New Pest Response Guidelines

Ralstonia solanacearum “race 3 biovar 2” 

Brown rot of potato

Potato wilting due to infection with R. solanacearum R3bv2 (Source: Juan Herrera)
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Contents

Figures and Tables.................................................................................................4

1. Introduction .....................................................................................................6

2. Pest Overview...................................................................................................7

3. Pest Identification and Damage....................................................................15

4. Delimitation Survey.......................................................................................22

5. Eradication and Control Options.................................................................28

6. Research Needs..............................................................................................32

7. Literature Cited.............................................................................................33

Appendix A: Environmental Compliance..........................................................42

Appendix B: USDA–APHIS Procedures for Submitting Samples...................46

8. Authors and Reviewers.................................................................................51

R.

solanacearum

R3bv2
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Figures and Tables

Figures

Tables

Cover Potato wilting due to infection with R. solanacearum R3bv2 ........... 1

Figure 2-1 Potatoes harvested acres for sale in the continental U.S.

in 2012.............................................................................................. 13

Figure 2-2 Tomatoes harvested acres for sale in the continental U.S.

in 2012.............................................................................................. 14

Figure 2-3 Combined host density of potato and tomato in the

continental U.S. in 2012................................................................... 14

Figure 3-1 Wilting on potato plant caused by infection with

R. solanacearum R3bv2................................................................... 17

Figure 3-2 Cut section of a tomato stem displaying vascular browning

caused by infection with R. solanacearum....................................... 18

Figure 3-3 Cut section of potato tubers displaying a brown

discoloration to the vascular ring caused by infection

with R. solanacearum R3bv2........................................................... 18

Figure 3-4 Bacterial ooze on geranium caused by infection with R.

solanacearum R3bv2 ....................................................................... 19

Figure 3-5 Left: Chlorosis on geranium plants caused by infection with

R. solanacearum R3bv2; Right: Healthy geranium ......................... 19

Figure 3-6 Early wilting symptoms on geranium caused by infection

with R. solanacearum R3bv2........................................................... 20

Table 2-1 List of reported plant hosts of R. solanacearum R3bv2........................ 9

Table 2-2 List of reported symptomless hosts of R. solanacearum

R3bv2.................................................................................................. 10

R.

solanacearum

R3bv2
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Table 2-3 List of hosts of R. solanacearum R3bv2 that can be asymptomatically

infected under artificial conditions...................................................... 10

Table 2-4 Reported worldwide distribution of R. solanacearum R3bv2............. 11

Table 4-1 Approved skin disinfectant active ingredients for R. solanacearum... 23

Table 4-2 Approved surface disinfectant active ingredients for

R. solanacearum.................................................................................. 23
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Introduction

Plant Protection and Quarantine (PPQ) develops New Pest Response Guidelines 

(NPRGs) in preparation for potential pest introductions. This document is based 

on the best information available at the time of development and may not reflect 

the latest state of knowledge at the time the pest is detected. In addition, the PPQ 

response must be tailored to the specific circumstances of each pest introduction 

event, which cannot be predicted. Therefore, this document provides only general 

guidelines that can be used as a basis for developing a situation-specific response 

plan at the time a new pest is detected.

Program managers of Federal emergency response or domestic pest control 

programs must ensure that their programs comply with all Federal Acts and 

Executive Orders pertaining to the environment, as applicable. Refer to the 

Environmental Compliance section in Appendix A for details.

This document provides information on the select agent Ralstonia solanacearum 

race 3 biovar 2. For more information, refer to the Federal Select Agent

Program website.

Chapter

1
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2 Pest Overview

Pest Summary

Ralstonia solanacearum race 3 biovar 2 (R3bv2), a causal agent of potato brown 

rot, is thought to have originated in the Andean highlands of Colombia and Peru 

(Champoiseau et al., 2009; Cook and Sequeira, 1991). This pathogen affects 

mainly solanaceous crops and weeds and is considered a select agent in the U.S. 

(Janse et al., 2004; USDA-APHIS-PPQ, 2018). A select agent is a biological 

agent that has the potential to pose a severe threat to plant health (USDA-APHIS, 

2017).

Key Information

♦ Can infect and cause disease at temperatures as low as 61 oF, but symptoms are 

most likely at 75–95 oF).

♦ Primarily spread through contaminated irrigation and surface runoff water, 

infested soil, tools and equipment.

♦ Major hosts are potato, tomato and geranium.

♦ Many native plant and weed species, such as bittersweet nightshade, can be 

infected but asymptomatic.

♦ Main symptoms are wilting, leaf chlorosis (yellowing), stunting, and vascular 

browning in stems.

♦ Symptoms of R. solanacearum R3bv2 can be mistaken for various other wilt 

pathogens on potato, tomato and geranium.

♦ Management in the field is very difficult due to strain variation, host range, 

pathogen persistence in asymptomatic hosts, infected soil and surface water, and 

lack of adequate chemical treatments.

♦ All potentially infected plants and planting material must be destroyed.

♦ Soil fumigation using chloropicrin has produced similar results to methyl 

bromide, but further studies are needed.

♦ Exclusion, use of certified seed, cultural practices and phytosanitation are the best 

control methods.

Chapter

2
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Taxonomy

Scientific Name

♦ Ralstonia solanacearum (Smith, 1896) Yabuuchi et al., 1996 race 3 biovar 2

♦ A revised classification system based on phylogenetic analysis of genome 

sequences places Ralstonia solanacearum R3bv2 strains in sequevars 1 and 2 of 

the phylotype II B subgroup (Allen et al., 2005; Prior and Fegan, 2005b).

Taxonomic Position

♦ Bacteria : Proteobacteria : Betaproteobacteria : Burkholderiales : 

Burkholderiaceae

Synonyms

♦ Burkholderia solanacearum (Smith, 1896) Yabuuchi et al.

♦ Pseudomonas solanacearum (Smith, 1896) Smith

Common Names

♦ Brown rot of potato

♦ Bacterial wilt of tomato

♦ Southern wilt of geranium

Biology and Ecology

Life Cycle

Ralstonia solanacearum R3bv2 is soil and waterborne and can survive for days to 

years in infected soil (at least 29 inches) (Champoiseau et al., 2009; Graham et al., 

1979), as well as surface irrigation water and weeds (Champoiseau and Momol, 

2009). This bacterium thrives in cool tropical highlands and in temperate zones.

Ralstonia solanacearum R3bv2 is highly virulent at temperatures between 66 oF 

and 82 oF (Huerta et al., 2015). Virulence decreases with temperatures above 95 

oF or below 60.8 oF (Champoiseau et al., 2009; Ciampi and Sequeira, 1980).

Additionally, Milling et al. (2009) found that the bacterium can survive for more 

than 4 months in sterile water at 39 oF. Other factors affecting disease 

development include soil type and structure, soil moisture, salt content and water 

pH (Champoiseau and Momol, 2009).
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Infection can occur through wounds in roots (points of emergence of lateral root), 

injury caused by soil-borne organisms (e.g. the root-knot nematode) and stem 

injuries caused by agricultural practices (Champoiseau and Momol, 2009; 

Swanson et al., 2005). Once the plant is infected, the pathogen spreads 

systemically through the xylem vessels causing wilting and death (Champoiseau 

et al., 2009; Genin, 2010). Symptomless infection is common, especially at cooler 

temperatures.

