Bulletin OEPP/EPPO Bulletin (2018) 48 (1), 32–63 ISSN 0250-8052. DOI: 10.1111/epp.12454

European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/21 (2)

Diagnostics Diagnostic

PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex)

Specific scope It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. This Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Specific approval and amendment Ralstonia solanacearum Species Complex (RSSC).1 Approved in 2003-09. First revised in 2018-02.

origin, whereas Phylotype III strains have evolved in the 1. Introduction African highlands and Phylotype IV strains in Indonesia. Ralstonia solanacearum (Smith) Yabuuchi et al. is included Their importance is compounded by the large range of in the EPPO A2 List of pests recommended for regulation economically important hosts, which include S. tuberosum, and in many EPPO members’ lists of regulated pests. It has S. lycopersicum, Solanum melongena (eggplant), Musa been described by Fegan & Prior (2005) as distributing into spp. (banana and plantain), Nicotiana tabacum (tobacco) four phylotypes in a species complex. Each phylotype com- and many ornamental plants. One genotype assigned to prises multiple phylogenetic and pathogenic variants differ- Phylotype IIB sequevar 1 (PIIB-1), formerly referred to as ing in barcoding genes (including ITS, hrpB, mutS and egl), race 3 biovar 2 and known as the causal agent of potato known as sequevars. Historically, the species complex has brown rot, is of particular importance to the EPPO region, been recognized as a number of phenotypically diverse having spread from South America with the movement of strains that were originally placed into five pathogenic races infected seed potato around the world. This genotype has and five biovars (Buddenhagen et al., 1962; Hayward, 1964). a lower optimum growth temperature (approximately Recently, it has been reclassified by Safni et al. (2014) into 27°C) and can cause wilting at lower temperatures than three distinct species: R. solanacearum (Phylotype II), most other genotypes, which have an optimal growth tem- Ralstonia pseudosolanacearum (Phylotype I and III) and perature of up to 35°C. It often occurs in latent (symp- Ralstonia syzygii (Phylotype IV). Ralstonia syzygii com- tomless) infections at high altitudes in the tropics and in prises 3 subspecies: subsp. syzygii found on clove, subsp. subtropical and temperate areas. This genotype is already celebesensis found on banana and subsp. indonesiensis found present in the EPPO region, usually with few occurrences on Solanum tuberosum (potato), Solanum lycopersicum or restricted distribution, but has the potential to spread. It (tomato), Capsicum annuum (chilli pepper) and Syzygium mostly attacks S. tuberosum, S. lycopersicum, occasionally aromaticum (clove). These three species are covered by this S. melongena and C. annuum, as well as some solana- diagnostic protocol under the taxonomy of phylotype/seque- ceous weeds such as Solanum nigrum (black nightshade) var, which clearly separate Phylotype I and III strains and and Solanum dulcamara (woody nightshade). In EPPO other epidemiologically distinct strains in Phylotype II (caus- temperate countries, symptomless infection of ing potato brown rot or Moko disease). S. dulcamara growing with roots in surface water is an Phylotype I strains are referenced to be of Asian origin, important factor in disease epidemiology (Elphinstone Phylotype II strains are referenced as of South American et al., 1998; Janse et al., 1998; Wenneker et al., 1999). It has also occasionally been found on non-solanaceaous 1Use of brand names of chemicals or equipment in these EPPO Stan- hosts, including Pelargonium zonale (horse-shoe pelargo- dards implies no approval of them to the exclusion of others that may nium), and has spread from Africa and Central America also be suitable. via the international trade in young plants (Strider et al.,

32 ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 33

1981; Janse et al., 2002). The reported host range of Taxonomic position: Bacteria, Gracilicutes, Proteobacteria PIIB-12 is listed in the EPPO Global Database (EPPO, b subdivision, Burkholderiales, Ralstoniaceae4 2015). EPPO Code: RALSSO Genotypes within Phylotype II sequevars IIA-6, IIA-24, Phytosanitary categorization: EPPO A2 List no. 58, EU IIA-41, IIA-53, IIB-3, IIB-4 and IIB-25, were described as Annex designation I/A2 Moko disease of Musa spp. and Heliconia. These occur mainly in South and Central America and the Caribbean, but Name: Ralstonia pseudosolanacearum Safni et al., 2014 are also reported on cooking banana genotypes ABB and Synonyms: Ralstonia solanacearum (Smith 1896) Yabuuchi BBB in the Philippines (causing so-called Bugtok disease). A et al. 1996; Burkholderia solanacearum (Smith 1896) Yabu- variant of R. solanacearum PIIB-4, but not pathogenic on uchi et al. 1992; Pseudomonas solanacearum (Smith 1896) banana, termed the PIIB-4NPB strain, was found to affect Smith 1914; many other synonyms in older literature cucurbits and Anthurium in Martinique and has also been Taxonomic position: Bacteria, Gracilicutes, Proteobacteria found in Brazil, Costa Rica and Trinidad (Wicker et al., b subdivision, Burkholderiales, Ralstoniaceae4 2007). Other susceptible hosts include S. lycopersicum, EPPO Code: RALSPS C. annuum, S. melongena, Impatiens hawkerii (New-Guinea Phytosanitary categorization: EPPO A2 List no. 401, EU impatiens), Heliconia caribaea (wild heliconia) and some Annex designation: not listed as such.5 weeds (Portulaca olaracea, Cleome viscose and Solanum americanum). Plantain could also be latently infected Name: Ralstonia syzygii (Roberts et al. 1990) Vanee- following inoculation. In Portugal, a virulent strain of choutte et al. 2004 emend. Safni et al., 2014 R. solanacearum Phylotype II (identified as biovar 1) was Synonyms: Ralstonia solanacearum (Smith 1896) Yabuuchi found on potato and caused rapid wilting of lower leaves, et al. 1996; Burkholderia solanacearum (Smith 1896) Yabu- yellowing and reduced growth of the whole plant and could uchi et al. 1992; Pseudomonas solanacearum (Smith 1896) also induce symptoms on S. lycopersicum, N. tabacum, Smith 1914; many other synonyms in older literature S. melongena, Pelargonium spp. and Eucalyptus globulus Taxonomic position: Bacteria, Gracilicutes, Proteobacteria (Cruz et al., 2008). Some Phylotype I strains behave similarly. b subdivision, Burkholderiales, Ralstoniaceae4 Strains that were previously designated as the broadest EPPO Code: RALSSY ‘race 1’ occur in tropical areas all over the world, attacking Phytosanitary categorization: EPPO A1 List no. 400. EU many hosts in over 50 plant families. Some of these strains Annex designation: not listed as such5 have occasionally been introduced into the EPPO region with ornamental/herbal plants or plant parts of tropical origin and 3. Detection can cause disease under greenhouse conditions in temperate climates, for example in Curcuma longa (turmeric), 3.1. Symptoms Anthurium, Epipremnum or more recently Rosa spp. (Tjou- Tam-Sin et al., 2016). Within R. pseudosolanacearum, some 3.1.1. Solanum tuberosum strains with specific sequevars affect Zingiber (ginger) and Foliar symptoms include rapid wilting of leaves and stems, Morus (mulberry). usually first visible in single stems (Fig. 3) at the warmest For the geographical distribution of the whole species time of day. Eventually, plants fail to recover and become complex see the EPPO Global Database (EPPO, 2015).3 yellow and then necrotic. As the disease develops, a streaky Flow diagrams describing the diagnostic procedure for brown discoloration of the stem may be observed on stems R. solanacearum, R. pseudosolanacearum and R. syzygii in above the soil line, and the leaves may have a bronze tint. plants (symptomatic and asymptomatic) and in water sam- Epinasty of the petioles may occur. A white, slimy mass of ples are presented in Figs 1 and 2, respectively. bacteria often exudes from cut or broken vascular bundles. On tubers, external symptoms may or may not be visible, depending on the state of development of the disease. 2. Identity Symptoms may be confused with those of ring rot due to Name: Ralstonia solanacearum (Smith 1896) Yabuuchi Clavibacter michiganensis subsp. sepedonicus (EPPO/ et al. 1996 emend. Safni et al., 2014 CABI, 1997). Infection eventually results in bacterial ooze Synonyms: Ralstonia solanacearum (Smith 1896) Yabu- emerging from the eyes and stolon end attachment of uchi et al. 1996; Burkholderia solanacearum (Smith 1896) infected tubers. Soil may adhere to the tubers at the eyes Yabuuchi et al. 1992; Pseudomonas solanacea- (Fig. 4). Cutting the diseased tuber will reveal a browning rum (Smith 1896) Smith 1914; many other synonyms in and eventual necrosis of the vascular ring and immediately older literature

4Ralstonia pickettii (a saprophyte or opportunistic clinical pathogen) is 2Referred to in EPPO Global Database as race 3 biovar 2 on 2017-06. the type species of the genus Ralstonia, which includes 16 species in 3On 2017-10 revision of the EPPO database according to current taxon- total. omy was in progress. 5Possibly covered as R. solanacearum.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 34 Diagnostics

Symptomaticor asymptomatic plant sample

Screening tests

Other screening tests Isolation Serological tests: IF (Appendix 2)

Molecular tests: Colonies with No typical for symptomatic samples only typical morphology colonies (1) & LAMP test (Appendix 3) (2) Conventional PCR test (Appendix 4) For critical cases Real-time PCR test (Appendix 5) Molecular identification tests When two tests other than isolation are performed they should be based on different biological principles or targeting different parts of the genome Perform at least two tests

LAMP (Appendix 3) Conventional PCR (Appendix 4,6) Two tests positive Inconsistent Test(s) negative Real-time PCR (Appendix 5) test results Barcoding RSCC not MLST/MLSA detected in the sample RSSC detected in Re-testing and/or RSSC not detected the sample re-sampling in the sample recommended

Tests positive Tests negative Tests with inconsistent results

RSCC identified in RSCC not identified in Retesting of colonies and/or culture culture resampling recommended

For critical cases RSSC = Ralstonia solanacearum, R pseudosolanacearum and R. syzygii (Ralstonia solanacearum Species Complex)

(1) When two serological or molecular screening tests are positive failure of isolation should trigger re-isolation Pathogenicity test (2) For symptomatic samples; when isolation is one of the screening test and no typical colonies are produced, perform a serological or a molecular screening test or re-isolate .

Fig 1 Flow diagram describing the diagnostic procedure for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii in plants (symptomatic and asymptomatic). [Colour figure can be viewed at wileyonlinelibrary.com]

Water sample

Selective isolation

Typical colonies No typical colonies

Molecular identification tests Perform at least two tests LAMP (Appendix 3) Conventional PCR (Appendix 4,6) Real-time PCR (Appendix 5) RSCC not detected Barcoding MLST/MLSA

Tests positive Tests with Tests negative inconsistent results

RSSC detected and RSSC not identified in Retesting of colonies and/or identified in culture culture resampling recommended

For critical cases

RSSC = Ralstoniasolanacearum, Rpseudosolanacearum and R.syzygii Pathogenicity test (Ralstonia solanacearum Species Complex)

Fig. 2 Flow diagram describing the diagnostic procedure for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii in water samples. [Colour figure can be viewed at wileyonlinelibrary.com]

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 35

3.1.3. Nicotiana tabacum One of the main symptoms is unilateral wilting and prema- ture yellowing (Fig. 9). Leaves on one side of the plant or even half a 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 sec- ondary roots may become brown to black.

3.1.4. Cucurbit plants Symptoms on cucurbits due to PIIB-4NPB develop rapidly from older to younger leaves that may wilt or not. Leaves turn yellow with necrotic lesions between or along major Fig. 3 Foliar symptoms on potato caused by Ralstonia solanacearum veins (as for Anthurium). Plants become flaccid and eventu- (Phylotype IIB-1). Courtesy Defra, Crown Copyright. ally collapse and die; there are no apparent symptoms on mature fruits (see Fig. 10).

3.1.5. Musa spp. Moko disease caused by R. solanacearum is easily confused with the 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 dis- ease. On young and fast-growing plants, the youngest leaves turn pale-green or yellow and wilt (Fig. 11). Within a week all leaves may collapse. Young suckers may be blackened, stunted or twisted. The pseudostems show brown vascular discoloration. It is now known that some Moko strains are also able to wilt solanaceous plants, producing the same symptoms as brown rot strains in this type of host.

3.1.6. Ornamental hosts Fig. 4 Bacterial ooze from potato eye. Courtesy J. Janse, PD Wageningen (NL). 3.1.6.1. Pelargonium (geranium). First symptoms are wilt- ing and subsequent chlorosis (often sectorial yellowing) of leaves (Fig. 12A). Stems may blacken and eventually surrounding tissues. A creamy fluid exudate usually appears become necrotic. Internally, vascular browning is often visi- spontaneously on the vascular ring of the cut surface a few ble. Leaves later become brown and necrotic as the whole minutes after cutting (Fig. 5). In the case of ring rot the plant desiccates, collapses and dies (Fig. 12B). tuber has to be squeezed in order to press out a mass of yellowish macerated vascular tissue and bacterial slime. 3.1.6.2. Curcuma longa. The first symptoms of Atypical symptoms (necrotic spots on the epidermis) have R. pseudosolanacearum (Phylotype I) in Curcuma are wilt- been described on potato, possibly caused after lenticel ing and subsequent chlorosis of leaves. Stems, including infection. Plants with foliar symptoms may bear healthy the flower stems (stalks), may acquire a brownish to black and diseased tubers, while plants that show no signs of discoloration and become necrotic (Fig. 13). Similar symp- wilting may sometimes produce diseased tubers. toms may be seen in the roots, including the rhizomes of the plant. Internal vascular browning is often visible. Under 3.1.2. Solanum lycopersicum and S. melongena favourable environmental conditions wilting of the whole The youngest leaves are the first to be affected and have a plant may follow rapidly. flaccid appearance, usually at the warmest time of day. Wilting of the whole plant may follow rapidly if environ- 3.1.6.3. Anthurium. Greasy, water-soaked lesions (on the mental conditions are favourable for the pathogen (Figs 6– lower leaf surface) turn necrotic with greasy margins (on 8). Under less favourable conditions, the disease develops the upper leaf surface). When the disease becomes sys- less rapidly, stunting may occur and large numbers of temic, these lesions, generally originating from the insertion adventitious roots are produced on the stem. The vascular point of the leaf with the petiole, develop following the tissues of the stem show a brown discoloration and, if the main and secondary veins in a full or partial glove-shape. stem is cut crosswise, drops of white or yellowish bacterial External infections (disseminated by water) may develop ooze may be visible (Fig. 6C). from any natural opening such as hydathodes. Leaves may

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 36 Diagnostics

(b) (a)

(c) (d)

Fig. 5 Symptoms of brown rot caused by Phylotype IIB-1 on cut tubers from early to advanced symptoms (A–D, respectively). (A) Courtesy J. van Vaerenbergh (ILVO, BE). (B), (D) Courtesy Defra, Crown Copyright. (C) Courtesy G. Cellier (Anses, FR).

(c) (a) (b)

Fig. 6 Wilting of tomato caused by (A) Ralstonia pseudosolanacearum and (B) Ralstonia solanacearum. (C) bacteria oozing from cut xylem vessels. (A) Courtesy G. Cellier (Anses, FR). (B), (C) Courtesy Defra, Crown Copyright.

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Fig. 7 Wilting of eggplant caused by Ralstonia solanacearum (Phylotype II). Courtesy M. Bergsma Vlami (NPPO-NL).

