A comprehensive comparison of assays for detection and identification of Ralstonia solanacearum race 3 biovar 2

Authors


Abstract

Aims

To determine the reliable combination of protocols for specific detection and identification of R. solanacearum race 3 biovar 2 (R3bv2) through a comprehensive comparison among currently available techniques.

Methods and Results

Sensitivity and specificity of the conventional isolation, bioassay, serological assays, conventional and real-time PCR and multiplex PCR were assessed for the detection of 25 strains of R. solanacearum biovars 1, 2 and 3 (Phylotypes I, II, III and IV) in spiked potato saps. Results indicated that all assays evaluated varied in complexity and sensitivity and should be applied strategically in indexing schemes to maximize efficiency of testing without compromising accuracy of the results.

Conclusions

The TaqMan PCR assay, with an internal reaction control and confirmation by melting curve and electrophoretic analysis, achieved best sensitivity at 102–10CFU ml−1 for all eighteen strains of R. solanacearum R3bv2. Selective enrichment on mSMSA medium plates enhanced the detection sensitivity up to 10–100 CFU ml−1 for the conventional PCR-based assays.

Significance and Impact of the Study

This is the first time nine different assays were compared side by side for their sensitivity and specificity in detection and identification of R. solanacearum R3bv2. The data accumulated here will provide basis for regulatory applications for low level detection and rapid identification of latently infections caused by R. solanacearum R3bv2.

Introduction

For more than a century, Ralstonia solanacearum (Smith, 1896) Yabuuchi et al. (1995) species complex has been one of the most economically important phytopathogenic bacteria because of its lethality, complex host profile, worldwide distribution and persistent survival in soil and waterways (Denny 2006). This bacterium causes vascular wilt in more than 200 plant species belonging to more than 54 families in tropical and subtropical regions (Hayward 1991; Cellier and Prior 2010). The spread of R. solanacearum race 3 biovar 2 (R3bv2), which causes potato brown rot, has become a major threat to the potato industry in some temperate regions. As a result, R. solanacearum R3bv2 is considered to be a quarantine pathogen in Europe and Canada and is listed as a select agent in the US Agroterrorism Protection Act of 2002.

Isolates of R. solanacearum were classified into five races and 6 biovars based on their host range (Buddenhagen et al. 1962; Buddenhagen and Kelman 1964) and ability to oxidize various disaccharides and hexose alcohols (Hayward 1964; He et al. 1983). However, there are no standard laboratory tests to determine the ‘race’ of R. solanacearum partially because host ranges are broad and often overlap, and the original research on race differentiation only traced back to an abstract published by Buddenhagen et al. (1962) with no further literature support due to the broader range of host specificity than initially observed (Denny 2006). Generally, interspecies characterization described in most of the literature is based on the biovar classification (Denny 2006). The race and biovar classifications do not correspond to each other, except that race 3 strains causing brown rot of potato (Solanum tuberosum L.) are generally equivalent to biovar 2, here thus referred to as race 3 biovar 2 (R3bv2) strains hereafter.

More recently, a new intraspecific classification scheme (Fegan and Prior 2005; Prior and Fegan 2005) based on multiplex PCR and nucleotide sequence analysis of three marker genes, intergenic spacer region of the rrn operon (ITS), endoglucanase (egl) and a transcriptional regulator (hrpB), was introduced to categorize R. solanacearum strains into four phylotypes that accommodate different sequevars as subgroups. The phylotype with sequevar classification is broadly consistent with the race and biovar system and, in some cases, gives an indication of the geographical origin and/or pathogenicity of the strains.

Ralstonia solanacearum R3bv2 strains, causing brown rot and bacterial wilt of potato, Southern wilt of geranium and bacterial wilt of tomato and other solanaceous crops, were classified as phylotype II sequevars 1 and 2. Different from other strains of the R. solanacearum species complex, R3bv2 strains have adapted to a temperate climate and have caused significant losses to the potato industry throughout Europe during the last decade. R. solanacearum R3bv2 was detected in the United States in geraniums imported from Kenya, Guatemala and Costa Rica (Swanson et al. 2005). Latently infected geranium cuttings from Kenya and Central America were believed to be the origin of the disease in Europe and the United States (Denny 2006). So far, the commercial movement of infected, generally asymptomatic, planting material represents the most significant route by which the pathogen has spread on a global scale. Eradication becomes difficult or impossible once the bacterium is established in local soil and irrigation systems. Strict quarantine regulations are applied in many countries for bacterial brown rot disease of potato caused by R. solanacerum R3bv2.

Various techniques such as serological methods utilizing monoclonal antibody and molecular assays based on PCR have been used to develop protocols for specific detection and identification of R. solanacearum at the species level. Additional methods have been developed for the detection and identification of specific biovars, phylotypes and sequevars, particularly of R. solanacearum R3bv2. However, there are no direct data available for a comprehensive comparison of the sensitivity of these methods, which becomes problematic for selecting standards to be used for regulatory actions. In this study, we carried out a comprehensive comparison of nine assays for 25 strains of R. solanacearum to determine the sensitivity and specificity of these methods for the detection and identification of R. solanacearum R3bv2.

