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Journal of Plant Pathology (2004), 86 (2), 147-155

Edizioni ETS Pisa, 2004

147

INTRODUCTION TO EUROPE OF RALSTONIA SOLANACEARUM BIOVAR 2, RACE 3 IN PELARGONIUM ZONALE CUTTINGS* J.D. Janse1, H.E. van den Beld1, J. Elphinstone2, S. Simpkins2, N.N.A. Tjou-Tam-Sin1 and J. van Vaerenbergh3 1Department

of Bacteriology, Plant Protection Service, P.O. Box 9102, 6700 HC, Wageningen, The Netherlands Health Group, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK 3Department of Crop Protection, Agriculture Research Center, Burg. Van Gansberghelaan 96, B-9820 Merelbeke, Belgium 2Plant

SUMMARY

INTRODUCTION

In December 1999, a sample of imported Pelargonium zonale cuttings was found to be infected by Ralstonia solanacearum in the UK. From September to December 2000, bacterial wilt was diagnosed in several Pelargonium nurseries in Belgium, Germany and the Netherlands. R. solanacearum biovar 2, race 3 was consistently isolated and identified. Surveys traced the origin of infections to cuttings produced in Kenya for the European market. Quarantine measures were imposed to prevent spread and persistence of the organism. Infections of R. solanacearum have not been found on imported cuttings or nurseries in European countries since then. The introduction of the pathogen in Pelargonium raised concern of spread to potato. In Kenya it was found that the contamination most probably occurred through contaminated surface (river) water used for irrigation in the nurseries. Infection was found in different cultivars. The nurseries in Kenya have taken strict hygiene and water disinfection measures and eliminated all infected and probable infected plant material. Since than no further infections have been found. The R. solanacearum isolates from Pelargonium were characterised as R. solanacearum biovar 2, race 3 on the basis of host range, morphology, serology, PCR (also using biovar-specific primers, REP-PCR, AFLP) and fatty acid patterns. Symptoms in Pelargonium, results of strain characterisation and a proposed testing method for latent infections are presented.

Infections of Ralstonia solanacearum biovar 2, race 3 have recently been found on potato and tomato in several countries of the European Union. These findings have resulted in a control directive that is implemented in the European Union (Elphinstone, 1996; Janse, 1996; Anonymous, 1999). Biovar 2, race 3 is usually restricted to the above-mentioned cultivated Solanaceae species, and has been reported occasionally on Solanum melongena (eggplant), Capsicum annuum (Martin and French, 1995, Caffier and Herve˙, 1996) and some solanaceous weeds (Table 1). A number of additional symptomless weed hosts have been reported (Table 1) which may enable biovar 2, race 3 to survive in a latent form or in their rhizosphere or some of which acted as hosts after artificial inoculation. Race 1 strains (biovar 1 and 3) have been found to occur on greenhouse ornamentals in the northern hemisphere. In a finding in 1979 of a strain of R. solanacearum from Pelargonium x hortorum (= P. zonale hybrids or geranium) in the USA, race and biovar identification was unclear but pathogenicity tests showed that the isolates from Pelargonium failed to cause disease on tobacco (Strider et al., 1981). More recently biovar 2, race 3 was diagnosed from Pelargonium zonale in Wisconsin (Hudelson, 1999; Hudelson et al., 2002). There are also some doubtful reports of findings of R. solanacearum in Pelargonium spp. from W. Australia (Pittman, 1933) and Tanganyika (Wallace, 1934). The bacterium was also reported in P. zonale from Ohio (Nameth, 1999) and Pennsylvania (Anonymous, 1999) in the USA. In these publications the race or biovar was not identified or reported, but there are indications that biovar 1, race 1 was involved (C. Hayward, pers. comm.). In one case of infected P. capitatum from Reunion island race 1, biovar 1 was involved (Hayward, 1964). Ralstonia solanacearum was detected for the first time in December 1999 in the UK on imported Pelargonium zonale cuttings produced in Kenya for the European market. The following year (September-December 2000) symptoms of bacterial wilt were observed in several Pelargonium nurseries in Belgium and Germany. R.