Fig. 8 Wilting of eggplant caused by Ralstonia pseudosolanacearum (Phylotype I). Courtesy G. Cellier (Anses, FR). turn yellow depending on the severity of the systemic inva- sion, and the stem may rot with abundant bacterial ooze. The plant eventually collapses and dies (Fig. 14).

3.1.6.4. Rosa spp.. Initial wilting of young shoots and flower stalks in Rosa spp. (Fig. 15) is followed by yellow- ing and early abscission of leaves, stunting, dieback with black necrosis of pruned branches, and in some cases puru- Fig. 9 Southern wilt of tobacco caused by Ralstonia solanacearum lent discharge of creamy white slime on cut wounds in the (Courtesy R. J. Reynolds Tobacco Company Slide Set, R. J. Reynolds stem (Fig. 16). Typical symptoms following heavy Tobacco Company, https://bugwood.org/).

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 38 Diagnostics

Fig 10 Symptoms caused by Phylotype IIB-4NPB on Cucumis melo, Cucumis sativus and Cucurbita moschata (from Wicker et al., 2002).

example Fusarium spp., Verticillium spp., Dickeya spp. and Clavibacter michiganensis. Rapid presumptive tests are described in Section 3.2.1.

3.2. Detection in symptomatic plant material 3.2.1 Rapid presumptive diagnostic tests Bacterial slime oozes spontaneously from the cut surface of infected stems when suspended in water (Fig. 17). Such oozing is generally not observed with other bacterial or fun- gal pathogens and this test is of presumptive diagnostic value in the field. Immunoassay field test kits, based on lat- eral flow device technology (Danks & Barker, 2000), are also available (see Appendix 1). Fig. 11 Symptoms caused by Moko strain Phylotype IIB-4 on young banana plants (control on the left). Courtesy G. Cellier (Anses, FR). 3.2.2. Isolation from symptomatic host plants infections of R. pseudosolanacearum (Phylotype I) include 3.2.2.1. Sample preparation for isolation. Sampling may necrosis of the stems and intense brown discoloration at the include removal of bacterial ooze or sections of discolored stem base (Tjou-Tam-Sin et al., 2016) (Fig. 16). tissue from the vascular ring in a potato tuber or from the vascular strands in stems/pseudostems of wilting host 3.1.7. Possible confusions plants. The sample should then be suspended in a small Wilting symptoms caused by Ralstonia spp. may be con- volume of sterile distilled water or phosphate buffer (PB; fused with those caused by other wilt pathogens, for 0.05 M) for 5–10 min.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 39

(a) (b)

Fig. 12 Wilting symptoms on Pelargonium infected with (A) Ralstonia solanacearum and (B) Ralstonia pseudosolanacearum (Phylotype III). (A) Courtesy Defra, Crown Copyright). (B) Courtesy G. Cellier (Anses, FR).

Fig. 13 Curcuma plant infected with Ralstonia pseudosolanacearum Phylotype I showing a brown discoloration of the base of the stem and of the roots. Courtesy M. Bergsma Vlami (NPPO-NL; left and middle) and D. J. Janse (right).

3.2.2.2. Isolation. Isolation from symptomatic material can widely used for isolation from symptomatic plant material. be performed using general non-selective nutrient media If the presence of banana Moko strains is suspected, media such as nutrient agar (NA), yeast peptone glucose agar should be amended with 1 g of yeast extract, as otherwise (YPGA), sucrose peptone agar (SPA) (Lelliott & Stead, these strains grow poorly. 1987) or Kelman’s tetrazolium medium (Kelman, 1954). Suspensions should then be prepared and aliquots (50– Some strains may produce a brown diffusible pigment on 100 lL) transferred to plates of a general nutrient medium some media (Fig. 18A). Since secondary infections and (NA, YPGA or SPA; Lelliott & Stead, 1987) and/or to Kel- high populations of saprophytic bacteria are often present, man’s tetrazolium medium (Kelman, 1954) and/or semi- isolation on a semi-selective medium may be necessary. selective SMSA/Sequeira medium (Appendix 2) by spread- The semi-selective medium as modified by Elphinstone ing or streaking, using an appropriate dilution plating tech- et al. (1996) (SMSA) or modified Sequeira medium (Gran- nique. Separate plates can be prepared with diluted cell ada & Sequeira, 1983; Poussier et al., 1999) have been suspensions of reference strains of the relevant Ralstonia

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 40 Diagnostics

Fig. 14 Symptoms caused by Ralstonia solanacearum on Anthurium spp. Courtesy Coranson-Beaudu and Wicker (Cirad, FR).

Fig. 16 Rosa sp. (tea rose) dieback with black necrosis of pruned branches and discharge of slime on cut wounds in the stem, Phylotype I. Courtesy N. Tjou-Tam-Sin (NPPO-NL).

On Kelman’s tetrazolium and SMSA (Fig. 18B)/Sequeira media (Fig. 18C), the whorls are blood red in colour. On these media avirulent forms of Ralstonia spp. form small, round, non-fluidal butyrous colonies which are entirely deep red and difficult to distinguish amongst other saprophytic bacteria which may be co-isolated. Typical colonies are shown in Fig. 18D. Note that colony morphology may vary with the matrix from which it is isolated.

3.2.3. Other screening tests 3.2.3.1. Immunofluorescence (IF) test. Instructions to per- Fig. 15 (left) Rosa flower stalk with necrotic wilted leaves Phylotype I. form an IF test are provided in the EPPO Standard PM 7/ Courtesy N. Tjou-Tam-Sin (NPPO-NL). 97 Indirect immunofluorescence test for plant pathogenic bacteria. Commercially available polyclonal antibodies may sp. as a positive control. The plates should be incubated for be subject to false-positive reactions. Sources of validated 2–6 days at 28°C. antibodies are given in Appendix 1. The IF test is usually performed on undiluted or concentrated (see Sections Colony characteristics (Fig. 18)—On general nutrient 3.3.1.1. and 3.3.1.2.) plant extracts and 10-fold dilutions of media, virulent isolates develop pearly cream-white, flat, these in 10 mM PB (pH 7.2). irregular and fluidal colonies (Fig. 18A) often with charac- teristic whorls in the centre. On these media avirulent forms 3.2.3.2. Molecular tests. Several molecular tests have been of Ralstonia spp. form small, round, non-fluidal butyrous developed for the detection and identification of all RSSC phy- colonies which are entirely cream-white. lotypes. For information on the phylotypes detected by each

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3.3. Detection in asymptomatic plant material Screening is performed using either an IF test, selective plating on SMSA/Sequeira medium and/or a molecular test. A positive result from any two of the screening tests above can be further supported by isolation (see Sec- tion 3.2.2.2.) and identification of the bacterium (see Sec- tion 4). Isolation should be performed in critical cases (EPPO, 2017). The procedures described in this protocol have been standardized and validated in many EU member states through interlaboratory comparisons (e.g. Van Vaer- enbergh et al., 2016), although mostly on potato tubers.

3.3.1. Sampling and sample preparation Screening methods have been validated based on a compos- ite sample size of 200 potato tubers or stems from potato or other host plants and pieces randomly collected from the population to be tested. More intensive sampling requires tests on additional sub-samples of 200 tubers. The maxi- mum number of tubers or stems that can be processed in one test is 200, as a higher number may lead to inhibition of the tests or difficultly in interpreting the results. The pro- cedure can be conveniently applied to samples of fewer than 200 tubers or stems.

Fig. 17 Bacterial ooze from cut stem suspended in water Courtesy 3.3.1.1. Potato tubers. Potato tubers can be first washed Defra, Crown Copyright. and air dried, if necessary, to remove any excess soil which may contain saprophytic/opportunistic bacteria that may cause false-positive results in the IF test. After removing a test see Table 1. The following tests, routinely used for screen- small area of peel with a sterile knife from the heel (stolon) ing plant material, are described in full in this Standard. end of each tuber, small cores (e.g. 0.2–0.5 g) of the • A LAMP (loop-mediated isothermal amplification) test exposed vascular tissue can be removed, keeping the (Lenarcic et al., 2014) was recently described for diag- amount of non-vascular tissue to a minimum. nosis of Ralstonia spp. in symptomatic plants and can After covering the 200 vascular tuber cores from each be used on site (see Appendix 3). sample in sterile 50 mM PB, pH 7.0 (see Appendix 2), the • The conventional PCR test, the most widely validated and bacteria can be extracted from the tissue by either: routinely used screening test for detection of (a)rotary shaking (50–100 rpm) for 4 h below 24°C or for R. solanacearum Phylotype II and R. pseudosolanacearum 16–24 h refrigerated, or Phylotype I in latently infected potato tubers (Pastrik et al., (b)mechanical homogenization in a sealed bag using a suit- 2002), is described in Appendix 4. able grinding apparatus (e.g. a Homex 6 homogenizer) • TaqMan real-time PCR (Weller et al., 2000) is or rubber mallet. described in Appendix 6 and can be used to detect all After decanting the supernatant, it can be clarified four phylotypes. either by slow speed centrifugation (at not more than The selection of the tests to be performed should be made 180g for 10 min at 4–10°C) or by vacuum filtration (40– taking into account the phylotype likely to be present on 100 lm), washing the filter with additional (approxi- the sample. mately 10 mL) extraction buffer. The bacterial fraction can then be concentrated by centrifugation at 7000g for 3.2.3.3. Bioassay in tomato. A bioassay is described in 15 min (or 10 000g for 10 min) at 4–10°C and discard- Appendix 7 (Janse, 1988). ing the supernatant without disturbing the pellet. After resuspending the pellet in 1.5 mL of 10 mM PB, pH 7.2 3.2.3.4. Other tests. A number of other tests have been (Appendix 2), a proportion (e.g. 500 lL) should be stored developed but are no longer commonly used. They are con- at 68 to 86°C by addition of 10–25% (v/v) sterile sequently not described in this protocol. These include tests glycerol in case repeat testing is later required. The based on fluorescent in situ hybridization (FISH; Wullings remainder of the resuspended pellet should be kept refrig- et al., 1998) and enzyme-linked immunosorbent assay erated (4°C) and used in the following screening tests, (ELISA; Caruso et al., 2002). which should be optimized before use to enable detection

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 42 Diagnostics

(A) (B)

(D) (C)

Fig. 18 Colonies of Phylotype IIB-1 strain growing on (A) SPA medium, (B) SMSA medium, (C) Sequeira medium and (D) typical/atypical colonies isolated from water samples. (A), (B), (D) Courtesy J. van Vaerenbergh (ILVO, BE). (C) Courtesy G. Cellier G (Anses, FR).

of 103–104 cells mL1 of a reference strain of the appro- Other plants—Detection of latent Ralstonia populations in priate Ralstonia sp. added to a negative sample of resus- other symptomless plants (e.g. potato, tomato, eggplant, pended pellet as a positive control. Pelargonium spp., Curcuma spp., Anthurium sp., Epipremnum spp., Rosa spp., S. dulcamara) is commonly 3.3.1.2. Other asymptomatic host plants. Plant material done on composite samples. For outdoor grown plants, should be preferably processed immediately or within 72 h pathogen detection will be most reliable during warm grow- if kept refrigerated. Stored stem/pseudostem samples should ing conditions (>15°C), although natural infections can be be refreshed prior to testing by a cross-section at each end detected all year round in the perennial S. dulcamara grow- to expose freshly cut xylem vessels. ing in watercourses in temperate regions. Screening tests can be applied to composite samples containing up to 200 Banana plants—Testing for banana plants is performed on stem segments, taken from a representative sample of the individual plants. The first rolled leaves should be plant population under investigation. Stem segments (ap- removed to avoid contamination. The sample to be tested proximately 1 cm long) are removed using a disinfected should consist of 2.0 g of freshly excised pseudostem knife from the base of each main stem or lowermost side- material. shoot just above the soil level. For S. dulcamara or other

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Table 1. Molecular tests for detection and identification of Ralstonia species and phylotypes

Ralstonia species and phylotypes iden- Reference Primers Details of test tified

Seal et al. (1992) PS96-H/PS96-I Conventional PCR: from subtractive R. solanacearum hybridization R. pseudosolanacearum (Phylotypes I and II) Seal et al. (1993) OLI-1/Y-2 Conventional PCR: 16S rRNA gene R. solanacearum sequence R. pseudosolanacearum R. syzygii (all phylotypes) Opina et al. (1997) 759/760 Conventional PCR: conserved IpxC R. solanacearum gene sequence R. pseudosolanacearum R. syzygii (all phylotypes) Fegan et al. (1998) 630/631 Conventional PCR: from subtractive R. solanacearum hybridization (Phylotypes IIB-1, IIB-2 and some other Phylotype IIB isolates of unde- termined sequevar) Boudazin et al. (1999) D2/B Conventional PCR: 16S rRNA gene R. solanacearum sequence R. syzygii (Phylotypes II and IV) Pastrik & Maiss (2000) PS1/PS2 Conventional PCR: 16S rRNA gene R. solanacearum sequence R. pseudosolanacearum (Phylotypes I and II) Weller et al. (2000) RS-I-F/RS-II-R/ Real-time TaqMan PCR: 16S rRNA R. solanacearum RS-P gene sequence P. pseudosolanacearum R. syzygii (all phylotypes) Weller et al. (2000) B2-I-F/B2-II-R/B2- Real-time TaqMan PCR: from R. solanacearum P subtractive hybridization (Phylotypes IIB-1, IIB-2 and some other Phylotype IIB isolates of unde- termined sequevar) Pastrik et al. (2002) RS-1-F/RS-1-R Conventional PCR: 16–23S rRNA R. solanacearum spacer sequence (Phylotype II) Pastrik et al. (2002) RS-1-F/RS-3-R Conventional PCR: 16–23S rRNA R. pseudosolanacearum spacer sequence (Phylotype I) Ozakman & Schaad, 2003; RSC-F/RSC-R/ Real-time TaqMan PCR: from R. solanacearum RSC-P subtractive hybridization (Phylotypes IIB-1, IIB-2 and some other Phylotype IIB isolates of unde- termined sequevar) Schonfeld€ et al. (2003) Rsol_fliC_for/ Conventional PCR: fliC gene sequence R. solanacearum Rsol_fliC_rev R. pseudosolanacearum R. syzygii (all phylotypes) Fegan & Prior (2005) Nmult21:1F/ Conventional PCR: 16-23S rRNA R. pseudosolanacearum Nmult22:RR spacer sequence (phylotype I) Fegan & Prior (2005) Nmult21:2F/ Conventional PCR: 16–23S rRNA R. solanacearum Nmult22:RR spacer sequence (Phylotype II) Fegan & Prior (2005) Nmult23:AF/ Conventional PCR: 16–23S rRNA R. pseudosolanacearum Nmult22:RR spacer sequence (Phylotype III) Fegan & Prior (2005) Nmult22:InF/ Conventional PCR: 16–23S rRNA R. syzygii Nmult22:RR spacer sequence (Phylotype IV) Prior & Fegan (2005) Mus20-F/Mus20-R Conventional multiplex PCR: from R. solanacearum Mus35-F/Mus35-R subtractive hybridization (Moko Phylotype IIB-3, IIB-4 and Mus06-F/Mus06-R IIA-6 strains pathogenic to Musa- Si28-F/Si28-R ceae) Guidot et al. (2009) Multiple primers Conventional PCR: from genome R. solanacearum (Phylotypes IIB-1 comparisons and IIB-2) Kubota et al. (2011) Various primers LAMP and conventional PCR: from in R. solanacearum (Phylotypes IIB-1 silico genome comparisons and IIB-2)