Materials and methods

Spiked plant sap

Initially, bacterial isolates for determining assay sensitivities included three strains of R. solanacearum R3bv2 (NCPPB909, NCPPB339 and B03167) and one strain each of biovar 1 (CEPD 7) and biovar 3 (NCPPB 790). For further exploration, additional fifteen R. solanacearum R3bv2 strains and 5 biovar 1 and 3 strains were included (Table 1) in the multiple assays. Bacterial suspensions of test strains were mixed with potato tuber sap to serve as contaminated test materials in different assays. The species and intraspecific identities of all strains were confirmed using the Biolog identification system as described previously (Li and Hayward 1992), an ImmunoStrip kit (Agdia, Elkhart, IN), and conventional PCR using species-specific primers targeting the flagellum subunit FliC protein gene (Schonfeld et al. 2003). The assignment of strains to biovars was verified according to the procedure described by Denny and Hayward (2001).

Table 1. Strains of Ralstonia solanacearum species complex tested and their interspecies and intraspecies classifications
CFIAStrainBiovarPhylotypea SequevarHostOrigin
  1. a

    Phylotype II has been divided into different subgroups by different research groups (Fegan and Prior 2005; Denny 2006; Castillo and Greenberg 2007; Cellier and Prior 2010) and confirmed using multiplex PCR published by Fegan and Prior (2005).

680CFBP14172IIB-1PotatoAustralia
682CFBP18102IIB-1PotatoHaiti
869CFBP45872IIB-1PotatoUnited Kingdom
152CMR442IIB-1PotatoCameroon
256PSS5252IIB-1PotatoTaiwan
687CFBP37852IIB-1PotatoPortugal
691CFBP38702IIB-1PotatoSouth Africa
860CFBP45782IIB-1PotatoEgypt
696CFBP37842IIB-1PotatoPortugal
160JT5162IIB-1PotatoReunion
909NCPPB9092IIB-1PotatoEgypt
339NCPPB3392IIB-1PotatoIsrael
3167B031672IIB-1GeraniumCosta Rica
945CFBP38572IIB-1PotatoNetherlands
859CFBP45772IIB-1PotatoEgypt
641CFBP48122IIB-1TomatoFrance
906JS9262IIB-1PotatoIndia
652LNPV23.432IIB-1AnthuriumGuadeloupe
7CEPD 71IIIN/AN/A
362EGBBC11381III-43PotatoGuinea
790NCPPB 7903I Physalis angulata Costa Rica
157PSS43I-15TomatoTaiwan
54GMI1003I-18TomatoFrench Guiana
70MAFF3015522NIV-8TomatoJapan
145CMR321III-29HuckleberryCameroon

Bacterial cells of the 25 R. solanacearum strains used for the sensitivity test were harvested from casamino acid-pepton-glucose (CPG) (Denny and Hayward 2001) agar plates and suspended in CPG broth at an optical density (OD) of 600 nm to provide stock suspensions. Each stock suspension was serially diluted 10-fold to 10−9 in potato tuber sap. Bacterial concentrations were determined by plating 50-μl suspensions on CPG agar plates in triplicates.

Potato tuber sap was extracted in a Bioreba extraction bag with a Hominex extractor (Bioreba Ag, Switzerland) from table stock potatoes (cv. Yukon Gold) purchased at a local grocery store, and the commercial tubers were free from any visible bacterial infection. One ml of the sap was mixed with various dilutions of bacterial suspensions in a 1·5-ml micro-tube followed by centrifugation at a low speed for 5 min to remove potato debris. The suspensions of spiked potato sap were stored at 4°C before DNA extraction, or at −20°C for long-term storage.

Detection with immunological kits

All 25 strains of R. solanacearum were tested using serology-based lateral flow devices (ImmunoStrip; Agdia, Elkhart, IN) according to the manufacturer's instructions. At all times, a freshly prepared cell suspension of strain NCPPB909 was used as the positive control and BEB1 sample extraction buffer (Agdia, Elkhart, IN) was used as the negative control in parallel with 100-μl aliquots of 10-fold dilutions of R. solanacearum-spiked potato sap samples in the immunoStrip assays. The actual assay took approx. 60 min for a single sample from sample preparation to obtain the ImmunoStrip result.

The sensitivity of the double-antibody sandwich (DAS) ELISA (Agdia, Elkhart, IN) was assessed using serially diluted R. solanacearum-spiked potato sap samples according to the manufacturer's instructions. A freshly prepared cell suspension of strain NCPPB909 was used as the positive control, and BEB1 sample extraction buffer was used as the negative control. The actual assay took approx. 5 h for a single sample with three duplicates (usually in 96-well format for 28 samples) from sample preparation to obtain the ELISA result.