Hosts

Ralstonia solanacearum R3bv2 has a broad host range (Table 2-1) that includes 

many asymptomatically infected native plant and weed species (Jones et al., 

2017). In Europe, numerous outbreaks of R. solanacearum R3bv2 have been 

attributed to bittersweet nightshade, a weed species that grows along waterways 

and in wet areas (Janse, 1996). Bittersweet nightshade is present in the U.S. and 

considered an invasive species (Waggy, 2009). Tables 2-2 and 2-3 list other 

species reported to be asymptomatic hosts.

Table 2-1 List of reported plant hosts of R. solanacearum R3bv2

Scientific name Common name References

Amaranthus viridis green amaranth Lin et al. (2015)

Capsicum annuum sweet pepper Martin and French (1995)

Capsicum spp. Pepper Lin et al. (2015)

Oxalis latifolia broadleaf wood-sorrel Khoodoo et al. (2010)

Pelargonium × hortorum zonal geranium Williamson et al. (2002)

Pelargonium spp. Geranium Ozakman and Schaad (2003)

Pelargonium zonale horseshoe pelargonium Janse et al. (2004)

Physalis angulate cut-leaf ground-cherry Swanepoel (1992)

Portulaca oleracea common purslane Lin et al. (2015)

Solanum americanum American nightshade Khoodoo et al. (2010)

Solanum betaceum

(=Cyphomandra betacea)

tree-tomato Martin and Nydegger (1982)

Solanum cinereum Narrawa-bur Graham and Lloyd (1978)

Solanum lycopersicum Tomato Mahbou Somo Toukam et al. (2009)

Solanum melongena Eggplant Caffier and Hervé (1996)

Solanum nigrum black nightshade Tomlinson and Guntber (1986)

Solanum pimpinellifolium

(=Lycopersicon 

pimpinellifolium)

currant tomato Khoodoo et al. (2010)

Solanum spp. Potato Allen et al. (2005)

Solanum tuberosum Potato Tahat and Sijam (2010)

Solanum tuberosum subsp. 

andigenum (=Solanum 

phureja)

Andean potato Ciampi and Sequeira (1980); Patil et 

al. (2012)
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Table 2-2 List of reported asymptomatic hosts of R. solanacearum R3bv2

Scientific name Common name References

Ageratum conyzoides Goatweed Tusiime et al. (1998)

Amaranthus spp. Pigweed Tusiime et al. (1998)

Bidens pilosa beggar-ticks Tusiime et al. (1998)

Dopatrium sp. Dopatrium Pradhanang and Momol (2001)

Drymaria cordata tropical chickweed Pradhanang et al. (2000)

Erigeron floribundus Bilbao fleabane Tusiime et al. (1998)

Galinsoga parviflora dumb-nettle Pradhanang and Elphinstone (1996); 

Tusiime et al. (1998)

Leucas martinicensis Whitewort Tusiime et al. (1998)

Monochoria vaginalis pickerel-weed Pradhanang and Momol (2001)

Nicotiana glutinosa NA Fernandez (1986)

Oxalis latifolia broadleaf wood-sorrel Tusiime et al. (1998)

Persicaria capitata

(=Polygonum capitatum)

Japanese knotweed Pradhanang and Elphinstone (1996); 

Pradhanang et al. (2000)

Persicaria nepalensis

(=Polygonum nepalense)

Nepal knotweed Tusiime et al. (1998)

Rumex abyssinicus Mekmeko Tusiime et al. (1998)

Solanum dulcamara bittersweet nightshade Wenneker et al. (1999)

Stellaria sennii NA Tusiime et al. (1998)

Spergula arvensis corn spurrey Tusiime et al. (1998)

Tagetes minuta Aztec marigold Tusiime et al. (1998)

Urtica dioica stinging nettle Wenneker et al. (1999)

Table 2-3 List of hosts of R. solanacearum R3bv2 that can be asymptomatically 

infected under artificial conditions

Scientific name Common name References

Brassica juncea brown mustard Pradhanang et al. (2000)

Brassica oleracea Cabbage Álvarez et al. (2008)

Brassica spp. wild mustard Álvarez et al. (2008)

Calibrachoa sp. Calibrachoa Janse et al. (2004)

Cerastium glomeratum mouse-ear chickweed Pradhanang et al. (2000)

Cichorium endivia Endive Álvarez et al. (2008)

Datura stramonium Jimsonweed Fernandez (1986)

Nicandra physalodes broadleaf-nightshade Pradhanang et al. (2000)

Nicotiana glutinosa knekt-tobak Martin and French (1995)

Nicotiana rustica Aztec tobacco Martin and French (1995)

Petunia × atkinsiana

(=Petunia × hybrida)

common garden 

petunia

Janse et al. (2004)

Physalis pubescens

(=Physalis floridana)

downy ground-cherry Fernandez (1986)

Salpiglossis sinuata painted-tongue Olsson (1976)

Solanum pseudocapsicum

(=Solanum capsicastrum)

false capsicum Fernandez (1986)

Solanum virginianum

(=Solanum xanthocarpum)

yellow-fruit nightshade Pradhanang et al. (2000)

Stellaria media Chickweed Pradhanang et al. (2000)

Tropaeolum majus garden nasturtium Pradhanang et al. (2000)
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Dispersal

Natural Movement

Ralstonia solanacearum R3bv2 can spread naturally from infected roots to 

healthy roots of neighboring plants (Kelman and Sequeira, 1965 as cited by 

Kelman, 1998) and through insect and possibly nematode damage (Champoiseau 

et al., 2009). Note that this pathogen does not spread from plant to plant aerially 

or through casual contact or water splashing (Swanson et al., 2005).

Human-Assisted Spread

This pathogen is primarily water and soilborne and can be dispersed by 

contaminated irrigation and surface runoff water, infected soil and plant material, 

soil transfer on machinery and equipment, and unsanitized handling 

(Champoiseau and Momol, 2009; Janse, 1996). Greenhouse spread may occur 

through transplanting infected plants, pinching buds off plants without sanitizing 

(Jones et al., 2017), using contaminated tools between cuttings (Janse et al., 2004) 

and irrigating with sub-irrigation or ebb-and-flow systems (Swanson et al., 2005). 

Vegetative propagation also plays a key role in spreading R. solanacearum R3bv2 

through asymptomatically infected seed tubers and cuttings of geranium and other 

ornamentals (Janse, 1996; Jones et al., 2017).

According to studies by Pasqua di Bisceglie et al. (2005), R. solanacearum can 

survive on poplar for 17 days, oak for 4 days, and on high-density polyethylene in 

cold storage at 39 °F and 80–90% relative humidity for 2 days. Survival of R. 

solanacearum on jute fabric dropped after 24 hours with the population reaching 

zero after 78 days.

