measures for Nurseries

Nursery Personnel

9-1-4 Phytophthora ramorum Manual/Biosecurity Measures for Nurseries 3/2024-07

1. Personnel should not have access to production areas of the nursery after 

entering the destruction radius on the same day

2. Place a disinfectant foot bath for use by personnel entering and exiting the 

quarantine area and entering and exiting the destruction radius at the 

infested nursery site, where the movement of soil or plant debris on 

footwear is likely. The foot bath must be filled with fresh disinfectant at 

least on a daily basis, or more frequently if contaminated with dirt or 

debris, in accordance with label directions.

3. Do not visit other nursery sites in potentially contaminated work clothing 

and footwear.

Boots

Wearing rubber boots is strongly encouraged when working in an infested 

area(s). Wear waterproof, smooth boots that can be disinfected. As a last resort, 

disposable waterproof boot covers can be worn over work boots. The rubber 

boots must be disinfected upon arrival, even if the boots were disinfected 

when leaving the last work site. At the conclusion of any inspection, clean the 

boots of soil and disinfect them between the infested block and other blocks, as 

well as prior to placing them in the vehicle area designated as a “clean area.” 

Dispose used boot covers by double bagging and placing them in the 

designated “dirty area” of the vehicle for proper disposal. After removing the 

boot covers, the soles of the work boots must be inspected for soil and, if soil 

is present, must be cleaned and treated with disinfectant.

Hands

Thoroughly wash hands with soap and water before entering and after leaving 

the work site. Wet hands with warm running water then lather with soap for at 

least 20 seconds before rinsing and drying hands. When sampling, wear gloves 

and use antiseptic rubs, gels, or rinses (containing a minimum of 70% ethyl 

alcohol) between samples. To avoid cross-contaminations, disinfect hands or 

change gloves after handling any plants or other contaminated matter in the 

infested area.

Equipment

Any equipment used (pruners, measuring tapes, clipboards, pens, etc.) at a 

work site must be disinfected prior to leaving the work site. Where practical, 

equipment should be disinfected as frequently as possible at each work site. 

Use a spray bottle containing a dilute (10%) bleach solution, a quaternary 

ammonium solution at labeled rates, or disinfectant spray (with ETOH) to treat 

all tools between samples. When equipment must leave the work site for 

disinfection, it must be double bagged and placed in the vehicle’s designated 

“dirty area.”
Biosecurity Measures for Nurseries

Nursery Personnel

3/2024-07 Phytophthora ramorum Manual/Biosecurity Measures for Nurseries 9-1-5

Sampling Supplies and Equipment Checklist

APHIS Lists of Proven Hosts of and Plants Associated with P. 

ramorum

Bigger collection bags in which to carry samples while inspecting and 

sampling

Box for mailing samples

Camera

Clipboard or PDA, PPQ Form 391 Specimens for Determination (or 

State equivalent) nursery inspection and sampling forms, paper, etc.

Cooler, coolant, and newspapers to keep samples cool until mailed

Disposable gloves

Flagging, pin flag, or label sticks to mark sampled plants/blocks

Foot bath bin

GPS (optional)

Hand sanitizer to sanitize gloves between samples

Larger bags for mailing samples (must arrive in the laboratory double 

bagged)

Nursery maps and nursery inventory

Permanent marking method

Pruners to sample twigs and branches

Quaternary ammonium solution or other approved disinfectant, at 

labeled rates 1” deep in bath

Rubber boots

Self-sealing plastic sample bags

Spray bottle of an approved disinfectant for P. ramorum

Toilet brush or other stiff brush for scrubbing dirt off shoes

Writing pen
03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-1

Chapter

10 Water Sampling and 

Processing Protocol

Contents

Introduction 10-1-1

In Vitro Water Sampling with Regulated Plant Material Leaf Baits—Bottle of 

Bait (BoB) Technique 10-1-2

Bait Selection 10-1-2

Sample Collection 10-1-2

Sample Processing in Receiving Laboratory 10-1-4

Bottle of Bait (BoB) Materials List 10-1-4

In Situ Water Sampling with Regulated Plant Material Leaf Baits 10-1-6

Bait Selection 10-1-6

Baiting Techniques 10-1-7

Sample Transport, Storage, Shipping, and Processing 10-1-10

Water Sampling for Filtration 10-1-11

Sample Collection 10-1-11

Sample Processing 10-1-11

Additional Guidance on Culture and Identification 10-1-13

Diagnostic Determination for Cultures 10-1-14

Disposing of Cultures After Diagnosis 10-1-14

Water Filtration Methods Materials List 10-1-16

Growing Media Formulae 10-1-17

PARPH-V8 Selective Medium: for Phytophthora Species 10-1-17

PAR-V8 Selective Medium: for Phytophthora Species 10-1-18

References 10-1-19

Introduction

Phytophthora ramorum is a soilborne plant pathogen well adapted to dispersal 

and movement via water. Described as a “water mold,” P. ramorum is more 

closely related to algae than fungi. For this reason, water samples collected 

from potentially infested nursery areas can be tested for the presence of P. 

ramorum spores.

Contact the National Plant Protection Laboratory Accreditation Program 

(NPPLAP)-approved laboratory if P. ramorum is suspected from any

samples
Water Sampling and Processing Protocol

In Vitro Water Sampling with Regulated Plant Material Leaf Baits—Bottle of Bait (BoB) Technique

10-1-2 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

Water filtration is best used in cases where you expect low inoculum 

levels

1. Bottle of bait or “BoB”—BoB relies on using regulated plant material for 

“baiting” collected water samples. After a water sample is collected in a 1-

liter bottle, an intact leaf and/or leaf pieces from a regulated plant leaf are 

placed in the bottle as soon as possible. Bottles are incubated at 18 °C to 22 

°C in the dark for 3 days, with the leaf pieces tested directly and the intact 

leaf incubated for symptom development, as described in Sample 

Processing in Receiving Laboratory on page 10-1-4. This is the preferred 

method of testing for Phytophthora spp. in most nursery settings.

2. Bait bags or bait stations—bait bags containing leaves of P. ramorum host 

plants (e.g., Rhododendron or Camellia) are used to attract Phytophthora

spp. This method is used for testing large holding ponds, lakes, streams, 

and rivers. This method is most effective when water temperatures are 

between 46 °F (8 °C) and 71 °F (22 °C). After exposure, leaf baits are held 

for a minimum of 7 to 10 days at 18 °C to 22 °C in a dark, moist chamber 

to promote disease development and symptom expression.

In Vitro Water Sampling with Regulated Plant Material Leaf Baits—

Bottle of Bait (BoB) Technique

Bait Selection

1. Collect healthy leaves from a population of P. ramorum host plants (e.g., 

Rhododendron or Camellia). Bait-source host plant material must not have 

been sprayed with fungicide within the last 3 months. Avoid using newly 

acquired plants for this reason. Bait-source plants should be sufficiently 

large, robust, and numerous enough to supply leaves during the entire 

duration of the survey.

2. Use healthy leaves that have been on the plant for at least 1 year and are as 

free as possible from insect and mechanical damage. Do not use succulent, 

newly formed leaves. Present-year leaf growth may be used after full leaf 

expansion and a period of hardening in the summer.

3. Place bait leaves in self-sealing plastic bags for refrigerated storage for no 

more than 14 days before use.

Sample Collection

1. The number of water samples collected is based on the number of water 

bodies present, availability of run-off water, and the overall size of the 

nursery. If this sampling method is selected, collect a minimum of one 800-

ml sample per sampling site. More samples will be needed for larger 

nurseries with more water and irrigation and drainage sources.
Water Sampling and Processing Protocol

In Vitro Water Sampling with Regulated Plant Material Leaf Baits—Bottle of Bait (BoB) Technique

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-3

2. Collect 800 ml of water from each sample point and record water 

temperature. If possible, sample should be collected from upper surface of 

the water; collect the cleanest sample possible by minimizing sediment 

disturbance, while avoiding plant and other floating debris. Use a 100-ml 

measuring cup or disposable paper cup to fill a 1-liter screw cap plastic 

bottle in increments rather than filling the container all at once. Collect 100 

ml of water from each sample point; use 8 different sample points, which 

would total 800 ml.

3. Record and mark the location of the sample site. If possible, record GPS 

coordinates or write a description of the sample collection location for each 

sample.

4. Affix identification labels (e.g., laboratory tape or masking tape) both to 

the screw cap and the outside of each water collection bottle using a 

waterproof marking method. Sufficiently code labels to correspond with 

datasheet entries for each nursery and water body and include date 

collected, water source (location), a unique sample number, and nursery 

(e.g., nursery name or license number).

5. When sampling drainage ditches or areas of moving water within nurseries, 

rinse bottles downstream with the water about to be sampled before water 

is collected.

6. Immediately bait each sample with 20 freshly punched leaf pieces, and 1 

healthy, intact host plant leaf. This must be done on site, using a hand-held 

hole punch to get uniform leaf pieces. Sometimes, the punched pieces can 

become too degraded or have too many other organisms growing on them 

to successfully isolate P. ramorum, if present. Because of this, the whole 

leaf is also used.

7. Maintain collected water samples on their side in a cooler (without ice if 

external temperatures are cool or with enough ice to gradually cool water 

samples) for transport to the sample processing laboratory. Pack and ship 

water samples with the host plant leaf baits via overnight mail with a cold 

pack or drive the samples directly to the laboratory.

8. Maintain a record of the water sample information. Assign a unique sample 

number to each bottle.

9. Wash each 100-ml measuring cup with warm, soapy water or use a new 

disposable cup between sample collections. Thoroughly and completely 

rinse each item. For best results, use an automatic dishwasher with a heated 

drying cycle or an autoclave to wash collection bottles. Contact the 

receiving laboratory to determine if they will provide samplers with clean 

1-liter water collection bottles.

10. Prepare enough cup/bottle sets for water collection at a number of sample 

sites per nursery. Use only clean, sanitized collection materials at each site 

and water source.
Water Sampling and Processing Protocol

In Vitro Water Sampling with Regulated Plant Material Leaf Baits—Bottle of Bait (BoB) Technique

10-1-4 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

Sample Processing in Receiving Laboratory

Email APHIS–PPQ–Plant Pathogen Confirmatory Diagnostics Laboratory at: 

PPQ.CPHST.Beltsville.Diagnostics@usda.gov for the latest version of the P. 

ramorum diagnostic protocols.

1. Place bottles containing water samples and leaf baits on their sides and 

incubate for 3 days at 18 °C to 22 °C in the dark.

2. Following incubation, remove baits (leaf pieces and whole leaf), rinse them 

in distilled water, and blot dry. Immediately process leaf pieces for 

detection, but place whole leaf in a moist chamber in the dark for a 

minimum of 7 to 10 days at 18 °C to 22 °C to promote disease 

development and symptom expression. In the event that no lesions 

develop, the sample can be considered negative and discarded.

3. If symptoms appear on the incubated whole leaf, process the leaf using the 

same methods as for nursery leaf samples. The approved molecular 

diagnostic work instructions must be followed for these regulatory 

samples.

4. Leaf pieces can be placed into selective PARPH-V8 medium to isolate P. 

ramorum and then tested using approved molecular diagnostics, or they 

can be tested directly using the USDA-approved PCR protocols. Leaf 

pieces that are to be tested directly can also be frozen and held for testing at 

a later time. The pieces from one sample can also be split with a portion of 

them being tested through PCR and the remainder either going to plating 

and/or freezing.

5. If symptoms appear on the incubated whole leaf, process the leaf using the 

same methods as for nursery leaf samples.

6. Transfer the isolation plates immediately to a State or Federally approved 

processing laboratory via overnight courier OR maintain the agar plates at 

20 °C in the dark for at least 3 days.

7. At regular time intervals, using an inverted or dissecting microscope under 

low magnification, check the plates for colonies with typical 

morphological characters of P. ramorum (e.g., coralloid hyphae, semipapillate sporangia, and large chlamydospores).

8. Contact laboratory personnel if P. ramorum is suspected from any samples.

Bottle of Bait (BoB) Materials List

Bait leaves in plastic bag kept in a cooler until needed

Hand-held paper hole punch (heart-shaped preferred)

Ice chest cooler (with a small amount of ice or other refrigerant if 

temperatures are warm outside)—isolate ice from sample with newspaper
Water Sampling and Processing Protocol

In Vitro Water Sampling with Regulated Plant Material Leaf Baits—Bottle of Bait (BoB) Technique

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-5

Multiple 100-ml plastic measuring cups or beakers and large-capacity 

(100 ml) syringe per water sample site—disposable paper cups are also 

suitable for collecting water

One-liter bottles per sample site

Figure 10-1-1 Bottle of Bait (BoB) Materials and Leaf Pieces Plated into PARPH Media1

1 Photos courtesy of Steve Oak, USDA–Forest Service, Southern Region FHP and Dr. Craig Webb, USDA–APHIS–PPQ.
Water Sampling and Processing Protocol

In Situ Water Sampling with Regulated Plant Material Leaf Baits

10-1-6 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

In Situ Water Sampling with Regulated Plant Material Leaf Baits

Bait Selection

1. Collect healthy leaves from a population of native or naturalized 

Rhododendron spp., Camellia spp., Viburnum spp., or other hosts that have 

susceptible responses to P. ramorum. Source-regulated plant material must 

not have been sprayed with fungicide within the last 3 months. Avoid using 

newly acquired regulated plants for this reason. Bait-source plants should 

be sufficiently large, robust, and numerous enough to supply leaves during 

the entire duration of the survey.