Genomic and metagenomic DNA preparation

Genomic DNA from bacterial cultures and metagenomic DNA from both spiked and nonspiked potato tuber sap were extracted using the modified procedure described by Smith et al. (2008) using Magnesil KF DNA extraction kit (Promega, Madison, WI). Initially, 500 μl of digest was mixed with an equal volume of lysis buffer and 100 μl of magnetic beads. The remaining kit components were used to wash and elute the DNA with a Kingfisher magnetic particle processor (Thermo Scientific, Waltham, MA) according to the kit instructions. The extracts were transferred into sterile, nuclease-free microfuge tubes and stored at −20°C or colder until analysed.

Detection by isolation followed by PCR identification

Ralstonia solanacearum-spiked potato sap samples serially diluted up to 10−9 were plated on mSMSA plates (Denny and Hayward 2001) in triplicates and incubated at 28°C for 48 h. Presumed R. solanacearum colonies with a creamy colony type were restreaked onto fresh plates while the remaining colonies were suspended in 50 μl of lysis buffer (1% Triton X-100, 20 mmol l−1 Tris-Cl pH 8·0, 2 mmol l−1 EDTA, pH 8·0) for PCR analysis. Bacterial suspensions were heated at 95°C for 10 min followed by centrifugation at 15 000 g for 10 min. A 4-μl aliquot of each supernatant fraction was subjected to conventional PCR using primers that targeted the flagellum subunit protein FliC gene (Schonfeld et al. 2003). The actual assay took approx. 75 h for a single sample from sample preparation, serial dilution, isolation and colony PCR to obtain the PCR result.

Detection by conventional PCR and BIO-PCR

A two-step PCR assay targeting the gene encoding the flagellum subunit FliC protein of R. solanacearum (Schonfeld et al. 2003) was evaluated for specificity by differentiating all 42 strains of R. solanacearum from other related organisms in a previous study (data not shown). Test sensitivity was determined using a dilution series of R. solanacearum-spiked potato sap samples with genomic DNA of R. solanacearum NCPPB909 as positive control and DNA extracted from healthy potato tubers as the negative control. Each PCR reaction was carried out in 25 μl volumes containing 1× PCR standard Taq buffer, 0·2 mmol l−1 of each dNTP, 2U of Taq DNA polymerase (New England Bio-Lab, Ipswich, MA, USA), 0·5 μmol l−1 of primer RsoL_fliC-F (5′-GAA CGC CAA CGG TGC GAA CT-3′) and 0·5 μmol l−1 of primer RsoL_fliC-R (5′- GGC GGC CTT CAG GGA GGT C-3′). Two μl of template DNA was used for each reaction. The two-step PCR protocol included an initial denaturing step at 94°C for 5 min, followed by 35 cycles at 94°C for 45 s, 68°C for 60 s and no final extension. Five μl of each PCR reaction was analysed by agarose gel (1·5%) electrophoresis. The actual assay took approx. 5 h for a single sample from sample preparation, DNA extraction and PCR to obtain the electrophoresis result.

To improve the sensitivity of the PCR assay, the concept of bio-PCR (Ozakman and Schaad 2003) was adopted by introducing a solid media enrichment step prior to conventional PCR analysis as described previously. For each dilution of the spiked potato sap samples, 100 μl was spread plated onto mSMSA media in triplicate and incubated at 28°C for 24–30 h, whereafter the microcolonies from each of the three plates were harvested in 1 ml sterile distilled water. The bacterial cell suspensions were boiled for 10 min and used as template in the two-step PCR assay as described above. Genomic DNA of R. solanacearum NCPPB909 was used as the positive control while DNA extracted from healthy potato tuber sap served as the negative control. The actual assay took approx. 52 h for a single sample from sample preparation, serial dilution, plating, DNA extraction and PCR to obtain the electrophoresis result.

Detection by a multiplex PCR assay

The phylotyping scheme, utilizing a multiplex PCR assay on pure cultures, was originally designed for interspecies classification of R. solanacearum strains, followed by sequence analysis for various sequevars (Fegan and Prior 2005). Phylotype-specific multiplex PCR was conducted using primers 759/760 (4 pmoles) specific to R. solanacearum species complex (Opina et al. 1997) in combination with the five phylotype-specific primers of Nmult:21:1F (CGT TGA TGA GGC GCG CAA TTT) (6 pmoles), Nmult:21:2F (AAG TTA TGG ACG GTG GAA GTC) (6 pmoles), Nmult:23:AF (ATT ACS AGA GCA ATC GAA AGA TT) (6 pmoles), Nmult:22:InF (ATT GCC AAG CAG AGA GAA GTA) (6 pmoles) and Nmult:22:RR (TCG CTT GAC CCT ATA ACG AGT A) (24 pmoles) designed by Fegan and Prior (2005) in a single PCR assay. Five μl of PCR products was examined by agarose gel (1·5%) electrophoresis.

