Approved Method(s)
| Method | Detail | NAPIS Survey Method |
|---|---|---|
| Visual | Collect symptomatic host tissue. | 3031 - General Visual Observation |
In Potatoes: Slice tubers and look for ooze, vascular discoloration (can incubate at 30oC 86oF for 3-4 weeks).
Survey Recommendations
The first visible symptoms are wilting of the youngest leaves during the hottest part of the day, often on just one side of a leaflet or on a single branch. Plant stunting is another common symptom associated with bacterial wilt. The entire plant may wilt quickly, leading to general wilting and yellowing of foliage and eventually plant death.
A longitudinal slice of infected stems or stolons will reveal vascular browning, visible as long, narrow, dark brown streaks (See Fig. 3 in the CAPS datasheet). In succulent young plants of highly susceptible varieties, the stem can collapse, and gray-white bacterial ooze may be visible on stem surfaces.
In geranium:
Symptoms of Southern wilt can be subtle and easily overlooked. Symptoms usually begin with chlorosis and wilting of the lower leaves, then progress to an upward curling of leaf margins. Under favorable conditions, the disease develops rapidly on geraniums and wilting may move upwards from older to younger leaves.
Wilted leaves often develop wedge-shaped areas of chlorosis that become necrotic. The leaf margins may also become chlorotic, then necrotic, and the whole plant may desiccate and die. In late stages of disease, stems may collapse. Vascular discoloration is visible in stems (especially at the root crown) and roots; these can blacken and eventually become necrotic.
In the early stages of disease in potato and geranium, the infected plants may appear to recover at night.
For perennial alternative or weedy hosts, survey can occur throughout the year, but pathogen detection will be more accurate when plants are growing at temperatures higher than 15°C
Walk through susceptible hosts in the survey area looking for symptomatic plants and take random samples to cover the entire area. Consider surveying during the day when temperatures are the hottest because this is when wilt symptoms are most obvious. Look for plants exhibiting symptoms such as wilting, yellowing leaves, stunted growth, and vascular browning evident in longitudinal stem slices.
Focus on potato, tomato, and geranium, including other cultivated and weedy hosts, as resources allow. Look for signs of wilting throughout the field, but especially in areas where water accumulates. Inspect plants, including weeds, near drainage canals or irrigation rigs.
To diagnose bacterial diseases (including R3 Bvr2) quickly and easily in the field, surveyors may test infected plants for bacterial streaming. To test suspected infected plants, place freshly cut petioles or stems in a tube of sterile water and let sit undisturbed. If a thready white bacterial cloud or viscous milky streaming appears within 20 minutes, it is possible the plant is infected.
Note: This is not a confirmatory diagnostic and may not be effective in early stages of the disease. This procedure only indicates that visible symptoms may be caused by a bacterial pathogen, which may help surveyors target plants for sample collection. See the Figures 1 & 5 in the CAPS datasheet to see an image of bacterial streaming.
- Collect a maximum of 200 samples from each location.
- For potato tubers, take 0.2-0.5 g tissue of the heel end of each tuber (washing off soil first).
- For stems, harvest 1-2 cm stem segments from the base of the stem above the ground.
- For aquatic plants (such as Solanum dulcamara), 1-2 cm sections are sampled from underwater stems or stolons with aquatic roots.
- Prepare samples immediately or within 72 hrs of sampling.
Approved Method(s)
States can identify samples to genus and species using immunostrips or ELISA, but confirmation is required by the Plant Pathogen Confirmatory Diagnostics Laboratory (PPCDL).
Preliminary Screening:
Immunostrip tests or ELISA kits can be used to identify this pathogen to genus and species. The following commercially available immunostrips are approved by APHIS for field level screening:
- Agdia ImmunoStrip® for Ralstonia solanacearum, AgDia, Inc. Elkhart, IN
- Potato Brown Rot Pocket TM Pocket Diagnostic, from Forsite Diagnostics Ltd, York, UK
Here is the link to the forms and guidance on how to complete the forms: APHIS/CDC Form 4a
This form should be completed by the appropriate persons and sent to the following e-mail address: DASAT@usda.gov or according to the instructions on the form and the guidance below. Questions about the Select Agent Program should be directed to the above e-mail address or call: 301-851-2070.
There are two choices for plant dispositions that show positive for R. solanacearum using the immunostrip or ELISA: A) destroy the sample, or B) send for further testing at the USDA-APHIS-PPQ-S&T-Plant Pathogen Confirmatory Diagnostics Laboratory (PPCDL, formerly the Beltsville Lab). Below are the criteria to assist in those choices:
- The sample may be destroyed according to the select agent regulations if they are not from a foreign source, are not unusual hosts, do not show unusual symptomatic reactions, or are from areas in the U.S. where R. solanacearum is known to occur.
- Send an APHIS/CDC Form 4a (Report of Identification) to the DASAT and the sample is destroyed by the diagnostic lab (keep destruction record for a minimum of 3 years). No further action required.
- For other domestic detections of R. solanacearum, forward samples to PPCDL, following the directions below.
