North Carolina State University
Ralstonia solanacearum
Pathogen profile created by Heather A. Olson
Requirement for PP 728 Soilborne Plant Pathogens, Spring 2005
Department of Plant Pathology


Introduction

        Ralstonia solanacearum (Smith) Yabuuchi et al. (formerly called Pseudomonas solanacearum), is a soilborne bacterial pathogen that is a major limiting factor in the production of many crop plants around the world.  This organism is the causal agent of brown rot of potato, bacterial wilt or southern wilt of tomato, tobacco, eggplant, and some ornamentals, and Moko disease of banana (1, 8). 


Host Range and Distribution

Ralstonia solanacearum is a widely distributed pathogen found in tropical, subtropical, and some temperate regions of the world (3).  The species as a whole has a very broad host range and infects hundreds of species in many plant families.  The majority of hosts are dicots with the major exception being bananas and plantains.  Most economically important host plants are found in the Solanaceae or nightshade family (8).

Specific host range and distribution of R. solanacearum depends on the race and to some degree the biovar of the pathogen (2).  These host ranges and distributions have been changing in recent years.  Race 1 is endemic to the southern United States.  Race 3 Biovar 2 is a USDA “select agent” listed on the Agricultural Bioterrorism Act of 2002 and is subject to regulatory actions including strict quarantines since potato is extremely sensitive to this race (5).  Table 1 illustrates the relationship of race, biovar, host range, and geographic distribution.


Isolation

Kelman’s tetrazolium chloride (TZC) agar or 2% sucrose peptone agar (SPA) should be used for isolation when R. solanacearum is suspected.  After two days on TZC medium, virulent wildtype colonies are large, elevated, fluidal, and either entirely white or with a pale red center; avirulent mutant colonies are butyrous, deep-red often with a bluish border.  On SPA, R. solanacearum colonies are white and fluidal with characteristic whorls (4).


Culture of virulent Ralstonia solanacearum
on TZC agar. 







(Photo courtsey of
T. Momol and S.M. Olson, University of Florida)

Direct isolation of Ralstonia solanacearum can be obtained from plant ooze and exudates.  The infected stems and/or petioles are cut using a sterile sharp knife or razor blade.  If bacterial ooze does not actively appear, the plant material is squeezed between two fingers.  A suspension of the ooze is prepared in sterile distilled water and then streaked onto either TZC or SPA plates.  Pure cultures are usually easily isolated (4).  R. solanacearum can also be isolated from water and soil using a modified Kelman's TZC medium (4).  

Isolates of R. solanacearum rapidly lose virulence when maintained on laboratory media; however, the organism can easily be maintained for years in sterile distilled water or on agar slants covered with sterile mineral oil and stored at room temperature (7).


Identification

Classification (from NCBI):

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Betaproteobacteria
Order: Burkholderiales
Family: Burkholderiaceae

Organism Characteristics:   

Ralstonia solanacearum is a gram-negative motile rod.  The organism grows aerobically and does not form endospores.  Cells are 0.5-0.7 X 1.5-2.0 µm and are non-encapsulated.  R. solanacearum is catalase positive, oxidase positive, and reduces nitrates.  The pathogen does not hydrolyze starch and does not readily degrade gelatin.  In broth culture, the organism is inhibited by concentrations of NaCl greater than 2% (8). 

Field Identification:

          For a quick field diagnostic identification of R. solanacearum and to distinguish bacterial wilt from vascular wilts caused by fungal pathogens, bacterial streaming from infected plant material can be used.  A stem section is cut from a plant with vascular discoloration using a sharp knife or razor blade.  The stem section is placed against the inside wall of a water-filled clear beaker or test tube so that the end of the section just touches the water surface.  Milky white strands containing bacteria and extracellular polysaccharide will stream from the cut ends of the xylem (7).  Quick serological tests are also now available for detection of the pathogen.

Bacterial streaming
from a cut infected
tomato stem.











(Photo courtsey of
T. Momol and S.M. Olson,
University of Florida)

Races and Biovars:

          The pathogen species is subdivided into races based on host range.  Currently, polymerase chain reaction (PCR) is the primary means of definitive identification of pathogen race.  Before PCR, a tobacco hypersensitivity test, developed by Lozano and Sequeira, was used to distinguish between Races 1, 2, and 3, the most economically important races.  The species is also subdivided into biovars based on the utilization of the disaccharides cellobiose, lactose, and maltose and oxidation of the hexose alcohols dulcitol, mannitol, and sorbitol (4).  Table 2 illustrates the classification into biovars based on this method.