(continued)

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 44 Diagnostics

Table 1 (continued)

Ralstonia species and phylotypes iden- Reference Primers Details of test tified

Kubota et al. (2011) BDB2400-F/ Conventional PCR: from genome R. syzygii subsp. celebesensis (Phylo- BDB2400-R comparisons type IV-10 banana blood disease strains) Ha et al. (2012) RRSL_2403F Real-time PCR with SYBR green R. solanacearum (Phylotype IIB-1 and RRSL_2403R detection: phage protein gene IIB-2) (RRSL_02403) sequence Massart et al. (2014) Multiraso-F/ Real-time PCR: 16–23S rRNA spacer R. solanacearum Multiraso-R/ sequence (Phylotype IIB + some Phylotype IIA Multiraso-P strains) Lenarcic et al. (2014) F3-Rs/B3-Rs/FIP- LAMP: egl gene sequence R. solanacearum Rs/Bip-Rs/ Floop-RS/Bloop- R. pseudosolanacearum RS R. syzygii (all phylotypes with the exception of one Phylotype IV isolate of R. syzygii from Australia) Stulberg et al. (2015) Various primers Conventional multiplex PCR: from R. solanacearum (Phylotypes IIB-1 genome comparisons and IIB-2 only) Cellier et al. (2015) 93F/93R & 5F/5R Conventional duplex PCR: putative Phylotype II Moko strains from Musa KfrA and chemotaxis-related protein and Phylotype IIB-4NPB sequences host plants growing in water, 1–2 cm sections are removed remaining extract with 10–25% (v/v) sterile glycerol at from underwater stems or stolons with aquatic roots. Visual 16 to 24°Corat68 to 86°C, in case further testing examination for internal symptoms (vascular staining or is required. bacterial ooze) can be done at this stage. Any stem seg- ments with symptoms should be set aside and tested sepa- 3.3.2. Screening tests rately (see Section 3.2 ). 3.3.2.1. Isolation. Serial dilutions of the re-suspended pel- lets or plant extracts should be prepared and aliquots (50– Extraction of the bacteria—Stem segments can be disin- 100 lL) spread on plates of semi-selective SMSA/Sequeira fected briefly with 70% ethanol and immediately blotted medium (Appendix 2). For bananas (or where the potential dry on absorbent paper. After covering the stem segments presence of R. solanacearum Moko strains is suspected), from each sample in sterile 50 mM PB, pH 7.0 (see isolation media should be amended with 1 g of yeast Appendix 2), the bacteria can be extracted from the tissue extract (see Section 3.2.2.2). The plates should be incubated by either: for 2–6 days at approximately 28°C. (a)rotary shaking (50–100 rpm) for 4 h below 24°C or for For colony description see Section 3.2.2.2. 16–24 h refrigerated or (b)mechanical homogenization in a sealed bag using a suit- 3.3.2.2. Other screening tests. All screening tests described able grinding apparatus (e.g. a Homex 6 homogenizer) or under Section 3.2.3. can be performed, with the exception rubber mallet, or of the LAMP test which has not yet been validated for (c)particularly for banana plants, samples can be ground in detection in asymptomatic plant material. a sterile mortar or chopped with sterile scalpel in 5 mL of extraction buffer, leaving for approximately 10 min 3.4. Detection in other matrices before performing the test. Further clarification of the extract or concentration of the 3.4.1. Surface or recirculation water, sewage/industrial bacterial fraction is not usually required but may be effluents achieved by filtration and/or centrifugation as described in Surface water should be ideally sampled when water tem- Section 3.3.1.1. peratures are at or above 15°C. At selected sampling points, The neat or concentrated sample extract should then be surface water can be collected by filling sterile tubes or bot- tested immediately, or within 2 h if kept at room tempera- tles, ideally at a depth below 30 cm and in the vicinity of ture. If necessary, the remaining extract can be stored at 4– any known host plants. For industrial or sewage effluents, 10°C during the testing period, although this may affect the samples should be collected from the point of effluent dis- reliability of pathogen isolation. Preferably store the charge.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 45

It is advisable to take duplicated samples of at least identification, including at least one test which is known to 30 mL on at least three occasions per sampling point. Sam- detect all three pathogen species. Choice of other PCR tests ples should be transported in cool dark conditions (4–10°C) will also depend on the level of infrasubspecific identifica- and tested preferably within 24 h. tion required. The following PCR tests for which validation Dilution plating on semi-selective SMSA medium or data is available are described in full in this protocol. Sequeira medium (Appendix 2) should ensure that any Molecular tests targeting all three species: background saprophytic colony-forming populations are • a LAMP test Lenarcic et al. (2014) described in diluted out. Plates should be spread with 50–100 lLof Appendix 3 sample for each dilution and incubated at 28°C. They • a real-time TaqMan PCR test (Weller et al., 2000), should then be observed for typical R. solanacearum colo- described in Appendix 5 nies after 48 h and daily thereafter up to 6 days. • a conventional PCR test (Opina et al., 1997) performed To improve the likelihood of detecting Ralstonia spp. in as part of a phylotype-specific multiplex PCR test, water, it is recommended to first concentrate bacterial popu- described in Appendix 6. lations using one of the following methods: PCR for sub-specific identification: (i) centrifugation of 30–50 mL sub-samples at 10 000g for • phylotype-specific multiplex conventional PCR tests 10 min (or 7000g for 15 min) preferably at 4–10°C, dis- (Fegan & Prior, 2005), described in Appendix 6 carding the supernatant and resuspending the pellet in • conventional PCR tests (Pastrik et al., 2002), for specific 1 mL sterile water identification of either R. solanacearum (Phylotype II) (ii) membrane filtration (1 L through a maximum pore or R. pseudosolanacearum (Phylotype I) strains, size of 0.45 lm) followed by washing the filter in 5– described in Appendix 4 10 mL pellet buffer and retention of the washings. This • a duplex PCR (Cellier et al., 2015) for R. solanacearum method is suitable for larger volumes of water containing Phylotype IIB-4NPB and Moko strains (sequevars IIB-3, low numbers of saprophytes. If the samples are turbid, IIB-4, IIA-6, IIA-24, IIB-25, IIA-41 and IIA-53) in this may block the filtration process and saturate the filter. Musa spp. described in Appendix 8. A 1/10 dilution of the sample in sterile water is then A microarray that allows a multiplex characterization of highly recommended. a given R. solanacearum strain within 17 major phyloge- Dilution plating of the concentrated samples can then be netic/ecotype groups has been developed (Cellier et al., performed as above (Section 3.3.2.1). 2017). This custom microarray represents a significant Concentration is usually not advisable for samples of improvement in the epidemiological monitoring of sewage/industrial effluents since increased populations of R. solanacearum strains worldwide, and it has the potential competing saprophytic bacteria will inhibit detection of to provide insights for phylogenetic incongruence of RSSC Ralstonia spp. strains based on the host from which the bacterium has been isolated and may be used to indicate potentially emer- 3.4.2. Detection in soil gent strains. Testing soil is not recommended as it is considered erra- tic due to the highly dispersed and low population of 4.1.2. Barcoding bacteria in soil. Testing known weed hosts or crop hosts Comparisons of sequenced PCR products amplified from and their volunteers growing in the soil is considered selected housekeeping gene loci allow accurate differentia- more reliable. tion of phylotypes and sequevars within the three Ralstonia species from their closest relatives. For example, the four Ralstonia phylotypes can be routinely distin- 4. Identification guished based on the sequences of their 16S–23S rRNA When isolation is performed, identification should be per- gene intergenic spacer (ITS) region and the hrpB, mutS formed using at least two tests, based on different biologi- and egl genes (Fegan & Prior, 2005; Guidot et al., 2007). cal principles or targeting two different parts of the genome Multilocus sequence analysis (MLSA) and multilocus for molecular tests. Relevant tests are described below. For sequence typing (MLST) approaches using 7 housekeeping critical cases (EPPO, 2017), when a positive identification genes (gdhA, MutS, ppsA, adk, leuS, rplB and gyrB) have is made it is recommended to perform a pathogenicity test distinguished phylogenetic relatedness amongst 89 refer- to confirm infection in the sample. ence isolates of R. solanacearum, R. pseudosolanacearum and R. syzygii (Wicker et al., 2012). Methods for routine barcoding using the mutS sequence and reference 4.1. Molecular methods sequences from 22 strains are available at www.Q-bank.e 4.1.1. Molecular tests u/Bacteria. General procedures for sequencing are Several molecular tests are available for rapid identification described in the EPPO Standard PM 7/129 DNA barcoding of the three Ralstonia species (see Table 1). It is recom- as an identification tool for a number of regulated pests mended to use more than one primer set for reliable Appendix 2 (EPPO, 2016).

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 46 Diagnostics

NCPPB 4029 = CFBP 4615 (R. pseudosolanacearum 4.2. Matrix-assisted laser desorption/ionization time of Phylotype I, (race 4, biovar 4) flight mass spectrometry (MALDI-TOF-MS) NCPPB 4011 = CFBP 4617 (R. pseudosolanacearum A MALDI-TOF method for proteomic analysis has been Phylotype I, (race 5, biovar 5) described by Prior et al. (2016). This allows rapid, reliable NCPPB 3446T = LMG 10661T = DSMZ(4) 7385T and robust identification of strains of the three Ralstonia (R. syzygii subsp. syzygii type strain (Phylotype IV) species. Database entries (mass spectra profiles, MSPs) LMG 27703T = DSMZ 27478T (R. syzygii subsp. specific for the three species were created prior to the rou- indonesiensis type strain (Phylotype IV) tine identification of isolates from plant and water samples. LMG 27706T = DSMZ 27477T (R. syzygii subsp. For their routine identification, all individual isolates were celebesensis type strain (Phylotype IV) included in duplicate by directly depositing harvested 2-day- (1) NCPPB, National Collection of Plant Pathogenic Bac- old bacterial cells from NA plates onto a stainless plate, teria, Fera, Sand Hutton, York YO411LZ, UK; www. without having to perform any extraction step. All spectra ncppb.fera.defra.gov.uk were obtained in linear positive-ion mode with an m/z range (2) CFBP, Collection Francßaise de Bacteries associees aux of 2000–20 000 Da. Validation of the database entries Plantes, CIRM, IRHS-INRA, 42 Rue Georges Morel, (MSPs) of reference isolates for the three Ralstonia and CS60057, 49071 Beaucouze Cedex, France; https://www6. related species is under development. Once available, this inra.fr/cirm/CFBP-Bacteries-associees-aux-Plantes method will have high potential for routine identification. (3) LMG, Belgian Co-ordinated Collections of Micro- organisms (BCCM)/LMG Bacteria Collection, Labora- torium voor Microbiologie, Universiteit Gent, K. L. 4.3. Other tests Ledeganckstraat 35, B-9000 Gent, Belgium; http://bcc 4.3.1. Genomic fingerprinting tests m.belspo.be/ Ralstonia strains can be reliably identified by matching (4) DSMZ, Leibniz Institute DSMZ–German Collection their unique BOX-PCR genomic fingerprints to those of ref- of Microorganisms and Cell Cultures, Inhoffenstraße erence strains of similar identity (see EPPO PM 7/100 rep- 7B, 38124 Braunschweig, Germany; www.dsmz.de/c PCR tests for identification of bacteria). ontact.html

4.3.2. Fatty acid analysis 6. Reporting and documentation Methods for identification using fatty acid methyl ester (FAME) analysis are described by Janse (1991) and in Guidance on reporting and documentation is given in EPPO Anonymous (2006). Standard PM 7/77 Documentation and reporting on a diagnosis. 4.3.3. General phenotypical characteristics Differential biochemical characteristics that can help to dis- 7. Performance criteria tinguish the plant pathogenic Ralstonia species and sub- species are described in Safni et al. (2014) and Prior et al. When performance criteria are available, these are provided (2016). with the description of the test. Validation data is also available in the EPPO Database on Diagnostic Expertise (http://dc.eppo.int), and it is recommended that this data- 4.4. Pathogenicity test base is consulted as additional information may be avail- The procedure for the pathogenicity test is described in able there (e.g. more detailed information on analytical Appendix 9. specificity, full validation reports, etc.).

5. Reference material 8. Further information NCPPB(1) 325T = CFBP(2) 2047T = LMG(3) 2299T Further information on this organism can be obtained from: • (R. solanacearum type strain, Phylotype IIA-7, (race 1, bio- J. G. Elphinstone, Fera, Plant Protection Programme, var 1) Sand Hutton, York, YO411LZ, UK; email john.elphin- NCPPB 4156 = CFBP 3857 (R. solanacearum Phylotype [email protected] • IIB-1, (race 3, biovar 2) M. Bergsma-Vlami, Plant Protection Service, PO NCPPB 2314 = CFBP 1412 (R. solanacearum Phylotype Box 9102, 6700 HC, Wageningen, Netherlands; email IIB-4, (race 2, biovar 1) [email protected] • NCPPB 1029T = LMG 9673T (R. pseudosolanacearum J. van Vaerenbergh, ILVO-Department of Crop Protec- type strain, Phylotype III, (race 1, biovar 1) tion, Section of Bacteriology, Van Gansberghelaan 96, – NCPPB 3996 = CFPB 3928 (R. pseudosolanacearum B 9820 Merelbeke, Belgium; email johan.vanvaeren- Phylotype I, (race 1, biovar 3) [email protected]