Molecular confirmation and race typing:
For other domestic detections of R. solanacearum from unusual hosts, with unique symptomatic reactions, and/or from areas in the country where R. solanacearum is not known to occur, forward samples to PPCDL. The usual cultivated hosts of R. solanacearum race 3 biovar 2 include eggplant, geranium, pepper, potato, and tomato.
- Refer to the Shipping instructions section below for samples requiring further testing at an approved laboratory.
- Contact PPCDL to inform them of the sample and receive guidance through the APHIS/CDC reporting process.
- Send an APHIS/CDC Form 4a (Report of Identification) to DASAT.
- PPCDL will initiate a Request to Transfer by submitting Form 2 Section 1. Once completed, Form 2 Section 2 will be sent to the submitter by DASAT for completion. After Form 4a (Report of Identification) and sections 1 and 2 of Form 2 (Request to Transfer) are approved, the sample may be shipped.
- If positive for R3bv2, PPCDL will submit an APHIS/CDC Form 4a (Report of Identification) to DASAT.
- Sample destroyed at both diagnostic and confirmatory labs.
- Sample retained: If registered for the agent, notify the DASAT of retention and put in inventory.
Shipping instructions for samples requiring further testing at an approved laboratory:
- For a positive R. solanacearum sample that requires further testing to confirm if it is race 3, biovar 2, the step-by-step procedures are summarized below.
- If identified as R. solanacearum, and further race/biovar determination is necessary at a Federal Select Agent Program registered lab because the plant is associated with a foreign source, or an unusual host and or from an area in the country where the organism is not known to occur:
- Contact PPCDL at APHIS- PPQCPHSTBeltsvilleSampleDiagnostics@usda.gov to inform them of the sample and receive guidance on sample submission.
- Complete sections A & B of the APHIS/CDC Form 4a (Report of Identification) and send to DASAT@usda.gov
- PPCDL will complete Form 2 (Request to Transfer) Section 1.
- Complete Form 2 (Request to Transfer) Section 2 and send to DASAT@usda.gov.
- Coordinate with PPQ to generate an Agriculture Risk Management (ARM) Diagnostic Request (DR) Routing Form.
- Complete a PPQ Form 391.
- Send the sample, a copy of the approved Form 2, ARM DR, and PPQ Form 391 in the shipment to PPCDL.
- PPCDL, will receive the sample, complete the APHIS/CDC Form 2 Section 3, and send the completed paperwork to DASAT.
- PPCDL, will then determine if it is R. solanacearum R3bv2. If positive for R3bv2, PPCDL, will complete the APHIS/CDC Form 4a (Report of Identification), and send the APHIS/CDC Form 4a to DASAT.
- PPCDL, will notify the Domestic Diagnostics Coordinator of the test outcome, who will then report the results according to the Pest Identification Notification Protocol.
Sample Preparation and Sending Procedures:
When sending the sample, you can send the whole plant including roots, but they must be free of soil. If rinsing with water, ensure sample is blotted dry prior to packing. At a minimum, enclose 0.035 ounces of symptomatic stem/crown tissue. Include completed hard copies of the PPQ form 391, the ARM DR routing form, and the APHIS/CDC Form 2 in the shipping container. Be sure to include cool packs in a sturdy insulated container and send by overnight carrier (do not send on a Friday) to the following address:
Plant Pathogen Confirmatory Diagnostics Laboratory
Bldg. 580, BARC-East
9901 Powder Mill Road
Laurel, MD 20708
Phone: (301) 504-7100, VOIP: (301) 313-9200
Group E-mail Address:
APHIS-PPQCPHSTBeltsvilleSampleDiagnostics@usda.gov
Once packaged and sent, please send an e-mail message to the group e-mail address above with a pdf of the completed PPQ form 391 and the overnight carrier tracking number.
Results Reporting
Results will be reported through the PPQ National Identification Services Domestic Diagnostics Coordinator and the National Survey Coordinator, according to the Pest Identification Notification to the States protocol. Sample submitters and originating laboratories will receive results through their State Plant Regulatory Officials (SPROs) or State Plant Health Directors (SPHDs).
Stephen W. Bullington, Ph.D.
Domestic Diagnostic Coordinator
National Identification Services
USDA-APHIS-PPQ
4700 River Road, Unit 52
Rm. 4D-04.35
Riverdale, MD 20737
301-851-2153
Aimee Hyten
Plant Pathologist
Agriculture Select Agent Services
4700 River Rd. Unit 2
Riverdale, MD 20737
301-851-3300, option 3
Other strains in the R. solanacearum species complex (RSSC), including non-Select Agent R. solanacearum, R. pseudosolanacearum and R. syzygii, Clavibacter michiganensis on potato, and Xanthomonas campestris pv. pelargonii (bacterial blight) on geranium. Bacterial blight may produce leaf spots in addition to the wilting symptom.
If you are unable to find a reference, contact STCAPS@usda.gov. See the CAPS Pest Datasheet for all references.