2. Use healthy leaves that have been on the plant for at least 1 year and are as 

free as possible from insect and mechanical damage. Do not use newly 

formed, succulent leaves. Present-year leaf growth may be used after full 

leaf expansion and a period of hardening in the summer.

3. If bait leaves are smaller in size than 8 cm x 3 cm (3.2” x 1.2”) at the 

widest point, use 8 leaves at each sampling location (1 in each mesh bait 

bag). If leaves are larger than this dimension, 4 leaves per site can be used.

4. Bait leaves may be stored wrapped in dry paper towels and sealed in selfsealing plastic bags for refrigerated storage for no more than 14 days 

before use. Dry towels help prevent leaves from breaking down during 

storage.

5. Place four to eight leaves with the petioles (the stalk-like tissue that 

attaches the leaf to a stem) attached into each container (depending on leaf 

size as discussed above) at each sampling site. Insert a uniquely 

numbered plastic tag into each bait bag for identification. On a 

datasheet, record the dates (when bait was established and when bait was 

collected), water source (location), nursery information (i.e., nursery 

license number), tag number, water temperatures (initial temperature when 

bait was established and final temperature when bait was collected), and 

GPS coordinates. If forwarding these samples to a confirmatory laboratory, 

this information will be necessary to complete a PPQ Form 391 for 

specimens for determination.
Water Sampling and Processing Protocol

In Situ Water Sampling with Regulated Plant Material Leaf Baits

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-7

Baiting Techniques

Bait Bags

Bait bags (approximately 12” x 12”) should be constructed of a durable, coarse 

nylon mesh material (e.g., non-wire window screening) and fastened together 

on 3 sides to allow sufficient overlapping material to seal bag edges (refer to 

Figure 10-1-2). Single-use bait bags can be fashioned from muslin. Bait bags 

must have a separate pocket for each leaf, even if several small leaves are 

being used. This maximizes the surface area in the water. Exact configuration 

is not crucial and any bag type that can be closed and securely fastened 

(drawstring, flapped, rolled, etc.) is sufficient. Once leaves and numbered 

plastic tag are placed into the bag, secure the bag so it cannot float out and 

away.

1. Firmly secure each bag by tethering it to a stake driven into the ground or 

by suspending it from a rope that spans the width of the watercourse or 

pond. Bags should float near or just below the water’s surface for 7 to 14 

days depending on water temperature (8 °C to 22 °C). Suspend baiting 

when water temperatures exceed 22 °C. If necessary, water can be tested 

using the BoB method. Place bait bags in an area where water flows more 

slowly and pools. Locate the bags such that the leaves remain submerged 

even if water levels fluctuate. Do not leave the bait in the water for more 

than 7 days if the water temperatures are at the higher range (22 °C) as the 

leaf tissue will degrade and baiting efficacy will be sharply reduced.

Figure 10-1-2 Example of a Bait Bag Construction

Table 10-1-1 Temperature Versus Days for Floating Baits in Water

If the temperature is: Then:

22 °C Leave in water for 7 days

> 8 °C, but < 22 °C Leave in water for 7 to 14 days
Water Sampling and Processing Protocol

In Situ Water Sampling with Regulated Plant Material Leaf Baits

10-1-8 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

2. When possible, choose shaded locations.

3. Record the water temperature.

4. When placing bait bags in retention ponds, give priority to inflow and 

outflow points, preferably in shaded areas. Deploy a minimum of two bait 

bags per pond.

Bait Stations

An alternative to bait bags is a bait station, which consists of an enclosure 

constructed from a PVC frame with plastic cable ties attaching half-inch 

plastic fencing material (refer to Figure 10-1-3 and Figure 10-1-4 for photo 

and construction details). Leaves are attached with binder clips secured to the 

bottom of the enclosure. The numbered plastic tag is placed inside the 

enclosure before sealing.

Figure 10-1-3 Pattern of Mesh Screen to Use for Constructing a Bait Station1

1 Photo courtesy of Dr. Steve Jeffers, Clemson University.
Water Sampling and Processing Protocol

In Situ Water Sampling with Regulated Plant Material Leaf Baits

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-9

1. Attach the station to a stake driven into the ground or by suspending it from 

a rope that spans the width of the watercourse or pond. Deploy the station 

for at least 7 days (refer to Table 10-1-1).

2. When possible, choose shaded locations.

3. When placing bait stations in retention ponds, give priority to inflow and 

outflow points located in shaded areas. Deploy a minimum of two bait 

stations per pond.

Bait Station Construction Details—Frame. 

1. Frame is made using 1/2-inch diameter PVC pipe and four elbow joints.

2. Cut four lengths of PVC—two 11 1/2-inch and two 10 1/2-inch lengths.

3. Glue pipes and joints into a rectangular shape using PVC primer and 

cement.

Bait Station Construction Details—Mesh. The mesh used is a plastic, 1/2-inch 

fence material (with 3/8-inch openings) available at any national home 

improvement chain store.

1. Dimensions are given both in inches and are also based on number of 

squares (refer to Figure 10-1-3).

2. Secure mesh to the frame using plastic cable ties.

3. Leave one side of mesh half secured until leaves are inserted.

4. Tie four small binder clips into mesh using plastic-covered twist ties; these 

clips hold leaves in place by the petioles. This maintains separation 

between bait leaves, allowing for maximum water flow exposure for each 

bait tissue (refer to Figure 10-1-4).

Figure 10-1-4 Completed Bait Station Construction1

1 Photo courtesy of Dr. Steve Jeffers, Clemson University.
Water Sampling and Processing Protocol

In Situ Water Sampling with Regulated Plant Material Leaf Baits

10-1-10 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

Bait Retrieval

1. After 7 to 14 days, depending on water temperature (8 °C to 22 °C), 

remove bait leaves (and the numbered tag) from each bag or station and 

rinse using water from the stream or pond, thereby reducing the foreign 

matter (organic and soil particles) on the bait leaves.

2. Wrap leaves in a 1-gallon, self-sealing plastic bag. Be certain to place the 

numbered plastic tag from each bait bag into the plastic bag of the 

corresponding leaf bait tissues. Double bag the samples to prevent 

contamination or desiccation in the event a bag ruptures.

3. Place all sample bags in an insulated cooler with cold packs for transport to 

the laboratory. Do not place bait samples directly on the ice or cold pack; 

cardboard can be used to separate the ice from the bait samples.

4. Do not leave bags exposed to direct sunlight or in hot conditions for an 

extended length of time before placing into a cooler. Clear plastic bags can 

solarize and ruin leaf bait samples if left in direct sunlight. Leaf bait 

samples not placed in a cooler first can also be negatively affected if left in 

the hot interior of a vehicle.

5. Record the date of bait retrieval as well as the water temperature at time of 

retrieval.

6. Following each use, clean bait bags with either 95% ethanol or a 10% 

household bleach solution. To decontaminate, bait bags should be either 

sprayed until runoff occurs or soaked. To ensure adequate time to 

decontaminate and dry, wait at least 4 hours prior to reuse. Upon 

completing the cleaning process, thoroughly rinse bags with chlorinated 

tap or sterile water. Check for signs of deterioration and bag failure and 

replace bags accordingly.

Sample Transport, Storage, Shipping, and Processing

1. Keep samples in a cooler with a cold pack or in a refrigerator until shipped. 

Do not permit the samples to freeze or dry out. Before shipping, contact 

laboratory personnel to coordinate sample reception and processing. Pack 

leaf bait samples just prior to shipping via overnight courier. Do not

prepare and store packed samples if they will not be shipped immediately.

2. Laboratory personnel should process bait samples using the same methods 

as for nursery leaf samples.
Water Sampling and Processing Protocol

Water Sampling for Filtration

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-11

Water Sampling for Filtration

Sample Collection1

1. Record and mark the location of the sample site. If possible, record GPS 

coordinates for each sample. When feasible, record the water temperature.

2. Affix identification labels (time tape or masking tape) both to the screw 

cap and the outside of each water collection bottle using a waterproof 

marking method. Sufficiently code labels to correspond with datasheet 

entries for each nursery and water body and include date collected, water 

source (location), a unique sample number, and nursery (e.g., nursery name 

or license number).

3. When sampling drainage ditches or areas of moving water within nurseries, 

rinse bottles downstream with the water about to be sampled before water 

is collected.

4. Maintain collected water samples in a cooler.

5. Maintain a record of the water sample information, assign a unique sample 

number to each bottle.

6. Between sample collection trips, wash each 100-ml measuring cup and 1-

liter bottle with warm, soapy water. Thoroughly and completely rinse each 

item. For best results, use an automatic dishwasher with a heated drying 

cycle. Care should be taken when using soap and/or bleach as the residue 

could potentially affect the retrieval process.

7. Prepare enough cup/bottle sets for water collection for a number of sample 

sites per nursery. Use only clean collection materials at each site and water 

source.

Sample Processing

Most water samples can be vacuum filtered through polycarbonate membrane 

filters with 3-μm pores. Turbid or muddy water samples will need to be filtered 

through membrane filters with 5-μm pores. Typically, a minimum of 10 plates 

and 10 filters will be required if 100 ml of water per filter are used. Surveyors 

should always take extra plates and filters to ensure they have enough in the 

field.

1 Water sampling for filtration is an optional water sample protocol.

NOTICE

Samples should be processed within 8 hours of collection to optimize detection of 

Phytophthora spores. Samples will begin to degrade or decline as a detection tool 

after storing for 12 hours or more.
Water Sampling and Processing Protocol

Water Sampling for Filtration

10-1-12 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

1. Place a filter funnel into a filter flask with a capacity of at least 1 liter and 

connect the flask to a vacuum source using plastic tubing; use a second 

filter flask as a trap between the flask with the funnel and the vacuum 

source (i.e., electric vacuum pump or a hand vacuum pump) (refer to 

Figure 10-1-6).

2. Wet the filter holder with distilled water and place a polycarbonate 

membrane with the shiny side up or a polyvinylidene fluoride membrane 

with smoothest side up. Ensure the paper between the filters has been 

removed, the filter is aligned over the perforated area of the funnel, and the 

filter is not wrinkled. Assemble the filter funnel and clamp it in place.

3. Thoroughly mix the water sample by inverting the plastic bottle and/or 

swirling. Pour 100 ml of sample into the funnel. If the water is highly 

turbid, 100 ml may not be completely filtered by a single filter and smaller 

volumes should be used per filter to complete the sample. Conversely, if 

the water is extremely clear, additional water (up to 200 ml) may be 

processed by a single filter. More than 1 filter will be necessary to 

completely filter the 1-liter sample.

4. Initially, turn on the vacuum source at a low setting to filter water 

subsamples and adjust as necessary. Turn off the air just prior to complete 

filtration of each subsample to avoid building up excessive vacuum 

pressure, which could damage Phytophthora spores.

5. Rinse the inner surface of the funnel with distilled water to wash down any 

spores onto the filter that may be on the funnel wall. Briefly apply the 

vacuum to remove excess water.

6. Gently remove sizable organic debris or soil particles trapped on the 

surface of the filter if it will prevent complete contact of the filter with the 

surface of PARPH-clarified V8 agar contained in petri plates. Using 

forceps, gently lift the filter from the filter funnel and invert it so the 

collection side contacts the media surface. Smooth the filter with the 

forceps to remove air bubbles that may have formed between the filter and 

the agar media surface (refer to Figure 10-1-6).

7. Repeat the above steps until the entire 1-liter water sample has been 

filtered. A minimum of 10 agar plates per collection bottle should be 

produced resulting from filtration if 100 ml of sample is used per filter. 

Always have additional filters and plates prepared as the number required 

per sample can vary.

8. Rinse the filter funnel assembly and forceps under hot, running tap water 

after each sample to avoid cross-contamination between samples. Do not

disinfest the funnel with a bleach solution or alcohol as any chemical 

residue may affect spore viability.

9. Place plates upside down in a plastic box or zip-to-close bag to prevent 

desiccation; incubate plates at 16 to 22 °C (64 to 71 °F) in the dark for 10 to 
Water Sampling and Processing Protocol

Water Sampling for Filtration

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-13

14 days. Because P. ramorum can grow slowly in culture, additional 

incubation up to 28 days is optional. A designated incubator works best, 

but a closed cabinet in an air-conditioned room can also be used.

10. Using a dissecting or inverted microscope, examine plates frequently 

(starting 2 days after baits have been plated) for colonies resembling P. 

ramorum—i.e., those with typical coralloid hyphae, large golden 

chlamydospores, and packets of semi-papillate sporangia on the surface 

(refer to Figure 10-1-6); mark these with a permanent, waterproof marking 

method.

11. P. ramorum hyphae are often visible 2 to 5 days after baits have been 

plated. However, because P. ramorum can grow slowly in culture, it may 

not be recognizable until several weeks after baits are plated. Patience, 

persistence, and good observational skills are often the keys to finding P. 

ramorum on the isolation plates. P. ramorum grows slower than most other 

species of Phytophthora commonly found in water, leaving the filters on 

the agar plates for a 3-day incubation period is critical for recovery of this 

species.