Geographic Distribution

World Distribution

Table 2-4 Reported worldwide distribution of R. solanacearum R3bv2

Country References

Argentina French (1988)

Australia Graham and Lloyd (1979); Stansbury et al. (2001)

Bangladesh Chakraborty and Roy (2016)

Belgium1 EPPO (2015b)

Bolivia Castillo and Plata (2016)

Brazil Almeida et al. (2003)

Burundi Autrique and Potts (1987)

Cameroon Mahbou Somo Toukam et al. (2009)

Chile Ciampi et al. (1997); van der Wolf et al. (2004)

China Wang et al. (2017)

Colombia Lebeau et al. (2011); Prior and Fegan (2005a)
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Country References

Costa Rica Gabriel et al. (2006); Williamson et al. (2002)

Czech Republic1 EPPO (2012a)

Egypt Farag et al. (1999)

Ethiopia Lemessa and Zeller (2007)

France1 EPPO (2014)

French Guiana Deberdt et al. (2014)

Georgia Lashkhi et al. (2018)

Germany1 EPPO (2012b)

Greece2 EPPO (2007)

Guadeloupe Prior and Steva (1990)

Guatemala Sanchez Perez et al. (2008)

Hungary2 EPPO (2002); Nemeth et al. (2002)

India Sagar et al. (2013)

Indonesia Horita and Tsuchiya (2001)

Iran Izadiyan and Taghavi (2013)

Japan Horita and Tsuchiya (2001)

Kenya Janse et al. (2004)

Korea, Republic of Jeong et al. (2007)

Malawi Zayamba Kagona (2008)

Mauritius Khoodoo et al. (2007)

Mexico Hernández-Romano et al. (2012); Perea Soto et al. (2011)

Nepal Pradhanang et al. (2000)

Netherlands EPPO (2016)

New Caledonia EPPO (2015c); IPPC (2015)

Nigeria Popoola et al. (2015)

Pakistan Begum (2011)

Papua New Guinea Tomlinson and Guntber (1986)

Peru Gutarra et al. (2017)

Philippines Natural et al. (2005)

Poland1 EPPO (2015a)

Portugal Cruz et al. (2012)

Reunion Nicole et al. (1998)

Russia Matveeva et al. (2003)

Rwanda Uwamahoro et al. (2018)

Slovakia1 EPPO (2004)

South Africa Cellier et al. (2012)

Spain Caruso et al. (2017)

Sri Lanka EPPO (1998)

Sweden1 EPPO (2010)

Taiwan Wu et al. (2011)

Tanzania Mwankemwa (2015)

Turkey Ustun et al. (2008)

Uganda Allen et al., 2005

Uruguay Siri et al. (2011)

Venezuela Garcia et al. (1999)

1 Under eradication

2 Under official control
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Potential Distribution in the United States

Ralstonia solanacearum R3bv2 is not known to be present in the U.S. However, 

nearly every state grows at least one of the major crops that can host R. 

solanacearum R3bv2. California, Colorado, Idaho, Maine, Michigan, Minnesota, 

North Dakota, Oregon, Washington and Wisconsin harvest the majority of 

potatoes (Fig. 2-1) (USDA–APHIS–PPQ–S&T Fort Collins Lab, 2018a), while 

California and Florida are the major producers of tomatoes (Fig. 2-2) (USDA–

APHIS–PPQ–S&T Fort Collins Lab, 2018b). Geraniums, sold as annual 

bedding/garden plants, are grown in California, Florida, Hawaii, Illinois, 

Maryland, Michigan, New Jersey, New York, North Carolina, Ohio, Oregon, 

Pennsylvania, South Carolina, Texas and Washington (USDA-NASS, 2016).

Figure 2-3 includes combination map of potato and tomato.

Figure 2-1 Potatoes harvested acres for sale in the continental U.S. in 2012 (Source: 

USDA–APHIS–PPQ–S&T Fort Collins Lab, 2018a)
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Figure 2-2 Tomatoes harvested acres for sale in the continental U.S. in 2012 (Source: 

USDA–APHIS–PPQ–S&T Fort Collins Lab, 2018b)

Figure 2-3 Combined host density of potato and tomato in the continental U.S. in 2012 

(Source: NCSU-CIPM, 2019)
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Pest Identification and 

Damage

Species Description/Morphology

Ralstonia solanacearum R3bv2 is a gram-negative, rod-shaped, motile, strictly 

aerobic bacterium that is 0.2–0.28 inches × 0.59–0.79 inches in size 

(Champoiseau et al., 2009; Smith, 1896; Yabuuchi et al., 1995).

Normal or virulent type colonies are irregularly shaped, white or cream-colored, 

opaque and highly fluidal, while the mutant or non-virulent type colonies appear 

consistently round, smaller, butyrous (buttery consistency) or dry (Champoiseau 

et al., 2009). Ralstonia solanacearum R3bv2 grows relatively slowly and is easily 

outcompeted by other microbes in culture; individual bacterial colonies that 

appear in less than 36 hours are not R. solanacearum (Allen, 2019).

ID/Diagnostic

Molecular

♦ Real-time, multiplex PCR assay, specifically an assay for biovar 2A, 

can detect all strains of R. solanacearum (USDA-APHIS-PPQCPHST, 2015; Weller et al., 2000).

♦ An additional multiplex reaction was developed for infected potato 

tissue that integrates a third primer set with an internal control 

(USDA-APHIS-PPQ-CPHST, 2015).

♦ If a sample tests positive for R. solanacearum, Select Agent Program 

guidelines must be followed. See Appendix B for further information.

Signs and Symptoms

Ralstonia solanacearum R3bv2 symptoms are identical to those from other R. 

solanacearum strains (Champoiseau, 2009). Additionally, under favorable 

conditions both geranium and potato plants may form asymptomatic infections 

(Swanson et al., 2005). Signs and symptoms of R. solanacearum R3bv2 include 

the following:

Chapter

3
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Potato and Tomato

♦ Early symptoms of wilting occur on the youngest leaves during the hottest 

time of the day. Wilting may be limited to the top portion of the plant on 

just one side of a leaflet or an individual branch. Plants may appear to 

recover following rain or when temperatures cool down at night (Fig. 3-1) 

(Champoiseau et al., 2009). Infected tomato plants often develop 

adventitious roots on the lower stem (Allen, 2019).

♦ The entire plant may decline rapidly under favorable conditions, starting 

with wilt, leaf chlorosis and ending with death. Leaves can wither, but 

dried leaves remain green (Champoiseau et al., 2009). In tomato, disease 

develops rapidly and plants may die within 4–7 days after the first 

appearance of wilt symptoms (Jones et al., 2017).

♦ Stunting is another common symptom, occurring at any stage of growth 

(Champoiseau et al., 2009).

♦ Infected stems may collapse, revealing vascular browning displayed as 

narrow, dark brown necrotic streaks with grey-white bacterial ooze (Fig.

3-2) (Champoiseau et al., 2009). When disease develops very rapidly, 

ooze may appear on the surface of intact stems (Allen, 2019).

