This standard describes a diagnostic protocol for Ralstonia solanacearum.
This standard describes a diagnostic protocol for Ralstonia solanacearum.
Approved in 2003-09.
Bacterial wilt caused by Ralstonia solanacearum was reported for the first time at the end of the 19th century on potato, tobacco, tomato and groundnut in Asia, southern USA and South America. The bacterium was described for the first time as Bacillus solanacearum by Smith (1896). In the years following, at least five pathogenic races and five biovars have been discriminated (Buddenhagen et al., 1962). Race 1 occurs in tropical areas all over the world and attacks tobacco, many other solanaceous crops and many hosts in other plant families. It has a high temperature optimum (35 °C, as do race 2, 4 and 5). Race 2 occurs mainly in tropical areas of South America and attacks bananas and Heliconia (causing so-called Moko disease), but also in the Philippines (causing so-called bugtok disease on plantains). Race 3, occurring at higher altitudes in the tropics and in subtropical and temperate areas attacks potato, tomato, occasionally Pelargonium zonale, aubergine and capsicum, some solanaceous weeds like Solanum nigrum and Solanum dulcamara. A number of non-solanaceous weed hosts have also been found to harbour race 3 infections, often asymptomatically (Pradhanang et al., 2000; Strider et al., 1981; Wenneker et al., 1999; Janse et al., 2004). This race has a lower temperature optimum (27 °C) and appears to be mostly biovar 2 A RFLP group 26 with a worldwide distribution (Cook & Sequeira, 1994), biovar 2 A RFLP group 27 (found in Chile and Colombia), or biovar 2T (sometimes also called 2 N, found in tropical areas in South America). Race 4 is particularly aggressive on ginger, race 5 (biovar 5) is specialized on Morus. Another recent classification of R. solanacearum, based on RFLP and other genetic fingerprinting studies (Hayward, 2000), is into Division I (biovars 3, 4 and 5 originating in Asia) and II (biovars 1, 2 A and 2T, originating in South America).
Within the EPPO region, the race which is now present and has potential for spread is race 3. This is the main race described in this protocol. Race 1 may be introduced with ornamental/herbal plants or plant parts of tropical origin and grown in glasshouses in temperate climates, like Curcuma longa (turmeric), Anthurium or Epipremnum. Worldwide, the most important hosts are: Arachis hypogaea (groundnut), Heliconia spp., Lycopersicon esculentum (tomato), Musa paradisiaca (banana and plantain), Nicotiana tabacum (tobacco), Solanum melongena (aubergine) and Solanum tuberosum (potato). For extensive host range, see Bradbury (1986). For host range of race 3, see Janse et al. (2004). For geographical distribution, see CABI (1999).
Name: Ralstonia solanacearum (Smith, 1896) Yabuuchi et al. (1995)
Synonyms: Pseudomonas solanacearum (Smith, 1896) Smith 1914; Burkholderia solanacearum (Smith, 1896) Yabuuchi et al. 1992; many other synonyms in Literature
Taxonomic position: Bacteria, Gracilicutes, Proteobacteriaβ subdivision. It belongs to rRNA homology group II (nonfluorescent) within the pseudomonads. R. pickettii (saprophyte or human facultative pathogen), Pseudomonas syzygii (causing Sumatra disease of cloves), and the so-called Blood Disease Bacterium (BDB, causing blood disease of banana in Indonesia) are closely related to R. solanacearum and may cross-react in serological and DNA-based detection methods. Subclassification of R. solanacearum, based on RFLP and other genetic fingerprinting studies (Hayward, 2000) is into Division I (biovars 3, 4 and 5 originating in Asia) and II (biovars 1, 2A and 2T, originating in S. America). Further taxonomic division mainly based on nucleic sequence analysis into phyllovars and sequevars has been proposed (Poussier et al., 2000; Taghavi et al., 1996)
Bayer computer code: PSDMSO (PSDMS3 for race 3)
Phytosanitary categorization: EPPO A2 list no. 58, EU Annex designation I/A2
Foliage symptoms include rapid wilting of leaves and stems (Web Fig. 3), usually first visible at the warmest time of day. Eventually, plants fail to recover, become yellow and brown necrotic and die. As the disease develops, a streaky brown discoloration of the stem may be observed on stems above the soil line, and the leaves may have a bronze tint. Epinasty of the petioles may occur. A white, slimy mass of bacteria exudes from vascular bundles, when broken or cut. This slime oozes spontaneously from the cut surface of a potato stem in the form of threads, when suspended in water. Such threads are not formed by other bacterial pathogens of potato. This test is of presumptive diagnostic value in the field. Under cool growing conditions, wilting and other foliar symptoms may not occur.