The R. solanacearum species complex specific primers 759/760 (Opina et al. 1997) and the five phylotype-specific primers designed by Fegan and Prior (2005) were used to detect the bacterium in dilutions of the spiked potato sap samples. PCR was carried out in 25 μl volumes containing 1× PCR standard Taq buffer, 0·2 mmol l−1 of each dNTP, 2U of Taq DNA polymerase (New England Bio-Lab) and 2 μl of template DNA. The PCR mix was heated to 96°C for 5 min and then cycled 30 times at 94°C for 15 s, 59°C for 30 s and 72°C for 30 s followed by a final extension for 10 min at 72°C. Five μl of PCR products was examined by agarose gel (1·5%) electrophoresis. Presence of bands at 144, 372, 91 and 213 bp designating phylotypes I, II, III and IV, respectively, was recorded as well as the 280-bp band generated from all strains of the R. solanacearum species complex (Fegan and Prior 2005). The actual assay took approx. 5 h for a single sample from sample preparation, DNA extraction and PCR to obtain the electrophoresis result.

Detection by real-time TaqMan PCR assays

The real-time TaqMan PCR assay of Smith and De Boer (2009) with internal control was used to compare with a real-time PCR assay originally described by Ozakman and Schaad (2003). Both assays are used for detecting R. solanacearum R3bv2 in asymptomatic potato tubers.

For the TaqMan PCR assay (Smith and De Boer 2009), the PCR mixture for each reaction consisted of SYBR green Jumpstart Taq Ready Mix, 2% Blotto, 0·3 μmol l−1 forward primer (BI-F 5′- TGG CGC ACT GCA CTC AAC), 0·3 μmol l−1 reverse primer (BI-R 5′- AAT CAC ATG CAA TTC GCC TAC G -3′), 0·2 μmol l−1 TaqMan probe (B-P Cy5- 5′- TTC AAG CCG AAC ACC TGC TGC AAG -3′) and 2 μl of DNA template in a 25-μl reaction mixture. Linearized reaction control plasmid pRB2C2 was also included in the reaction mix at a final concentration of 100 copies per reaction. Amplification conditions were set at 95°C for 2 min followed by 40 cycles of 95°C for 15 s, 64°C for 60 s and 72°C for 45 s. The final extension was 3 min at 72°C. Fluorescence generated by the TaqMan probe was captured in the Cy5 channel, and SYBR green fluorescence was captured in the FAM channel. The default instrument settings were used to generate the melt curves.

In the real-time PCR assay of Ozakman and Schaad (2003), PCR mixture for each reaction consisted of the following: l× PCR buffer, 5 mmol l−1 MgC12, 200 μmol l−1 each of the dNTPs, 1 μmol l−1 forward primer (RSC-F 5′- TTC ACC GCA AAC AGC G -3′), 1 μmol l−1 reverse primer (RSC-R 5′- TAC GCC CCA GCA GAT G -3′), 400 nmol l−1 probe (RSC-P 5′- TTC GCC GAT GCT TCC CA-3′); 1·25 units of Taq DNA polymerase (Perkin Elmer Applied Biosystems, Foster City, CA), l  ×  additive reagent containing bovine serum albumin at 1 mg ml−1, 150 mmol l−1 trehalose, Tween 20 at 1% (v/v) and 2 μl of DNA template in a 25 μl reaction mixture. PCR was carried out in a Rotor-gene cycler (Qiagen, Valencia, CA). Optimized amplification conditions were a denaturing step at 95°C for 5 min and 40 repeats cycling at 95°C for 30 s, 58°C for 30 s and 72°C for 45 s.

In both assays, genomic DNA of R. solanacearum NCPPB909 was used as the positive control while DNA extracted from a healthy potato tuber served as the negative control. The actual assay took approx. 3·5 h for a single sample from sample preparation, DNA extraction and PCR to obtain the real-time PCR result.

Cool temperature bioassay for the verification of the virulence of Ralstonia solanacearum R3bv2 on tomato plant

Bioassay was carried out on tomato (Solanum esculentum var. Money Maker) plants of 5–6 leaf stage in a plant containment facility maintained at a constant temperature of 20°C with a 16/8 h light/dark cycle. Plants were inoculated in duplicate by injecting 200 μl of bacterial suspension into the stem at one of the top 2–3 leaf nodes using a 1-cc syringe. To determine disease progression, symptom development was rated daily on a scale of 1–6 beginning 2 days postinoculation (dpi) until 31 dpi.