♦ In symptomatic potato tubers the vascular ring turns a grey-brown color 

that may extend into the pith or cortex as the infection progresses. When 

infected potatoes are cut open, they ooze a milky-white sticky exudate. 

Visible threads may form from the ooze when the two sides of a cut potato 

are pressed together and then pulled apart. This ooze may also cause dirt

to adhere to the tuber (Fig. 3-3) (Champoiseau et al., 2009).

♦ A common diagnostic sign is bacterial streaming, which occurs when 

freshly cut stems from infected plants are placed in water. Fine, milky 

white strands of a viscous white slime containing bacteria often run from 

the cut end of the stem within 15 minutes (Fig. 3-4) (Jones et al., 2017).

♦ Bacterial streaming and oozing may not be visible in the early stages of 

disease development (Jones et al., 2017).

Geranium

♦ Early wilt symptoms in geranium are subtle and sometimes unnoticeable. 

Symptoms begin with chlorosis and wilt in the lower leaves and progress 

into upward curling of leaf margins (Figs. 3-5 and 3-6) (Champoiseau et 

al., 2009).

♦ Like potato and tomato, geraniums may appear to recover in the cooler 

night temperatures (Champoiseau et al., 2009).

♦ The disease develops rapidly as wilting moves upward from older to 

younger leaves (Champoiseau et al., 2009).
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♦ Wilted leaves often have wedge-shaped areas of chlorosis that become 

necrotic (Champoiseau et al., 2009).

♦ In later stages of disease, stem collapse can occur. Stems (particularly at 

the root crown) and roots exhibit vascular discoloration, which can 

blacken and eventually become necrotic (Champoiseau et al., 2009).

♦ Bacterial streaming can also occur in geranium (Champoiseau and Momol, 

2009).

Figure 3-1 Wilting on potato plant caused by infection with R. solanacearum R3bv2 

(Source: Amilcar Sanchez Perez)
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Figure 3-2 Cut section of a tomato stem displaying vascular browning caused by 

infection with R. solanacearum (Source: Clemson University - USDA Cooperative 

Extension Slide Series, Bugwood.org)

Figure 3-3 Cut section of potato tubers displaying a brown discoloration to the vascular 

ring caused by infection with R. solanacearum R3bv2 (Source: Caitilyn Allen, University 

of Wisconsin-Madison)
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Figure 3-4 Bacterial ooze on geranium caused by infection with R. solanacearum R3bv2 

(Source: Margery Daughtrey, Cornell University, Bugwood.org)

Figure 3-5 Left: Chlorosis on geranium plants caused by infection with R. solanacearum 

R3bv2; Right: Healthy geranium (Source: Caitilyn Allen, University of WisconsinMadison)
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Figure 3-6 Early wilting symptoms on geranium caused by infection with R. 

solanacearum R3bv2 (Source: Jean L. Williams-Woodward, University of Georgia, 

Bugwood.org)

Similar Species

Other diseases that produce symptoms that can be mistaken for R. solanacearum

R3bv2 include the following:

♦ Other R. solanacearum strains that are endemic in the southern U.S. 

produce indistinguishable symptoms in the same hosts (Jones et al., 2017).

♦ Xanthomonas hortorum pv. pelargonii, the causal agent of bacterial blight 

of geranium, produces wilting symptoms similar to R. solanacearum 

R3bv2 (Champosiseau et al., 2010).

♦ Fusarium oxysporum f. sp. lycopersici, the causal agent of Fusarium wilt 

on tomato, causes wilting and chlorosis on one side of the plant and dark 

brown streaks in the stem (Cook and Sequeira, 1991; Jones et al., 1991).

♦ Verticillium albo-atrum and V. dahliae, the causal agent of Verticillium 

wilt on potato and tomato (Cook and Sequeira, 1991; Jones et al., 2017), 

causes leaf wilting during the day, which reverses at night (tomato), 

chlorosis on one side of the plant (potato) and vascular browning in 

infected stems (potato and tomato) (Jones et al., 1991; Stevenson et al., 

2001).

♦ Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring
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rot, produces a vascular ring containing bacterial ooze and causes leaf 

wilting during the hottest period of the day and looks like the plant 

recovers overnight (Stevenson et al., 2001).

Abiotic stresses, such as drought, mechanical damage to the root or nutrient 

deficiency can also produce symptoms easily mistaken for R. solanacearum 

R3bv2. Therefore diagnosis should not be based solely on symptoms (Jones et al., 

2017).
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Delimitation Survey

Delimitation Area

Delimitation surveys determine the extent of the infected area after an infection 

has been confirmed. Since each survey location has different characteristics and 

agency resources are unknown until a response begins, PPQ develops situationspecific surveys after pests are detected. To prepare response teams for the 

delimitation survey, we provide survey considerations below.

Survey Techniques for Delimitation

Survey Preparation, Sanitization and Clean-Up

1. Prior to beginning a survey, determine whether there have been recent 

pesticide applications that would render it unsafe to inspect the plants and 

soil. Contact the property owner or manager and ask if there is a re-entry 

period in effect due to pesticide application. Look for posted signs 

indicating recent pesticide applications, particularly in commercial fields or 

nurseries.

2. Determine whether quarantines for other pests or crops are in effect for the 

survey area. Comply with all quarantine requirements.

3. When visiting the area to conduct surveys or take samples, take strict 

measures to prevent contamination by R. solanacearum R3bv2 or other 

pests between properties during inspections.

4. Designate a clean area where transport vehicles can park. Make sure this 

area is not located near infected fields.

5. Important: Use disposable protective clothing, gloves and footwear, and 

change them before entering each site. Disinfectants available for use on 

skin are listed in Table 4-1.

6. Disinfect all potentially contaminated surfaces (i.e. benches, flats, 

walkways, footbaths, drainage areas under benches, footwear) and 

equipment near an infected greenhouse or area that may have come in 

contact with infected material (Table 4-2).

a. Clean any soil or media adhering to the surface.

b. Change the disinfectant in footbath reservoirs at least twice daily 

Chapter

4
Last update 17JAN2024 R. solanacearum R3bv2 23

(USDA-APHIS, 2007).

c. When taking plant samples, disinfect tools with an approved surface 

disinfectant prior to and between uses (Table 4-2). Submerge the entire 

blade or portion of that tool that makes is in contact with soil or plant 

material.

d. Important: To disinfect large pieces of equipment, storage areas or 

bins, use a high-pressure delivery system, such as a steam pressure wash 

system, at 212 oF.

7. Drain and clean all recirculating irrigation systems, sub irrigation systems 

and any systems that do not prevent backflow of water from infected 

greenhouses.

a. Clean all parts, sumps, and pumps, with approved disinfectant solutions. 

To be effective against R. solanacearum R3bv2, systems must have 

ozonation with 0.4 ppm residual ozone for four minutes with ultraviolet 

(UV) light of at least 300 j/m2 (1.36 ft-lb/in2) at >50% transmission 

(USDA-APHIS, 2007).