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• G. Cellier, Anses – Plant Health Laboratory, Unite Dams EL, Hendriks L, Van de Peer Y, Neefs JM, Smits G, Ravageurs et Agent Pathogenes Tropicaux, Pole^ de Pro- Vandenbempt I et al. (1988) Compilation of small ribosomal subunit – tection des Plantes, 7 Chemin de l’Irat – Ligne Paradis, RNA sequences. Nucleic Acids Research 16(Suppl), R87 R173. Danks C & Barker I (2000) On-site detection of plant pathogens using 97410 St Pierre, France – Reunion Island; e-mail gilles.- lateral flow devices. Bulletin OEPP/EPPO Bulletin 30, 421–426. [email protected] Elphinstone JG, Hennessey JK, Wilson JK & Stead DE (1996) Sensitivity of different methods for the detection of Pseudomonas solanacearum (Smith) Smith in potato tuber extracts. EPPO Bulletin 9. Feedback on this diagnostic protocol 26, 663–678. If you have any feedback concerning this Diagnostic Elphinstone JG, Stanford HM & Stead DE (1998) Detection of Protocol, or any of the tests included, or if you can provide Ralstonia solanacearum in potato tubers, Solanum dulcamara, and associated irrigation water. In Bacterial Wilt Disease: Molecular and additional validation data for tests included in this protocol Ecological Aspects (eds Prior P, Allen C & Elphinstone J), pp. 133– that you wish to share please contact [email protected] 139. Springer publishing, Berlin (DE). EPPO (2015) EPPO Global Database (available online). https://gd.eppo. int [accessed on 01-June-2017] 10. Protocol revision EPPO (2016) PM 7/129 (1) DNA barcoding as an identification tool for – A periodic review process is in place to identify the need for a number of regulated pests. Bulletin OEPP/EPPO Bulletin 46, 501 537. revision of Diagnostic Protocols. Protocols identified as EPPO (2017) PM 7/76 (4) Use of EPPO diagnostic protocols Bulletin needing revision are marked as such on the EPPO website. OEPP/EPPO Bulletin (version under revision - to check for the latest version see http://archives.eppo.int/EPPOStandards/diagnostics.htm) Acknowledgements 47,7–9. EPPO/CABI (1997) Clavibacter michiganensis subsp. sepedonicus.In This revised protocol was drafted by: J. G. Elphinstone, Fera Quarantine Pests for Europe (eds Smith IM, McNamara DG, Scott Science Ltd, United Kingdom; M. Bergsma-Vlami, Plant PR & Harris KM), 2nd edn, pp. 1071–1081. CAB International, Protection Service, Netherlands; J. van Vaerenbergh, ILVO Wallingford (GB). Fegan M, Holloway G, Hayward AC & Timmis J (1998) Development Belgium; G. Cellier, Anses, France – Reunion Island; F. of a diagnostic test based on the polymerase chain reaction to identify Poliakoff, Anses, France strains of R. solanacearum exhibiting the biovar 2 genotype. In Bacterial Wilt Disease: Molecular and Ecological Aspects (eds Prior P, Allen C & Elphinstone J), pp. 34–43. Springer Verlag, Berlin (DE). References Fegan M & Prior P (2005) How complex is the Alvarez AM, Berestecky J, Stiles JI, Ferreira SA & Benedict AA “Ralstonia solanacearum species complex”. In Bacterial Wilt (1993) Proc. Bacterial Wilt Intl. Conf (28 -31 Oct. 1992, Kaohsiung, Disease and the Ralstonia solanacearum Species Complex (eds Allen – Taiwan), Serological and molecular approaches to identification of C, Hayward AC & Prior P), pp. 449 461. American Pseudomonas solanacearum strains from Heliconia, eds Hartman Phytopathological Society, St Paul, MN (US). G.L., Hayward A.C. pp 62–69. Granada GA & Sequeira L (1983) A new selective medium for – Anonymous (2006) Commission Directive 2006/63/CE of 14 July 2006 Pseudomonas solanacearum. Plant Disease 67, 1084 1088. amending Annexes II to VII to Council Directive 98/57/EC on the Guidot A, Elbaz M, Carrere S, Siri MI, Pianzzola MJ, Prior P et al. control of Ralstonia solanacearum (Smith) Yabuuchi et al.. Official (2009) Specific genes from the potato brown rot strains of Journal European Communities no. L 206,36–106. Ralstonia solanacearum and their potential use for strain detection. – Boudazin G, Le Roux AC, Josi K, Labarre P & Jouan B (1999) Design Phytopathology 99, 1105 1112. of division specific primers of Ralstonia solanacearum and Guidot A, Prior P, Schoenfeld J, Carrere S, Genin S & Boucher C application to the identification of European isolates. European (2007) Genomic structure and phylogeny of the plant pathogen Journal of Plant Pathology 105, 373–380. Ralstonia solanacearum inferred from gene distribution analysis. – Buddenhagen IW, Sequeira L & Kelman A (1962) Designation of races Journal of Bacteriology 189, 377 387. of Pseudomonas solanacearum. Phytopathology 52, 726. Ha Y, Kim J-S, Denny TP & Schell MA (2012) A rapid, sensitive Caruso P, Gorris MT, Cambra M, Palomo JL, Collar J & Lopez MM assay for Ralstonia solanacearum race 3 biovar 2 in plant and soil (2002) Enrichment double-antibody sandwich indirect enzyme-linked samples using magnetic beads and real-time PCR. Plant Disease 96, – immunosorbent assay that uses a specific monoclonal antibody for 258 264. sensitive detection of Ralstonia solanacearum in asymptomatic potato Hayward AC (1964) Characteristics of Pseudomonas solanacearum. – tubers. Applied and Environmental Microbiology 68, 3634–3638. Journal of Applied Bacteriology 27, 265 277. Cellier G, Arribat S, Chiroleu F, Prior P & Robene I (2017) Tube-wise Janse JD (1988) A detection method for Pseudomonas solanacearum in diagnostic microarray for the multiplex characterization of the symptomless potato tubers and some data on its sensitivity and – complex plant pathogen Ralstonia solanacearum. Frontiers in Plant specificity. EPPO Bulletin 18, 343 351. Science 8, 821, https://www.frontiersin.org/articles/10.3389/fpls.2017. Janse JD (1991) Infra- and intraspecific classification of Pseudomonas 00821/full [accessed on 1 June 2017] solanacearum strains, using whole cell fatty acid analysis. Systematic – Cellier G, Moreau A, Chabirand A, Hostachy B, Ailloud F & Prior P and Applied Microbiololgy 14, 335 345. (2015) A duplex PCR assay for the detection of Ralstonia solanacearum Janse JD, Arulappan FAX, Schans J, Wenneker M & Westerhuis W phylotype II strains in Musa spp. PLoS ONE 10, e0122182. (1998) Experiences with bacterial brown rot Ralstonia solanacearum Cruz L, Eloy M, Quirino F & Carrinho H (2008) Ralstonia solanacearum biovar 2, race 3 in The Netherlands. In Bacterial Wilt Disease: biovar 1 associated with a new outbreak of potato brown rot in Molecular and Ecological Aspects (eds Prior P, Allen C & – Portugal. Phytopathologia Mediterranea 47,87–91. Elphinstone J), pp. 146 152. Springer publishing, Berlin (DE).

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Janse JD, van den Beld HE, Elphinstone J, Simpkins S, Tjou-Tam-Sin complex: proposal to amend the descriptions of NNA & van Vaerenbergh J (2002) Introduction of Ralstonia solanacearum and Ralstonia syzygii and reclassify current Ralstonia solanacearum biovar 2/race 3 in Pelargonium zonale R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., cuttings. In: Proceedings 3rd International Bacterial Wilt R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. Symposium, Nelspruit, S.A., 4–8 February 2002 (in press). indonesiensis subsp. nov., banana blood disease bacterium strains as Kelman A (1954) The relationship of pathogenicity of Pseudomonas Ralstonia syzygii subsp. celebesensis subsp. nov. and solanacearum to colony appearance on a tetrazolium medium. R. solanacearum phylotype I and III strains as Ralstonia Phytopathology 44, 693–695. pseudosolanacearum sp. nov. International Journal of Systematic and Korner€ UA, Kroll U & Hilgert S (2017) False positive Ralstonia Evolutionary Microbiology 64, 3087–3103. solanacearum real-time PCR results in routine potato tuber samples Schonfeld€ J, Heuer H, Van Elsas JD & Smalla K (2003) Specific and caused by Advenella species: adaptations to avoid these cross- sensitive detection of Ralstonia solanacearum in soil on the basis of reactions. EPPO Bulletin 47,33–40. https://doi.org/10.1111/epp.12366 PCR amplification of fliC fragments. Applied Environmental Kubota R, Schell MA, Peckham GD, Rue J, Alvarez AM, Allen C Microbiology 69, 7248–7256. et al. (2011) In silico genomic subtraction guides development of Seal SE, Jackson LA & Daniels MJ (1992) Use of tRNA consensus highly accurate DNA-based diagnostics for Ralstonia solanacearum primers to indicate subgroups of Pseudomonas solanacearum by race 3 biovar 2 and blood disease bacterium. Journal of General polymerase chain reaction amplification. Applied Environmental Plant Pathology 77, 182–193. Microbiology 58, 3759–3761. Lelliott RA & Stead DE (1987) Methods for the Diagnosis of Bacterial Seal SE, Jackson LA, Young JPW & Daniels MJ (1993) Detection of Diseases of Plants, p. 216. Blackwell Scientific Publications Ltd., Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas Oxford (GB). pickettii and Blood Disease Bacterium by partial 16S rRNA Lenarcic R, Morisset D, Pirc M, Llop P, Ravnikar M & Dreo T (2014) sequencing: construction of oligonucleotide primers for sensitive Loop-mediated isothermal amplification of specific endoglucanase detection by polymerase chain reaction. Journal of General gene sequence for detection of the bacterial wilt pathogen Microbiology 139, 1587–1594. Ralstonia solanacearum. PLoS ONE 9(4), e96027. https://doi.org/10. Strider DL, Jones RK & Haygood RA (1981) Southern bacterial wilt of 1371/journal.pone.0096027 geranium caused by Pseudomonas solanacearum. Plant Disease 65, Massart S, Nagy C & Jijakli MH (2014) Development of the 52–53. simultaneous detection of Ralstonia solanacearum race 3 and Stulberg MJ, Shao J & Huang Q (2015) A multiplex PCR assay to Clavibacter michiganensis subsp. sepedonicus in potato tubers by a detect and differentiate select agent strains of Ralstonia multiplex real-time PCR assay. European Journal of Plant Pathology solanacearum. Plant Dis 99, 333–341. http://dx.doi.org/10.1094/ 138,29–37. PDIS-05-14-0483-RE Opina N, Tavner F, Holloway G, Wang J-F, Li TH, Maghirang R et al. Tjou-Tam-Sin NNA, de van Bilt JLJ, Westenberg M, Bergsma-Vlami (1997) A novel method for development of species and strain- M, Korpershoek HJ, Vermunt AMW et al. (2016) First report of specific DNA probes and PCR primers for identifying Burkholderia bacterial wilt caused by Ralstonia solanacearum in Ornamental solanacearum (formerly Pseudomonas solanacearum). Asia-Pacific Rosa sp. doi.org.ezproxy.library.wur.nl/10.1094/PDIS-02-16-0250- Journal of Molecular Biology and Biotechnology 5,19–30. PDN Ozakman M & Schaad NW (2003) A real-time BIO-PCR assay for Van Vaerenbergh J, Muller€ P, Elphinstone JG, Vreeburg RAM & detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic Janse JD (2016) EUPHRESCO Inter-Laboratory Comparison potato tubers. Canadian Journal of Plant Pathology 25, 232–239. (2009–2012) on detection of Clavibacter michiganensis subsp. Pastrik KH, Elphinstone JG & Pukall R (2002) Sequence analysis and sepedonicus and Ralstonia solanacearum in potato tubers: Proposal detection of Ralstonia solanacearum by multiplex PCR amplification to include TaqMan real-time PCR as a primary (core) screening of 16S-23S ribosomal intergenic spacer region with internal positive test in EU/EPPO standard methods. Bulletin OEPP/EPPO Bulletin control. European Journal of Plant Pathology 108, 831–842. 47,24–32. Pastrik KH & Maiss E (2000) Detection of R. solanacearum in potato Vreeburg RAM, Bergsma-Vlami M, Bollema RM, de Haan EG, tubers by polymerase chain reaction. Journal of Phytopathology 148, Kooman-Gersmann M, Smits-Mastebroek L et al. (2016) 619–626. Performance of real-time PCR and immunofluorescence for the Pirc M, Ravnikar M, Tomlinson J & Dreo T (2009) Improved fireblight detection of Clavibacter michiganensis subsp. sepedonicus and diagnostics using quantitative real-time PCR detection of Erwinia Ralstonia solanacearum in potato tubers in routine testing. Bulletin amylovora chromosomal DNA. Plant Pathology 58, 872–881. OEPP/EPPO Bulletin 46, 112–121. Prior P, Ailloud F, Dalsing BL, Remenant B, Sanchez B & Allen C Weller SA, Elphinstone JG, Smith N, Stead DE & Boonham N (2000) (2016) Genomic and proteomic evidence support the division of the Detection of Ralstonia solanacearum strains using an automated and plant pathogen Ralstonia solanacearum into three species. BMC quantitative flourogenic 50 nuclease TaqMan assay. Applied and Genomics 17, 90. Environmental Microbiology 66, 2853–2858. Poussier S, Vandewalle P & Luisetti J (1999) Genetic diversity of Wenneker M, Verdel MSW, Van Beuningen AR, Derks JHJ & Janse African and worldwide strains of Ralstonia solanacearum as JD (1999) Ralstonia (Pseudomonas) solanacearum race 3 (biovar 2) determined by PCR-Restriction Fragment Length Polymorphism in surface water and natural weed hosts: first report on stinging analysis of the hrp gene region. Applied and Environmental nettle (Urtica dioica). European Journal of Plant Pathology 105, Microbiology 65, 2184–2194. 307–315. Prior P & Fegan M (2005) Diversity and molecular detection of White TJ, Bruns T, Lee S & Taylor J (1990) Amplification and direct Ralstonia solanacearum race 2 strains by multiplex PCR. In sequencing of fungal ribosomal RNA genes for phylogenetics. In Bacterial wilt Disease and the Ralstonia solanacearum Species PCR Protocols: A Guide to Methods and Applications (eds Innis Complex (eds Allen C, Prior P & Hayward AC), pp. 405–414. APS MA, Gelfand DH, Sninsky JJ & White TJ), pp. 315–322. Academic Press, Madison, WI (USA). Press, Inc, New York, US. Safni I, Cleenwerck I, De Vos P, Fegan M, Sly L & Kappler U (2014) Wicker E, Grassart L, Coranson-Beaudu R, Mian D, Guilbaud C, Fegan Polyphasic taxonomic revision of the Ralstonia solanacearum species M et al. (2007) Ralstonia solanacearum strains from Martinique