12. After the incubation period, remove the filters with sterile forceps and 

gently rinse the surface of the agar medium with running tap water to wash 

off small particles and bacterial colonies that may interfere with 

microscopic observation. Filters and rinse water should be treated and 

properly discarded to eliminate any risk associated with P. ramorum.

13. Under low magnification and using an inverted or dissecting microscope, 

check the plates at regular intervals for colonies with typical morphological 

characteristics of P. ramorum (e.g., coralloid hyphae, semi-papillate 

sporangia, and large chlamydospores).

14. Subculture isolates to fresh PARPH-V8 and then to PAR-V8.

15. If morphology or PCR testing does not indicate a positive P. ramorum by 

14 days of culture, the sample is considered negative for regulatory 

purposes. The culture can be disposed of in the manner described in 

Treatments and Disinfectants on page 12-1-1.

Additional Guidance on Culture and Identification

1. Be sure to culture mostly green tissue at the margin of the lesion, and not

too much brown, dead tissue.

2. If the initial culture media is PARP-H, take care to adjust the concentration 

of Hymexazole. Adding too much Hymexazole to the media will inhibit P. 

ramorum growth.

3. If a surface sterilization step is added, do not oversterilize leaf pieces, 5% 

bleach solution for 30 seconds is generally sufficient for this purpose.

4. The culture conditions should be at a constant temperature between 16 and 

22 °C (64 to 71 °F) with inverted plates placed in the dark.
Water Sampling and Processing Protocol

Water Sampling for Filtration

10-1-14 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

5. Cultures should be checked up to 10 to 14 days for hyphal growth and 

morphological identification. Real-time PCR can be attempted on hyphal 

growth suspected to be P. ramorum.

6. Additional steps and procedures can be carried out in attempts to isolate P. 

ramorum, but this is not required for regulatory purposes. However, if P. 

ramorum is morphologically identified using these extra procedures, the 

sample is a presumptive positive (refer to below).

Diagnostic Determination for Cultures

1. If the bait or mycelia growing from the bait is tested positive by PCR in a 

NPPLAP-accredited laboratory and it qualifies as a Potentially Actionable 

Suspect Sample (PASS), the DNA can be forwarded to the PPQ–S&T Plant 

Pathogen Confirmatory Diagnostics Laboratory. If it is a non-PASS, the 

sample is considered positive for regulatory purposes.

2. If the culture is morphologically identified as P. ramorum, regardless of 

PCR results, the sample is determined as presumptive positive. If the 

original sample is a PASS, the culture must be confirmed at the PPQ–S&T 

Plant Pathogen Confirmatory Diagnostics Laboratory according to the 

instructions in this program manual.

3. If the key morphological features of P. ramorum are clearly observed, 

attempt to transfer to a fresh plate for singular isolation. If no morphology 

is observed, PCR can be attempted.

Disposing of Cultures After Diagnosis

1. Please contact the P. ramorum program via email at P.ramorum@usda.gov

for instructions on the final disposition of non-PASS cultures. These 

cultures may be found useful for regulatory research or methods 

development needs. If the required PPQ permit is in place, arrangements 

can be made to forward cultures to the research and method development 

scientific community. If not needed for this purpose, the cultures can be 

disposed of as instructed in Treatments and Disinfectants on page 12-1-1.

2. If morphology or PCR testing does not indicate a positive P. ramorum by 

14 days of culture, the sample is considered negative for regulatory 

purposes. The culture can be disposed of as instructed in Treatments and 

Disinfectants on page 12-1-1.

NOTICE

If the initial P. ramorum identification is not made at the laboratory, transfer the 

plates immediately to a State or Federally approved processing laboratory via overnight courier. Do not permit the samples to freeze or dry out at any time. Before shipping, contact laboratory personnel to coordinate sample reception and processing. If 

you do not have contact with an appropriate laboratory, notify the S&T representative (refer to Contact Information for the Phythopthora ramorum Program on page 

A-1-1) to arrange for culture forwarding and identification.
Water Sampling and Processing Protocol

Water Sampling for Filtration

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-15

3. Place plates upside down in a plastic box or zip-to-close bag to prevent 

desiccation. Incubate plates at 15 to 20 °C (59 to 68 °F) in the dark for up 

to 28 days. A designated incubator works best, but a closed cabinet in an 

air-conditioned room can also be used.

Figure 10-1-5 Characteristic Structure of P. ramorum1

1 Photos courtesy of Dr. Steve Jeffers, Clemson University.
Water Sampling and Processing Protocol

Water Sampling for Filtration

10-1-16 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

Water Filtration Methods Materials List

1-liter bottles for sample collection (Nalgene® preferred)

47-mm diameter polycarbonate membrane filters with 3-μm pores (e.g., 

Sterlitech SKU No. PCT3047100)

47-mm diameter polyvinylidene fluoride (Durapore®) membrane filters 

with 5-μm pores (e.g., Fisher Scientific #SVLP04700)

100-ml plastic measuring cup or beaker per sample site; or 5-ounce paper 

cups

Bent-tip forceps

Clamp-type filter funnel (Nalgene® preferred)

Disposable pipette or syringe for shallow water collection

Electric vacuum pump or hand-operated vacuum pump

Ice chest cooler (with a small amount of ice or other refrigerant if 

temperatures are warm outside)

Inverted or dissecting microscope

Plastic tubing

Sterile PARPH-clarified V8 selective medium (refer to PARPH-V8 

Selective Medium: for Phytophthora Species on page 10-1-17) in 

disposable petri plates; 10 plates per collection bottle; media plates can be 

stored in sealed plastic sleeves or bags in a refrigerator for 2 months 

before use

Squeeze bottle containing distilled water

Figure 10-1-6 Vacuum Apparatus for Water Filtration and Filter Plated onto PARPH-V8 Plate1

1 Photos courtesy of Dr. Steve Jeffers, Clemson University.
Water Sampling and Processing Protocol

Growing Media Formulae

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-17

Thermometer (water-resistant type preferred)

Two 1- or 2-liter filtering flasks (plastic or glass)

Growing Media Formulae

PARPH-V8 Selective Medium: for Phytophthora Species2

Directions

1. Add ingredients for basal medium to a 2-liter flask; thoroughly mix on a 

magnetic stirrer with a large stir bar in the flask.

2. Autoclave for 20 minutes at 121 °C and 15 psi; turn waterbath on to ~50 

°C.

3. Add each amendment to a sterile water blank [5 ml distilled water in a 16-

mm test tube]; vortex to mix.

4. Cool medium in waterbath.

5. Slowly stir medium with a magnetic stirrer in laminar flow hood.

6. Vortex each amendment thoroughly and add to mixing basal medium.

2 Adapted from Jeffers and Martin, 1986; Ferguson and Jeffers, 1999.

Table 10-1-2 PARPH-V8 Selective Medium: for Phytophthora Species

Ingredient:

Amount per:

1.0 liter 0.5 liter

Basal medium

Clarified V8 Concentrate1 50 ml 25 ml

Distilled water 950 ml 475 ml

Difco Bacto agar 15 g 7.5 g

Amendments

Delvocid [50% pimaricin] 10 mg 5 mg

Ampicillin sodium 250 mg 125 mg

Rifamycin-SV [sodium salt] 10 mg 5 mg

Terraclor [75% PCNB] 66.7 mg 33.4 mg

Hymexazol 50 mg 25 mg

1 Clarified V8 Concentrate is made from buffered V8 Juice (1.0 g CaC)3/100 ml V8 Juice) clarified in one of three ways:

Centrifugation at 4000 RPM for 20 minutes followed by filtration using 2 layers of Whatman No. 1 filter paper under vacuum

Centrifugation at 7000 RPM for 10 minutes; then filtration is not necessary

Vacuum filtration alone through a 1- to 2-cm deep layer of Celite

Clarified V8 should be frozen at -20 °C in 50-ml aliquots (e.g., in disposable 50-ml centrifuge tubes). Pentachloronitrobenzene (PCNB) and hymexazol are optional and can be omitted (e.g., to make PAR, PARP, and PARH):

PCNB is useful to inhibit soilborne fungi on soil dilution plates

Hymexazol inhibits most species of Pythium while allowing most species of Phytophthora to grow, although they may grow 

more slowly
Water Sampling and Processing Protocol

Growing Media Formulae

10-1-18 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

7. Use one additional sterile water blank to sequentially rinse all amendment 

tubes and then add rinse water to the medium; continue mixing medium.

8. Pour plates relatively thin (i.e., about 15 ml/plate = 60 plates/liter); pour 

molten medium so it does not quite cover the entire plate; thus, plates will 

need to be swirled gently to evenly distribute medium before it hardens.

9. Cool plates at room temperature.

10. Store plates inverted in self-sealing plastic bags in the dark in a refrigerator.

11. Use plates within 30 days.

PAR-V8 Selective Medium: for Phytophthora Species3

Directions

1. Add ingredients for basal medium to a 2-liter flask; thoroughly mix on a 

magnetic stirrer with a large stir bar in the flask.

2. Autoclave for 20 minutes at 121 °C and 15 psi; turn waterbath on to ~50 

°C.

3. Add each amendment to a sterile water blank [5 ml distilled water in a 16-

mm test tube]; vortex to mix.

4. Cool medium in waterbath.

5. Slowly stir medium with a magnetic stirrer in laminar flow hood.

6. Vortex each amendment thoroughly and add to mixing basal medium.

3 Adapted from Ferguson and Jeffers, 1999.

Table 10-1-3 PAR-V8 Selective Medium: for Phytophthora Species

Ingredient:

Amount per:

1.0 liter: 0.5 liter:

Basal medium

Clarified V8 concentrate1 50 ml 25 ml

Distilled water 950 ml 475 ml

Difco Bacto agar 15 g 7.5 g

Amendments

Delvocid [50% pimaricin] 10 mg 5 mg

Ampicillin sodium 250 mg 125 mg

Rifamycin-SV [sodium salt] 10 mg 5 mg

1 Clarified V8 Concentrate is made from buffered V8 Juice (1.0 g CaC)3/100 ml V8 Juice) clarified in one of three ways:

Centrifugation at 4000 RPM for 20 minutes followed by filtration using 2 layers of Whatman No. 1 filter paper under vacuum

Centrifugation at 7000 RPM for 10 minutes; then filtration is not necessary

Vacuum filtration alone through a 1- to 2-cm deep layer of Celite

Clarified V8 should be frozen at -20 °C in 50-ml aliquots (e.g., in disposable 50-ml centrifuge tubes).
Water Sampling and Processing Protocol

References

03/2024-07 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 10-1-19

7. Use one additional sterile water blank to sequentially rinse all amendment 

tubes and then add rinse water to the medium; continue mixing medium.

8. Pour plates relatively thin (i.e., about 15 ml/plate = 60 plates/liter); pour 

molten medium so it does not quite cover the entire plate; thus, plates will 

need to be swirled gently to evenly distribute medium before it hardens.

9. Cool plates at room temperature.

10. Store plates inverted in self-sealing plastic bags in the dark in a refrigerator.

11. Use plates within 30 days.

References

Anon. 2006. Detection of Phytophthora ramorum in water courses. Dept. for 

Environment, Food and Rural Affairs (Defra). March 2006 report.

Erwin, D.C. and Ribeiro, O.K. 1996. Phytophthora Disease Worldwide. 

American Phytopathological Society Press, St. Paul, MN.

Ferguson, A.J. and Jeffers, S.N. 1999. Detecting multiple species of 

Phytophthora in container mixes from ornamental crop nurseries. Plant Dis. 

83:1129-36.

Ghimire, S.R., Richardson, P.A., Moorman, G.W., Lea-Cox, J.D., Ross, D.S., 

and Hong, C.X. 2009. An in-situ baiting bioassay for detecting Phytophthora

species in irrigation runoff containment basins. Plant Pathol. Doi. 10.1111/

j.1365-3059.2008.02016.x.

Hwang, J., Oak, S.W., and Jeffers, S.N. 2007. Detecting Phytophthora 

ramorum and other species of Phytophthora in streams in natural ecosystems 

using baiting and filtration methods. Pages 55-58 in: Proceedings of the 

Sudden Oak Death Third Science Symposium. 5-9 Mar. 2007. Santa Rose, CA.

Jeffers, S.N. and Martin, S.B. 1986. Comparison of Two Media Selective for 

Phytophthora and Pythium Species. Plant Dis. 70:1038-43.

Oak, S.W., Hwang, J., Jeffers, S.N., and Tkacz, B.M. 2007. 2007 Pilot survey 

for Phytophthora ramorum in forest streams in the USA. Pages 59-63 in: 

Proceedings of the Sudden Oak Death Third Science Symposium. 5-9 March 

2007. Santa Rose, CA.
Water Sampling and Processing Protocol

References

10-1-20 Phytophthora ramorum Manual/Water Sampling and Processing Protocol 03/2024-07

Oak, S.W., Hwang, J., Jeffers, S.N. 2012. Comparison of in situ and in vitro 

baiting assays for Phytophthora ramorum survey of waterways in the 

southeastern USA. Pages 51-52 in: Proceedings of the Sudden Oak Death Fifth 

Science Symposium. 19-22 June, 2012. Petaluma, CA.