Key words: Ralstonia solanacearum biovar 2, race 3, hosts, detection method, strain characterisation, latent infection.

* Paper presented at the 3rd International Bacterial Wilt Symposium, White River, South Africa, 4-8 February 2002. Corresponding author: J.D. Janse Fax: 0031.317.421701 E-mail: [email protected]

Labiatae Capparadidaceae Compositae Solanaceae Solanaceae Polygonaceae Solanaceae Urticaceae Chenopodiaceae

Salvia reflexa

Natural latent hosts Cleome monophylla Galinsoga ciliata Nicotiana glutinosa

Nicotiana rustica Polygonum capitata Solanum sisymbriifolium Urtica dioica

Beta vulgaris

Solanaceae

Solanum nigrum (black nightshade)

Cucurbitaceae Geraniaceae Leguminosae Portulacaceae

Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae

Solanaceous weeds Cyphomandra betaceae Datura stramonium* Physalis sp*. Physalis angulatum Solanum carolinense Solanum cinereum Solanum dulcamara (bittersweet)

Compositae Cruciferae Chenopodiaceae

Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae

Solanaceous crops Capsicum annuum Lycopersicon esculentum Solanum tuberosum Solanum melongena (eggplant) Solanum phureja

Non-solanaceous natural host plants Bidens pinnata* Brassica rapa Chenopodium spp. Melampodium perfoliatum Momordica charantia (bitter gourd) Pelargonium zonale (= P. x hortorum) Phaseolus vulgaris (string bean) Portulaca oleracea

Family

Natural host

Sweden

S. America Nepal Brazil Netherlands

Harris, 1976 Pradhanang et al., 1996, 2000 Martin and French, 1995; Moraes, 1947, when high inoculum pressure in soil, also symptoms may be produced Martin and French, 1995; as for N. glutinosa Pradhanang et al., 1996, 2000 Gillings and Fahy, 1993, host for biovar 2T Wenneker et al., 1999, rare, when roots exposed to high inoculum pressure from surface water, wilting under high temperature conditions in greenhouse. Olsson, 1976b

Dukes et al., 1965 Singh, 1992 Pradhanang et al., 1996, 2000 Jackson et al., 1979 Valdez, 1986 Hudelson, 1999; Hudelson et al., 2002 Valdez, 1986 Gillings and Fahy, 1993 personal observation by senior author Pradhanang et al., 1996, 2000 Hayward, 1975, under high inoculum pressure

Martin and Nydegger, 1982 Dukes et al., 1965 Dukes et al., 1965 Swanepoel, 1992 Dukes et al., 1965 Graham and Lloyd, 1978 Elphinstone et al. 1996; Elphinstone et al., 1998; Janse, 1996; Janse et al., 1998; Olsson, 1976a Hayward and Fegan, 2001; Tomlinson and Gunther, 1986

Martin and French, 1995 many references many references Martin and French, 1995; Caffier and Hervé, 1996 Ciampi and Sequeira, 1980

Reference and remarks

Ralstonia solanacearum biovar 2, race 3 in Europe

Kenya Nepal S. America

rare, Georgia, USA India Nepal Costa Rica Philippines USA Philippines Kenya Egypt Nepal Australia

widespread

2T? rare, Colombia rare, Georgia (USA) rare, Georgia, USA rare, S. Africa rare, Georgia, USA Australia only NW Europe

rare, S. America widespread widespread rare, S. America, France rare Colombia

Occurrence

Table 1. Hosts of Ralstonia solanacearum biovar 2, race 3 reported in literature. Root colonization (N=natural, A= after artificial inoculation) or host with or without symptoms after artificial inoculation (S= through inoculum present in soil; I= stem inoculation) only.