8. When ponds, outdoor soil or holding areas have become contaminated 

during plant storage or runoff, contact your PPQ regional office and APHIS 

State Plant Health Director (SPHD) about possible environmental 

consideration and treatment options.

Table 4-1 Approved skin disinfectant active ingredients for R. solanacearum. Follow 

product label instructions prior to using any product (USDA-APHIS, 2007)

Active Ingredient

Alcohols (>60% ethanol)

Chlorhexidine (0.5%-4.0%)

Chloroxylenol (0.3%-3.75%)

Iodine and iodophors (7.5%-10.0% povidone-iodine)

Quaternary ammonium

Triclosan (0.2%-2.0%)

Table 4-2 Approved surface disinfectant active ingredients for R. solanacearum. Follow 

product label instructions prior to using any product (USDA-APHIS, 2007)

Active Ingredient Use Sites

Chlorine Greenhouse hard non-porous surfaces (i.e. 

floor, walls, benches, counter tops, buckets, 

coolers)

Shoe wash

Iodine Greenhouse hard non-porous surfaces (i.e. 

floor, walls, benches, counter tops, buckets, 

coolers)

Shoe wash

Quaternary ammonium (ammonium 

chloride)

Outer clothing, field harvesting equipment, 

greenhouse packing areas

Greenhouse hard non-porous surfaces (i.e. 

floors, walls, benches, counter tops, buckets, 

coolers, tools, metal and plastic surfaces, 

knobs, handles, railings, glass)

Walkways
Last update 17JAN2024 R. solanacearum R3bv2 24

Visual Inspection

Conduct a visual inspection by searching for plants with typical wilting symptoms 

(see Signs and Symptoms). The absence of symptoms, however, does not confirm 

an absence of R. solanacearum R3bv2 in the inspected area. Some infected plants 

may be asymptomatic, even when continually exposed to ideal temperatures.

Other areas to survey include the following:

♦ Look for signs of wilting in areas where water accumulates or throughout 

the field. Ralstonia solanacearum R3bv2 is readily carried in irrigation 

water and can spread rapidly.

♦ Inspect plants, including weeds, near drainage canals or irrigation rigs.

Delimitation Survey

Survey timing should depend upon the pathogen life cycle, the plant growth stage 

when infection is likely to occur, and ecological parameters that support pathogen 

dispersal. Other considerations include logistics and available resources; available 

resources can vary based on the time and location of detection and the pest 

species.

Prior to collecting samples:

♦ Define the site to be sampled on an acre basis.

♦ For whole plant sampling use the following or current surveying 

procedures already set in place in your state or county:

o Walk down rows and collect symptomatic plants.

o For example: Use the following to determine sampling rows 

(NDSU, 2018):

 If plants are widely spaced, 36 inches apart, walk down 

every 6th row looking at all plants in adjacent rows up to 20 

feet out.

 If they are tightly spaced, 12 inches apart, do the same 

every 20th row.

o Take random samples to represent the entire acre.

Sample Collection

Surveyors visiting sites to place holds or obtain samples should collect the
Last update 17JAN2024 R. solanacearum R3bv2 25

following information:

♦ Date of collection or observations.

♦ Collector’s name.

♦ Grower’s field identification numbers (grower can provide this 

information).

♦ Full name of business, institution, or agency.

♦ Full mailing address, including county.

♦ Type of property (commercial nursery, natural field, residence).

♦ GPS coordinates of the survey site.

♦ Host plant species and specific crop plant variety, if applicable.

♦ Presence or absence of the pest.

♦ Observations of signs and symptoms.

♦ Percentage of the field displaying disease symptoms.

♦ General conditions or any other relevant information.

Test symptomatic plants for the presence of R. solanacearum bacteria using 

ImmunoStrip® available from commercial providers. Positive reactions will 

indicate the presence of R. solanacearum, but will not distinguish the R3bv2 

subgroup (USDA-APHIS-PPQ-CPHST, 2018). If a sample is positive, follow the 

USDA Select Agent Process listed in Appendix B:

♦ Collect entire plants (including roots) showing wilting symptoms and 

wash the soil from them.

♦ At a minimum, enclose 0.04 ounce of symptomatic stem/crown tissue.

♦ Double bag the samples and keep cool. Do not refrigerate. Samples may 

be held at room temperature if less than or equal to 60 oF.

Tuber Sampling

Sampling is not required for fields or facilities with a direct link to positive 

testing seed lots or fields. Tubers in this category are automatically considered 

positive and are subject to control (quarantine) actions without testing.

1. Sampling for subsequent testing is required for tubers:

a. In adjacent fields.

b. On a shared water source.

c. Connected by a history of shared harvesting machinery.

2. Follow these guidelines for sample collection:

a. Samples include potato tubers, stems from potato or other host 

plants, and seed pieces (Elphinstone et al., 2018).

b. Repeat the sampling for each field, warehouse or storage unit at a 

given location.
Last update 17JAN2024 R. solanacearum R3bv2 26

c. Collect a maximum of 200 samples from each location 

(Elphinstone et al., 2018).

d. Collect samples throughout the entire building or storage area.

e. If tubers are in bags, collect samples from each bag.

f. Double bag the samples and keep cool. Do not refrigerate. 

Samples may be held at room temperature if less than or equal to 

60 oF.

g. Prepare samples within 72 hours of collecting the sample.

Tomato and Other Solanaceous Hosts

1. Collect entire plants showing wilting symptoms, including roots, and wash 

them free of soil. Bare root plants are ideal. Since the pathogen is 

concentrated in the lower stem, leaf and partial stem samples are inadequate 

for testing.

a. Double bag the samples and keep cool. Do not refrigerate. Samples 

may be held at room temperature if less than or equal to 60 oF.

b. Submit entire plants, not sub‐samples. Samples must include lower 

stems with the leaves and petioles removed. Samples that are dead or 

fermented upon arrival cannot be tested and will be rejected.

Sampling of Water Sources

1. Test irrigation and water sources near positive fields or processing plants to 

limit the bacterium’s spread. At present, there is no test that can be used in the 

field. Therefore, all water samples must be sent to a diagnostics lab.

2. When taking samples:

a. Sterilize bottles.

b. Collect samples of approximately 0.50 quarts.

c. Collect water samples at a depth of 12 inches.

d. Keep samples cool and in a dark location and perform tests within 24 

hours of collection.

e. For best results, conduct sampling when water temperatures exceed 59 

oF and populations of bacterium are highest in water( Jones et al., 

2017).

Sample Shipping

See Appendix B for USDA-APHIS procedures for submitting domestically 

detected suspect R. solanacearum positives based on immunostrip testing.
Last update 17JAN2024 R. solanacearum R3bv2 27

Timing of Surveys

In general, survey for this pathogen should take place during the day when 

temperatures are the hottest and symptoms of wilt, if present, are most obvious.
Last update 17JAN2024 R. solanacearum R3bv2 28

Eradication and Control 

Options

Overview

This information can be used by PPQ decision-makers after a detection to assess 

the suitability of potential actions to eradicate, contain, or suppress R. 

solanacearum R3bv2. Although eradication should always be prioritized, its 

success will depend on invasion factors unique to this pathogen.