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(French West Indies) exhibiting a new pathogenic potential. Applied exclusivity with 73 reference strains from 5 pathovars of and Environmental Microbiology 73, 6790–6801. Pseudomonas syringae, 10 strains of other Pseudomonas Wicker E, Grassart L, Mian D, Coranson Beaudu R, Dufeal D, spp. and 24 strains of four other genera (Alvarez et al., Guilbaud C et al. (2002) Cucumis melo, Cucumis sativus, Cucurbita 1993). Data from Agdia. moschata, and Anthurium spp, new hosts of Ralstonia solanacearum in Martinique (French West Indies). Bacterial Wilt Newsletter 17, 20–21. Diagnostic sensitivity. Wicker E, Lefeuvre P, de Cambiaire J-C, Lemaire C, Poussier S & • Pocket diagnostic (PD51119) combined sensitivity of Prior P (2012) Contrasting recombination patters and demographic 100% compared with real-time TaqMan PCR of Weller histories of the plant pathogen Ralstonia solanacearum inferred from et al. (2000), tested on healthy (n = 21) and infected – MLSA. The ISME Journal 6, 961 974. (n = 18) stems of tomato, eggplant and Solanum Wullings BA, Van Beuningen AR, Janse JD & Akkermans ADL dulcamara (1998) Detection of Ralstonia solanacearum, which causes brown and potato tuber samples (200 tubers each) with rot of potato, by fluorescent in situ hybridization with 23S rRNA- (n = 4) and without (n = 19) latent infections. Data from targeted probes. Applied and Environmental Microbiology 64, Fera. 4546–4554. Diagnostic specificity. • Pocket diagnostic (PD51119) combined specificity of Appendix 1 – Validated antibodies for 87.2% (5 false-positive results with tomato, eggplant and S. dulcamara stems). Data from Fera. serological tests • ImmunoStrip (ISK 33900) diagnostic specificity unknown but no cross-reaction reported with potato, 1. Field test kits tomato or Pelargonium tissues. Unreliable for testing crown and root tissue of Helleborus. Data from Validated lateral flow device (LFD) field test kits for rapid Agdia. presumptive field diagnosis include: Pocket Diagnostic (PD51119; Abingdon Health, York, GB) with monoclonal antibody Y9 raised against 2. Immunofluorescence Ralstonia solanacearum (Phylotype II) reference strain Procedures for performing the immunofluorescence test NCPPB 4156 from potato. are described in full in EPPO Standard PM 7/97 ImmunoStrip (ISK 33900; Agdia Inc., Elkhart, ID, US) Indirect immunofluorescence test for plant pathogenic with monoclonal antibody Ps-1 raised against bacteria. Antibodies for use in this test, which have been Ralstonia pseudosolanacearum reference strain A3380 validated in EUPHRESCO interlaboratory comparisons from peanut. (Van Vaerenbergh et al., 2016), include polyclonal anti- bodies from: Performance criteria available • Loewe Biochemica, Sauerlach, DE (07356) Analytical sensitivity. • Plant Print Diagnostics S.L., Valencia, ES (1546-H/ • Pocket diagnostic (PD51119) detected down to (1.0– IVIA). 5 1 3.0) 9 10 cfu mL of 5 reference strains of • Prime Diagnostics, Wageningen, NL (B-Rsol_C) R. solanacearum (Phylotype II) in water. Data from Fera. Available performance criteria • ImmunoStrip (ISK 33900) detection limit in plant • Loewe Biochemica (07356): 5 1 extract = 5 9 10 cfu mL . Data from Agdia. Manufacturers’ analytical specificity data shows: 100% inclusivity (based on testing of 45 isolates of Analytical specificity. R. solanacearum and 5 isolates of • Pocket diagnostic (PD51119) showed 100% inclusivity R. pseudosolanacearum) and 100% exclusivity (based with 29 reference strains of R. solanacearum (Phylotype on testing of 24 isolates of 7 other plant pathogenic, II), 16 reference strains of R. pseudosolanacearum (Phy- including 12 isolates of pathogens which can affect lotypes I and III) and 6 reference strains of R. syzygii potato, tomato or Pelargonium). (Phylotype IV) and 100% exclusivity with 15 reference Vreeburg et al. (2016) obtained the following perfor- strains of related Ralstonia spp. (n = 3), other bacterial mance data using Loewe Biochemica goat polyclonal pathogens of potato (n = 7) or bacteria known to cross- antibodies (07356) on spiked extracts prepared from 200 react with polyclonal antibodies to R. solanacearum healthy potato heel end cores: (n = 5). Data from Fera. • Monoclonal antibody Ps-1 used in ImmunoStrip (ISK Analytical sensitivity: 100% accurate down to 105 cfu 33900) showed 100% inclusivity with 73 reference mL–1 but only 50% and 33% accurate at 104 and 103 cfu strains belonging to R. solanacearum (Phylotype II) and mL–1, respectively. No detection at 102 cfu mL–1. R. pseudosolanacearum (Phylotype I) and 100% Repeatability: 100% (n = 8).

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 50 Diagnostics

Reproducibility: 100% (n = 8). 2. Generic media Diagnostic sensitivity: 100%, based on testing 187 rou- tine potato samples (200 tubers each) of which 14 had 2.1. Nutrient agar (NA) (Lelliott and Stead, 1987) tested positive and 173 had tested negative according to the full procedure annexed to EU Directive 2006/63/EC. Peptone 5.0 g Yeast extract 3.0 g Diagnostic specificity: 98.3% based on the same samples NaCl 0.5 g as described above. Microbiological-grade agar 15.0 g • Plant Print Diagnostics S.L. (1546-H/IVIA): Distilled water 1.0 L Manufacturers’ analytical specificity data shows: 100% pH is adjusted to 7.2. inclusivity (based on testing 71 isolates of R. solanacearum and R. pseudosolanacearum) and 2.2. Sucrose peptone agar (SPA) (Lelliott and Stead, 1987) 100% exclusivity (based on testing of 56 isolates of 7 genera of other plant pathogenic bacteria). Sucrose 20.0 g • Prime Diagnostics (B-Rsol_C): Peptone 5.0 g Manufacturer’s analytical specificity data shows: 100% K2HPO4 7.0 g inclusivity (based on testing 28 isolates of Microbiological-grade agar 12.0 g Distilled water 1.0 L R. solanacearum or R. pseudosolanacearum) and 100% exclusivity (based on testing 1 isolate each of 4 other pH is adjusted to 7.2. potato pathogens and 1 isolate of Burkholderia cepacia). No false-positive results were found when 2.3. Yeast peptone glucose agar (YPGA) (Lelliott and testing healthy plant extracts including potato leaves or Stead, 1987) tuber peel, Pelargonium leaves or stems and rose Yeast extract 7.0 g leaves or stems. Bacto Peptone 7.0 g Glucose 7.0 g Microbiological-grade agar 18.0 g Appendix 2 – Buffers and media Distilled water 1.0 L All media are sterilized by autoclaving at 121°C for pH is adjusted to 7.2. 15 min, except when stated otherwise. When expecting problems with fungal contamination it is recommended to 3. Semi-selective isolation media add antifungal compounds (such as cycloheximide 50– 100 mg L 1 or nystatin 35 mg L 1). 3.1 SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996) 1. Buffers Bacto Peptone (BD*) 10.0 g Phosphate buffer (PB; 50 mM, pH 7.0) for the extraction Glycerol 5 mL * and dilution of bacteria from infected tissues Bacto Agar (BD ) 15.0 g Casamino acids (BD*) 1.0 g Yeast extract (BD*) 1.0 g Na2HPO4 4.26 g Deionized distilled water 1 L KH2PO4 2.72 g Distilled water 1000 mL After autoclaving, cool to 45–50°C and add filtered sterilized Additional components may be useful, as follows: aqueous stock solutions of the following ingredients to obtain the Lubrol flakes (deflocculant) 0.5 g L1 specified final concentrations: DC silicone anti-foam (anti-foam agent) 1.0 mL L1 2,3,5-Triphenyl-2H-tetrazolium chloride 0.050 g tetrasodium pyrophosphate (anti-oxidant) 1.0 g L1 (Sigma-Aldrich T8877) polyvinylpyrrolidone-40000 (PVP-40; Crystal violet (Sigma-Aldrich C0775) 0.005 g 1 binding of PCR inhibitors) 50 g L Chloramphenicol (water soluble; Sigma-Aldrich C3175) 0.005 g Penicillin G (benzylpenicillin sodium salt; 825 U Phosphate buffer (10 mM PB, pH 7.2) for resuspension Sigma-Aldrich P8431) of pelleted extracts Polymyxin B (sulphate salt; Sigma-Aldrich P1004) 600 000 U Bacitracin (Sigma-Aldrich B0125) 1250 U

Na2HPO412H2O 2.70 g Final pH is usually about 6.5 and does

NaH2PO42H2O 0.40 g not need adjusting. Distilled water 1.0 L *BD, Becton, Dickinson and Company.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 51

Note: 1 For isolation of Ralstonia solanacearum Moko strains or the 2,3,5-Triphenyl-2H-tetrazolium chloride 0.025 g Phylotype II sequevar 4 (NPB) strain, 1 g of yeast extract Crystal violet 0.002 g Chloramphenicol 0.005 g should be added to the basal medium before autoclaving. Penicillin G 20 U 2 Oxoid Agar No. 1 can be used in place of Bacto Agar (Bec- Polymyxin B 0.01 g (60 000 U) ton, Dickinson and Company). In this case growth of Tyrothricin 0.02 g R. solanacearum will be slower or may not occur. Typical Adjust pH to 7.2 colonies of Ralstonia spp. may take 1–2 days longer to form The medium should be kept at a temperature of <20°C in dark and the red coloration may be lighter and more diffuse than conditions for no more than 3 months. on Bacto Agar. 3 Increasing the Bacitracin concentration to 2500 U L–1 may reduce populations of competing bacteria without affecting Appendix 3 – Loop-mediated isothermal growth of the Ralstonia spp. (Elphinstone et al.,1996). amplification (LAMP) (Lenarci c et al., 2014) 4 Store media and stock solutions of antibiotics at 4°C in the dark and use within 1 month. 5 If alternative sources of antibiotics or crystal violet are used it 1. General Information may be necessary to first dissolve in small quantities of etha- nol or methanol prior to making up stock solutions with dis- 1.1 The following LAMP test is used for on-site detection tilled water. as well as laboratory detection of R. solanacearum in 6 Plates should be free from surface condensation before use. symptomatic plant material. The test can be applied to 7 Avoid excess drying of plates. bacterial exudates of symptomatic plant material after 8 Quality control can be performed after preparation of each 2-min boiling without further DNA purification. The new batch of medium by plating a suspension of a reference test also performed well on a limited set of artificially culture of a relevant Ralstonia species and observing forma- and naturally contaminated samples of potato tubers tion of typical colonies after incubation at 28°C for 2–6 prepared according to the EC Directive 2000/29/EC, 5 days. detecting 10 cells per mL of potato tuber extract; however, further data is needed to confirm its reliabil- Performance criteria available. Matrices tested: potato ity for latent testing. tubers, Pelargonium petioles, surface water. 1.2 The test was developed by Lenarcic et al. (2014) Analytical sensitivity: From potato tuber samples 1.3 The test targets the endoglucanase (egl) gene 2 1 1 1 3 9 10 cfu mL , from water samples 3 9 10 cfu mL 1.4 Oligonucleotides: primer sequences are given in 50–30 1 and from Pelargonium petioles 6.5 9 102 cfu mL . orientation with positions on the egl sequence of the Analytical specificity: of 25 strains of different race/bio- GMI100 strain (GenBank accession number var combinations, all formed typical colonies on SMSA. Of DQ657595) in brackets: 37 non-target bacteria found on potato tubers, water and F3_RS_egl:50-GAGCAACTACATCTATCCGTC-30 ornamental plants, some growth was observed on SMSA by (330–350) Burkholderia andropogonis, Burkholderia cepacia and B3_RS_egl:50-CATCAGCCCGAAGATGAC-30 (637– Ralstonia pickettii; however, colony morphology was not 654) typical (all 37 bacteria gave negative results). FIP_RS_egl:50-ACAGCTCGTTCGCGTCGACGACA Repeatability: 100% GCGCGACCTACTA-30 (354–371, 446–463) Reproducibility: 100% BIP_RS_egl:50-GGTTCGTCAACGCCGTGAGATCA Diagnostic sensitivity: 100% (cf. TaqMan PCR of CGTTGCCG TAGTAG-30 (476–493, 540–558) Weller et al., 2000). FLoop_RS_egl:50-GTTCATGCCCTTGTTCTTG-30 Diagnostic specificity: 100% (cf. TaqMan PCR of (372–390) Weller et al., 2000). BLoop_RS_egl:50-GCTCGATCCGCACAACTA-30 (516–533) 3.2. Sequeira modified semi-selective medium (Granada The validation data has been produced using standard and Sequeira, 1983; Poussier et al., 1999) purified primers; however, HPLC-purified primers are now generally advised for LAMP. Bacto Peptone 11.0 g Glycerol 6.3 g 1.5 The test has been successfully performed on three dif- Bacto Agar 18.0 g ferent machines: SmartCycler (Cepheid, Sunnyvale, Yeast extract 1.0 g CA, US), Genie II (Optigene Ltd, Horsham, GB) and Deionized distilled water 1 L Roche Light Cycler 480 (Roche Applied Science) After autoclaving, let the medium cool to 45–50°Candaddfilter with comparable results. Because of the differences in sterilised sterilized aqueous stock solutions of the following ingredi- melting curve analysis among the instruments, the ents to obtain the specified final concentrations: melting temperature (Tm) range can differ and was

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 52 Diagnostics

observed to be approximately 1°C lower in GenieII Table (continued) compared with SmartCycler and LightCycler 480. 1.6 Software allowing fluorescence acquisition in real- Working Volume per Final time and melting curve analysis should be used. The Reagent concentration reaction (lL) concentration specific instrument manual should be consulted. Note: F3_RS_egl 100 lM 0.05 0.2 lM software for cycling (real-time PCR devices) should B3_RS_egl 100 lM 0.05 0.2 lM be programmed to measure the fluorescence during FIP_RS_egl 100 lM 0.5 2 lM the amplification and the melting curve analysis. BIP_RS_egl 100 lM 0.5 2 lM FLoop_RS_egl 100 lM 0.5 2 lM BLoop_RS_egl 100 lM 0.5 2 lM 2. Methods Subtotal 22.5 2.1 Nucleic acid extraction and purification Genomic 2.5 DNA sample 2.1.1 The test can be applied to pure cultures, bacterial (boiled exudates and extracts from small pieces of symp- bacterial tomatic plant material after boiling (see point suspension or 2.1.2), and to plant material after a DNA extrac- DNA isolated tion (see 2.1.3 below). from plant 2.1.2 Heating. A heating time of 2 min at 95°C was extract) repeatedly successful for the confirmation of tuber Total 25 infections (six tubers tested) and other symp- *Molecular-grade water should be used preferably, or prepared, purified tomatic plant material. For heating, collect (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and approximately 2–10 lL of bacterial exudates or nuclease-free water. tuber vascular tissue or a piece of stem from 2.2.1 LAMP conditions: 60°C for 30 min; melting curve –1 symptomatic host plant (e.g. a 1-cm piece cut ver- analysis: 98°Cto80°C, 0.05°Cs . tically or in small pieces). Put the material into 100 lL of sterile water or phosphate buffer and 3. Essential procedural information briefly vortex. Heat in a waterbath or heat in an incubator set at 95–98°C for 2 min. Inoculation 3.1 Controls loops can be used at all stages of preparation For a reliable test result to be obtained, the following (ex- 2.1.3 DNA extraction from plants. For on-site use on ternal) controls should be included for each series of symptomatic tubers no DNA extraction is per- nucleic acid extraction (not applicable when crude homoge- formed. Magnetic-bead-based QuickPick SML nates are tested) and amplification of the target organism and target nucleic acid, respectively: Plant DNA kits (Bio-Nobile, Turku, FI) automated • with the KingFisher mL system (Thermo Labsys- Negative isolation control (NIC) to monitor contamina- tem) can be used to extract and purify DNA from tion during nucleic acid extraction: nucleic acid extrac- extracts from 200 potato tubers and symptomatic tion and subsequent amplification of a sample of clean extraction buffer. plants of tomato, eggplant and pelargonium. The • extraction is done from 100-lL extracts according Positive isolation control (PIC) to ensure that nucleic to Pirc et al. (2009), with a minor modification acid of sufficient quantity and quality is isolated: nucleic (440 lL lysate is used for purification). acid extraction and subsequent amplification of the tar- 2.1.4 DNA should preferably be stored at approxi- get organism or a matrix sample that contains the target mately 20°C. organism (e.g. naturally infected host tissue or host tis- sue spiked with the target organism). • 2.2. LAMP Negative amplification control (NAC) to rule out false Master mix positives due to contamination during the preparation of the reaction mix: amplification of molecular-grade water that was used to prepare the reaction mix. Working Volume per Final • Reagent concentration reaction (lL) concentration Positive amplification control (PAC) to monitor the effi- ciency of the amplification: amplification of nucleic acid of Molecular- N.A. 7.9 N.A. the target organism. This can include nucleic acid extracted * grade water from the target organism, total nucleic acid extracted from 9 9 Isothermal 2 12.5 1 infected host tissue, whole-genome amplified DNA or a master mix synthetic control (e.g. cloned PCR product). The PAC (Optigene Ltd.) should preferably be near to the limit of detection. (continued) For on-site applications only NAC and PAC are used.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 53