Sutton, W., Hansen, E.M., Reeser, P.W., and Kanaskie, A. 2009. Stream 

monitoring for in detection of Phytophthora ramorum Oregon tanoak forests. 

Plant Dis. 93:1182-1.
3/2024-07 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 11-1-1

Chapter

11

Soil and Container Mix 

Sampling and Processing 

Protocol

Contents

Introduction 11-1-1

Sampling Soils and Container Mixes 11-1-2

Sample Collection 11-1-2

Sample Transport, Storage, Shipping, and Processing 11-1-4

Procedure for Baiting Soil and Container Mix Samples 11-1-4

Additional Guidance on Culture and Identification 11-1-8

Diagnostic Determination for Cultures 11-1-8

Disposing of Cultures After Diagnosis 11-1-8

Second Baiting of Soil and/or Container Mix Samples 11-1-9

Materials and Supplies for the Soil and Container Mix Protocol 11-1-9

Growing Media Formulae 11-1-10

PARPH-V8 Selective Medium: for Phytophthora Species 11-1-10

PAR-V8 Selective Medium: for Phytophthora Species 11-1-11

References 11-1-12

Introduction

Soil or container mix sampling is a preferred method of collecting samples 

from symptomatic plants grown in pots or grown on, or adjacent to, soil. Soil 

sampling is not required during a detection survey. Soil or container mix 

infested with Phytophthora ramorum appears to the unaided eye exactly the 

same as noninfested soil or container mix. Therefore, all soil and container mix 

samples must be handled carefully.
Soil and Container Mix Sampling and Processing Protocol

Sampling Soils and Container Mixes

11-1-2 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 3/2024-07

Sampling Soils and Container Mixes

Sample Collection

1. Record the physical location (address) of the nursery site along with GPS 

reference coordinates.

2. Prepare or secure from the facility manager/owner a diagram of the nursery 

or sampling area, which includes row or block numbers and plant species/

cultivars. If possible, collect reference GPS coordinates for each block of 

plants.

3. Each 1-L (1-qt) composite sample should consist of a minimum of 15 

subsamples collected from soil or container mix within the targeted area. 

Collect subsamples in zigzag transects according to the pattern in the 

diagram below. Collect subsamples from underneath positive plants. If 

plants are on benches or gravel, take a composite from each pot comprising 

one liter of soil thoroughly mixed. 

4. Referencing Table 11-1-1 collect composite samples from both soil and 

container mix for each block of plants. An exception to this would be if all 

plants (including container mix and pots) were destroyed or the plants are 

not on a soil substrate (e.g., concrete or asphalt). Each sample should 

contain approximately 1 L/1 qt (volume) of soil or container mix and be 

placed in a 4-L (1-gal) size zip-to-close plastic bag. The number of 

composite samples collected will depend on the size of the block of plants 

being sampled.

NOTICE

If a GPS unit is not available, many cellular phones have GPS capabilities. Also 

Google Earth can be used to obtain coordinates using an address.

Table 11-1-1 Number of Composite Samples to Collect Per Block

Survey area size: Composite samples of 

soil:

Composite samples of 

container mix: (m2

) (ac)

<1,000 <0.25 5 5

1,000 to 2,000 0.25 to 0.5 10 10

2,000 to 4,000 0.5 to 1 20 20

>4,000 >1 30 30

NOTICE

If the soil surface is covered with gravel with a large amount of plant debris on top, 

collect as much debris as practical. If the gravel has little plant debris on top, collect 

subsamples from the soil beneath the gravel. If water-permeable weed block (landscape cloth) is present, either covered with gravel or under gravel, make small slits 

in the cloth to allow for sample collection.
Soil and Container Mix Sampling and Processing Protocol

Sampling Soils and Container Mixes

3/2024-07 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 11-1-3

A. Soil

a. With a trowel, collect a representative composite sample 

(approximately 1 L (1 qt)) from the surveyed area (e.g., a block of 

plants, a nursery bed, a shade house, etc.) to a depth of 

approximately 5 to 10 cm (2” to 4”).

b. Collect samples from around and under pots containing plants 

suspected of being infested or infected with P. ramorum or from 

areas where diseased plants were previously located. This may 

require scraping soil from on or under nursery cloth or anything else 

on which pots are or were located.

c. Place each composite sample into an individual plastic bag; if the 

soil is wet or saturated from rain or excessive irrigation, double bag 

the slurry to avoid leaks.

B. Container mix

a. Collect a representative composite sample from each block of plants 

using a wide-bore soil tube (highly recommended); one core from 

each or every other pot in the block of plants is sufficient depending 

on the number of pots present.

5. For each composite sample, break up clods and root masses, then 

thoroughly mix the sample in the bag; this can be done in the field or 

laboratory.

6. If it appears dry, moisten the sample with distilled water, as desiccation 

will severely affect the ability to recover P. ramorum from a soil sample.

7. Disinfest sampling tools and soles of shoes (e.g., 10% bleach, quaternary 

ammonium at the labeled rate, or full-strength disinfectant spray (with 

ETOH)) between samples to prevent potential dissemination of the 

pathogen. Next, thoroughly rinse tools with distilled water to remove all 

disinfestation product residues or allow tools to dry.

NOTICE

If soil is loosely packed, a plastic spoon can be used to collect the sample. The 

spoon can then be sealed in the corresponding sample’s plastic bag for easy disposal in the laboratory. This method is not recommended for sampling container mix 

because substrate at the bottom of pots cannot be sampled.

NOTICE

Rinsing off disinfestation residues and allowing the tool to dry prevents possible sterilization of your next sample. Distilled water can be purchased at most grocery and 

big-box stores.
Soil and Container Mix Sampling and Processing Protocol

Procedure for Baiting Soil and Container Mix Samples

11-1-4 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 3/2024-07

Sample Transport, Storage, Shipping, and Processing

1. Place all samples in a cooled, insulated ice chest for transport to the 

laboratory or until samples are shipped. If samples cannot be shipped 

immediately, hold them in a refrigerator or cold room (4 to 10 °C/39 to 50 

°F) for a maximum of 2 days.

2. Before shipping, double bag samples using gallon-size zip-to-close, selfsealing plastic bags, ensuring each bag is clearly labeled using a 

permanent, waterproof pen. To further protect samples, each sample can 

then be placed inside a 2-L (2-qt) disposable storage container that is also 

clearly labeled using a permanent, waterproof marking method. 

Completely fill out PPQ Form 391 and place inside a separate zip-to-close 

bag and place in the same box as the samples.

3. Contact laboratory personnel before shipping to advise them that a sample 

will be arriving. Ship samples via overnight courier. Avoid shipping on 

Fridays and prior to holidays to avoid shipping delays that may 

compromise the quality of the sample.

4. Send samples to a qualified State laboratory or a USDA–APHIS–PPQ 

regional laboratory at:

Bliss M. Coffin

USDA–APHIS–PPQ

Dep’t of Plant Pathology

Kansas State University

4024 Throckmorton Plant Sciences

Manhattan, KS 66506-5502

(785) 532-1349

bliss.betzen@usda.gov

Procedure for Baiting Soil and Container Mix Samples

1. Once1 samples arrive in the laboratory, protect them from exposure to 

direct sunlight near windows. If samples cannot be processed immediately, 

store them in a refrigerator or cold room (4 to 10 °C/39 to 50 °F). Do not

permit the samples to freeze or dry out.

2. If the soil or container mix sample is desiccated when it arrives in the 

laboratory, moisten with distilled water, reseal the bag, and allow the 

moistened sample to sit for 40 to 72 hours before processing.

3. Thoroughly mix the 1-L (1-qt) sample within the bag, breaking up any 

clods. Divide the sample equally into two 500-ml (17-oz.) aliquots, placing 

one sample into a new sealed zip-to-close bag or container then placing it 

in cold storage (4 to 10 °C/39 to 50 °F) for a minimum of 30 days.

1 These procedures are provided by S.N. Jeffers, Clemson University, 2010.
Soil and Container Mix Sampling and Processing Protocol

Procedure for Baiting Soil and Container Mix Samples

3/2024-07 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 11-1-5

4. Prepare and label 3 containers (e.g., small 0.5-L (1-pt.) plastic containers, 

self-sealing plastic 1-liter (1-qt) bags, etc.) for each composite sample to be 

baited.

5. Thoroughly mix the remaining sample, then place an aliquot approximately 

1 to 2 cm (0.5” to 1”) deep into each of the 3 containers; soil deeper than 

this may inhibit zoospores from swimming to the surface.

6. Add distilled water to a depth of 2.5 cm (1”) above the soil surface; stir the 

mixture and allow it to settle.

7. Bait leaves should be free of blemishes, damage, disease, and pesticides. 

Rhododendron catawbiense and/or Camellia japonica are recommended 

bait types (if both bait types are available it is recommended to use both). 

Using a standard hole-punch or scissors, prepare enough leaf pieces (~10 

per container) to bait all containers (refer to Figure 11-1-1). Leaf pieces cut 

with scissors should be approximately 5 mm (<0.25”) across.

8. Using sterile forceps, add 8 to 10 leaf pieces of each bait type (or 15 to 20 

leaf pieces if only using one bait type) to each container. Baits should float 

on the water surface. If some of the baits sink, do not remove them, 

instead, add additional baits. Cover containers to avoid evaporation and 

desiccation.

9. Store containers at 18 to 22 °C/64 to 72 °F for 3 days (an incubator 

maintained at 20 °C/68 °F or a closed cabinet works best).

10. For each container, remove 6 baits of each regulated plant type (or 12 baits 

of 1 regulated plant type) with sterile forceps and blot dry on a clean paper 

towel. Dispose of paper towels after each sample.

11. Place the 6 bait pieces of each regulated plant type from 1 container on a 

separate plate of PARPH-V82

 medium (e.g., 1 plate with 6 Rhododendron

leaf pieces and 1 plate with 6 Camellia leaf pieces, or alternatively, 2 plates 

of same regulated plant tissue baits) so they are embedded completely in 

the agar. Leaf pieces placed on the agar surface will dry out and curl up. 

There should be 6 plates and 36 baits from each composite sample: 3 

containers x 2 plates/container x 6 baits/plate (refer to Figure 11-1-1).

NOTICE

Organic debris may continue floating.

NOTICE

Use different-shaped leaf pieces to differentiate between bait types if two types of 

bait are being used.

2 Refer to Growing Media Formulae on page 11-1-10.
Soil and Container Mix Sampling and Processing Protocol

Procedure for Baiting Soil and Container Mix Samples

11-1-6 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 3/2024-07

12. Place plates upside down in a plastic box or zip-to-close bag to prevent 

desiccation; incubate plates at 18 to 22 °C (64 to 71°F) in the dark for 10 to 

14 days. Because P. ramorum can grow slowly in cultures, additional 

incubation up to 28 days is optional. A designated incubator works best, 

but a closed cabinet in an air-conditioned room can also be used.

13. Using a dissecting or inverted microscope, examine plates frequently 

(starting 2 days after baits have been plated) for colonies that resemble P. 

ramorum—i.e., those with typical coralloid hyphae, large golden 

chlamydospores, and packets of semi-papillate sporangia on the surface 

(refer to Figure 11-1-2); mark these with a permanent, waterproof marking 

method.

14. P. ramorum hyphae are often visible 2 to 5 days after baits have been 

plated. However, patience, persistence, and good observational skills are 

often the keys to finding P. ramorum on the isolation plates; P. ramorum

NOTICE

Baits can be vertically inserted into the medium, which prevents shadowing during 

microscopic examination. To prevent media tearing, a scalpel can be used to make 

small incisions where baits are going to be placed. Vertical placement may require 

slightly thicker agar or smaller bait pieces, however, the places no longer have to be 

read on both sides.

Figure 11-1-1 Baiting Soil Samples and Plating Baits1

1 Photos courtesy of Grace O’Keefe, PPQ and Jennifer Falacy, Washington State Department of Agriculture. A) Preparing soil 

samples for baiting; B) baiting soil samples using Rhododendron and Camellia leaves; C) plate with baits inserted vertically; 

and D) plate with baits inserted horizontally.
Soil and Container Mix Sampling and Processing Protocol

Procedure for Baiting Soil and Container Mix Samples

3/2024-07 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 11-1-7

may be recovered from only 1 of the 36 bait pieces and may not be 

recognizable until several weeks after baits are plated.

15. Subculture isolates to fresh PARPH-V8 and then to PAR-V83.

16. If morphology or PCR testing does not indicate a positive P. ramorum by 

14 days of culture, the sample is considered negative for regulatory 

purposes. The culture can be disposed of in the manner described in 

Treatments and Disinfectants on page 12-1-1.

NOTICE

P. ramorum grows and sporulates better in the absence of hymexazol (i.e., on PARV8). It is best to subculture from suspect colonies early, before these colonies 

become overgrown by fast-growing organisms.

3 Refer to Growing Media Formulae on page 11-1-10.

Figure 11-1-2 Characteristic Structure of P. ramorum1

1 Photos courtesy of Dr. Steve Jeffers, Clemson University.
Soil and Container Mix Sampling and Processing Protocol

Procedure for Baiting Soil and Container Mix Samples

11-1-8 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 3/2024-07

Additional Guidance on Culture and Identification

1. Be sure to culture mostly green tissue at the margin of the lesion, and not

too much brown, dead tissue.