Results

Species-specific assays for detection of Ralstonia solanacearum strains

Some of the test results of seven species-specific assays for detecting 25 R. solanacearum strains are shown in Table 2. Isolation of R. solanacearum using selective and differential media (mSMSA with TTC) followed by colony PCR using primers targeting the flagellum subunit FliC protein gene was successful for plant sap samples spiked with bacteria at densities equal or less than 102 CFU ml−1 (Table 2). However, the PCR assay could not differentiate R. solanacearum R3bv2 from strains belonging to other subgroups of the same species, and the process of isolation followed by conventional PCR confirmation took 75 h to complete. It would take several additional days to complete the traditional identification of the bacterial isolates by testing the metabolism of various disaccharides and hexose alcohols for biovar assignment (Hayward 1964).

Table 2. The sensitivity of diagnostic methods for detecting Ralstonia solanacearum strains in potato sap samples spiked with bacterial suspensions at concentrations ranging from nondetected up to 1010 CFU ml−1
Ralstonia solanacearum R3bv2 strain NCPPB 909
Concentration (CFU ml−1)Isolation followed by PCR confirmationaSerological assaysPCR targeting fliC geneMultiplex PCR for phylotypingBioassay on tomato
ImmunoStripELISAConventional PCRBio-PCRb
2·3 × 109+++++++
2·3 × 108+++++++
2·3 × 107+++++++
2·3 × 106+++/−+++/−+
2·3 × 105++++
2·3 × 104+++
2·3 × 103+++
2·3 × 102+++
23++
2+
Negative control
Time took for completion (h)7515552510 days
Ralstonia solanacearum biovar 1 strain CEPD 7
Concentration (CFU ml−1)Isolation followed by PCR confirmationaSerological assaysPCR targeting fliC geneMultiplex PCR for phylotypingBioassay on tomato
ImmunoStripELISAConventional PCRBio-PCRb
3·3 × 108++++++
3·3 × 107++++++
3·3 × 106++++++
3·3 × 105++++++/−
3·3 × 104++++
3·3 × 103++/−+
3·3 × 102++
33++
3
0
Negative control
Time took for completion (h)7515552530 days
Ralstonia solanacearum biovar 3 strain NCPPB 790
Concentration (CFU ml−1)Isolation followed by PCR confirmationaSerological assaysPCR targeting fliC geneMultiplex PCR for phylotypingBioassay on tomato
ImmunoStripELISAConventional PCRBio-PCRb
1·9 × 1010+++++++
1·9 × 109+++++++
1·9 × 108+++++++
1·9 × 107+++++++
1·9 × 106+++++
1·9 × 105+++/−+
1·9 × 104+++
1·9 × 103++
1·9 × 102++
19
Negative control
Time took for completion (h)7515552516 days
Ralstonia solanacearum R3bv2 strain B03167
Concentration (CFU ml−1)Isolation followed by PCR confirmationaSerological assaysPCR targeting fliC geneMultiplex PCR for phylotypingBioassay on tomato
ImmunoStripELISAConventional PCRBio-PCRb
6·8 × 108++++++N/A? Check
6·8 × 107++++++N/A
6·8 × 106++++++N/A
6·8 × 105++++++/−N/A
6·8 × 104+++N/A
6·8 × 103+++N/A
6·8 × 102++N/A
68+N/A
7N/A
0N/A
Negative controlN/A
Time took for completion (h)75155525N/A
Ralstonia solanacearum R3bv2 strain NCPPB339
Concentration (CFU ml−1)Isolation followed by PCR confirmationaSerological assaysPCR targeting fliC geneMultiplex PCR for phylotypingBioassay on tomato
ImmunoStripELISAConventional PCRBio-PCRb
  1. a

    Isolation is made on selective and differential medium (mSMSA with TTC) followed by colony PCR using primers targeting the flagellum subunit FliC protein gene.

  2. b

    DNA extracted from enriched mSMSA plate and tested in conventional PCR assay using primers targeting the flagellum subunit FliC protein gene.

8·6 × 108++++++N/A
8·6 × 107++++++N/A
8·6 × 106++++++N/A
8·6 × 105++++++/−N/A
8·6 × 104++++N/A
8·6 × 103++/−+N/A
8·6 × 102++N/A
86++N/A
9+N/A
0N/A
Negative controlN/A
Time took for completion (h)75155525N/A

Commercial ELISA and ImmunoStrip kits were equally efficient for confirming the identity of R. solanacearum strains with 100% accuracy at the species level, but could not differentiate R. solanacearum R3bv2 from strains belonging to other subgroups of the same species (Tables 2). The detection sensitivity of the ImmunoStrip kit was consistently in the 105–10CFU ml−1 range for all 25 strains belonging to the 3 biovars (four phylotypes), whereas the detection limit of ELISA varied significantly among different biovars. The detection limit of ELISA for biovar 1-spiked samples reached 103 CFU ml−1, while the detection limit for biovar 2 and biovar 3-spiked samples was in the range of 104–10CFU ml−1 (Table 2).