148 Journal of Plant Pathology (2004), 86 (2), 147-155

Cruciferae Cruciferae Solanaceae Caryophyllaceae Chenopodiaceae Cucurbitaceae Caryophyllaceae Geraniaceae Compositae Compositae Leguminosae Compositae Compositae Graminae Solanaceae Convolvulaceae Solanaceae Solanaceae Solanaceae Leguminosae Leguminosae Solanaceae Leguminosae Polygonaceae Scrophulariaceae Caryophyllacae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Compositae Asteraceae Tropaeolaceae Verbenaceae Leguminosae Leguminosae

Brassica juncea., B. napus Brassica rapa Browallia speciosa Cerastium glomeratum Chenopodium abrosioides, C. amaranticolor, C. paniculatum Cucurbita pepo Drymaria cordata Erodium moschatum Eupatorium cannabinum Galinsoga parviflora Glycine max Gnaphalium elegans Helianthus annuus Hordeum vulgare Hyoscyamus niger Ipomea sp. Lycopersicon chilense Nicandra physaloides Nicotiana alata Phaseolus vulgaris

Phaseolus multiflorus Physalis floridana Pisum sativum Rumex spp. Salpiglossis sinuata Spergula arvensis Solanum capsisastrum Solanum caripense Solanum luteum Solanum sarrachoides Solanum xanthophyllum Soliva anthemidifolia Tagetes sp. Tropaeolum majus Verbena brasiliensis Vicia faba Vigna sinensis

I Chile I I S I Chile, S Colombia, I S Chile Nepal, I, A I Peru S I I I

Nepal I, A, Sweden, S I I Nepal, S, A I Japan Nepal, S I S Nepal, N, A I I I A I Peru I A, I I S, I

Occurrence

* Based on data presented in publication, not entirely clear if biovar 2, race 3 was involved.

Family

Natural host Olsson, 1976b, Pradhanang et al., 1996, 2000 Belalcazar et al., 1968 Olsson, 1976b Pradhanang et al., 1996, 2000 Belalcazar et al., 1968 Melo and Takatsu, 1998 Pradhanang et al., 1996, 2000 Belalcazar et al., 1968 Olsson, 1976b Pradhanang et al., 1996, 2000 Olsson, 1976b Belalcazar et al., 1968 Moraes, 1947, showing dwarf growth when grown in inoculated soil Pradhanang et al., 1996, 2000 Olsson, 1976b Zambrano, 1990 Olsson, 1976b Olsson, 1976b, Pradhanang et al., 1996, 2000 Olsson, 1976b Moraes, 1947; Olsson, 1976b, showing also dwarf growth when planted in infected soil Moraes, 1947, as for P. vulgaris Fernandez, 1986 Olsson, 1976b Belalcazar et al., 1968; Zambrano, 1990 Olsson, 1976a Belalcazar et al., 1968 Fernandez, 1986; Olsson, 1976b Belalcazar et al., 1968 Olsson, 1976a Fernandez, 1986 Pradhanang et al., 1996, 2000 Belalcazar et al., 1968 Zambrano, 1990 Olsson, 1976a Belalcazar et al., 1968 Olsson, 1976b Melo and Takatsu, 1998

Reference and remarks

Journal of Plant Pathology (2004), 86 (2), 147-155 Janse et al. 149

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solanacearum biovar 2, race 3 was consistently isolated and identified. Surveys in Belgium, Germany and the Netherlands (where only latent infections were found) showed that the origin of the infection was cuttings produced in Kenya by several nurseries for export to associated companies in Belgium, Germany, the Netherlands and the UK for further propagation. The results of surveys to trace the origin of infection and the control measures imposed to prevent spread and persistence of the organism both in Europe and Kenya will be presented. R. solanacearum isolates from Pelargonium were characterised on the basis of morphology, serology, PCR, RFLP, AFLP and fatty acid patterns. Symptoms in Pelargonium, results of isolate characterisation and a proposed testing method for latent infections are also presented.