Recently, it has been suggested that Ralstonia solanacearum should be considered a “species complex.”  Also, some consider the current race and biovar system inadequate since it is based on phenotypic measures and propose switching to a phylogenetically-based hierarchical system.  This scheme is based upon phylogenetic analysis of sequence information using RFLP typing, 16S rDNA phylogeny, and hrpB PCR-RFLP data.  The system divides the species complex into phylotypes (phylogenetic grouping of strains), sequevars (group of strains with endoglucanase or mutS gene sequenes diverging by <1%), and clones (group of strains exhibiting the same genomic fingerprint) (3). 


Symptoms

Tomato
Tobacco
Geranium
Potato
Banana






Ecology and Life Cycle

High temperatures and high soil moisture generally favors Ralstonia solanacearum, the exception being certain Race 3 strains that are pathogenic on potato and are able to grow well at lower temperatures (8).  The pathogen is found in many different soil types and over a wide range of soil pH.  The organism survives in infected plant material, vegetative propagative organs, wild host plants, and soil.  Alternate hosts, especially latently infected weed species, are thought to play a major role in the overwintering ability of the organism in temperate regions (5, 8).  Bittersweet nightshade (Solanum dulcamara), a common aquatic weed, has been implicated as the source of several brown rot outbreaks (8).

 Sources of inoculum for agricultural fields and methods of spread include irrigation and surface water, aquatic weeds, infested soil and field weeds, contaminated planting material, latently infected vegetative propagative material, and contaminated farm tools and equipment.  Once established in a field, plant-to-plant spread may occur when bacteria move from roots of infected plants to roots of healthy plants (5, 8). 

Potential Sources of Inoculum
(Photos courtsey of T. Momol and S.M. Olson,
University of Florida)


Contaminated irrigation pond and equipment

Infested soil

Latently infected weeds in and around fields
Contaminated farm equipment and/or
planting material




R. solanacearum enters the plant through wounds in the roots from cultivating equipment, nematodes, insects, and through cracks where secondary roots emerge (1).  The bacteria reach the large xylem elements and are spread into the plant, where they multiply.  Once established in the xylem vessels, the bacteria are able to enter the intercellular spaces of the parenchyma cells in the cortex and pith in various areas of the plant.  Here, R. solanacearum is able to dissolve the cell walls and create slimy pockets of bacteria and cell debris.  Production of highly polymerized polysaccharides increases the viscosity of the xylem, which results in plugging (7).


Additional Links
PPQ Pest Alert
USDA Action Plan for R2B3 found in nursery facilities
PPQ Ralstonia Data Sheet


References

1.       Agrios, G.N.  1997.  Plant Pathology, 4th Edition.  Academic Press, San Diego, CA.

2.       Daughtrey, M.  2003.  Southern bacterial wilt, caused by Ralstonia solanacearum.  Society of American Florists’ 19th Annual Conference on Insect and Disease Management on Ornamentals.

3.       Fegan, M. and P. Prior.  2004.  Recent developments in the phylogeny and classification of Ralstonia solanacearum.  Presentation at the 1st International Tomato Symposium.  Orlando, FL. 

4.       French, E.B., L. Gutarra, P. Aley, and J. Elphinstone.  1995.  Culture media for Ralstonia solanacearum isolation, identification, and maintenance.   Fitopatologia 30(3):126-130.

5.       Ji, P., T. Momol, S.M. Olson, J. Hong, P. Pradhanang, A. Narayanan, and J.B. Jones.  2004.  New Tactics for Bacterial Wilt Management on Tomatoes in the Southern US. 

6.       Jones, J.B, J.P. Jones, R.E. Stall, and T.A. Zitter, Eds.  1991.  Compendium of Tomato Diseases.  APS Press, St. Paul, MN.

7.       Shew, H.D. and G.B. Lucas, Eds.  1991.  Compendium of Tobacco Diseases.  APS Press, St. Paul, MN.

8.       Stevenson, W.R., R. Loria, G.D. Franc, and D.P. Weingartner, Eds.  2001.  Compendium of Potato Diseases, 2nd Ed.  APS Press, St. Paul, MN.



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