2. If the initial culture media is PARP-H, take care to adjust the concentration 

of Hymexazole. Adding too much Hymexazole to the media will inhibit P. 

ramorum growth.

3. If a surface sterilization step is added, do not oversterilize leaf pieces, 5% 

bleach solution for 30 seconds is generally sufficient for this purpose.

4. The culture conditions should be at a constant temperature between 16 and 

22 °C (64 to 71 °F) with inverted plates placed in the dark.

5. Cultures should be checked up to 10 to 14 days for hyphal growth and 

morphological identification. Real-time PCR can be attempted on hyphal 

growth suspected to be P. ramorum.

6. Additional steps and procedures can be carried out in attempts to isolate P. 

ramorum, but this is not required for regulatory purposes. However, if P. 

ramorum is morphologically identified using these extra procedures, the 

sample is a presumptive positive (refer to below).

Diagnostic Determination for Cultures

1. If the bait or mycelia growing from the bait is tested positive by PCR in a 

NPPLAP-accredited laboratory and it qualifies as a Potentially Actionable 

Suspect Sample (PASS), the DNA can be forwarded to the PPQ–S&T Plant 

Pathogen Confirmatory Diagnostics Laboratory. If it is a non-PASS, the 

sample is considered positive for regulatory purposes.

2. If the culture is morphologically identified as P. ramorum, regardless of 

PCR results, the sample is determined as presumptive positive. If the 

original sample is a PASS, the culture must be confirmed at the PPQ–S&T 

Plant Pathogen Confirmatory Diagnostics Laboratory according to the 

instructions in this program manual.

3. If the key morphological features of P. ramorum are clearly observed, 

attempt to transfer to a fresh plate for singular isolation. If no morphology 

is observed, PCR can be attempted.

Disposing of Cultures After Diagnosis

1. Please contact the P. ramorum program via email at P.ramorum@usda.gov

for instructions on the final disposition of non-PASS cultures. These 

cultures may be found useful for regulatory research or methods 

development needs. If the required PPQ permit is in place, arrangements 

can be made to forward cultures to the research and method development 

scientific community. If not needed for this purpose, the cultures can be 

disposed of as instructed in Treatments and Disinfectants on page 12-1-1.
Soil and Container Mix Sampling and Processing Protocol

Materials and Supplies for the Soil and Container Mix Protocol

3/2024-07 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 11-1-9

2. If morphology or PCR testing does not indicate a positive P. ramorum by 

14 days of culture, the sample is considered negative for regulatory 

purposes. The culture can be disposed of as instructed in Treatments and 

Disinfectants on page 12-1-1.

3. Remove composite samples from cold storage and hold at room 

temperature (22 to 24 °C/72 to 75 °F) for 3 days to acclimate before baiting 

begins.

Second Baiting of Soil and/or Container Mix Samples

1. Remove composite samples from cold storage and hold at room 

temperature (22 to 24 °C/72 to 75 °F) for 3 days to acclimate before baiting 

begins.

2. Bait samples again as described above in Procedure for Baiting Soil and 

Container Mix Samples on page 11-1-4.

3. After samples have been baited a second time, destroy or sterilize any 

remaining soil and/or container mix using an appropriate method (e.g., 

autoclaving).

Materials and Supplies for the Soil and Container Mix Protocol

4-L (1-gal) self-sealing plastic bags (at least 4 mil in thickness); avoid 

the bags with the “zipper” mechanism

70% alcohol and flame for sterilizing laboratory utensils

Baits—use Rhododendron and/or Camellia leaves that have been on 

the plant for at least 1 year; leaf pieces should be 5 x 5 mm squares 

(<0.25”) or 5 mm (<0.25”) in diameter disks

Disinfesting solution (10% bleach, quarternary ammonium at the 

labeled rate, or full-strength disinfectant spray (with ETOH))

Disposable gloves

Distilled water

Forceps and scalpel

Insulated ice chest (with ice in bags or blue ice if external 

temperatures are above 21 °C/70 °F)

PAR-V8 selective medium (as needed for subcultures)

PARPH-V8 selective medium; two plates per baited container

Paper towels

Permanent, waterproof marking method

Plastic or glass containers with lids; square, wide-bottom containers 

work best (e.g., 0.5 L (1 pt) freezer boxes) (refer to Figure 11-1-1)
Soil and Container Mix Sampling and Processing Protocol

Growing Media Formulae

11-1-10 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 3/2024-07

Single-hole punches, scissors, or razor blades

Trowel or other soil-sampling tool

Wide-bore soil tube (2.5 cm/1” or larger)

Growing Media Formulae

PARPH-V8 Selective Medium: for Phytophthora Species4

Directions

1. Add ingredients for basal medium to a 2-L flask; thoroughly mix on a 

magnetic stirrer with a large stir bar in the flask.

2. Autoclave for 20 minutes at 121 °C and 15 psi; turn waterbath on to ~50 

°C.

3. Add each amendment to a sterile water blank [5 ml distilled water in a 16-

mm test tube]; vortex to mix.

4. Cool medium in waterbath.

4 Adapted from Jeffers and Martin, 1986; Ferguson and Jeffers, 1999.

Table 11-1-2 PARPH-V8 Selective Medium: for Phytophthora Species

Ingredient:

Amount per:

1.0 liter 0.5 liter

Basal medium

Clarified V8 concentrate1 50 ml 25 ml

Distilled water 950 ml 475 ml

Difco Bacto agar 15 g 7.5 g

Amendments

Delvocid [50% pimaricin] 10 mg 5 mg

Ampicillin sodium 250 mg 125 mg

Rifamycin-SV [sodium salt] 10 mg 5 mg

Terraclor [75% PCNB] 66.7 mg 33.4 mg

Hymexazol 50 mg 25 mg

1 Clarified V8 concentrate is made from buffered V8 Juice (1.0 g CaC)3/100 ml V8 Juice) clarified in one of three ways:

Centrifugation at 4000 RPM for 20 minutes followed by filtration using 2 layers of Whatman No. 1 filter paper under 

vacuum

Centrifugation at 7000 RPM for 10 minutes; then filtration is not necessary

Vacuum filtration alone through a 1- to 2-cm deep layer of Celite

Clarified V8 should be frozen at -20 °C in 50-ml aliquots (e.g., in disposable 50-ml centrifuge tubes). PCNB and hymexazol 

are optional and can be omitted (e.g., to make PAR, PARP, and PARH):

PCNB is useful to inhibit soilborne fungi on soil dilution plates

Hymexazol inhibits most species of Pythium while allowing most species of Phytophthora to grow, although they may 

grow more slowly
Soil and Container Mix Sampling and Processing Protocol

Growing Media Formulae

3/2024-07 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 11-1-11

5. Slowly stir medium with a magnetic stirrer in laminar flow hood.

6. Vortex each amendment thoroughly and add to mixing basal medium.

7. Use one additional sterile water blank to sequentially rinse all amendment 

tubes and then add rinse water to the medium; continue mixing medium.

8. Pour plates relatively thin (i.e., about 15 ml/plate = 60 plates/liter); pour 

molten medium so it does not quite cover the entire plate; therefore, plates 

will need to be swirled gently to evenly distribute medium before it 

hardens.

9. Cool plates at room temperature.

10. Store plates inverted in self-sealing plastic bags in the dark in a refrigerator.

11. Use plates within 30 days.

PAR-V8 Selective Medium: for Phytophthora Species5

Directions

1. Add ingredients for basal medium to a 2-L flask; thoroughly mix on a 

magnetic stirrer with a large stir bar in the flask.

2. Autoclave for 20 minutes at 121 °C and 15 psi; turn waterbath on to ~50 

°C.

3. Add each amendment to a sterile water blank [5 ml distilled water in a 16-

mm test tube]; vortex to mix.

5 Adapted from Ferguson and Jeffers, 1999.

Table 11-1-3 PAR-V8 Selective Medium: for Phytophthora Species

Ingredient:

Amount per:

1.0 liter: 0.5 liter:

Basal medium

Clarified V8 concentrate1 50 ml 25 ml

Distilled water 950 ml 475 ml

Difco Bacto agar 15 g 7.5 g

Amendments

Delvocid [50% pimaricin] 10 mg 5 mg

Ampicillin sodium 250 mg 125 mg

Rifamycin-SV [sodium salt] 10 mg 5 mg

1 Clarified V8 concentrate is made from buffered V8 Juice (1.0 g CaC)3/100 ml V8 Juice) clarified in one of three ways:

Centrifugation at 4000 RPM for 20 minutes followed by filtration using 2 layers of Whatman No. 1 filter paper under 

vacuum

Centrifugation at 7000 RPM for 10 minutes; then filtration is not necessary

Vacuum filtration alone through a 1- to 2-cm deep layer of Celite

Clarified V8 should be frozen at -20 °C in 50-ml aliquots (e.g., in disposable 50-ml centrifuge tubes).
Soil and Container Mix Sampling and Processing Protocol

References

11-1-12 Phytophthora ramorum Manual/Soil and Container Mix Sampling and Processing Protocol 3/2024-07

4. Cool medium in waterbath.

5. Slowly stir medium with a magnetic stirrer in laminar flow hood.

6. Vortex each amendment thoroughly and add to mixing basal medium.

7. Use one additional sterile water blank to sequentially rinse all amendment 

tubes and then add rinse water to the medium; continue mixing medium.

8. Pour plates relatively thin (i.e., about 15 ml/plate = 60 plates/liter); pour 

molten medium so it does not quite cover the entire plate; therefore, plates 

will need to be swirled gently to evenly distribute medium before it 

hardens.

9. Cool plates at room temperature.

10. Store plates inverted in self-sealing plastic bags in the dark in a refrigerator.

11. Use plates within 30 days.

References

Ferguson, A.J. and Jeffers, S.N. 1999. Detecting multiple species of 

Phytophthora in container mixes from ornamental crop nurseries. Plant Dis. 

83:1129-1136.

Jeffers, S.N. and Martin, S.B. 1986. Comparison of two media selective for 

Phytophthora and Pythium species. Plant Dis. 70:1038-1043.
3/2024-07 Phytophthora ramorum Manual/Treatment and Disinfection Options 12-1-1

Chapter

12

Treatments and 

Disinfectants

Contents

Treatment Options 12-1-1

Infected Plants and Associated Potting Mix and Containers 12-1-1

Incineration (Burning to Ash) 12-1-2

Deep Burial 12-1-2

Steam Sterilization 12-1-2

Disinfestation Options 12-1-3

Disinfesting Nonporous Surfaces 12-1-3

Disinfesting Water 12-1-4

Disinfesting Soil and Container Mix 12-1-4

Treatment Options

The following techniques have been determined to effectively control P. 

ramorum in nurseries with sample site confirmed positives. Any of these 

options may be required to mitigate P. ramorum infestations, providing the 

options are appropriately labeled for use in the State. A systems approach to 

treatment should be considered rather than relying on just one method. Always

follow label directions when applying any chemical treatment.

Infected Plants and Associated Potting Mix and Containers

SAFETY

Do not place regulated plant material, including leaf litter, in compost piles or 

remove regulated plant material from the nursery site as trash or with debris 

removal. Regulated plant material should be collected and incinerated, double 

bagged and deep buried in a site approved by USDA–APHIS or delegated regulatory authority, or steam sterilized. Properly disinfest all tools or materials used for 

cleanup or material movement. To prevent unintentional inoculum movement, all

personnel involved with cleanup should follow appropriate procedures. To prevent 

contamination of other areas and methods of conveyance, follow appropriate procedures concerning all material movement on the nursery site.
Treatments and Disinfectants

Infected Plants and Associated Potting Mix and Containers

12-1-2 Phytophthora ramorum Manual/Treatment and Disinfection Options 3/2024-07

Incineration (Burning to Ash)

Infected plants, associated growth media, associated containers (e.g., pots and 

trays1), all leaf debris in and around the area where plants were stored may be 

incinerated at a facility or other location (e.g., on site). The facility or other 

location must be approved by USDA and permitted within State and 

municipal statutes or regulations. Off-nursery (off-site) movement must be 

properly safeguarded and every effort taken to prevent plant debris or soil from 

being dislodged from the plants prior to incineration. Incineration may be 

through open burning or in an incinerator.

Deep Burial

Infected plants, associated growth media, associated containers (e.g., pots and 

trays1), all leaf debris in and around the area where plants were stored may be 

double bagged using plastic bags of 2-mil thickness or greater and buried to a 

depth of no less than 2 m. The material must be buried at a USDA-approved 

site, on site, or a municipal landfill, where it is expected to remain undisturbed. 

Take every effort to prevent plant debris or soil from being dislodged from the 

plants.

Steam Sterilization

Infected plants, associated growth media, associated containers (e.g., pots and 

trays1), all leaf debris in and around the area where plants were stored may be 

treated with steam sterilization or dry heat commonly heated to internal 

temperatures of 176 °F (80 °C) for 60 minutes (steam) or 120 minutes (dry 

heat), or as otherwise detailed in the USDA Treatment Manual Schedule T521 

for plant pathogenic fungi and bacteria.