MATERIALS AND METHODS

Bacterial strains. Strains PD (PD = Culture Collection Plant Protection Service, Wageningen, NL) 4027, 4052 isolated from Pelargonium zonale in The Netherlands, strains PD 4116-4118 and 4124 isolated from P. zonale in Belgium were used in all identification and diagnostic tests mentioned below, except AFLP. Strain PD 2762 (biovar 2, race 3) from potato served as a positive control strain. Strain PD 4027 was used in all host studies performed by the PPS, Wageningen, NL. In fatty acid analysis and BOX PCR a larger number of biovar 2, race 3 strains isolated from potato, tomato, water, Solanum dulcamara and surface water as well as some biovar 3, race 1 and biovar 1, race 2 strains present in the PD collection were included for comparison. For AFLP similar control strains as well as strains NCPPB (National Collection of Plant Pathogenic Bacteria, CSL, York, UK) 4211 and 4212 isolated from P. zonale in Kenya and NCPPB 4213 and 4215 isolated from surface water in Kenya were included. Isolation and identification. Methods for isolation and identification, including pathogenicity tests were those described by Wenneker et al. (1999) using antiserum 2679/BE 21129 (Loewe Biochimika, Sauerlach, Germany) for indirect immuno-fluorescence test (IF). Fatty Acid Analysis (FAA) made use of the race specific library developed at the PPS, Wageningen, NL (Janse, 1991). For routine PCR the primers of Seal et al. (1993) were used. For biovar-specific PCR the primer pair and method of Fegan et al. (1998) was used. BOX-PCR was performed according to Rademaker and De Bruijn (1997). Quantitative PCR was also performed on isolates obtained in the UK and Kenya using the Taqman assay of Weller et al. (1999) with R. solanacearum-specific and biovar 2-specific probes. BOX-PCR was performed according to Rademaker and De Bruijn (1997). The AFLP


method of Vos et al. (1995) used the primer combination EcoRI+C/MseI+0 with analysis by the UPGMA method with Bionumerics software (Applied Maths, St-MartensLatem, Belgium). Host range tests. Young plants, raised from cuttings of selected host plant species mentioned in Table 1 were grown at 75% RH, 25-23°C day/night air temperature during a 14h photoperiod in a quarantine greenhouse. Inoculation was carried out by i) micro-injection of 10 µl into the lower stem or ii) pouring 35 ml bacterial suspension (107 cfu ml-1) of a 48 h NA culture per plant (n=6) in soil around uninjured plants, according to the methods used by Wenneker et al. (1999) for stinging nettle. Negative control plants were inoculated or drenched with sterile 0.01 M PB only. Surveys and methods for detection of latent infections. Surveys were performed by sampling 200 stem parts (1 cm in length) aseptically removed from the lower part of the stem or petiole of the lowest leaf per lot of Pelargonium cuttings. The samples were analysed the same day in the laboratory. Stem parts were crushed in plastic bags (if necessary following a pre-wash and short decontamination in 70% alcohol and blotted dry). Thereafter 50 ml phosphate buffer (PB, 0.05 M) was added and the homogenate incubated for 30 min at 100 rpm in a shaker at room temperature. The extract was then concentrated by centrifugation for 15 min at 7000 g. Subsequent testing followed the methods described in Anonymous (1998) and Wenneker et al. (1999).

RESULTS

Symptoms on pelargonium. Early symptoms were wilting of the lower leaves with rolling of the leaf margins (Fig 1). Subsequently leaves showed sectorial chlorosis (Fig. 2) and eventually papery brown necrosis (Fig. 3). Some stems showed brown to black discoloration (especially at soil/air level) externally and when cut, brown watery discoloration of the vascular tissues. Sometimes only one part of the stem showed wilting symptoms. Isolation and identification. All isolates formed R. solanacearum-typical pearly white, flat, irregular and fluidal colonies on YPGA and milky-white, flat, irregular and fluidal colonies with red coloured whorls in the centre on SMSA. All isolates from Pelargonium proved to be R. solanacearum in biochemical tests, R. solanacearumspecific PCR with all primer sets used, IF with R. solanacearum-specific sera, HR-reaction in tobacco ‘White Burley’ and pathogenicity tests on tomato ‘Moneymaker’. All isolates from Pelargonium proved to be consistently biovar 2 in FAA, biovar 2-specific PCR and


Fig. 1. Pelargonium zonale showing wilting and rolling leaves, 7 days after inoculation with Ralstonia solanacearum biovar 2, race 3 strain PD 4027 from Pelargonium zonale.