3.2 Interpretation of results • On infected symptomatic plants: In order to assign results from PCR-based tests the follow- All symptomatic material tested gave positive results. ing criteria should be followed: The following symptomatic material was tested: Plants with wilting symptoms (2–4 days after inocula- Verification of the controls tion): bacterial ooze at the site of inoculation was col- • NIC and NAC (and if relevant NIC) should produce no lected using a sterile plastic inoculation loop, and fluorescence. suspended in 100 lL of sterile water. • The PAC (and if relevant PIC and IC) amplification Bacterial ooze from seven symptomatic tubers was curve should be exponential. T should be characteristic m tested. of the Ralstonia species (see below). 4.2 Analytical specificity data When these conditions are met The test was 99% accurate (1 isolate of R. syzygii from • A test will be considered positive if it produces an expo- Australia gave a false-negative result, no false positives). nential amplification curve and has a characteristic T . m Tested on 88 strains of target Ralstonia spp., 13 non-target Note that the determined T values vary among phylo- m and potentially cross-reacting strains listed in EU Council genetically distinct strains. Also, T values depend on m Directive 2009/29/EC and 13 other bacterial pathogens that the instruments and temperature profile used to generate can be present on Ralstonia spp. host plants. melting curve analysis. Consequently, the specific Tm should be verified in each laboratory. In the amplifica- 4.3 Data on repeatability tion of strains using SmartCycler (Cepheid) the T val- m Repeatability: 100% detection for dilutions with at least 104 ues were: 94.6°C(0.2) for Phylotype I (Asia), 94.5°C copies of rS DNA or more. The test was repeated 10 times (0.4) for Phylotype III (Africa), 93.8°C(0.2) for – on R. solanacearum GBBC1172 strain at 108 cells mL 1. Phylotype IIA and 93.7°C(0.2) for Phylotype IIB. Time of positivity (Tp) was 12.7 0.49 min, with Tm of Positive samples of exudates from infected potato tubers – 94.1 0.32°C. Lower dilution (104 cells mL 1) was (R3Bv2 strain) analysed on GenieII exhibited a T of m repeated three times, with times to positivity for strain 92.1 0.11°C. GBBC1172 as follows: 23.2, 23.0 and 16.2 min. • A test will be considered negative if it produces no fluo- rescence or the T is not characteristic of the Ralstonia m 4.4 Data on reproducibility species. The test was performed successfully on three different • Tests should be repeated if any contradictory or unclear machines: SmartCycler (Cepheid), Genie II (Optigene Ltd), results are obtained. and Roche Light Cycler 480 (Roche Applied Science). Results are comparable. However, due to different ways of

4. Performance criteria available obtaining Tm by the GenieII machine, the range of Tm is about 1°C lower than the T obtained with the SmartCycler 4.1 Analytical sensitivity data m and LightCycler 480. • On pure cultures: Sensitivity was assessed in triplicates on pure culture 4.5 Analytical selectivity suspension on a single strain that represents each phy- No cross-reactivity of different hosts/cultivars/tissues was lotype: observed. DNA from healthy plant extracts was tested: Phylotype I (GBBC1172), limit of detection at potato (43 samples/21 cultivars), tomato (4), eggplant (3), 104 cells mL–1 pelargonium (6) and Solanum dulcamara (6). Phylotype IIA (RUN 30), limit of detection at 106 cells mL–1 4.6 Diagnostic sensitivity Phylotype IIB (GBBC729), limit of detection at All 50 samples tested with the egl LAMP test showed 105 cells mL–1 results in agreement with the diagnostic status of the sam- Phylotype III (LMG 2296), limit of detection at ple (samples above the limit of detection of the method). 104 cells mL–1 Seven real diagnostic samples that were previously con- Phylotype IV (RUN 71), limit of detection at 106 cells firmed to be positive for R. solanacearum were positive mL–1 using the egl LAMP test. • On spiked potato tubers: 105 cells per mL of plant extract, based on three serial 4.7 Diagnostic specificity dilutions of R. solanacearum in extracts from potato Forty-three samples of healthy potato extract (confirmed by tubers, which had previously tested negative for real-time PCR and immunofluorescence) were negative Ralstonia. using the egl LAMP assay.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 54 Diagnostics

Extracts of healthy plants of other hosts [tomato (4), egg- amplifiable target DNA from a known negative plant plant (3), pelargonium (6) and S. dulcamara (6)] were all extract to which has been added between 103 and 104 cfu negative. mL–1 of a reference strain of R. solanacearum as a posi- tive control. PCR tests can be performed on undiluted and diluted DNA extracts. Testing diluted DNA (1/2 or 1/5) Appendix 4 – Conventional PCR tests for may help to avoid false-negative PCR results due to poly- detection and identification of specific merase inhibitors co-extracted from plant samples. DNA Ralstonia solanacearum (Phylotype II) and may be prepared in advance and stored frozen prior to Ralstonia pseudosolanacearum (Phylotype I) use. (Pastrik et al., 2002) For crude DNA extraction from presumptive Ralstonia isolates, suspend single colonies from each isolate and l 1. General information from cultures of reference strains in 100 L of sterile dis- tilled water. Heat in closed microvials at 95°C for 1.1 The test can be used for detection of Ralstonia spp. 12 min. Transfer heated suspensions to ice and pulse cen- (Phylotypes I and II only) in potato tuber tissue trifuge after cooling. extracts and for identification of Ralstonia isolates in DNA should preferably be stored at approximately Phylotypes I and II. Primer RS-1-F is generic for all 20°C. strains, whereas the specificity of the reverse primers was selected for amplification of R. solanacearum 2.2. Polymerase chain reaction phylotype II strains (RS-1-R) only or 2.2.1 Multiplex reaction with internal PCR control R. pseudosolanacearum phylotype I strains (RS-3-R) 2.2.1.1 Master mix only. 1.2 The test was developed by Pastrik et al. (2002). 1.3 The test targets 16–23S rRNA intergenic spacer (ITS) Volume per sequences. Primers NS-5-F and NS-6-R (Dams et al., Working reaction Final l 1988; White et al., 1990) amplify 18S rRNA target Reagent concentration ( L) concentration DNA co-extracted from plant samples and can be Molecular-grade water* N.A. 12.625 N.A. used in multiplex or simplex as an internal positive PCR buffer1 with 109 2.5 19

control (IPC). 15 mM MgCl2 1.4 Oligonucleotides: (e.g. Invitrogen) Forward primer RS-1-F 50-ACTAACGAAGCAGAGAT dNTPs (e.g. Boehringer, 20 mM 0.125 0.1 mM Mannheim, DE2) GCATTA-30 BSA (fraction V) 10% 0.25 0.1% Reverse primer RS-1-R 50-CCCAGTCACGGCAGAG 0 (e.g. Serva, Germany) ACT-3 Forward primer (RS-1-F) 10 lM 2 0.8 lM 0 Reverse primer RS-3-R 5 -TTCACGGCAAGATCGC Reverse primer (RS-1-R) 10 lM 2 0.8 lM TC-30 Forward primer 10 lM 0.15 0.06 lM 3 Forward primer NS-5-F 50-AACTTAAAGGAATTGA (NS-5-F ) 0 Reverse primer 10 lM 0.15 0.06 lM CGGAAG-3 3 0 (NS-6-R ) Reverse primer NS-6-R 5 -GCATCACAGACCTGTTA 1 l 1 0 Polymerase (Gibco )5UL 0.2 1 U TTGCCTC-3 . Subtotal 20 1.5 With primers RS-1-F and RS-1-R an amplicon of Genomic DNA 5 718 bp is generated; with primers RS-1-F and extract RS-3-R an amplicon of 716 bp is generated; with Total 25 primers NS-5-F and NS-6-R an amplicon of 310 bp *Molecular-grade water should be purified (deionized or distilled), is generated. sterilized (autoclaved or 0.45-lm filtered) and nuclease-free. 1The above test was originally optimized for the MJ Research PTC 200 thermocycler with Gibco BRL Taq Polymerase and buffer. 2. Methods Further optimization is recommended for use with other reagents and 2.1 Nucleic acid extraction systems. 2 Isolate DNA from potato tuber tissue extracts and DNA Provider as indicated in the publication, but this company does not exist anymore. Further optimization is recommended for use with other extraction controls using, for example, an Invitrogen Easy dNTPs. TM DNA kit according to the manufacturer’s instructions and 3Concentrations of primers NS-5-F and NS-6-R were optimized for with the addition of 1 mg per 100 lL of lysozyme in the potato heel end core extraction and DNA purification using the lysis buffer and heating to 37°C for 30 min, as validated by methods according to Pastrik & Maiss (2000). Re-optimization of Pastrik & Maiss (2000). If alternative extraction kits are reagent concentrations will be required if extraction by shaking or other used they should first be shown to reliably extract DNA isolation methods is used.

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2.2.1.2 PCR conditions: an initial 5-min incubation at Table (continued) 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 58°C and 45 s at 72°C, followed by a Volume final extension of 5 min at 72°C. Working per reaction Final 2.2.2 Phylotype II-specific PCR Reagent concentration (lL) concentration 2.2.2.1 Master mix Genomic DNA 5 extract Total 25 Volume Working per reaction Final *Molecular-grade water should be used preferably, or prepared purified Reagent concentration (lL) concentration (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and nuclease-free water. * Molecular-grade water N.A. 12.925 N.A. 1The above method was originally optimized for a MJ Research PTC 9 9 PCR buffer with 15 mM 10 2.5 1 200 thermocycler with Gibco BRL Taq Polymerase and buffer. Further 1 MgCl2 (e.g. Invitrogen) optimization is recommended for use with other reagents and systems. 2 dNTPs (e.g. Boehringer ) 20 mM 0.125 0.1 mM 2Provider as indicated in the publication but this company does not BSA (fraction V) 10% 0.25 0.1% exist anymore. Further optimization is recommended for use with other (e.g. Serva, Germany) dNTPs. Forward primer (RS-1-F) 10 lM 2 0.8 lM Reverse primer (RS-1-R) 10 lM 2 0.8 lM 2.2.3.2 PCR conditions: an initial 5-min incubation at Polymerase (Gibco 1)5UlL 1 0.2 1 U ° ° Subtotal 20 95 C followed by 35 cycles of 30 s at 95 C, Genomic DNA extract 5 30 s at 58°C and 45 s at 72°C, followed by a Total 25 final extension of 5 min at 72°C.

*Molecular-grade water should be used preferably, or prepared purified (deionized or distilled), sterilized (autoclaved or 0.45-lm filtered) and 3. Essential procedural information nuclease-free water. 1The above method was originally optimized for a MJ Research PTC 3.1 Controls 200 thermocycler with Gibco BRL Taq Polymerase and buffer. For a reliable test result to be obtained, the following (ex- Further optimization is recommended for use with other reagents and ternal) controls should be included for each series of systems. nucleic acid extraction and amplification of target nucleic 2 Provider as indicated in the publication, but this company does not acid, respectively: exist anymore. Further optimization is recommended for use with • Negative isolation control (NIC) to monitor contamina- other dNTPs. tion during nucleic acid extraction: nucleic acid extrac- 2.2.2.2 PCR conditions: an initial 5-min incubation at tion and subsequent amplification, preferably of a 95°C followed by 35 cycles of 30 s at 95°C, sample of uninfected matrix or if not available clean ° ° extraction buffer. 30 s at 58 C and 45 s at 72 C, followed by a • final extension of 5 min at 72°C. Positive isolation control (PIC) to ensure that nucleic 2.2.3 Phylotype I-specific PCR acid of sufficient quantity and quality is isolated: nucleic 2.2.3.1 Master mix acid extraction and subsequent amplification of the tar- get organism or a matrix sample known to contain the target organism (e.g. naturally infected host tissue or Volume host tissue spiked with the target organism). Working per reaction Final • Negative amplification control (NAC) to rule out false Reagent concentration (lL) concentration positives due to contamination during the preparation of the reaction mix: amplification of molecular-grade water Molecular-grade water* N.A. 14.925 N.A. PCR buffer with 15 mM 109 2.5 19 that was used to prepare the reaction mix. 1 • Positive amplification control (PAC) to monitor the effi- MgCl2 (e.g. Invitrogen) dNTPs (e.g. Boehringer2) 20 mM 0.125 0.1 mM ciency of the amplification: amplification of nucleic acid BSA (fraction V) 10% 0.25 0.1% of the target organism. This can include nucleic acid (e.g. Serva, Germany) extracted from the target organism, total nucleic acid l l Forward primer 10 M 1 0.4 M extracted from infected host tissue, whole-genome ampli- (RS-1-F) fied DNA or a synthetic control (e.g. cloned PCR pro- Reverse primer 10 lM 1 0.4 lM (RS-3-R) duct). For PCRs not performed on isolated organisms, the Polymerase (Gibco1)5UlL1 0.2 1 U PAC should preferably be near to the limit of detection. Subtotal 20 The multiplex PCR (2.2.1 above) includes an additional IPC involving amplification of 18S rDNA co-extracted from (continued) the host plant.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 56 Diagnostics