1 Associated containers (e.g., pots and trays) may be treated for reuse. Guidelines for safeguarding 

should be determined and approved to prevent movement or potential contamination of P. ramorum

to noninfested areas of the nursery. All potting mix should be removed to the extent possible prior

to treating. Dispose of the potting mix using one of the above methods. Containers may be sterilized 

by treatment with steam at or above 122 °F (50 °C) for 30 minutes or more (most containers will 

withstand 140 °F to 160 °F) or other approved disinfestant methods for the correct contact time.
Treatments and Disinfectants

Disinfestation Options

3/2024-07 Phytophthora ramorum Manual/Treatment and Disinfection Options 12-1-3

Disinfestation Options

Disinfesting Nonporous 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, 

regular disinfection using an appropriate disinfectant for controlling P. 

ramorum is recommended prior to removing from the quarantine area. 

Table 12-1-1 examines the effects of different classes of disinfectants on 

microbial populations. This table is for explanation and information only. Few 

disinfectants are specifically labeled for Phytophthora species and are shown 

in the table in bold type.

Strictly adhere to all labels for the disinfectants listed in Table 12-1-1 for 

maximum efficacy and environmental and worker safety. The contact time for 

the products must be followed to ensure efficacy. If the surface dries before the 

contact time is reached, re-wet the surface until the contact time is achieved.

Table 12-1-1 Summary of Disinfestant Activities1

Disinfectant: Trade name(s): Comments: Contact time:

Alcohols 

(ethyl and 

isopropyl)

(60% to 85%)

 Clorox Disinfecting Spray Evaporates quickly meaning adequate contact time 

may not be achieved, high concentrations of organic 

matter diminish effectiveness; flammable

10 to 15 minutes

Phenolics

(0.4% to 5%)

 Pheno-cen  Phenol penetrates latex gloves; eye/skin irritant; 

remains active upon contact with organic soil; may 

leave residue

10 to 15 minutes

Quaternary 

ammonium

(0.1% to 

1.5%)

 Consan Triple 

Action 20

 Physan 20

 Green-Shield 

20

 Effective for nonporous surfaces sanitation (floors, 

walls, benches, pots); low odor and low irritation; use 

according to labels

10 to 15 minutes

Chlorine

(100 to 1,000 

ppm)

 10% Clorox

 10% bleach

 Inactivated by organic matter; prepare fresh solutions 

of hypochorite (Clorox) every 8 hours or more frequently if exposed to sunlight; corrosive; irritating to 

eyes and skin; exposure to sunlight further reduces 

hypocholrite efficacy; keep solution in opaque container

10 to 15 minutes

1 Modified table from Columbia Research Environmental Health and Safety (EH&S).
Treatments and Disinfectants

Disinfestation Options

12-1-4 Phytophthora ramorum Manual/Treatment and Disinfection Options 3/2024-07

Disinfesting 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 2 ppm or 2 mg/liter or greater has been correlated with the 

control of Phytophthora spp. in recirculated irrigation systems. For irrigation 

purposes, recirculated, nonmunicipal 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.

Other systems that can be approved for treating water can include one or a 

combination of the following: bromine; chlorine; sodium hypochlorite; 

calcium hypochlorite; chlorine dioxide; ozone; activated peroxygen; ultraviolet 

radiation; copper ionization; heat treatment/pasteurization; and filtration.

Disinfesting Soil2 and Container Mix

Container Mix

Container mix must be heated so the temperature in the center of the load 

reaches at least 60 °C (140 °F) for 30 minutes. Heat treatment must be 

conducted in the presence of an inspector.

Fumigation may be the most efficacious and economical option to disinfest 

container mix.

Soil/Container Mix in Pots

Soil must be heated so the temperature in the center of the load reaches at 

least 60 °C (140 °F) for 30 minutes. Heat treatment must be conducted in the 

presence of an inspector.

2 Soil treatment through the use of solarization is being developed. If you are considering solarization 

treatment for infested nursery beds, consult the regulatory agencies in your State for further 

information and guidance.
Treatments and Disinfectants

Disinfestation Options

3/2024-07 Phytophthora ramorum Manual/Treatment and Disinfection Options 12-1-5

Soil in Situ or in Nursery Beds3

Nursery beds can be treated with steam following specific protocols to ensure 

soil heating to 50 °C (122 °F) for 30 minutes. Check soil water content prior

to steaming to avoid uneven heating. Monitor treatment with thermocouples 

placed in locations throughout the bed (15 cm deep4

) with sensors placed in 

locations most likely to reach the correct temperature the slowest. Place a 

steam sock on the nursery bed and cover with a tarp sealed on the edges with 

sand snakes. Concrete blocks can be used to support the tarp off of the bed 

surface. Treatment timing begins when the last sensor reaches the target 

temperature of 50 °C (122 °F). Steam treatment must be conducted in the 

presence of an inspector.

3 Schweigkofler, W., Kosta, K., Huffman, V., Sharma, S., Suslow, K., and Ghosh, S. 2014. Steaming 

inactivates Phytophthora ramorum causal agent of sudden oak death and ramorum blight, from 

infested nursery soils in California. Plant Health Progress doi: 10.1094/PHP-RS-13-0111.

4 Dart, N., Chastagner, G., Rugarber, E., and Riley, K. 2007. Recovery frequency of Phytophthora 

ramorum and other Phytophthora spp. in soil profiled of ornamental retail nurseries. Plant Dis. 

91:1419-1422.

Figure 12-1-1 Steam Sock in Place on Soil Surface
Treatments and Disinfectants

Disinfestation Options

12-1-6 Phytophthora ramorum Manual/Treatment and Disinfection Options 3/2024-07

Figure 12-1-2 Concrete Blocks Used to Support Tarp from Surface to Allow Steam Distribution from 

Steam Sock
Treatments and Disinfectants

Disinfestation Options

3/2024-07 Phytophthora ramorum Manual/Treatment and Disinfection Options 12-1-7

Fumigation may be the most efficacious and economical option to disinfest 

soil. 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 Phytophthora cinnamoni in 

citrus groves. However, many of the compounds currently in use have been 

implicated in human and environmental risks. Solarization is currently being 

evaluated as an option for soil treatment.

Figure 12-1-3 Tarp with Sand Snakes in Place to Secure Edges

NOTICE

Soil treatment through solarization is being developed. If you are considering solarization treatment of infested nursery beds, consult the regulatory agencies in your 

State for further information and guidance.
Treatments and Disinfectants

Disinfestation Options

12-1-8 Phytophthora ramorum Manual/Treatment and Disinfection Options 3/2024-07

Summary of Labeled Soil Fumigants

All fumigants are restricted use and must be applied according to labels by a 

licensed applicator. Any pesticide used in any manner not listed on the label is 

unlawful.

Physical Soil Treatment

Mitigating infested soil can be achieved by installing permanent impermeable, 

nonporous barriers consisting of cement, concrete, or asphalt. These barriers 

must be constructed such that no native soil within the block is visible. Grade 

the barriers such that no surface water can be observed. When soil treatment is 

absolutely impossible due to human health concerns, certain soil hydrologic 

conditions, or due to city, county, or State regulations, nurseries under an 

agreement may use avoidance and exclusionary methods on a case-by-case 

basis.

Equipment and Personnel (Inspectors and Employees)

Rubber boots or other very smooth and crevice-free waterproof boots are 

strongly encouraged as other footwear is too porous. When feasible, limit 

access to infested areas and hold areas to officials and necessary employees. 

Everyone entering and leaving the nursery site must scrape off loose pieces of 

soil into the infested site and disinfest footwear. Those working with or in 

contact with suspected infested material (including plants) must wear gloves 

and remove them or rub/wash them with an approved disinfectant between 

samples and between blocks. Currently, there are no products labeled for use 

on porous materials for Phytophthora control.

Table 12-1-2 Labeled Soil Fumigants

Fumigant: Trade names: Comments:

Chloropicrin  Chlor-O-Pic

 Metapicrin

 Timberfume

 Tri-Clor

 Often used in combination with methyl bromide due to its ability to be 

detected in small quantities

Dazomet  Basamid  Methyl isothyocyanate (MITC) breaks down into cyanide gas

 Requires careful soil preparation and incorporation into soil

 Water-activated granular formulation

 All applications must be made in accordance with labeling

Metamsodium Busan 1020

 Busan 1180

 Busan 1236 Metam

 Vapam

 Metam-sodium can be applied through irrigation

 Tarping can increase efficacy

 All applications must be made in accordance with labeling

Methyl bromide Tri-Con

 Terr-O-Gas

 Preplant Soil Fumigant

 Pic-Brom

 Colorless and odorless

 Use is restricted due to ozone depletion potential

 Usually combined in various concentrations with Chloropicrin (tear 

gas)
Treatments and Disinfectants

Disinfestation Options

3/2024-07 Phytophthora ramorum Manual/Treatment and Disinfection Options 12-1-9

Place a disinfectant foot bath near the exit to the destruction-radii and 

quarantine-radii. Because of the higher potential of footwear coming in contact 

with infested soil or plant debris, all personnel entering and exiting the infested 

site must use the foot bath. The foot bath must be filled with fresh disinfectant 

at least daily (or more frequently if contaminated with soil or organic debris) 

and must be in accordance with label directions.

Vehicle tires (or other vehicle parts in contact with soil or plants (e.g., truck 

beds) 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, 

do not allow vehicles in the infested site at all. Any product labeled for use on 

nonporous surfaces may be used on tires or vehicle undercarriages.

Do not visit other nursery sites in potentially contaminated work clothing and 

footwear. If it is necessary for a visitor to enter the nursery, the nursery should 

ensure every precaution is taken to prevent movement, by the visitor, of 

infected and/or contaminated plants, soil, or debris.

Dispose of wood surfaces suspected of P. ramorum contamination (refer to 

Infected Plants and Associated Potting Mix and Containers on page 12-1-1). 

There is no effective way to test or treat wood surfaces for contamination.
3/2024-07 Phytophthora ramorum Manual/Compliance Agreement A-1-1

Appendix

A

Resources

Contents

Contact Information for the Phythopthora ramorum Program A-1-1

U.S. State and Territory Plant Health Directors A-1-2

APHIS List of Phytophthora ramorum-Regulated Plants A-1-2

Phytophthora ramorum Nursery Questionnaire A-1-2

Contact Information for the Phythopthora ramorum Program

1. William D. Wesela, National Policy Manager

USDA–APHIS–PPQ

4700 River Rd.

Riverdale, MD 20737

(301) 851-2229

FAX (301) 734-8584

william.d.wesela@usda.gov

2. Betsy Randall-Schadel, National Operations Manager

USDA–APHIS–PPQ Field Operations

920 Main Campus Drive, Suite #500

Raleigh, NC 27606

(919) 855-7544

betsy.randall-schadel@usda.gov

3. Patrick J. Shiel, S&T Representative

920 Main Campus Drive, Suite #500

Raleigh, NC 27606

(919) 855-7416

FAX (919) 855-7480

patrick.j.shiel@usda.gov

4. Bliss M. Coffin, Plant Pathologist

USDA–APHIS–PPQ

Department of Plant Pathology

Kansas State University

4024 Throckmorton Plant Sciences Center

Manhattan, KS 66506-5502

(785) 532-1349

bliss.betzen@usda.gov
Resources

U.S. State and Territory Plant Health Directors

A-1-2 Phytophthora ramorum Manual/Compliance Agreement 3/2024-07

For questions regarding diagnostic work instructions, contact:

USDA–APHIS–Plant Pathogen Confirmatory Diagnostics Laboratory

BARC-East Building 580 

9901 Powder Mill Road

Laurel, MD 20708

(301) 313-9204

APHIS-PPQCPHSTBeltsvilleSampleDiagnostics@usda.gov

For the National Plant Pathogen Laboratory Accreditation Program 

(NPPLAP), contact Dr. Patrick Shiel at (919) 855-7416 or 

patrick.j.shiel@usda.gov.

Please send incoming samples to the following email address: 

PPQ.OpsKS.Manhattan.Lab@usda.gov.

U.S. State and Territory Plant Health Directors

For an up-to-date list of all U.S. State and Territory Plant Health Directors, 

please visit the U.S. State Plant Health Directors website.

APHIS List of Phytophthora ramorum-Regulated Plants

Consult the latest list of regulated plants prior to beginning any survey, 

inspection, or delimitation. A current list can be found at the APHIS–PPQ P. 

ramorum website.