Eradication Options

Quarantine and Regulatory Procedures

Remove all suspected or potentially infected plants (i.e., potato, tomato, 

geranium, and weeds (to include aquatic)) (see Host Removal).

Dispose of all host plant material on any property that tests positive or has

positive‐associated fields, including cull piles and other plant debris.

Leave the field fallow for two years and irrigate to promote volunteer sprouting. 

During the two years, sample, test, cull and bury under the volunteer crop during 

the growing season every four weeks to eliminate host material. Spray weed hosts 

in the field and along the edges with efficacious labeled herbicides to eliminate 

them from the area.

Following the two-year period, plant fields with non‐host crops; irrigate to 

promote volunteer potato sprouting, and cull and bury any volunteer potatoes as 

sprouting occurs. Test fields semi‐annually for four years after an initial positive 

find.

No seed production can occur in the field for at least five years after detection of

R. solanacearum R3bv2. Sample fields with susceptible hosts (potatoes or 

tomatoes) for two seasons prior to any new seed production on the property.

Fields adjacent to positive testing or associated fields, or those on a shared water

Chapter

5
Last update 17JAN2024 R. solanacearum R3bv2 29

supply may not grow host crops nor seed potatoes for two years. Any volunteer 

potato or weed hosts must be tested and destroyed. Maintain strict sanitation of all 

vehicles entering and leaving infested fields.

Quarantine storage facilities on properties with positive testing tubers until all 

potentially infested tubers are tested and found to be negative or destroyed. Clean 

and disinfect storage facilities with approved disinfectants in strict accordance 

with labeling.

Host Removal

Place all potentially infected plants and planting material, as determined by the 

delimiting survey, in double plastic bags and seal for disposal or destruction. If 

large inventories must be destroyed, then use of plastic bags may not be 

reasonable. Alternatives include a dumpster with double layers of plastic lining 

that can be folded over the top and sealed to prevent debris from escaping during 

transport or storage. Contact your regional PPQ office.

Incineration and steam sterilization are the preferred methods of disposal; 

however, these options may not be practical for large amounts of waste. In these 

cases, use an approved landfill.

Incinerate all plant material and media to the point of ash. If plastic pots are not 

accepted at the incinerator, remove them and properly disinfect or send to an 

approved landfill (see below).

Soil fumigation with an approved fumigant—such as methyl bromide or metam

sodium—at the labeled rate will greatly reduce populations of the bacteria in 

infested fields. However, fields with plant residue including tubers, roots and 

stems may still harbor R. solanacearum R3bv2 for up to two years. Before 

fumigation, remove and destroy as much host plant material from the fields as 

possible.

Fumigation of the soil may not totally eliminate the pathogen due to the presence 

of bacterial reservoirs in buried plant residue at the time of fumigation. Continue 

monitoring the soil for the presence of R. solanacearum R3bv2. Select agent 

regulations need to be followed if R. solanacearum is detected.

If an approved landfill is used, bury the double bagged material from nurseries or 

potato cropping systems under at least 6.6 feet or more of soil (Ebbels, 2003).
Last update 17JAN2024 R. solanacearum R3bv2 30

Alternative Control Techniques

Chemical Control

Managing R. solanacearum in the field is very difficult due to strain variation, 

broad host range and lack of adequate chemical treatments (Jones et al., 2017). 

Listed below are three chemical control options shown to be effective against R. 

solanacearum R3bv2 experimentally, but they have not been tested on a larger 

scale.

DL-3-aminobutyric acid (BABA) is an abiotic compound reported to induce 

resistance in tomato plants. When applied by soil drenching to tomato plants at

0.05 quarts per pot, it reduced the vascular browning index by 69.9% and leaf 

wilting index by 75.3% (Hassan and Abo-Elyousr, 2013).

Stable bleaching powder (SBP) applied two weeks before planting at a rate of

22.3 pounds per acre suppressed R. solanacearum R3bv2 by 76.94% and 88.89% 

for tuber infection and 66.96% and 71.87% for plant infection in greenhouse and 

field studies, respectively (Dhital et al., 1997).

During field trials on ginger, Mao et al. (2014) tested for alternatives to methyl 

bromide against infection with a phylotype I (Asian) R. solanacearum strain. 

Chloropicrin was injected at 1.47 ounces per yard2 and covered with either a 

0.002-inch polyethylene film (PE) or a 0.002-inch totally impermeable film (TIF), 

while methyl bromide was applied at a rate of 1.18 ounce per yard2 in situ under a 

PE sheet. Both treatments of chloropicrin produced similar efficacies to, or in 

some cases slightly lower than, methyl bromide in terms of controlling R. 

solanacearum R4bv4 infection.

Labeling

Although a proposed formulation may be approved by APHIS as an effective 

eradication or chemical control program, it may not be labeled at the time of 

pest detection for the specific use site or rate required. If a formulation is not 

labeled for the necessary use, there may be several options. One can request a 

quarantine exemption from the EPA under section 18 of FIFRA, or a Section 

24c from the state where the product will be applied. The prescribed 

formulation must be labeled both for use on the site at which it is to be applied 

and at the desired rate and must be registered for use in the state in which the 

eradication program is occurring. All applicable label directions must be 

followed, including but not limited to requirements for personal protection 

equipment, maximum treatment rates, storage and disposal.
Last update 17JAN2024 R. solanacearum R3bv2 31

Cultural Control and Sanitary Measures

Cultural practices and phytosanitation are the best control measures for R. 

solanacearum R3bv2 in the field( Jones et al., 2017).

Crop rotation every two or three years provides the best control in areas where R. 

solanacearum is already established and widespread (Jones et al., 2017). A twoseason rotation with bean and cereal crops in potato fields in East Africa reduced

R. solanacearum to less than 50% compared to the controlled monocrop which 

was higher than 80% (Lemaga et al., 2005).

Any contaminated irrigation water and wastewater from potato processing 

facilities must be disinfected. Adding 100 ppm of hydrogen peroxide to irrigation 

water can be sufficient to eliminate R. solanacearum (van Bueningen et al., 2005). 

A similar study conducted by Yao et al. (2010) using 1.3 ppm chlorine dioxide 

obtained >99% efficacy in inhibiting growth of R. solanacearum.

Other management techniques that can be used to prevent introduction and spread 

of R. solanacearum R3bv2 include:

♦ Plant healthy (certified) seed potatoes and propagative material in 

pathogen free soil (Janse, 2012; Jones et al., 2017).

♦ Use cover crops to reduce weeds and possibly nematodes (Momol et al., 

2005).

♦ Use well-drained and leveled fields (Momol et al., 2005).

♦ Important: Use strict sanitation practices with equipment, tools, 

transplanting, irrigation water, storage facilities, packing materials, etc. 

(Janse, 2012; Momol et al., 2005) (see Survey Preparation, Sanitization

and Clean-Up).

♦ Plow under crop residue immediately following harvest (Momol et al., 

2005).