3.2 Interpretation of results polymerase and thermocyclers (Van Vaerenbergh et al., Verification of the controls. 2016). • NIC and NAC should produce no amplicons. • PIC, PAC should produce amplicons of the (relevant) 4.5 Diagnostic specificity size (depending on the target or whether endogenous Diagnostic specificity was 100%. All 13 potato samples or exogenous nucleic acid is used). naturally latently infected with R. solanacearum (con- firmed by IF, isolation and tomato bioassay) tested When these conditions are met. positive with multiplex PCR using primers RS-1-F/RS-1-R • A test will be considered positive if amplicons of 718 and NS-5-F/NS-6-R. All 4300 healthy potato samples bp (RS-1-F and RS-1-R), 716 bp (RS-1-F and RS-3- from 143 potato cultivars tested negative (Pastrik et al., R) and 310 bp (NS-5-F and NS-6-R) are produced 2002). • A test will be considered negative if it produces no band or a band of a different size. Appendix 5 – Real-time TaqMan PCR tests • Tests should be repeated if any contradictory or for detection and identification of strains of unclear results are obtained. all Ralstonia solanacearum, Ralstonia pseudosolanacearum and 4. Performance criteria available Ralstonia syzygii (Phylotypes I–IV) and 4.1 Analytical sensitivity R. solanacearum Phylotype IIB sequevar 1 The minimum population of R. solanacearum NCPPB1331 (Weller et al., 2000) detected following serial dilution in sterile water or potato extracts was 0.5 cfu mL–1 after DNA purification and con- centration (Pastrik et al., 2002) whether performed as sim- 1. General information plex (RS-1-F/RS-1-R primers only) or as multiplex with 1.1 The test is used for the detection and identification of all NS-5-F/NS-6-R primers as an IPC. strains of R. solanacearum and R. pseudosolanacearum and for specific detection and identification of 4.2 Analytical specificity R. solanacearum Phylotype II sequevar 1 (race 3) Testing was done on 29 NCPPB reference strains of 3 only. Ralstonia phylotypes in the species complex from multiple 1.2 The test was developed by Weller et al. (2000). hosts and countries worldwide. RS-1-F/RS-1-R primers gave 1.3 Real-time PCR primers and probes RS-I-F, RS-II-R and positive results with all 16 isolates of R. solanacearum (Phy- RS-P target specific 16S rRNA gene sequences con- lotype II) and negative results with all 8 isolates of served within all R. solanacearum, R. pseudosolanac- R. pseudosolanacearum (Phylotype I) and 5 isolates of earum and R. syzygii strains (Weller et al., 2000). R. syzygii . RS-1-F and RS-3-R primers gave negative results Improved specificity for detection of all strains was R. solanacearum with all 16 isolates of (Phylotype II) and 5 demonstrated by Vreeberg et al. (2016) using a R. syzygii isolates of and gave positive results with all 8 iso- TaqMan probe modified from the original probe R. pseudosolanacearum lates of (Phylotype I). described by Weller et al. (2000). Modified primers Negative results were obtained with both primer sets after RS-I-FmAK/RS-II-RmAK (Korner€ et al.,2017)were Ralstonia eutrophus testing 25 non-target bacteria including also developed to improve the specificity of this test. (3 isolates), Ralstonia pickettii (3 isolates), Burkholderia Real-time PCR primers and probes B2-I-F, B2-II-R andropogonis (3 isolates), Burkholderia caryophylli (3 iso- and B2-P target specific prophage sequence specific to lates), Burkholderia cepacia (4 isolates), Burkholderia gladioli strains of R. solanacearum Phylotype IIB-1 (race 3, (1 isolate), Burkholderia glumae (2 isolates), Burkholderia biovar 2) (Fegan et al., 1998). Real-time PCR plantarii (2 isolates) and Clavibacter michiganensis subsp. primers COX-F, COX-R and COX-P target plant cyto- sepedonicus (4 isolates). chrome oxidase gene sequences co-extracted from plant samples and are used as positive amplification control 4.3 Repeatability (PAC). Not available. 1.4 Oligonucleotides: RS-I-F 50-GCATGCCTTACACATGCAAGTC -30 4.4 Reproducibility RS-II-R 50-GGCACGTTCCGATGTATTACTCA-30 In three EUPHRESCO interlaboratory comparisons, 29, *RS-I-FmAK 50-CATGCCTTACACATGCAAGTC-30 23 and 39 laboratories obtained accurate results in an *RS-II-RmAK 50-CACGTTCCGATGTATTACTCA-30 average of 97.5%, 98.1% and 98.6% of a total of 475 B2-I-F 50-TGGCGCACTGCACTCAAC-30 tests performed on spiked, naturally infected and healthy B2-II-R 50-AATCACATGCAATTCGCCTACG-30 potato tuber samples using a variety of sources of Taq COX-F 50-CGTCGCATTCCAGATTATCCA-30

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COX-R 50-CAACTACGGATATATAAGAGCCAAA 2.2. Polymerase chain reaction ACTG-30. 2.2.1 Master mix *The use of modified primers RS-I-FmAK/RS-II-RmAK in place of RS-I-F/RS-II-R was found to reduce/eliminate false-positive results with high Ct values, sometimes found Working Volume per Final l to occur due to co-extraction from potato tuber tissue of Reagent concentration reaction ( L) concentration € DNA from bacteria of the genus Advenella (Korner et al., Molecular-grade water* N.A. 12.875 N.A. 2017). Real-time (RT-)PCR 109 2.5 19 Probes: buffer RS-P 50-[FAM]-AGCTTGCTACCTGCCGGCGAGT (Applied Biosystems) 0 G-[TAMRA]-3 MgCl2 (Applied 25 mM 3.5 3.5 mM Biosystems) B2-P 50-[VIC]-TTCAAGCCGAACACCTGCTGCAA dNTPs (Applied 2.5 mM 2 0.2 mM G-[TAMRA]-30 0 Biosystems) COX-P 5 -[VIC]-TGCTTACGCTGGATGGAATGCC Forward primer 10 lM 0.75 0.3 lM 0 CT-[TAMRA]-3 (RS-I-F; B2-I-F; **RSP-55T 50-[FAM]-AGCTTGCTACCTGCCGG-[N COX-F) FQ-MGB]-30. Reverse primer 10 lM 0.75 0.3 lM **A modification of probe RS-P, containing a minor (RS-II-R; B2-II-R; COX-R) groove binding unit (mgb), was described by Vree- Probe 1 (RS-P; B2-P; 5 lM 0.5 0.1 lM burg et al. (2016) and results in elimination of occa- COX-P; RSP-55T) sional false-positive results with high Ct values due to AmpliTaq Gold 5UlL1 0.125 0.625 U non-specific binding to non-target DNA extracted Polymerase† (Applied from potato tuber tissue. Biosystems) 1.5 The test was originally optimized for the Applied Bio- Subtotal 23 sytems AmpliTaqTM and buffer with the ABI 7700 and DNA dilution 2 Total 25 7900 sequence detector TaqMan systems and the Cepheid Smartcycler system. Validation data from the *Molecular-grade water should be used preferably, or prepared purified article is under these conditions. (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and nuclease-free water. †Method originally optimized for Applied Biosytems AmpliTaq GoldTM and buffer with the ABI 7700 and 7900 sequence detector TaqMan 2. Methods systems and the Cepheid Smartcycler system. Further optimization will be required for use with other reagents and systems. 2.1 Nucleic acid extraction Isolate DNA from potato tuber tissue extracts and DNA 2.2.2 PCR conditions: an initial 10-min incubation at extraction controls using, for example, an Invitrogen Easy 95°C followed by 40 cycles of 15 s at 95°C and DNATM kit according to the manufacturer’s instructions 60 s at 60°C. Annealing time can be reduced to and with the addition of 1 mg per 100 lL of lysozyme in 30 s if using the Cepheid Smartcycler system. the lysis buffer and heating to 37°C for 30 min, as vali- dated by Pastrik & Maiss (2000). If alternative extraction 3. Essential procedural information kits are used they should first be shown to reliably extract amplifiable target DNA from a known negative plant 3.1 Controls extract to which has been added between 103 and 104 cfu For a reliable test result to be obtained, the following exter- mL–1 of a reference strain of the appropriate Ralstonia nal controls should be included for each series of nucleic species as a positive control. PCR tests can be performed acid extractions and amplifications: on undiluted and diluted DNA extracts. Testing diluted • Negative isolation control (NIC): 50 mM phosphate buf- DNA (1/2 or 1/5) may help to avoid false-negative PCR fer (pH 7.0) used for extraction of the bacteria from results due to polymerase inhibitors co-extracted from plant tissue. plant samples. • Positive isolation control (PIC): 50 mM phosphate buf- For crude DNA extraction from presumptive Ralstonia fer (pH 7.0) spiked with a reference strain of isolates, suspend single colonies from each isolate and from R. solanacearum. cultures of reference strains in 100 lL of sterile distilled • Negative amplification control (NAC): molecular-grade water. Heat in closed microvials at 95°C for 12 min. Trans- water used in the PCR mix. fer heated suspensions to ice and pulse centrifuge after • Positive amplification control (PAC): DNA extract of cooling. R. solanacearum cells added to molecular-grade water DNA should preferably be stored at approximately or negative tissue extract at a concentration above the – 20°C. detection threshold (103–104 cells mL 1).

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3.2. Interpretation of results not when using the alternative RSP-55T probe (R. Gotts- Verification of the controls. berger, personal communication). • The PIC and PAC amplification curves should be B2-I-F/B2-II-R/B2-P assay: 100% inclusivity when exponential. tested at Fera against all 20 R. solanacearum Phylotype • NIC and NAC should give no amplification II sequevar 1 (race 3) isolates collected worldwide. There was 100% exclusivity with 17 (non-race 3) isolates of When these conditions are met. R. solanacearum, 17 isolates of R. pseudosolanacearum,6 • A test will be considered positive if it produces an isolates of R. syzygii and with 30 non-target bacteria exponential amplification curve. including serologically cross-reacting strains listed in EU • A test will be considered negative if it does not pro- Council Directive 2009/29/EC and other bacterial duce an amplification curve or if it produces a curve pathogens that can be present on Ralstonia host plants which is not exponential. and other closely related Ralstonia and Burkholderia • Tests should be repeated if any contradictory or species. unclear results are obtained. 4.3 Repeatability 4. Performance criteria available Vreeburg et al. (2016) demonstrated 100% repeatability (n = 8) when the RS-I-F/RS-II-R/RS-P assay was used in 4.1 Analytical sensitivity conjunction with a different but equivalent DNA extraction When tested on pure cultures (4 isolates of method to that described under 2.1 above. R. solanacearum and 1 isolate of R. pseudosolanacearum) diluted in water, the lowest populations detected were between 103 and 104 cfu mL–1 using the RS-I-F/RS-II-R/ 4.4. Reproducibility RS-P assay and 105 cfu mL–1 using the B2-I-F/B2-II-R/B2- In two EUPHRESCO interlaboratory comparisons, 19 P assay (Weller et al., 2000). and 24 laboratories obtained accurate results in an aver- When tested on 1:100 dilutions of extract from 200 age of 95.4% and 93.5% of a total of 310 tests per- potato heel-end cores after spiking with R. solanacearum, formed on spiked, naturally infected and healthy potato the lowest populations detected were 104 cfu mL–1 using tuber samples using a variety of sources of TaqMan the RS-I-F/RS-II-R/RS-P assay and 105 cfu mL–1 using the master mixes and thermocyclers (Van Vaerenbergh B2-I-F/B2-II-R/B2-P assay (Weller et al., 2000). et al., 2016). Vreeburg et al. (2016) demonstrated 100% reproducibility. 4.2 Analytical specificity RS-I-F/RS-II-R/RS-P test: 100% inclusivity when tested 4.5. Diagnostic sensitivity at Fera on 60 NCPPB reference strains of Weller et al. (2000) reported that blind testing of extracts R. solanacearum species complex from multiple hosts and from 200 tuber samples successfully detected 4 known pos- countries worldwide, including 37 isolates of itive samples randomly distributed amongst 20 healthy sam- R. solanacearum (Phylotype II), 17 isolates of ples of various varieties. All wilted plants of each host, R. pseudosolanacearum (Phylotypes I and III) and 6 iso- which developed following inoculation with one of three lates of R. syzygii. There was 100% exclusivity with 30 isolates of R. solanacearum (NCPPB 325T, NCPPB 909 or non-target bacteria including serologically cross-reacting NCPPB 4536), tested positive. strains listed in EU Council Directive 2009/29/EC and Weller et al. (2000) reported that no false-positive results other bacterial pathogens that can be present on Ralstonia were obtained from the healthy samples or from stem sam- host plants and other closely related Ralstonia and ples (10 plants each) of healthy tomato, eggplant, Burkholderia species. One isolate obtained from sugar- Pelargonium or Solanum dulcamara. cane in Jamaica (NCPPB 928), identified as Ralstonia Vreeburg et al. (2016) demonstrated a diagnostic sp., tested positive. sensitivity of 100% using either the RS-I-F/RS-II-R/RS-P In addition, Lenarcic et al. (2014) detected all 84 iso- test or the RS-I-F/RS-II-R/RSP-55T test when testing 276 lates tested of the R. solanacearum species complex (in- routine potato tuber samples previously confirmed by cluding 41 R. solanacearum,35R. pseudosolanacearum IF. and 8 R. syzygii isolates) with the exception of 1 geneti- cally distinct isolate of R. syzygii (RUN 14) from Aus- 4.6. Diagnostic specificity tralia. Of 26 non-target bacteria, all tested negative with Vreeburg et al. (2016) demonstrated increased diagnostic the exception of 1 isolate of Ralstonia mannitolilytica specificity from 92.7% using the RS-I-F/RS-II-R/RS-P test (CFBP 6737). False-positive results have also been to 100% using the RS-I-F/RS-II-R/RSP-55T test due to the obtained with an isolate identified as Advenella elimination of false positives when testing 276 routine kashmirensis from rose when using the probe RS-P but potato tuber samples

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Appendix 6 – Multiplex conventional PCR 2. Methods tests for identification of Phylotypes I–IV of 2.1 Nucleic acid extraction and purification Ralstonia solanacearum, For crude DNA extraction from presumptive Ralstonia iso- Ralstonia pseudosolanacearum and lates, suspend single colonies from each isolate and from Ralstonia syzygii (Opina et al., 1997; Fegan cultures of reference strains in 100 lL of sterile distilled & Prior, 2005) water. Heat in closed microvials at 95°C for 12 min. Trans- fer heated the suspensions to ice and pulse centrifuge after cooling. 1. General information DNA should preferably be stored at approximately 1.1 This multiplex conventional PCR is suitable for the 20°C. identification of Ralstonia solanacearum (Phylotype II), R. pseudosolanacearum (Phylotype I or III) and 2.2. Multiplex PCR R. syzygii (Phylotype IV). It has not been validated 2.2.1 Master mix for direct detection in plant extracts. 1.2 The test is based on the publications of Fegan & Prior (2005) and Opina et al. (1997). Volume 1.3 The target sequences for primers 759 and 760 (Opina per et al., 1997) are those of the IpxC gene, coding for an Working reaction Final Reagent concentration (lL) concentration in planta expressed protein (unique to strains of all four phylotypes). The other primers are all targeted in Molecular-grade water* N.A. 15.25 N.A. the ITS intergenic region between the 16S and 23S PCR buffer 59 519 rRNA genes (Fegan & Prior, 2005). The reverse pri- MgCl2 25 mM 1.5 1.5 mM mer Nmult22:RR is common to all four phylotypes dNTPs (Promega) 10 mM 0.5 0.2 mM Forward primer (760) 100 lM 0.04 0.16 lM whereas the forward primers Nmult21:1F, Reverse primer (759) 100 lM 0.04 0.16 lM Nmult21:2F, Nmult23:AF and Nmult22:InF are speci- Forward primer 100 lM 0.06 0.24 lM fic to Phylotypes I, II, III and IV, respectively. (Nmult21:1F) 1.4 Amplicon sizes in base pairs are: Forward primer 100 lM 0.06 0.24 lM 280 bp or 282 bp for primers 759/760 (Nmult21:2F) l l 144 bp for primers Nmult21:1F/Nmult22:RR Forward primer 100 M 0.18 0.72 M 372 bp for primers Nmult21:2F/Nmult22:RR (Nmult23:AF) Forward primer 100 lM 0.06 0.24 lM 91 bp for primers Nmult23:AF/Nmult22:RR (Nmult22:InF) 213 bp for primers Nmult22:InF/Nmult22:RR Reverse primer 100 lM 0.06 0.24 lM 1.5 The primer sequences are: (Nmult22:RR) GoTaq G2 DNA 5UlL1 0.25 1.25 U polymerase (Promega)† Primer Sequence Specificity Subtotal 23 759 50-GTCGCCGTCAA All 4 phylotypes Target DNA 2 CTCACTTTCC-30 Total 25 760 50-GTCGCCGTCAGCAATG All 4 phylotypes *Molecular-grade water should be used preferably, or prepared purified CGGAATCG-30 (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and Nmult21:1F 50-CGTTGATGAGGCG Phylotype I nuclease-free water. CGCAATTT-30 †Further optimization will be required for use with other reagents and Nmult21:2F 50-AAGTTATGGACGGTG Phylotype II 0 systems. GAAGTC-3 ° Nmult23:AF 50-ATTACGAGAGCAAT Phylotype III 2.2.2 PCR conditions: 96 C for 5 min followed by 30 CGAAAGATT-30 cycles of (94°C for 15 s, 59°C for 30 s, 72°C for Nmult22:InF 50-ATTGCCAAGACGAG Phylotype IV 30 s) and a final step of 72°C for 10 min. AGAAGTA-30 Nmult22:RR 50-TCGCTTGACCCTAT All 4 phylotypes AACGAGTA-30 3. Essential procedural information 3.1 Controls 1.6 The multiplex PCR has been validated on a GeneAmp For a reliable test result to be obtained, the following (ex- PCR System 9700 thermal cycler (Thermo Fisher Sci- ternal) controls should be included for each series of entific) and a PCR Express thermocycler (Hybaid, nucleic acid extraction and amplification of the target Teddington, GB). organism and target nucleic acid, respectively:

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• Negative isolation control (NIC) to monitor contamina- (R. pseudosolanacearum) and 2 strains of Phylotype IV tion during nucleic acid extraction: nucleic acid extrac- (R. syzygii). tion and subsequent amplification preferably of a sample of uninfected matrix or, if not available, clean extraction 4.2. Additional performance criteria from Anses, FR (2008, buffer. formerly LNPV) for primers 759–760 • Positive isolation control (PIC) to ensure that nucleic Diagnostic sensitivity (evaluated with known reference cul- acid of sufficient quantity and quality is isolated: nucleic tures): 93.3% (three strains not detected). acid extraction and subsequent amplification of the tar- Diagnostic specificity (evaluated with known reference get organism or a matrix sample known to contain the cultures): 96.7% (one strain not detected). target organism (e.g. naturally infected host tissue or host tissue spiked with the target organism). • Negative amplification control (NAC) to rule out false Appendix 7 – Bioassay test in tomato based positives due to contamination during the preparation of upon Janse (1988) the reaction mix: amplification of molecular-grade water that was used to prepare the reaction mix. • Positive amplification control (PAC) to monitor the effi- Test plants ciency of the amplification: amplification of nucleic acid Ten test plants of a susceptible tomato cultivar (e.g. of the target organism. This can include nucleic acid Moneymaker or a cultivar with equivalent susceptibility as extracted from the target organism, total nucleic acid determined by the testing laboratory) at the third true leaf extracted from infected host tissue, whole-genome ampli- stage for each sample should be used. Plants are produced fied DNA or a synthetic control (e.g. cloned PCR product). in pasteurized potting soil. Prior to inoculation plants are For PCRs not performed on isolated organisms, the PAC not watered for 1 day. should preferably be near to the limit of detection.

3.2. Interpretation of results Inoculation In order to assign results from this multiplex PCR-based One hundred microlitres of sample extract are distributed test the following criteria should be followed: among the ten test plants. Two methods of inoculation are described below: Verification of the controls. • Syringe inoculation: inoculate the plant stems just above NAC should produce no amplicon. the cotyledons using a syringe fitted with a hypodermic • NIC and NAC should produce no amplicons. • needle (not less than 23G). Distribute the sample between PIC, PAC should produce amplicons of the relevant size. the test plants. Slit inoculation: holding the plant between two fingers, When these conditions are met. pipette a drop (approximately 5–10 lL) of the suspended • A test will be considered positive if amplicons of the pellet on the stem between the cotyledons and the first leaf. expected size(s) are produced. Using a sterile scalpel, make a diagonal slit, about 1.0 cm • A test will be considered negative if it produces no band long and approximately 2/3 of the stem thickness deep, or bands of different sizes. starting the cut from the pellet drop. Seal the cut with ster- • Tests should be repeated if any contradictory or unclear ile Vaseline from a syringe. results are obtained.

4. Performance criteria available Controls 4.1. Analytical specificity data Inoculate by the same technique five seedlings with an Compared with barcoding using 16S rRNA, mutS and egl aqueous suspension of 105–106 cells mL–1 prepared from a gene targets, Fegan & Prior (2005) used multiplex PCR to 48-h culture of a virulent strain of Ralstonia solanacearum, correctly identify 55 of 56 (98.2%) Ralstonia reference R. pseudosolanacearum or R. syzygii as a positive control strains representing the known diversity, including 47 strains and with buffer (Appendix 2) as a negative control. Sepa- of Phylotype II (R. solanacearum), 3 strains each of Phylo- rate positive and negative control plants from the other types I and III (R. pseudosolanacearum) and three strains of plants to avoid cross-contamination. Phylotype IV (R. syzygii). The Phylotype IV strain ACH0732 isolated from tomato in Australia was not identified using this Conditions of growth test. Wicker et al. (2007) similarly correctly identified 100% of 77 isolates and reference strains, including 14 strains of The test plants are grown for up to 4 weeks at 25–30°C, Phylotype I (R. pseudosolanacearum), 57 strains of Phylo- 10 000 lx during a 16-h photoperiod and high relative type II (R. solanacearum), 4 strains of Phylotype III humidity (70–80%). Appropriate watering should be

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 61 ensured to avoid both waterlogging and wilting through Appendix 8 – Duplex PCR test for the water deficiency. To avoid contamination, positive control identification of Ralstonia solanacearum and negative control plants should be incubated on clearly Phylotype II strains of the 4NPB and Moko separated benches in a glasshouse or growth chamber or, if ecotypes (Cellier et al., 2015) space is limited, strict separation between treatments should be ensured. If plants for different samples must be incubated close together they should be separated with 1. General information appropriate screens. When fertilizing, watering, inspecting and carrying out any other manipulations care should be 1.1 This PCR is suitable for the identification of taken to avoid cross-contamination. It is essential to keep R. solanacearum Phylotype II strains of the 4NPB glasshouses and growth chambers free of all insect pests and Moko ecotypes. It has only been validated so far since they may transmit the bacterium from sample to on cultures isolated from plant tissue and not for sample. direct detection in plant extracts. 1.2 The test is based on Cellier et al. (2015). 1.3 The target sequences are those of the Symptoms RALUWv1_4260003 gene, coding for a putative KfrA Observe the plants regularly during the period of growth protein (shared by strains of both the 4NPB and Moko for symptoms of wilting, epinasty, chlorosis and/or stunt- ecotype), and the RAL70v1_1150031 gene (only found ing. in the IIB-4NPB ecotype), coding for a chemotaxis- related protein. 1.4 The amplicon size is 477 bp for RALUWv1_4260003 Isolation from plants showing symptoms gene (primers 93F/R) and 661 bp for Isolation from plants showing symptoms should be per- RAL70v1_1150031 gene (primers 5F/R). formed (Section 3.2.2.2) and purified cultures of presump- 1.5 The forward primer 93F sequence is 50-CGC TGC tive Ralstonia solanacearum, R. pseudosolanacearum or GCG GCC GTT TCA C-30; the reverse primer 93R R. syzygii should be identified (Section 4). sequence is 50-CGG TCG CGG CAT GGG CTT GG- If no symptoms are observed after 3 weeks, IF/PCR/Iso- 30; the forward primer 5F sequence is 50-GCG CGC lation should be performed on a composite sample of 1-cm GAG GCT GGT GAT GT-30; the reverse primer 5R 0 stem sections of each test plant taken above the inoculation sequence is 5 -TGG GTT CGC AGG CGG ACA GC- 0 site. If the test is positive dilution plating should be per- 3 formed (Section 3.2.2.2). Identification of cultures should 1.6 The PCR amplification has been validated on a Ver- be performed (Section 4). iti thermal cycler (Applied Biosystems) and a Gen- eAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific). Interpretation of the bioassay test results Valid bioassay test results are obtained when positive con- 2. Methods trol plants show typical symptoms; the bacteria can be re- isolated from these plants and no symptoms are found on 2.1 Nucleic acid extraction and purification the negative controls. 2.1.1 Tissue source: bacterial cell culture, which can The bioassay test is negative if test plants are not be performed on plant extracts of Musa 9 infected and provided that R. solanacearum, paradisiaca (banana), Musa 9 paradisiaca (plan- R. pseudosolanacearum or R. syzygii are detected in posi- tain) cultivars and Solanaceae tissues, but further tive controls. validation is needed. The bioassay test is positive if the test plants are infected 2.1.2 For crude DNA extraction from presumptive by R. solanacearum, R. pseudosolanacearum or R. syzygii. Ralstonia isolates, suspend single colonies from each isolate and from cultures of reference strains in 100 lL of sterile distilled water. Heat Performance criteria in closed microvials at 95°C for 12 min. Trans- Analytical sensitivity: 103–104 cfu mL–1 added to sample fer heated suspensions to ice and pulse cen- extracts that previously tested negative. trifuge after cooling. The highest analytical sensitivity can be expected when 2.1.3 DNA should preferably be stored at approxi- using freshly prepared sample extract and optimal growth mately 20°C. conditions. However, the method can also be successfully applied to extracts that have been stored under glycerol at 2.2 Duplex PCR 68 to 86°C. 2.2.1 Master mix

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 62 Diagnostics

3.2 Interpretation of results Working Volume per Final In order to assign results from PCR-based tests the follow- Reagent concentration reaction (lL) concentration ing criteria should be followed:

Molecular-grade N.A. 13.88 N.A. water* Duplex PCR tests. Verification of the controls— G2 Hot Start Mix 59 519 • NIC and NAC should produce no amplicon. (Promega) • PIC, PAC (and if relevant IC) should produce amplicons

MgCl2 25 mM 1.5 1.5 mM of the relevant size (depending on the target, endoge- dNTPs 10 mM 0.5 0.2 mM nous or exogenous nucleic acid is used). Forward primer 100 lM 0.5 (each) 2 lM (93F, 5F) l l When these conditions are met— Reverse primer 100 M 0.5 (each) 2 M • (93R, 5R) A test will be considered positive if amplicons of 477 G2 Hot Start 5UlL1 0.125 0.625 U and/or 661 bp are produced. polymerase • A test will be considered negative, if it produces no (Promega) band or a band of a different size. Subtotal 23 • Tests should be repeated if any contradictory or unclear Homogenized 2 results are obtained. plant material Total 25 4. Performance criteria available *Molecular-grade water should be used preferably, or prepared purified (deionized or distilled), sterile (autoclaved or 0.45-lm filtered) and Performance criteria were obtained with bacterial cultures. nuclease-free water. ° 2.2.2 PCR conditions: 96 C for 5 min followed by 35 4.1 Analytical sensitivity data ° ° ° cycles of (94 C for 15 s, 70 C for 30 s, 72 C for Sensitivity calculated as 105 CFU mL1 for both 93F/93R ° 30 s) and a final step of 72 C for 10 min. and 5F/5R pairs; and also for the duplex 93F/93R, 5F/5R PCR. 3. Essential procedural information 4.2 Analytical specificity data 3.1 Controls Evaluated on 111 strains (40 targets strains and 71 non-tar- For a reliable test result to be obtained, the following (ex- get strains). ternal) controls should be included for each series of Inclusivity calculated as 92% for both 93F/93R and 5F/ nucleic acid extraction and amplification of the target 5R pairs. organism and target nucleic acid, respectively: Exclusivity calculated as 100% for both 93F/93R and 5F/ • Negative isolation control (NIC) to monitor contamina- 5R pairs. tion during nucleic acid extraction: nucleic acid extrac- Accuracy calculated as 96% for the 93F/93R pairs and tion and subsequent amplification, preferably of a 100% for the 5F/5R. sample of uninfected matrix or, if not available, clean extraction buffer. 4.3 Repeatability • Positive isolation control (PIC) to ensure that nucleic Repeatability calculated as 95% for 93F/93R pairs and acid of sufficient quantity and quality is isolated: nucleic 100% for 5F/5R pairs. acid extraction and subsequent amplification of the tar- get organism or a matrix sample known to contain the 4.4 Reproducibility target organism (e.g. naturally infected host tissue or Different PCR machines, operators, time and reagents were host tissue spiked with the target organism). used for validation, showing no difference in the results. • Negative amplification control (NAC) to rule out false positives due to contamination during the preparation of the reaction mix: amplification of molecular-grade water Appendix 9 – Pathogenicity test that was used to prepare the reaction mix. Pathogenicity of isolates is usually confirmed on the same • Positive amplification control (PAC) to monitor the effi- host from which they were isolated, although tomato is usu- ciency of the amplification: amplification of nucleic acid ally also susceptible to those strains that are known to have of the target organism. This can include nucleic acid a wide host range. Young healthy seedlings or cuttings extracted from the target organism, total nucleic acid should be raised in a greenhouse and preferably used at the extracted from infected host tissue, whole-genome ampli- second to fourth leaf stage. Prior to inoculation, plants fied DNA or a synthetic control (e.g. cloned PCR product). should not be watered for 1–2 days in order to increase the For PCRs not performed on isolated organisms, the PAC uptake and translocation of the inoculated bacterial suspen- should preferably be near to the limit of detection. sion in the plant vascular tissues. It is recommended that

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63 PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex) 63 plants are kept in the same environmental conditions (25– taken to avoid the temperature falling below 24°C, as 30°C, 70% RH and 14-h photoperiod) before and after symptoms may be significantly delayed or may not appear inoculation, in order to avoid stress which could adversely at all. Symptoms usually appear between the 8th and 12th affect the plant’s response to pathogen inoculation. days after inoculation for potatoes but between the 3rd The inoculum is prepared by culturing the bacteria for and 7th days after inoculation for tomato. Symptoms 24–48 h at 28°C on sucrose peptone agar or YPGA prior to include partial or total wilting of leaflets and leaves, start- suspending and diluting to the required concentration in ing from those nearest to the inoculation cut. Epinasty is sterile, distilled water. Bacterial density can be measured also a common symptom, with or without leaf wilting. spectrophotometrically, adjusting the cell suspension to an Symptom development in Musa spp. may take longer than optical density (OD) of 0.1 (k = 660 nm), corresponding to 3weeks. a bacterial concentration of about 108 cells mL1 and dilut- As soon as symptoms appear the bacterium should be re- ing 1:100 to 106 cells mL1. isolated from plants by taking a stem, pseudostem or peti- Plants are inoculated by making a vertical cut, approxi- ole section above the inoculation point, placing it in a small mately 0.5 cm long, to the centre of the stem between the volume (1–2 mL) of sterile distilled water or 50 mM phos- cotyledons and the first true leaves (or in the base of the phate buffer and soak for 10–15 min. A loopful (10–20 lL) pseudostem in the case of monocotyledonous hosts), with a of the resulting suspension should then be streaked on sterile scalpel. A drop (10–20 lL) of the pathogen suspen- YPGA and/or SMSA medium and plates observed for the sion is then injected into the wound with a sterile syringe typical colonies (see Section 3.2.2). or pipette. After inoculating, the cut is sealed with sterile In the case of symptom development on both the positive petroleum jelly. A minimum of 5 (up to 10) plants per iso- control and at least one of the test plants, pathogenicity of late are inoculated. Five additional plants are inoculated an isolate is confirmed. In the case of no symptom develop- with sterile water as a negative control and five other plants ment in any of the inoculated plants and the positive con- are inoculated with a reference strain of the appropriate trol is symptomatic, pathogenicity of isolates is not Ralstonia species at the same concentration and with the confirmed. In the case of no symptom development on both same procedure as described above (as positive control). the test plants and the positive control, the pathogenicity Plants should be grown on for 21–28 days and inspected assay is invalid and should be repeated, taking care to use regularly for the development of symptoms. Care should be a virulent reference strain as a control.

ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 32–63