The sensitivity of conventional PCR assay using primers targeting the flagellum subunit FliC protein gene fluctuated in the range of 103–10CFU ml−1 among biovars 1, 2 and 3 strains (Table 2 and supporting materials). However, after enrichment of the spiked plant sap on mSMSA medium plates for 48 h, the BIO-PCR assay using the same primer set consistently offered a detection limit of 10–100 CFU ml−1 for all 25 strains of R. solanacearum, 100–1000 times more sensitive than conventional PCR without enrichment (Table 2). However, the entire BIO-PCR protocol took 52 h to complete, compared to 5 h for the conventional PCR assay without enrichment. Neither assay could be used to differentiate R. solanacearum R3bv2 strains from strains belonging to other subgroups of the species.

Multiplex PCR for phylotyping was successfully applied to DNA samples extracted from potato sap spiked with strains from various biovars (Gel images see supporting materials). The presence of a common amplicon of 280 bp in size was a signature band for R. solanacearum species. Spiked samples with both biovar 1 (CEPD7, EGBBC1138 and CMR32) and biovar 2 (NCPPB339, NCPPB909, B03167 and other sixteen) strains had an amplicon of 372 bp (phylotype II) while sample spiked with biovar 3 strains (NCPPB790, PSS4 and GMI100) had an amplicon of 91 bp (phylotype III). The maximum sensitivity achieved was 106 –10CFU ml−1 for all 25 strains representing biovar 1, 2 and 3 (Tables 1 and 2). It is demonstrated that the multiplex PCR for phylotyping can be used directly for screening environmental samples which may contaminate with R. solanacearum cells.

Specific assays for detection of Ralstonia solanacearum R3bv2 strains

Two different real-time PCR assays were used in parallel for detecting R. solanacearum R3bv2 in spiked potato tuber sap samples. Both assays successfully detected three strains of R. solanacearum R3bv2 in the spiked samples (Fig. 1, Tables 3 and 4), while they failed to reveal the presence of biovars 1 and 3 strains, in which positive controls were clearly showed at Ct value of 30 for Bv 3 (NCPP790) and 28 for Bv 1 (CEPD7) (Fig. 1: Florescence kinetic curves), respectively. However, the two assays differed in sensitivity (Table 3). The assay based on TaqMan chemistries with internal control (Pastrik et al. 2000; Smith and De Boer 2009) showed the highest sensitivity (Tables 3 and 4, gel images see supporting materials), detecting spiked concentration of 68, 86 and 120 CFU ml−1 for R3bv2 strains NCPPB339, B0167 and NCPPB909, respectively. The detection limit for 15 other R3bv2 strains was at a similar range. In the contrary, the real-time PCR protocol developed by Ozakman and Schaad (2003) had a detection limit of 105–107 CFU ml−1 for all the R3bv2 strains tested. Based on the regression curve of the cycle threshold (Ct) value of the dilution series of all the R3bv2 strains, Ct values of 30–34 could be confidently used for the determination of positive amplification in the TaqMan PCR assay (Table 4 and Fig. 1a). Figure 1b indicates that the melting temperature of real-time PCR amplicons generated from spiked potato sap samples agreed well with the melting temperature of amplicons generated from positive control samples of pure cultures (Table 4). Confirmation of amplicon identity by melting curve analysis provided further assurance of test specificity, and analyses of the internal reaction control amplicon prevented any possible false-negative results due to the presence of PCR inhibitors. In addition, it was also possible to achieve optional confirmation by electrophoresis using a 4% agarose gel to distinguish the amplicons of 68 bp from R. solanacearum R3bv2 strains and the amplicon of 94 bp from the internal reaction control (Smith and De Boer 2009).

Table 3. Comparison of two real-time PCR assays for sensitivity
Concentration (CFU ml−1)aTaqman real-time PCR of Smith and De Boer (2009)Real-time PCR of Ozakman and Schaad (2003)
Bv 2Bv 1Bv 3Bv 2Bv 1Bv 3
NCPPB 909NCPPB339B0167CEPD7NCPPB 790NCPPB 909NCPPB339B0167CEPD7NCPPB 790
  1. a

    The exact concentration for each strain is listed in Table 2.

109++++++
108++++++
107++++++
106++++
105+++
104+++
103+++
102+++
10
1
Neg. control
Hour took for the test3
Table 4. Plot profile of cycle threshold (Ct) values in Taqman PCR (Smith and De Boer 2009) detection of dilution series of potato sap spiked with five Ralstonia solanacearum strains
Concentration (CFU ml−1)Race 3 biovar 2Biovar 1Biovar 3
NCPPB 909NCPPB339B0167CEPD7NCPPN790
Cy5 CtMelt PeakCy5 CtMelt PeakCy5 CtMelt PeakCy5 CtMelt PeakCy5 CtMelt Peak
  1. R. solanacearum R3bv2 melting peak range: 82·49–83·71°C, Internal control melting peak range: 89·01–90·03°C.