♦ Store and grade potatoes in the original place of production to avoid 

spreading R. solanacearum R3bv2 (Janse, 2012).

♦ Avoid use of ebb-and-flow and flooding irrigation systems in greenhouses 

(Jones et al., 2017).
Last update 17JAN2024 R. solanacearum R3bv2 32

Research Needs

New technology, research or assessment is needed to:

♦ Improve the sensitivity and reliability of detection methods for R3bv2, 

especially methods that can distinguish R3bv2 strains from other R. 

solanacearum strains.

♦ Develop resistant plant varieties that are commercially viable.

♦ Develop and/or screen additional chemical control products.

♦ Study the effects of cover crops, crop rotation and mulches on pathogen 

dynamics and disease incidence.

♦ Determine which chloropicrin treatment provides better results.

Chapter

6
Last update 17JAN2024 R. solanacearum R3bv2 33

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Environmental Compliance

Introduction

Overview

Use Environmental Compliance as a guide to environmental regulations pertinent 

to Ralstonia solanacearum R3bv2.

Program managers of Federal emergency response or domestic pest control 

programs must ensure that their programs comply with all Federal Acts and 

Executive Orders pertaining to the environment, as applicable. Two primary 

Federal Acts, the National Environmental Policy Act (NEPA) and the Endangered 

Species Act (ESA), often require the development of significant documentation 

before program actions may commence. Environmental and Risk Analysis 

Services (ERAS), a unit of APHIS’ Policy and Program Development Staff 

(PPD), is available to provide guidance and advice to program managers and 

prepare drafts of applicable environmental documentation. In preparing draft 

NEPA documentation ERAS may also perform and incorporate assessments that 

pertain to other Acts and Executive Orders, described below, as part of the NEPA 

process. The Environmental Compliance Team (ECT), a part of PPQ’s 

Emergency Domestic Programs (EDP), assists ERAS in development of 

documents and implements any environmental monitoring. Program leadership is 

strongly advised to consult with ERAS and/or ECT early in the development of a 

program in order to conduct a preliminary review of applicable environmental 

statutes and to ensure timely compliance.

Environmental monitoring of APHIS pest control activities may be required as 

part of compliance with environmental statutes, as requested by program 

managers, or as suggested to address concerns with controversial activities.

Monitoring may be conducted with regards to worker exposure, pesticide quality 

assurance and control, off-site chemical deposition, or program efficacy. Different 

tools and techniques are used depending on the monitoring goals and control 

techniques used in the program. Staff from ECT will work with the program 

manager to develop an environmental monitoring plan, conduct training to 

implement the plan, provide day-to-day guidance on monitoring, and provide an

Appendix

A
Last update 17JAN2024 R. solanacearum R3bv2 43

interpretive report of monitoring activities.

The following is list of pertinent laws and Executive Orders:

National Environmental Policy Act (NEPA) – NEPA requires all Federal 

agencies to examine whether their actions may significantly affect the quality of 

the human environment. The purpose of NEPA is to inform the decision-maker 

prior to taking action and to inform the public of the decision. Actions that are 

excluded from this examination, actions that normally require an Environmental 

Assessment, and actions that normally require Environmental Impact Statements 

are codified in APHIS’ NEPA Implementing Procedures located in 7 CFR 372.5.

The three types of NEPA documentation are:

1. Categorical Exclusion

Categorical exclusions are classes of actions that do not have a significant effect 

on the quality of the human environment and for which neither an environmental 

assessment (EA) nor an environmental impact statement (EIS) is required. 

Generally, the means through which adverse environmental impacts may be 

avoided or minimized have actually been built into the actions themselves (see 7 

CFR 372.5(c)).

2. Environmental Assessment (EA)

An EA is a public document that succinctly presents information and analysis for

the decision-maker of the proposed action. An EA can lead to the preparation of 

an environmental impact statement (EIS), a finding of no significant impact 

(FONSI), or the abandonment of a proposed action.

3. Environmental Impact Statement (EIS)

In the event that a major Federal action may significantly affect the quality of the 

human environment (adverse or beneficial), or, the proposed action may result in 

public controversy, an EIS is prepared.

Endangered Species Act (ESA) – This statute requires that programs consider 

their potential effects on federally protected species. The ESA requires programs 

to identify protected species and their habitat in or near program areas and 

documentation of how adverse effects to these species will be avoided. The 

documentation may require review and approval by the U.S. Fish and Wildlife 

Service and the National Marine Fisheries Service before program activities can 

begin. Knowingly violating this law can lead to criminal charges against 

individual staff members and program managers.

Migratory Bird Treaty Act – This statute requires that programs avoid harm to
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over 800 endemic bird species, eggs, and their nests. In some cases, permits may 

be available to capture birds, which require coordination with the U.S. Fish and 

Wildlife Service.

Clean Water Act – This statute requires various permits for work in wetlands

and for potential discharges of program chemicals into water. This may require 

coordination with the Environmental Protection Agency, individual states, and the 

Army Corps of Engineers. Such permits would be required even if the pesticide 

label allows for direct application to water.

Tribal consultation – This Executive Order requires formal government to 

government communication and interaction if a program might have substantial 

direct effects on any federally-recognized Indian Nation. This process is often 

incorrectly included as part of the NEPA process, but must be completed prior to 

general public involvement under NEPA. Staff should be cognizant of the conflict 

that could arise when proposed federal actions intersect with tribal sovereignty.

Tribal consultation is designed to identify and avoid such potential conflict.

National Historic Preservation Act – This statute requires programs to consider 

potential impacts on historic properties (such as buildings and archaeological 

sites) and requires coordination with local State Historic Preservation Offices.

Documentation under this act involves inventorying the project area for historic 

properties and determining what effects, if any, the project may have on historic 

properties. This process may require public involvement and comment prior to the 

start of program activities.

Coastal Zone Management Act – This statute requires coordination with states 

where programs may impact Coastal Zone Management Plans. Federal activities 

that may affect coastal resources are evaluated through a process called “federal 

consistency”. This process allows the public, local governments, Tribes, and state 

agencies an opportunity to review the federal action. The federal consistency 

process is administered individually by states with Coastal Zone Management 

Plans.

Environmental Justice – This Executive Order requires consideration of 

program impacts on minority and economically disadvantaged populations. 

Compliance is usually achieved within the NEPA documentation for a project. 

Programs are required to given consider if the actions might disproportionally 

impact minority or economically disadvantaged populations, and if so, how such 

impact will be avoided.

Protection of Children –This Executive Order requires federal agencies to 

identify, assess, and address environmental health risks and safety risks that may 

disproportionately affect children. If such a risk is identified, then measures must
Last update 17JAN2024 R. solanacearum R3bv2 45

be described and implemented to minimize such risks.
Last update 17JAN2024 R. solanacearum R3bv2 46

USDA–APHIS Procedures 

for Submitting Samples

Symptomatic, i.e., wilting, plants can be tested for the presence of Ralstonia 

solanacearum bacteria using immunostrips available from commercial providers 

listed below, or by ELISA testing. Positive reactions will only indicate the 

presence of R. solanacearum, but not what race is present. R. solanacearum 

R3bv2 is considered a federal select agent and is not known to occur in the United 

States. R. solanacearum race 1 is considered endemic to the southern United 

States and is not a federal quarantine pest or select agent.