On tubers, external symptoms may or may not be visible, depending on the state of development of the disease. Symptoms may be confused with those of ring rot due to Clavibacter michiganensis subsp. sepedonicus (EPPO/CABI, 1997). R. solanacearum can be distinguished by the bacterial ooze that often emerges from the eyes and stolon-end attachment of infected tubers. Soil may adhere to the tubers at the eyes (Web Fig. 4). Cutting the diseased tuber will reveal a browning and eventual necrosis of the vascular ring and immediately surrounding tissues. A creamy fluid exudate usually appears spontaneously on the vascular ring of the cut surface a few minutes after cutting (Web Fig. 5). In the case of ring rot, the tuber has to be squeezed in order to press out a mass of yellowish macerated vascular tissue and bacterial slime. Atypical symptoms have been described on potato (necrotic spots on the epidermis), possibly caused after lenticel infection. Plants with foliar symptoms caused by R. solanacearum may bear healthy and diseased tubers, while plants that show no signs of the disease may sometimes produce diseased tubers.
The youngest leaves are the first to be affected and have a flaccid appearance, usually at the warmest time of day. Wilting of the whole plant may follow rapidly if environmental conditions are favourable for the pathogen. Under less favourable conditions, the disease develops less rapidly, stunting may occur and large numbers of adventitious roots are produced on the stem. The vascular tissues of the stem show a brown discoloration and, if the stem is cut crosswise, drops of white or yellowish bacterial ooze may be visible.
First symptoms are wilting and subsequent chlorosis (often sectorial yellowing) of leaves. Stems may blacken and eventually become necrotic. Internal vascular browning is often visible. In a later stage, leaves become brown necrotic and the whole plant desiccates and dies. In final stages, plants collapse totally.
One of the main symptoms is unilateral wilting and premature yellowing. Leaves on one side of the plant or even a half leaf may show wilting symptoms. In severe cases, leaves wilt without changing colour and stay attached to the stem. As in tomato, the vascular tissues show a brown discoloration when cut open. The primary and secondary roots may become brown to black.
Moko disease, caused by R. solanacearum, is easily confused with Panama disease caused by Fusarium oxysporum f.sp. cubense. A clear distinction is possible when fruits are affected: brown dry rot is seen only in the case of Moko disease. On young and fast-growing plants, the youngest leaves turn pale-green or yellow and collapse. Within a week, all leaves may collapse. Young suckers may be blackened, stunted or twisted. The pseudostems show brown vascular discoloration.
R. solanacearum may easily occur in a latent form and in temperate European countries infected but symptomless Solanum dulcamara growing along waterways with its roots in surface water is an important factor in epidemiology (Elphinstone et al., 1998; Janse et al., 1998; Wenneker et al., 1999). Several selective media for isolation from latently infected material or difficult substrates like soil, waste, or surface water have been described. SMSA medium as modified by Elphinstone et al. (1996) has been used successfully in Europe (e.g. Elphinstone et al., 1998; Wenneker et al., 1999). Isolation from symptomatic material can easily be performed using YPGA non-selective medium or Kelman's tetrazolium medium. In some cases when secondary infections are present, isolation on selective media is necessary. A presumptive test in the field can be the water-streaming test as described under disease symptoms or a serological agglutination test using a field kit in the form of a lateral flow device (Danks & Barker, 2000). Many standard methods for detection (of latent infection), identification and preparation of media for R. solanacearum, used in official EU testing schemes, can be found in EU (1998), Lelliott & Stead (1987) and OEPP/EPPO (1990). Detection of latent infection is by performing an immuno-fluorescence test and/or selective plating on SMSA medium eventually combined with optional PCR assays, ELISA or fluorescent in situ hybridization tests which can be performed following enrichment procedures for added sensitivity. Details of these methods and some recently developed techniques can be found in Boudazin et al. (1999); Caruso et al. (2002); Pastrik & Maiss (2000); Pastrik et al. (2002); Seal et al. (1993); Weller et al. (2000); Wullings et al., 1998). Standard samples should consist of 200 tubers per 25 tons. Many test methods have been standardized and ring-tested in EU Member States in an EU project (SMT CT97-2197) in which 20 laboratories co-operated. Figures 1 and 2 provide flow schemes for detection (of latent infection also) and identification. Correct application of these methods may require training from one of the laboratories involved in the above-mentioned project (EU, 1998; Elphinstone et al., 2000).