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Fig. 3. Pelargonium zonale showing severe wilting and necrosis caused by natural infections by Ralstonia solanacearum biovar 2, race 3.

of which was not apparent (Fig. 4). In AFLP analysis, Kenyan strains from Pelargonium and surface water clustered with other reference strains belonging to biovar 2, race 3. Some other surface water strains clustered with biovar 3 strains and not with strains belonging to other biovar/race combinations. Host range tests. Results of host tests are presented in Table 2. After challenge by stem inoculation or soil drenching (without wounding plants), Pelargonium, tomato and Portulaca oleracea showed typical wilting symptoms. Petunia and Calibrachoa showed latent populations of 105 cfu ml-1 of tissue extract 28 days after stem inoculation and 103 cfu ml-1 of root extracts 28 days after soil drenching.

Table 2. Results of host tests by soil drenching (D) soil (10 ml) and stem inoculation (SI) with 106 cells ml-1 of strain PD 4027 from Pelargonium and PD 2762 from potato.

Fig. 2. Pelargonium zonale showing wilting, sectorial yellowing and necrosis, 20 days after inoculation with R. solanacearum biovar 2, race 3 strain PD 325 from potato. Plants kept at 21ºC.

biochemical tests. Strains PD 4116-4118, 4124 and NCPPB 4211, 4212, 4213 and 4215 were also pathogenic to eggplant (Solanum melongena ‘Black Beauty’), S. nigrum and potato. These strains were not pathogenic to tobacco after stem inoculation. Control strains of biovars 3 and 4 (race 1) and biovar 1 (race 2) always tested accordingly. In BOX-PCR, biovar 2 strains from Pelargonium clustered with those isolated from other sources. Only one strain showed a strong extra band, the significance

Host plant

D

SI

Pelargonium zonale

+

+

Tomato ‘Moneymaker’

+

+

Portulaca oleracea

+

+

Petunia hybrid

latent

latent

Calibrachoa ‘Million Bells’

latent

latent

Begonia sp.

-

-

Impatiens sp.

-

-a

Poinsettia sp.

-

-a

Saintpaulia sp.

-

-a

a a

Very Very low low residual residual populations populations detected detected at at site site of of inoculation inoculation (after 28 days). (after 28 days).

152


Fig. 4. Photograph and resulting dendrogram (based on single linkage Piersson correlation) of BOX-PCR patters of Ralstonia solanacearum of different biotype/race and origin. Pelargonium strains group with known biovar 2, race 3 strains of different hosts and origin in one tight cluster. B = strain from bittersweet (Solanum dulcamara); NC = negative control, buffer solution only; P = strain from Pelargonium; PT = strain from potato (Solanum tuberosum); T = strain from tomato (Lycopersicon esculentum) 1 strain excluded; W = strain from surface water; number or B with number indicates to which biovar the strain belongs. Lanes 1, 10 and 27 in photograph indicate reference DNA ladder.

Surveys and results of control measures. Immediately after the finding, in December 2000, of R. solanacearum biovar 2 in two nurseries in Belgium re-