10917·3683·3615·983·2915·9883·3434·2789·4134·9389·37
10819·8883·3719·6383·4519·5583·4235·0789·5832·4989·76
10723·2783·424·583·3823·4883·3135·6289·3635·489·35
10626·9283·4928·0283·4726·6483·6836·1389·64089·67
10530·1383·4730·1383·1530·0883·534·1389·5234·9589·8
10434·2383·2232·9282·9633·9783·44089·4635·289·77
10335·283·3235·582·9534·2383·3234·7389·4434·5289·8
10235·9383·5534·1883·2235·8183·4934·9789·8134·9489·75
1035·4289·636·6489·8536·4289·7733·4789·5535·0689·64
135·0889·934·6689·8439·5189·8334·3389·5735·1589·56
Neg. control37·2189·7734·9589·7236·319035·9989·5135·7689·9
Figure 1.

TaqMan PCR detection of dilution series of potato sap spiked with Ralstonia solanacearum strains. Left and bottom: The plot profile of TaqMan PCR fluorescence kinetic curves. Dilution series of potato sap samples spiked with various concentrations of each strain in CFU ml−1 were listed in the legend and Table 2; Right: The melting temperature profiles for potato sap sample spiked with selected strains, including two R3bv2 strains (NCPPB 909 and B03167) and biovar 3 strain (NCPPB 790).

Bioassay for the verification of cool temperature virulence of Ralstonia solanacearum R3bv2 strains in tomato plant

Biovar 1, 2 and 3 strains of R. solanacearum displayed a significant variation in virulence in inoculated tomato plants at a cool temperature (Table 2). Maintained at 20°C in the greenhouse in parallel, the biovar 2 strain (NCPPB 909) at a concentrations of 2·3 × 102–2·3 × 109 CFU ml−1 was lethal to all tomato plants 24 dpi. Inoculated with bacterial suspension at a concentration of 2·3 × 109 CFU ml−1, tomato plants started to show wilting symptom in 5 days and were dead by day 10. Plants inoculated with bacterial suspension at a concentration of 2·3 × 102 demonstrated wilting symptoms at day 20 and were dead by day 24. For the biovar 3 strain NCPPB 790, on the other hand, concentration <1·9 × 106 CFU ml−1 did not affect inoculated tomato plants at 20°C even 30 dpi. Wilting symptom was detected at day 3 for tomato plants inoculated at a concentration of 1·9 × 1010 CFU ml−1, and plants were dead by day 7. But lower concentrations (1·9 × 107–1·9 × 109 CFU ml−1) of biovar 3 strains caused tomato plant wilt by day 13, but recovered from wilting and remained healthy until day 30. Interestingly, the biovar 1 strain CEPD 7 at higher concentration (3·8 × 108 CFU ml−1) did cause stem wilting, possibly caused by toxicity to tomato plants, but plants recovered after 2 weeks, and none of the other plants showed any sign of disease during the remainder of the 30-day period. For the remaining strains virulence tested at 20°C, we have eliminated the original dilution to avoid the toxicity effects. Majority of the regulated strains R3bv2 displayed a strong virulence on tomato at 20°C with a single strain JS 926 as an exception. JS 926, originally isolated from potato in India, showed no virulence on tomato at 20°C, but was still virulent to potato at normal growth conditions.

It was concluded that tomato plants maintained at 20°C can serve as an effective and selective bioassay for detection of R. solanacearum R3bv2, with a sensitivity of 102 CFU ml−1 in 3 weeks. It is a problem still that the bioassay may require up to 30 days to be completed and is too protracted for most certification and trade-related applications.

Discussion

Our study showed that various methods can be used successfully for a reliable detection and identification of R. solanacearum R3bv2 in plant samples. The methods vary in complexity and sensitivity and should be applied strategically in indexing schemes to maximize efficiency of testing without compromising accuracy of the results. A flow diagram (Fig. 2) depicts a practical approach to screening plant material using specific tests to index, confirm and verify the presence of R. solanacearum R3bv2 in consignments of crops such as potato and tomato.

Figure 2.

Flow chart for the detection and characterization of Ralstonia solanacearum R3bv2 in potato and tomato samples.

Indexing has been defined as the process of assessing or screening plant materials, especially vegetative plant propagulum, for the presence of a pest or pathogen (Stead et al. 1976). It involves a visual assessment or survey for a disease or pathogen based on symptomology followed by a laboratory test using one or more of the methods we evaluated to confirm the initial assessment or, for asymptomatic material, to detect the pathogen for possible latent infection. Indexing for viral, bacterial or nematode pathogens is currently being carried out in various countries to reduce disease incidence in domestic potato production, to access foreign markets or to eradicate a particular disease (De Boer et al. 1996). At the initial indexing or screening stage, both serological and conventional PCR would be sufficient to provide adequate sensitivity for detecting R. solanacearum at the species level. However, the specificity of these detection methods is such that they will not achieve identification beyond the species level unless a real-time PCR assay which specifically targets R. solanacearum R3bv2 is employed.