Gram-negative rods with a polar tuft of flagella, non-fluorescent but diffusible brown pigment often produced. Polyhydroxybutyrate (PHB) is accumulated as cellular reserve and can be detected by Sudan Black staining on nutrient-rich media or the Nile Blue test, also in smears from infected tissues (EU, 1998); levan not formed from sucrose; gelatin hydrolysis negative or weak; starch and esculin not hydrolysed, nitrate reduced by nearly all strains, many produce gas (denitrification), oxidative metabolism of glucose only, no growth at 4° or 40 °C; growth is weak at pH 8 with no growth at pH 4 or 9; oxidase and catalase positive; arginine dihydrolase, lecithinase (egg yolk) and lipase (Tween 80) negative. Most strains produce tyrosinase, the main exceptions being those isolated from the family Musaceae. Growth in 1% NaCl broth but little or no growth in 2% NaCl. This species belongs to rRNA group II. It is readily distinguished from other members of the group by failure to grow at 40 °C (Table 1). For carbon compounds utilized, see Bradbury (1986), Saddler (1994). Table 1 provides discriminating characters for R. solanacearum and some related plant pathogens, Tables 2 and 3 for biovars, and Table 4 for races. Media used are specified in EU (1998). Methods for identification using IF, ELISA, PCR, fatty-acid analysis, FISH, RFLP, REP-PCR and AFLP fingerprinting have been described in detail or sources are indicated in EU (1998) and are described in Janse (1991); Smith et al. (1998); Poussier et al. (1999); Poussier & Luisetti (2000); Poussier et al. (2000); van der Wolf et al. (1998).
|Test||Ralstonia solanacearum||Burkholderia cepacia||Burkholderia gladioli||Burkholderia caryophylli||Pseudomonas corrugata||Acidovorax avenae|
|Growth at 41 °C||–||V||V||+||–||+|
|Glycerol||+ W||–||–||+ W|
|Tobacco cv. White Burley plants (stem inoculation)||Wilting||No reaction||No reaction|
|Tobacco cv. White Burley leaves (hypersensitivity test)||Necrosis (48 h) and wilting (7–8 days)||HR (12–24 h)||Chlorosis (2–8 days)|
|Musa acuminata||No reaction||Wilting||No reaction|
Pathogenicity can be determined by inoculating a suspension (106 cell mL−1) of a 48-h nutrient agar/YPGA culture into 5–10 susceptible tomato (e.g. cv Moneymaker) or aubergine (e.g. cv Black Beauty) plants at, preferably, the third true leaf stage or slightly older. Incubation should be for up to two weeks at 25–28 °C under high relative humidity conditions. Symptoms are wilting and/or epinasty, chlorosis, sometimes only stunting. The bacterium should be reisolated from plants by taking a stem or petiole section above the inoculation point and placing it in a small volume of sterile distilled water or 50 mm phosphate buffer, plating on YPGA and/or SMSA medium, and observing for typical colonies (EU, 1998). Colonies can be used for confirmation by IF, PCR or FISH. For race determination, also tobacco (e.g. cv. White Burley) and Musa acuminata can be inoculated and incubated as described above. Symptom development in M. acuminata may take longer than two weeks.
Reference strains at the National Collection of Plant Pathogenic Bacteria, CSL, York (GB) are: UK NCPPB 325 (Type strain, biovar 1); NCPPB 4156 (biovar 2), NCPPB 3996 (biovar 3), NCPPB 4029 (biovar 4), NCPPB 4011 (biovar 5).
For discrimination of R. solanacearum from some related plant pathogenic species, see Table 1. Symptoms of bacterial wilt/brown rot caused by R. solanacearum may be confused with other wilting symptoms caused by, e.g. Fusarium spp., Verticillium spp., Erwinia chrysanthemi and Clavibacter michiganensis subsp. sepedonicus (see under Symptoms). The water-streaming test, described under symptoms, and the observation of spontaneous slime drop formation after cutting tubers/rhizomes are often of good value for a preliminary diagnosis. Serological field kits, which incorporate highly specific monoclonal antibodies, are commercially available (e.g. from CSL) for rapid on-site diagnosis. Definitive diagnosis should always be obtained through laboratory tests on a pure culture.
The procedures for detection and identification described in this protocol, and the decision scheme in Fig. 1 or 2, should have been followed. When the organism is diagnosed for the first time, or in critical (import/export) cases, the following are required:
A report on the execution of the protocol should include:
The original sample (with labels, if applicable), sample extract, IF-slide and pure culture should be kept under proper conditions (for at least two months) with correct registration and should be available in case of doubt.
Further information on this organism can be obtained from: Department Bacteriology, Plant Protection Service, PO Box 9102, 6700 HC, Wageningen, the Netherlands, Fax 0031-317 421701, E-mail: firstname.lastname@example.org; Plant and Environmental Bacteriology Team, Plant Health Group, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK
The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org.
This protocol was originally drafted by: J.D. Janse, Plant Protection Service, Wageningen (NL).