Bacterial wilt; Southern bacterial wilt - Ralstonia solanacearum race 3 biovar 2
Effective: April 6, 2018
Taxonomic Position: Burkholderiales : Ralstoniaceae
Pest Type: Bacteria
Pest Code (NAPIS): FGZTPUU
This pest is a member of the following surveys: Solanaceous Hosts
These Approved Methods are appropriate for: 2024, 2023, 2022, 2021, 2020, 2019, 2018, 2017, 2016, 2015
||NAPIS Survey Method
||Collect symptomatic host tissue.
||3031 - General Visual Observation
In Geranium: Collect stems from symptomatic plants (wilting; yellowing of lower leaves). Check for bacterial exudation by placing a piece of stem from a symptomatic plant into water, look for viscous streaming.
In Potatoes: Slice tubers and look for ooze, vascular discoloration (can incubate at 30oC 86oF for 3-4 weeks).
The following are recommendations for executing the survey using the approved methods for pest surveillance. The recommendations are developed through literature review and consultation with subject matter experts.
Bacterial ooze or streaming may occur.
On potatoes and other solanaceous hosts: 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. Another common symptom associated with bacterial wilt in the field is plant stunting.
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. 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 that is very characteristic.
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.
Key Diagnostic or Identification
Methods to confirm Ralstonia solanacearum: States can identify to genus and species by one of these methods but race/biovar testing and confirmation is required at the CPHST-Beltsville Laboratory:
1. Serological: test kits/ELISA kits can be used to identify to genus and species. Commercially available in the form of Immunostrips from Agdia and Lateral Flow Devices (Pocket Diagnostics) from Fera (formerly CSL).
2. Culture: To identify to genus and species.
a. mSMSA semi-selective medium - 28-30oC for 48 hours; R. solanacearum appears mucoid with a whitish colony.
b. TZC selective medium - 28-30oC for 36-48 hours. R. solanacearum appears as mucoid, whitish colonies that produce a pink pigmented centers (that do not diffuse into the medium).
3. Molecular: Weller et al. (2000) developed a real-time, multiplex PCR assay to detect all known strains of R. solanacearum and a specific assay for the biovar 2A genotype (both reactions in a single tube). Another multiplex reaction that incorporates a third primer set, with an internal control, was developed for detection of R. solanacearum in infected potato tissue.
PPQ CPHST has validated work instructions available upon request for:
a. Conventional PCR detection of R. solanacearum and biovar 2 (Fegan, 1998a; Fegan, 1998b).
b. Real-time PCR that is a modification of the real-time PCR assay provided from CSL based on Weller et al. (2000).
For race and biovar testing: Once Ralstonia solanacearum has been confirmed, samples or cultures are sent to CPHST- Beltsville laboratory for race and biovar confirmation.
Biovar Plate Assay: A plate assay that differentiates biovar based on pure culture utilization of carbohydrates and sugar alcohol utilization is available. A work instruction from PPQ CPHST is available.
In Progress / Literature-based Diagnostics:
Tomato bioassay: Using 2-3 week-old tomato seedlings to display symptoms. Wilting usually develop within a 1-3 weeks of inoculation when inoculated in a greenhouse kept at 25-28°C.
Molecular: DNA probe: Cooke and Sequeira (1991) developed a DNA probe based test to identify Pseudomonas solanacearum race 3.
Real-time BIO-PCR: Ozakman and Schaad (2003) developed a real-time BIO-PCR (includes a 24-hr enrichment phase) to detect R. solanacearum race 3, biovar 2 in asymptomatic potato tubers.
PCR: Opina et al. (1997) used PCR primers developed from cloned RAPD fragments to identify Burkholderia solanacearum. Fegan (1998a) developed a PCR based assay to detect the R. solanacearum biovar 2. There was a little cross-reactivity with three isolates of biovar 1 from potato. Pastrik and Maiss (2000) developed a new PCR assay for detection of R. solanacearum in potato tubers. Ten different DNA extraction techniques were compared and Easy-DNA, SDS, and CTAB were considered highly reliable.
Poussier and Luisetti (2000) developed a nested PCR-RFLP protocol that detected biovars of Ralstonia solanacearum in plant tissue. A PVPP/BSA treatment is advised prior to PCR, because some plant compounds inhibited amplification.
Pradhanang et al. (2000) compared detection methods in soil, including nested PCR and conventional PCR. Both methods worked better when first enriching the infested soil sample in SMSA broth.
Glick et al. (2002) developed a multiplex PCR that could simultaneously detect R. solanacearum and Xanthomonas campestris pv. pelargonii, the two major bacterial pathogens of geranium.
Schonfeld et al. (2003) developed a PCR assay based on filC fragments to detect R. solanacearum in soil.
Co-operational PCR (Co PCR): Caruso et al. (2003) developed a Co PCR reaction (based on the simultaneous and co-operational action of three primers) for detection of R. solanacearum in water. This method was utilized by Marco-Noales et al. (2008) to detect nonculturable cells of R. solanacearum in asymptomatic geranium.
Loop-mediated amplification (LAMP) technique: Kubota et al. (2008) were able to detect R. solanacearum using this technique and indicated it may be useful for identifying R. solanacearum from the soil.
Immunocapture and PCR (IC-PCR): Dittapongpitch and Surat (2003) combined immunocapture and PCR to detect R. solanacearum in soil and weeds. Soil detection required an enrichment step in nutrient broth.
Other races and biovars within R. solanacearum, Clavibacter michiganensis on potato, and Xanthomonas campestris pv. pelargonii (bacterial blight) on geranium. Bacterial blight can also produce leaf spots in addition to the wilting symptom.