The following commercially available immunostrips are approved by APHIS for 

field level screening:

1. https://orders.agdia.com/agdia-immunostrip-for-rs-isk-33900, AgDia, Inc. 

Elkhart,IN

2. Potato Brown Rot Pocket TM Pocket Diagnostic, from Forsite Diagnostics 

Ltd, York, UK

Because of changes in the Federal Select Agent Program (FSAP) regulations, all 

races and biovars are considered select agents until proven otherwise. Once a 

sample is determined to be positive for R. solanacearum, the sample must be 

handled according to FSAP’s regulations. There is a form from the Division of 

Agricultural Select Agents and Toxins (DASAT), which is necessary to use 

depending on your course of action to A) destroy the sample or B) transfer the 

sample to a registered entity for further testing. The appropriate form needs to be 

completed and approved by DASAT. See below which provides the specific 

requirements on how to handle R. solanacearum positive samples and forms.

Here are links to the forms: APHIS/CDC Form 4a info is here for Reporting the 

Identification of a Select Agent or Toxin from a Clinical/Diagnostic Specimen and

Guidance on How to Complete APHIS/CDC Form 4:

https://www.selectagents.gov/form4.html

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.

Appendix

B
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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:

A. 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. No 

further action required.

B. Samples fitting the following criteria will require further testing at 

PPCDL, approved for confirmation and select agent handling:

1) Positive testing (R. solanacearum) samples with a connection to a 

foreign source (i.e., imported plants)

♦ Forward the sample(s) to the USDA–APHIS–PPQ– S&T–PPCDL 

to be tested for R. solanacearum R3bv2. 

♦ Refer to the “Shipping instructions for samples requiring

further testing at an approved laboratory” section for 

instructions.

♦ Follow the select agent regulations listed below. 

a. Contact PPCDL to inform them of the sample and receive 

guidance through the APHIS/CDC reporting process.

b. Send an APHIS/CDC Form 4a (Report of Identification) to 

DASAT. DASAT approval with an approved Form 2 

(Request to Transfer) from the receiving lab is required 

before sample shipping.

c. PPCDL will initiate a Form 2 (Request to Transfer) Section 1. 

Once completed, Section 2 will be sent to the submitter by 

DASAT for completion. After the Form 4a (Report of 

Identification) and Form 2 (Request to Transfer) Sections 1 and 2 

are approved, the sample may be shipped.

d. If positive for R3bv2, PPCDL will submit an APHIS/CDC Form 

4a (Report of Identification) to DASAT.
Last update 17JAN2024 R. solanacearum R3bv2 48

e. Sample destroyed at both diagnostic and confirmatory labs.

f. Sample retained:

i. If registered for agent, notify the DASAT of retention and 

put in inventory.

2) For other domestic detections of R. solanacearum, forward samples 

to PPCDL from unusual hosts, unique symptomatic reactions, and/or 

from areas in the country where R. solanacearum is not known to 

occur. The usual cultivated hosts of R. solanacearum race 3 biovar 2 

include eggplant, geranium, pepper, potato, and tomato.

a. Refer to the “Shipping instructions for samples requiring further

testing at an approved laboratory” section for instructions.

b. Contact PPCDL to inform them of the sample and receive 

guidance through the APHIS/CDC reporting process.

c. Send an APHIS/CDC Form 4a (Report of Identification) to 

DASAT. DASAT approval with an approved Form 2 (Request to 

Transfer) from the receiving lab is required before sample 

shipping.

d. PPCDL will initiate a Form 2 (Request to Transfer) Section 1. 

Once completed, Section 2 will be sent to the submitter by 

DASAT for completion. After the Form 4a (Report of 

Identification) and in the Form 2 (Request to Transfer) sections 1 

and 2 are approved, the sample may be shipped.

e. If positive for R3bv2, PPCDL will submit an APHIS/CDC Form 

4a (Report of Identification) to DASAT.

f. Sample destroyed at both diagnostic and confirmatory labs.

g. Sample retained:

i. If registered for the agent, notify the DASAT of retention 

and put in inventory.

For samples being destroyed, meeting the stated criteria:

♦ If identified as R. solanacearum, and the plant is not associated with a foreign 

source, not an unusual host, and from an area of thecountry where the organism is 

known to occur, no further race/biovar determination is required at an approved 

lab.

1. Complete an APHIS/CDC Form 4a (Report of Identification).

2. Send Form 4a to DASAT by email, fax or mail as indicated on the form.

3. Destroy the sample.

4. No further action is required (destruction records for the pathogen must be 

kept and maintained for a minimum of three years).
Last update 17JAN2024 R. solanacearum R3bv2 49

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:

1. Contact PPCDL at APHISPPQCPHSTBeltsvilleSampleDiagnostics@usda.gov to inform 

them of the sample and receive guidance on sample submission.

2. Complete sections A & B of the APHIS/CDC Form 4a (Report of 

Identification) and send to DASAT@usda.gov.

3. PPCDL will complete Form 2 (Request to Transfer) Section 1.

4. Complete Form 2 (Request to Transfer) Section 2 and send to 

DASAT@usda.gov.

5. Coordinate with PPQ to generate an Agriculture Risk Management 

(ARM) Diagnostic Request (DR) Routing Form.

6. Complete a PPQ Form 391.

7. Send the sample, a copy of the approved Form 2, ARM DR, and 

PPQ Form 391 in the shipment to PPCDL.

8. PPCDL, will receive the sample, complete the APHIS/CDC Form 2 

Section 3, and send the completed paperwork to DASAT.

9. 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.

10. 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:
Last update 17JAN2024 R. solanacearum R3bv2 50

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

stephen.w.bullington@usda.gov

Aimee Hyten 

Plant Pathologist

Division of Agricultural Select 

Agents and Toxins

4700 River Rd. Unit 2

Riverdale, MD 20737

301-851-3300, option 3 

DASAT@usda.gov
Last update 17JAN2024 R. solanacearum R3bv2 51

8 Authors and Reviewers

Author

Jennifer C. Cook, Ph.D., NCSU–CIPM

Editor

Rosemary Hallberg, M.A., NCSU–CIPM

Reviewer

Maria Acevedo-Sanchez, USDA–APHIS–PPQ

Caitilyn Allen, Ph.D., University of Wisconsin-Madison 

Damon Crook, Ph.D., USDA–APHIS–PPQ

David Bednar, Ph.D., USDA–APHIS–PPQ 

Kathryn E. Bronsky, USDA–APHIS–PPQ 

Peter Hertl, Ph.D., USDA–APHIS–PPQ 

Julie F. Orr, USDA–APHIS–PPQ

Jaap van Kretschmar, Ph.D., NCSU–CIPM 

Ricardo Valdez, USDA–APHIS–PPQ

Contributors

Global Pest and Disease Database Team

R.

solanacearum

R3bv2