ceiving cuttings from a nursery in the Netherlands, which in turn had received its cuttings from a daughter nursery in Kenya, more than one hundred samples of 200 stem or petiole pieces were tested for latent infection. In one case the bacterium was found. Early in 2001, other Belgian and Dutch nurseries receiving cuttings from the Dutch nursery were checked for visual symptoms and latent infection. No further infections were found in Belgium, whereas one latent infected plant was found in one nursery in the Netherlands. On all other Dutch nurseries importing cuttings from Kenya directly no (latent) infections were found. In Kenya early in 2001, all mother material and cuttings from infected nurseries were checked and any infections found destroyed. Usually infected plants, in the nurseries involved, showed symptoms and the number of latently infected plants was low. A survey in the surroundings of one Kenyan nursery led to the detection of R. solanacearum biovars 2 and 3 in river water used for irrigation of Pelargonium. No infected weed hosts were found among several potential host species present. In the course of 2001 and 2002, following control measures mentioned below, no further infections were found upon visual inspections and testing for latency, in Europe or Kenya. The following control measures were imposed in Europe: • destruction of all plants of infected cultivars by burning or deep burial; • disinfestations of contaminated greenhouse (compartments), machines, tools and improved hygienic protocols; • officially controlled delivery of all potentially infected lots, after testing, to local market only; • tracing of all nurseries receiving material from Kenya; • sampling and testing of all Pelargonium plants on contaminated nurseries and nurseries receiving propagating material from Kenya; • prohibition to use surface water for irrigation not only for potato and tomato, but also for Pelargonium, Portulaca, aubergine and S. sisymbriifolium in known contaminated surface water areas. In Kenya, plants of infected cultivars were destroyed by burning or controlled dedicated waste disposal areas. Contaminated greenhouses, machines and tools were disinfected and hygienic protocols were strengthened. Nurseries changed to the use of borehole water or disinfected surface water for irrigation (using combinations of filtration, UV irradiation, chlorine dioxide and/or hydrogen peroxide with peracetic acid). Furthermore the complete production stock was discarded and the mother stock tested (monitored) by IF or ELISA. Drainage systems were improved and discussions were held with the Plant Protection Service in Kenya with a view to official monitoring of the control measures.

Journal of Plant Pathology (2004), 86 (2), 147-155 DISCUSSION

Results of this study clearly show that Pelargonium zonale is a host for Ralstonia solanacearum biovar 2, race 3. Strains from Pelargonium matched perfectly with other strains of biovar 2, race 3 isolated from different hosts and habitats. In an early study on isolates from Pelargonium in the USA race and biovar identification was unfortunately unclear but pathogenicity tests showed that the isolates from Pelargonium failed to cause disease on tobacco (Strider et al., 1981). The bacterium was also reported in P. zonale from Ohio (Nameth, 1999) and Pennsylvania (Anonymous, 1999) in the USA. In all these cases the race or biovar was not identified, but there are indications that biovar 1, race 1 was involved (C. Hayward, pers. comm.). A more detailed report is from Hudelson (Hudelson, 1999) and Hudelson et al. (2002) from Wisconsin USA where introduction of R. solanacearum biovar 2, race 3 apparently took place through importation of Pelargonium cuttings from Guatemala. The situation in Wisconsin is thus comparable to that in Europe (see below). In our study, strains from potato were pathogenic to Pelargonium and vice versa. Pelargonium appears not to be a very susceptible host. On contaminated nurseries in Kenya, spread from plant to plant only occurred in badly drained greenhouse compartments and most infected plants showed symptoms. Only very few plants were found to be latently infected. It was again demonstrated that Portulaca oleracea is a host of R. solanacearum biovar 2, race 3, showing symptoms (for other reports see Table 2). Petunia and Calibrachoa were also found to act as latent hosts for this bacterium. Calibrachoa has never been reported before as a latent carrier of R. solanacearum. For Petunia hybrids there is only one report from the USA for race 3 (Smith, 1939). Results also showed that the origin of the infection was contaminated surface water used for irrigation in Pelargonium nurseries producing cuttings in Kenya for the European market. The water may have become contaminated from infected potato crops known to be situated upstream. R. solanacearum (both race 1 and race 3) are widespread in Kenya (Smith et al., 1998). Unrooted cuttings from Kenya then contaminated a few nurseries in Europe. Following measures on the Kenyan nurseries to stop using contaminated surface water for irrigation, to destroy infected production stocks and to improve hygienic protocols no further infections have been found in propagation material.

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knowledged as well as Dr. Chris Hayward, University of Queensland, Department of Microbiology & Parasitology, Queensland, Australia for carefully reading the manuscript and helpful suggestions.

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Received 4 May 2004 Accepted 14 June 2004

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