Phytophthora ramorum Nursery Questionnaire

The P. ramorum Nursery Questionnaire (refer to Figure A-1-1) is a tool 

available to gather data about a positive nursery. The Questionnaire is most 

useful on the first visit to a nursery, but is also useful for verifying if nursery 

information has changed on subsequent visits. Information required for the 

Phytophthora ramorum program is marked with an asterisk. Additional 

information is helpful to understanding the nursery operation. Other data 

required by the P. ramorum program is listed in the core data document. To 

obtain the core data document and a fillable PDF of the P. ramorum Nursery 

Questionnaire, please contact the NOM.
Resources

Phytophthora ramorum Nursery Questionnaire

3/2024-07 Phytophthora ramorum Manual/Compliance Agreement A-1-3

Figure A-1-1 P. ramorum Nursery Questionnaire (page 1 of 5)

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Resources

Phytophthora ramorum Nursery Questionnaire

A-1-4 Phytophthora ramorum Manual/Compliance Agreement 3/2024-07

Figure A-1-2 P. ramorum Nursery Questionnaire (page 2 of 5)

WŚLJƚŽƉŚƚŚŽƌĂƌĂŵŽƌƵŵ^ƵƌǀĞLJYƵĞƐƚŝŽŶŶĂŝƌĞ

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Ύ^ŝƚĞƌĂŝŶĂŐĞ

ΎƌĂŝŶĂŐĞĞƐĐƌŝƉƚŝŽŶ͗
Resources

Phytophthora ramorum Nursery Questionnaire

3/2024-07 Phytophthora ramorum Manual/Compliance Agreement A-1-5

Figure A-1-2 P. ramorum Nursery Questionnaire (page 3 of 5)


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Resources

Phytophthora ramorum Nursery Questionnaire

A-1-6 Phytophthora ramorum Manual/Compliance Agreement 3/2024-07

Figure A-1-2 P. ramorum Nursery Questionnaire (page 4 of 5)

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zĞƐ EŽ

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Resources

Phytophthora ramorum Nursery Questionnaire

3/2024-07 Phytophthora ramorum Manual/Compliance Agreement A-1-7

Figure A-1-2 P. ramorum Nursery Questionnaire (page 5 of 5)

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zĞƐ EŽ


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3/2024-07 Phytophthora ramorum Manual/Glossary Glossary-1-1

Phytophthora 

ramorum 

Glossary

Introduction

Use this glossary to find the meaning of specialized words, abbreviations,

acronyms, and terms used in this manual. To locate where in the manual a

given definition, term, or abbreviation is mentioned, refer to the index.

Definitions, Terms, and Abbreviations

aliquot. volume of substrate from a composite sample that is placed into a 

container and assayed; usually 3 aliquots (approximately 50 to 150 ml or 2 to 5 

oz.) from each composite sample are baited

APHIS. Animal and Plant Health Inspection Service

associated plants. naturally infected plants from which P. ramorum has been 

cultured and/or detected using Polymerase Chain Reaction (PCR); for each of 

these plants, traditional Koch’s postulates have not yet been completed or 

documented and reviewed; a current list of associated plants is available at 

APHIS Lists of Proven Hosts of and Plants Associated with Phytophthora 

ramorum.

block. within a nursery, this is a contiguous block of regulated plant material; 

the block will be considered contiguous until there is a two-meter (6.5 foot) 

break of either no plants or non-regulated plant material

BMP. best management practices

biosecurity measures. actions taken to reduce or mitigate the potential 

introduction or spread of P. ramorum from one area or site to another area or 

site of a nursery; refer to Biosecurity Measure for Nurseries on page 9-1-2

CCP. critical control point

CNP. confirmed nursery protocol

CFR. Code of Federal Regulations
Glossary

Definitions, Terms, and Abbreviations

Glossary-1-2 Phytophthora ramorum Manual/Glossary 3/2024-07

composite sample. mixture of subsamples that are physically combined to 

form a single representative sample from a designated area

compost pile. mixture of decomposed organic matter of different origins, 

including plant debris, soil residues, as well as other substances found in the 

nursery; the finished product is used as a potting media component

confirmed positive. the presence of Phytophthora ramorum is confirmed by 

an APHIS-accredited diagnostic laboratory, only using current APHIS P. 

ramorum diagnostic work instructions. This may include plant, soil, and/or 

water samples. A final determination of a positive sample allows for Federal 

regulatory action. 

container mix. substrates without soil (also referred to as growing media) and/

or other materials placed in containers and used to grow plants, usually 

consisting of bark and peat but also may contain soil, slow-release fertilizer, 

sand, vermiculite, perlite, etc.

cull pile. an area in which discarded plant material is deposited; this area may 

also be known as a waste pile; may contain any combination of plants, plant 

material, water, container mix, compost, or soil. 

delimitation survey. survey to determine the extent of the infestation(s) 

within a nursery site; the quarantine period begins when all delimitation 

sampling and testing is completed

destruction radii. two meters from the edge (plant drip line or canopy) of the 

known positive plant(s); one meter around the periphery of positive water; 

destruction radii is surrounded by the quarantine radii/radius on page 

Glossary-1-5

destruction radius. area of plants to be destroyed; within a nursery, for 

purposes of the retail protocol, the destruction radius is defined as all P. 

ramorum-infected regulated plant material and all other regulated plant 

material within 2 meters of any infected regulated plant material

Emergency Action Notification (EAN). PPQ Form 523 or equivalent State 

document used to specify regulatory requirements and actions within a nursery

Federal confirmatory authority. authority to make a final determination on a 

regulatory sample1

final determination. last diagnostic result(s) necessary for a regulatory 

sample indicating whether P. ramorum is present or not; the final 

determination test(s) are conducted by analysts/laboratories with Federal 
Glossary

Definitions, Terms, and Abbreviations

3/2024-07 Phytophthora ramorum Manual/Glossary Glossary-1-3

confirmatory authority—if the final determination is positive, regulatory action 

may commence (refer to confirmed positive on page Glossary-1-2)

free of. without pests (or a specific pest) in numbers or quantities that can be 

detected by the application of phytosanitary procedures (ISPM Pub. No. 5, 

2007)

high-priority target plants. any regulated plant material that originated in the 

destruction radius at the infested (source) nursery; these plants are to be 

identified using the best-available information and to the lowest-available 

taxonomy (e.g., if high-priority target plants can be identified to cultivar, then 

trace forward activities may be conducted at the cultivar level); all domestic 

and international shipments of the high-risk regulated plant genera: Camellia; 

Rhododendron; Pieris; Viburnum; and Kalmia and regulated plant shipments 

of the infected plant species within the six months prior to the first positive 

detection of P. ramorum at the nursery as per the protocol

high-risk genera. Camellia, Rhododendron, Pieris, Viburnum, and Kalmia

HR. high risk

IES. Investigative and Enforcement Services

infected plants. officially confirmed plants verified as being infected with P. 

ramorum based on APHIS-approved diagnostics and following the PASS 

system (refer to Potentially Actionable Suspect Sample (PASS) on page 

Glossary-1-4)

lot. set of plants that can be identified or grouped by shipment, cultivar, or 

production unit

NDPN. National Plant Diagnostic Network

non-PASS. in nonregulated areas, once APHIS confirms a nursery is positive 

by during a given calendar year, all subsequent samples are considered “nonPASS,” meaning an NPPLAP-accredited laboratory outside of APHIS can 

1 

National Plant Protection Laboratory Accreditation Program (NPPLAP) accredited APHIS 

laboratories have authority to make a final determination on any P. ramorum regulatory sample

State NPPLAP accredited laboratories in the three regulated States (CA, OR, and WA) have 

authority to make the final determination on any regulatory sample in a previously positive 

nursery

State or National Plant Diagnostic Network (NPDN) NPPLAP accredited laboratories outside the 

regulated States have authority for subsequent samples to the initial APHIS confirmed positive 

for a given nursery during the EAN period; once the EAN period has lapsed and a new positive 

is detected, it must be forwarded to APHIS for final determination
Glossary

Definitions, Terms, and Abbreviations

Glossary-1-4 Phytophthora ramorum Manual/Glossary 3/2024-07

make the final determination using APHIS NPPLAP diagnostic work 

instructions; in the regulated areas, for a nursery that has been historically 

positive, all samples are “non-PASS” if tested by the NPPLAP-accredited State 

laboratory; if the diagnostic result is positive, the sample is confirmed positive 

and reported to the APHIS–PPQ operations manager within 24 hours; 

regulatory action can commence based on that positive sample

NPPLAP. National Plant Pathogen Laboratory Accreditation Program

nursery/facility. any location in which nursery stock is grown, propagated, 

stored, or sold; or any location from which nursery stock is distributed; 

locations that grow trees to be sold without roots (i.e., Christmas trees) and 

locations at which such trees are stored or distributed are also considered 

nurseries

nursery dealer. nurseries that are resellers—wholesale or retail—of nursery 

plants

nursery site. geographically separate location of a nursery/facility on page 

Glossary-1-4 that has a distinct physical address and appropriate biosecurity 

measures (refer to Biosecurity Measures for Nurseries on page 9-1-1) to 

prevent the movement of P. ramorum between locations

nursery site quarantine period. period of time during which regulated plants 

will not be moved within or out of the quarantine radii (refer to Schematic of 

Destruction and Quarantine Radii of Positive Plants on page 3-1-10); this 

period begins when the nursery delimitation survey is completed and last 

for 90 days during which proscribed activities must occur

nursery stock. any plants, including houseplants, propagative material grown 

in a nursery and tree seedlings for reforestation

parallel quarantine. quarantine or regulation imposed by a State or local plant 

regulatory authority essentially the same as a federally promulgated 

quarantine; these regulations can be more restrictive for intrastate movement 

and internal controls

PCR. polymerase chain reaction

Potentially Actionable Suspect Sample (PASS). presumptive positive P. 

ramorum sample that requires confirmatory testing by an official APHIS 

Laboratory due to the nature of the plant sampled and the necessity for Federal 

confirmation (for more information, refer to PASS System Policy

PPQ. Plant Protection and Quarantine
Glossary

Definitions, Terms, and Abbreviations

3/2024-07 Phytophthora ramorum Manual/Glossary Glossary-1-5

presumptive positive. preliminary diagnostic test result from a laboratory 

indicating P. ramorum is present; a final determination (the confirmatory test) 

is the next and final step

quarantine period. minimum of 90 days beginning when the nursery 

delimitation survey is completed and lasting until both plant parts and climatic 

conditions conducive to disease expression have occurred; plants, water, or 

other articles in quarantine hold radii remain on hold during this period; 

regulatory officials will inspect plants in the quarantine radii/radius on page 

Glossary-1-5 and all regulated plant on page Glossary-1-6 in the nursery a 

minimum of two additional times, once about halfway through the anticipated 

quarantine period and once near enough to the end to have test results coincide 

with the end of the quarantine period—all symptomatic plants during these 

surveys must be sampled and tested (the second inspection can be considered 

the quarantine release survey on page Glossary-1-5 at the discretion of the 

inspector)

quarantine radii/radius. for plant positives, quarantine radii is a two-meter 

radius around the destruction radii (refer to Schematic of Destruction and 

Quarantine Radii of Positive Plants on page 3-1-10) designed to determine if P. 

ramorum has spread beyond the destruction radius; use of this term 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. 5, 2007); also known as “Q-radii”

quarantine release survey. second of the two quarantine period inspections 

occurring near the end of the quarantine period; this survey includes inspection 

of plants in the quarantine radii/radius on page Glossary-1-5 and all regulated 

plant on page Glossary-1-6 within the nursery; sample and test any unhealthy 

plant tissue—for quarantine hold areas involving positive plants only, those 

areas can be released from quarantine if plant inspection, sampling, and testing 

reveal no further P. ramorum detection; for water and other regulated article 

positives, refer to Table 3-1-4 on page 3-1-12 for quarantine release 

instructions. Soil and surface or nonrecycled pond water may take longer than 

90 days to remediate; an avoidance/exclusion mitigation plan for these positive 

areas is written into Appendix D (refer to Example of PPQ Form 519, 

Compliance Agreement on page A-1-3) of the agreement prior to the end of the 

quarantine period

regulated establishment. nursery confirmed positive for Phytophthora 

ramorum, not located in a quarantined area, that ships regulated, restricted, or 

associated articles interstate
Glossary

Definitions, Terms, and Abbreviations

Glossary-1-6 Phytophthora ramorum Manual/Glossary 3/2024-07

regulated plant. listed on the official APHIS Lists of Proven Hosts of and 

Plants Associated with Phytophthora ramorum—naturally infected plants 

verified with completion, documentation, review, and acceptance of traditional 

Koch’s postulates

retail nursery dealer. nursery (e.g., big-box stored) that is a reseller of 

nursery plants to the end user

retail nursery/facility. nursery (e.g., big-box store) whose business is the sale 

of plants to the end user, typically a home owner

sample. referring to a single bag of P. ramorum leaves; the goal is to have at 

least two square inches of symptomatic (unhealthy) plant tissue per sample for 

the diagnostician to test, therefore, each sample will contain approximately 

five symptomatic leaves from medium leaf species and five to twelve leaves 

for small leaf species; for species with twig dieback as a symptom, include the 

terminal three inches of a symptomatic branch including one inch of live stem; 

regulatory action may commence on one sample confirmed positive. In P. 

ramorum diagnostics, sample may refer to a 25-microliter aliquot of DNA or a 

culture derived from one plant sample; for container mix and water baiting 

samples, refer to Soil and Container Mix Sampling and Processing Protocol on 

page 11-1-1

SITC. Smuggling, Interdiction, and Trade 

soil. loose surface material of the earth usually consisting of disintegrated rock 

with an admixture of organic material; the reference to soil in this manual is the 

surface or substrate under plant containers, the bare ground, and/or gravel; 

often with plant debris, peat, and bark fines are washed from plant containers

SOP. standard operating procedure

State Plant Health Director (SPHD). lead APHIS contact in each State 

responsible for overseeing all PPQ activities in that State

State Plant Regulatory Official (SPRO). primary person responsible for 

plant health programs in each State; a list of SPROs is available at the National 