ELISA serves as a rapid test well suited for screening a large numbers of samples. In the ELISA kit (Agdia, Elkhart, IN) used in this study, a monoclonal antibody targeting an epitope in the EPS I which is present in all the EPS-positive R. solanacearum strains (Alvarez et al. 1993). The 1000-fold difference in detection limit between biovars 1, 2 and 3 strains (Table 2) may be the result of variations in secretion of EPS I by these strains.

Ralstonia solanacearum R3bv2 is most readily detected in symptomatic plant material because sampling can be targeted and symptomatic tissue which usually contains a greater concentration of bacteria compared to latent infections in asymptomatic tissues. ELISA with a sensitivity of 105–106 CFU ml−1 serves as a rapid technique well suited to testing diseased samples on large scale in screening programs. Similar to ELISA, ImmunoStrip relies on monoclonal antibodies for rapid detection and identification of R. solanacearum at the species level and can be used to screen individual plant samples exhibiting symptoms of R. solanacearum infection, as well as for identification of purified bacterial isolates.

When consignments of plant materials do not show any symptom or sign of disease, the possible presence of latent infections can be tested by indexing a random sample of plant tissues at harvest or during storage. In the general screening programs, conventional PCR has been used to detect R. solanacearum in soil with a sensitivity of 105 cells per gram of soil (Schonfeld et al. 2003). With the aid of a nonradioactive probe in southern hybridization, the detection rate in soil was significantly enhanced (Schonfeld et al. 2003). On the other hand, the BIO-PCR assay demonstrated great sensitivity (101–102 CFU ml−1) and may serve as the best choice for detection of R. solanacearum in situations where a rather low density of pathogens is anticipated. This may be particularly important where there is a threat of incursion of the bacterium into new regions or areas currently free of the disease.

The real-time TaqMan PCR assay, highly specific for R. solanacearum R3bv2, achieved a sensitivity of 103 CFU ml−1 with confirmation by melting curve and electrophoretic analysis. The advantage of this procedure is that it also includes an internal reaction control consisting of a plasmid in which an unrelated DNA sequence flanked by the primer sequences is used to guard against false-negative results that may occur if PCR inhibitors are present in the sample (Smith and De Boer 2009). Furthermore, selective enrichment on mSMSA medium plates (Ozakman and Schaad 2003) enhances the detection sensitivity up to 10–100 CFU ml−1 for the TaqMan PCR assay (Smith and De Boer 2009) as confirmed in this study.

Confirmation of a positive detection of the target bacterium is essential to avoid any chance for a false-positive test and should use a technique that represents an alternative detection strategy to eliminate any possibility of a false-positive result being attributable to cross-reaction of the initial indexing method used. This could involve the use of both a serological and a PCR-based method in a single test strategy or two PCR-based tests targeting different genomic targets. Data obtained in this study indicated that isolation followed by biotyping or phylotyping was practical and provides the required specificity to ensure that false results are avoided. The data supported previous studies that isolation from symptomatic plant materials could be achieved using nonselective (SPA and CPG), selective (mSMSA) and differential (TTC) media (Lelliott and Stead 1987) at a detection sensitivity of up to 10CFU ml−1 (Marco-Noales et al. 2008). Because bioassay, pathogenicity and host specificity tests are a time-consuming process, isolation and characterization of a pathogen may only be recommended as a final verification step if it is necessary to prove the presence of R. solanacearum R3bv2 in new geographical areas, in hosts undescribed previously or when its detection would result in major economic consequences or trade disruption.

Furthermore, biovars 1, 2 and 3 strains of R. solanacearum behaved quite differently from one another in inoculated tomato plants at a lower temperature (Table 2). The virulence test of R3bv2 strains in tomato plants maintained at 20°C can serve as an effective and selective bioassay for detecting R. solanacearum R3bv2, with a sensitivity of 102 CFU ml−1 in 3 weeks. However, it is problematic still that the bioassay may require up to 30 days to be completed and is too protracted for most certification and trade-related applications.

Finally, the probability of detecting R. solanacearum in a crop field is limited by sample size, pathogen incidence and diagnostic methodology. For postharvest testing of potatoes, for example, the sample should be a minimum of 400 tubers or stems randomly collected from the consignment in question to provide a 0·99 probability of detecting a 1·5% incidence of infected units in a given population (De Boer et al. 1996; Priou et al. 2001).

Acknowledgements

Appreciation is expressed to Dr. Philippe Prior and Mr., Stéphan Brière for providing authentic R. solanacearum strains. The technical assistance of Alison Jenkense is greatly acknowledged.

Conflicts of Interest

No conflict of interest declared.

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