Plant Board (NPB) website

subsamples. small amounts of soil or container mix that are combined to form 

a single, composite sample; collection subsamples increased the changes of 

finding P. ramorum if it is present

surface water. water that collects on the surface of the ground. 
Glossary

Definitions, Terms, and Abbreviations

3/2024-07 Phytophthora ramorum Manual/Glossary Glossary-1-7

suspect plant material. plants with visible symptoms of P. ramorum

infection; and/or regulated plant material that are a part of destruction or 

quarantine radii; and/or plants that have tested positive using PCR or culturing, 

but have not been confirmed positive for P. ramorum by APHIS

trace back (TB) plants. all plants of the same taxon (i.e., genus, species, 

hybrid, variety, or cultivar) of the infected plant regardless of size, location, or 

lot, back to the original propagation source (if it still exists)

trace back (TB) site. any location that shipped high-priority target plants on 

page Glossary-1-3 to a confirmed positive nursery, residence, or commercial 

landscapes

trace forward (TF) plants. list of high-priority target plants on page 

Glossary-1-3 that were shipped within six months prior to detection at the 

nursery; list includes the shipment date(s), quantities, and destination nursery

trace forward (TF) site. any location that received high-priority target plants

on page Glossary-1-3 from a confirmed infested source nursery; including 

residential or commercial landscapes

USDA. United States Department of Agriculture

wholesale nursery. any place of production at which nursery stock is grown, 

propagated, stored, sold, or distributed to other wholesale or retail nurseries or 

landscapers
9/2023-06 Phytophthora ramorum Manual Index-1-1

Phytophthora 

ramorum 

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

Index

A

aliquot

definition of Glossary-1-1

ampicillin sodium 10-1-17, 10-1-18, 11-1-10, 11-1-

11

Animal and Plant Health Inspection Service

(APHIS) Glossary-1-1

APHIS

refer to Animal and Plant Health Inspection

Service

APHIS list of regulated hosts and associated plants

with P. ramorum 2-1-2, 2-1-3, 8-1-4, 9-1-5

associated mitigations

examples of 3-1-23

associated plants

definition of Glossary-1-1

B

bait bags 10-1-7

construction 10-1-7

bait selection 10-1-2, 10-1-6

bait stations 10-1-8

construction of 10-1-9

baiting soil

soil and container mix sampling 11-1-4

baiting techniques 10-1-7

Basamid

trade name for dazomet 12-1-8

biosecurity measures for nurseries

communications 9-1-2

definition of Glossary-1-1

nursery personnel 9-1-3

equipment 9-1-4

hands 9-1-4

nursery personnel access 9-1-3

nursery personnel boots 9-1-4

sampling supplies and equipment checklist 9-

1-5

vehicles 9-1-2

avoidance 9-1-2

cleaning 9-1-3

block

definition of Glossary-1-1

of plants Glossary-1-1

BMPs

refer to business management practices 3-1-

23

BoB

refer to bottle of bait

boots 9-1-4

bottle of bait (BoB)

materials list 10-1-4

Busan 1020

trade name for metam-sodium 12-1-8

Busan 1180

trade name for metam-sodium 12-1-8

Busan 1236 Metam

trade name for metam-sodium 12-1-8

business management practices (BMPs)

examples of 3-1-23

C

Camellia 2-1-5, 3-1-15, 10-1-2, 10-1-6, 11-1-5, 11-1-9, Glossary-1-3

Camellia japonica 11-1-5

camera 8-1-4, 9-1-5

cankers on boles and branches 2-1-5

CCP

refer to critical control point 3-1-23

CFR

refer to Code of Federal Regulations

checklists

sampling supplies and equipment 8-1-4

chlorine 12-1-3

Chlor-O-Pic

trade name for chloropicrin 12-1-8

chloropicrin 12-1-8

clarified V8 concentrate 10-1-17, 10-1-18, 11-1-

10, 11-1-11

Clorox

trade name for chlorine 12-1-3

Code of Federal Regulations (CFR) Glossary-1-2

communications

for biosecurity measures 9-1-2

composite sample

definition of Glossary-1-2

compost pile

definition of Glossary-1-2

confirmed nursery protocol (CNP) procedures

disinfest the nursery 3-1-11

quarantine period 3-1-13

confirmed positive

definition of Glossary-1-2

Consan Triple Action 20

trade name for a quarternary ammonium 12-1-

3

container mix

definition of Glossary-1-2

disinfesting 12-1-4

containers

sampling 2-1-9

treating 12-1-1

critical control point (CCP) 3-1-11

assessment procedures

assessment day 3-1-21

information needed 3-1-21
Index 

Index-1-2 Phytophthora ramorum Manual 9/2023-06

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

planning of 3-1-21

post-assessment 3-1-22

examples of 3-1-23

cull piles

definition of Glossary-1-2

sampling 2-1-10

D

dazomet 12-1-8

deep burial 12-1-2

delimitation sampling for plant positives and sample

handling and submission protocol

sample handling and submission protocol 8-1-

1

delimitation survey

definition of Glossary-1-2

Delvocid 10-1-17, 10-1-18, 11-1-10, 11-1-11

destruction radii

definition of Glossary-1-2

destruction radius

definition of Glossary-1-2

Difco Bacto agar 10-1-17, 10-1-18, 11-1-10, 11-1-

11

distilled water 10-1-17, 10-1-18, 11-1-10, 11-1-11

dust abatement 12-1-4

E

Emergency Action Notification (EAN)

definition of Glossary-1-2

equipment

of nursery personnel 9-1-4

equipment cleaning 12-1-4

F

Federal confirmatory authority

definition of Glossary-1-2

filtration

water sampling 10-1-11

final determination

definition of Glossary-1-3

fire suppression 12-1-4

footwear 9-1-4

free from

definition of Glossary-1-3

fumigants

soil 12-1-8

G

glossary Glossary-1-1

Green-Shield 20

trade name for a quarternary ammonium 12-1-

3

growing media formulae 10-1-17, 11-1-10

H

hands 9-1-4

high-risk genera

definition of Glossary-1-3

Hymexazol 10-1-17, 11-1-10

I

in situ water sampling with host material leaf baits

10-1-6

bait retrieval 10-1-10

bait selection 10-1-6

baiting techniques 10-1-7

in vitro water sampling and processing

bait selection 10-1-2

BoB technique 10-1-2

sample collection 10-1-2

sample processing in receiving laboratory 10-

1-4

incineration 12-1-2

infected plants

definition of Glossary-1-3

interstate shipping propagation wholesale nurseries

3-1-2

intrastate shipping wholesale nurseries 3-1-2

irrigation 12-1-4

irrigation water 2-1-9

K

Kalmia Glossary-1-3

L

labeled soil fumigants 12-1-8

laboratory notification 2-1-4

leaf lesions 2-1-5

leaf spots 2-1-5

Loropetalum 2-1-5

lot

definition of Glossary-1-3

M

materials list

for bottle of bait (BoB) 10-1-4

for water filtration 10-1-16

metam-sodium 12-1-8

Metapricrin

trade name for chloropicrin 12-1-8

methyl bromide 12-1-8

N

National Operations Manager 3-1-18

National Plant Diagnostic Network (NPDN) 8-1-2

National Plant Protection Laboratory Accreditation

Program (NPPLAP) 2-1-3

non-PASS

definition of Glossary-1-4

nonporous surfaces

disinfesting 12-1-3

nonrecycled retention ponds 2-1-9

NPDN

refer to National Plant Diagnostic Network

NPPLAP

refer to National Plant Protection Laboratory

Accreditation Program

nurseries

regulatory protocol for 3-1-1

wholesale

intrastate shipping 3-1-2

nursery

block Glossary-1-4

dealer Glossary-1-4

definition of Glossary-1-4

personnel

biosecurity measures 9-1-3

site Glossary-1-4

quarantine period Glossary-1-4

stock Glossary-1-4
Index 

9/2023-06 Phytophthora ramorum Manual Index-1-3

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

nursery inspection and sampling 2-1-1, 2-1-6

guidelines to 2-1-1

preparation 2-1-4

preparation for 2-1-4

timing 2-1-3

O

Official Regulatory Protocol for Nurseries Containing Plants with Phytophthora ramorum

disclaimers for 3-1-3

introduction to 3-1-1

other articles

sampling of 2-1-10

P

P. ramorum

plant symptoms 2-1-4

parallel quarantine

definition of Glossary-1-4

PARPH-V8 selective medium 10-1-17, 11-1-10

PAR-V8 selective medium 10-1-18, 11-1-11

PCR

refer to polymerase chain reaction

Pheno-cen

tradename for a phenolic 12-1-3

phenolics 12-1-3

Physan 20

trade name for a quarternary ammounium 12-

1-3

Phytophthora ramorum Inspection and Sampling

Protocol for Nurseries 2-1-1

Pic-Brom

trade name for methyl bromide 12-1-8

Pieris Glossary-1-3

Plant Protection and Quarantine (PPQ) Glossary-1-

5

plant symptom type 2-1-5

cankers on boles and branches 2-1-5

leaf spots 2-1-5

lesions 2-1-5

twig dieback 2-1-5

plant symptoms of P. ramorum 2-1-4

plants

treating 12-1-1

polymerase chain reaction (PCR) Glossary-1-4

ponds

nonrecycled retention 2-1-9

pots

sampling 2-1-9

potting mix

treating 12-1-1

PPQ

refer to Plant Protection and Quarantine

Preplant Soil Fumigant

trade name for methyl bromide 12-1-8

presumptive positive

definition of Glossary-1-5

Q

quarantine period

definition of Glossary-1-5

quarantine radii/radius

definition of Glossary-1-5

quarantine release survey

definition of Glossary-1-5

quaternary ammonium 12-1-3

R

regulatory protocol for nurseries 3-1-1

retail nursery

definition of Glossary-1-6

Rhododendron 10-1-5, 10-1-6, 11-1-5, 11-1-9, Glossary-1-3

Rhododendron catawbiense 11-1-5

Rifamycin-SV 10-1-17, 10-1-18, 11-1-10, 11-1-11

S

sample

definition of Glossary-1-6

sample collection

for BoB 10-1-2

soil and container mix 11-1-2

sample handling information 8-1-1

sample numbers

minimum 2-1-8, 3-1-16

sample processing in receiving laboratory 10-1-4

sample submission information 8-1-2

sampling

by symptom type 2-1-5

guidelines 2-1-1

sampling supplies and equipment checklist 8-1-4, 9-1-5

SME

refer to subject matter expert

soil Glossary-1-6

disinfesting 12-1-4

in nursery beds 12-1-5

in situ 12-1-5

physical treatment 12-1-8

sampling 2-1-9

soil and container mix sampling

baiting soil 11-1-4

growing media formulae 10-1-17, 11-1-10

sample collection 11-1-2

transport, storage, shipping, and processing

11-1-4

SPHD

refer to State Plant Health Director

SPRO

refer to State Plant Regulatory Official

State Plant Health Director (SPHD)

definition of Glossary-1-6

State Plant Regulatory Official (SPRO)

definition of Glossary-1-7

steam sterilization 12-1-2

subject matter expert (SME) 3-1-20

subsamples

definition of Glossary-1-7

surface water 2-1-9

suspect plant material

definition of Glossary-1-7

symptom type

sampling of 2-1-5

symptoms of P. ramorum

plant 2-1-4

T

Terraclor 10-1-17, 11-1-10

Terr-O-Gas

trade name for methyl bromide 12-1-8
Index 

Index-1-4 Phytophthora ramorum Manual 9/2023-06

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

Timberfume

trade name for chloropicrin 12-1-8

trace back (TB)

plants Glossary-1-7

protocol 5-3-1

site Glossary-1-7

trace forward (TF)

plants Glossary-1-7

protocol 5-2-1

site Glossary-1-7

tracing protocols

trace back (TB) protocol 5-3-1

trace forward (TF) protocol 5-2-1

transport, storage, shipping, and processing

soil and container mix sampling 11-1-4

treatments and disinfectants 12-1-1

deep burial 12-1-2

disinfection options 12-1-3

disinfesting nonporous surfaces 12-1-3

disinfesting soil and container mix 12-1-4

disinfesting water 12-1-4

equipment and personnel 12-1-8

incineration 12-1-2

infected plants and associated potting mix and

containers 12-1-1

physical soil treatment 12-1-8

soil in nursery beds 12-1-5

soil in situ 12-1-5

steam sterilization 12-1-2

treatment options 12-1-1

Tri-Clor

trade name for chloropicrin 12-1-8

Tri-Con

trade name for methyl bromide 12-1-8

twig dieback 2-1-5

U

United States Department of Agriculture (USDA)

Glossary-1-7

USDA

refer to United States Department of Agriculture

V

Vapam

trade name for metam-sodium 12-1-8

vehicles 9-1-2

Viburnum 10-1-6, Glossary-1-3

W

water

disinfesting 12-1-4

irrigation 2-1-9

surface 2-1-9

water filtration materials list 10-1-16

water sampling and processing 2-1-9

water sampling for filtration 10-1-11

sample collection 10-1-11