The RSSC is a well studied group of phytopathogenic bacteria because of its potential economic consequences in high-value crops planted worldwide. The R3bv2 potato brown rot pandemic lineage is strictly regulated as a Select Agent in the United States and a quarantine pathogen in Europe. The taxonomy of bacterial wilt pathogens has been revised multiple times to better reflect the evolutionary relationships among the strains (Paudel et al, 2020). Because related species in the RSSC can infect the same hosts, a robust user-friendly diagnostic tool that can identify and differentiate the major groups is urgently needed for quarantine decisions and further characterization.
Numerous detection tools are available for the detection of RSSC and the R3bv2 pandemic lineage. Serological detection methods, immunostrips or Lateral Flow Assays (LFA) are rapid tools for detection of RSSC strains but are known to give false-positive results and they cannot differentiate the major groups (Stulberg et al. 2015). The primer pair 630/631 for the real-time detection of R3Bv2 strains can also yield false positive and false negative results (Fegan et al. 1998; Ji et al. 2007). The multiplex PCR assay developed by Stulberg and colleagues (2015) accurately distinguishes other Rsol strains from phylotype IIB-1 sequevar 1 and 2 strains (which operationally define Select Agents by USDA-APHIS), but it does not detect strains in the Rsyz nor Rpseu genomospecies.
Rapid determination of the genomospecies and differentiation of the high consequence Select Agent lineage in a single reaction is not only be cost and time effective for diagnostics, but it is a valuable tool for epidemiological studies. To our knowledge, no detection tools were available that differentiated the three genomospecies and the strictly regulated R3bv2 Select Agent strains in a single reaction. We developed and validated a robust multiplex assay to fill this gap. In this study, we developed an endpoint multiplex PCR assay based on GoTaq Green Master Mix for the convenient and simultaneous detection of three genomospecies and the Select Agent strains belonging to R3bv2. The assay was validated with 110 strains from different hosts and geographic origins. None of the outgroups yielded bands and amplification of target bands was specific across two PCR kits. The developed PCR assay also accurately detected the pathogen from naturally and artificially infected plant material with no cross-amplification among the groups.
Robust primer design is a crucial consideration in diagnostic assays (Arif et al, 2021). With the decreased cost of whole genome sequencing and the availability of more sequencing resources in the public database, it is important to encompass the diversity of strains while selecting the region. We analyzed whole-genome sequences of Ralstonia strains using multiple comparative genomics software and web interface platforms to ensure a unique target region was selected for the five different groups. Multiple in-silico analyses suggested that the five selected target regions are unique and highly conserved among the available genome sequences. Several published RSSC diagnostic assays target genes from a potential mobile element or phage related sequence in the R3bv2 lineage (Guidot et al. 2009; Kubota et al. 2011) and the Banana Blood Disease lineage (Rincon-Florez et al. 2022). The instability and mobility of these elements potentially reduce the stability and specificity of the diagnostic assay. Thus, selection of a unique and conserved target region is a critical step in the development of a reliable, robust, and specific diagnostic tool (Dobhal et al. 2020; Arif et al, 2021; Arizala et al. 2022).
Primer optimization is another important step to develop an efficient diagnostic assay (Arif and Ochoa-Corona et al, 2013). The use of AT-rich flap sequences at the 5’ end has improved the primer thermodynamics and reduced secondary structure formation (Arif and Ochoa-Corona, 2013). Also, addition of these flaps increases the sensitivity and reaction efficiency (Larrea-Sarmiento et al. 2019). The uniformity in the GC content and melting temperature by adding about 10–12 AT-rich bases to the primer increases the PCR amplicon intensity and equilibrate competition among primer sets in a multiplex PCR reaction (Arif and Ochoa-Corona, 2013; Larrea-Sarmiento et al, 2019; Dobhal et al. 2020; Ramachandran et al, 2021; Arizala et al. 2022).
The primer set RsolP2WF2/R2 amplified all 47 Rsol strains. Surprisingly, one strain of Cupriavidus necator (a sister genus of Ralstonia) yielded a band with the Rsol primer set. However, the strain did not amplify with the RSSC primer set. Despite being related to the RSSC group, no species of C. necator (formerly Ralstonia eutropha) is known as a plant pathogen. The Rsol primer set targets a gene encoding a DUF692 domain-containing protein. DUF692-containing proteins are within the the AP2Ec Superfamily that contains enzymes that target phosphodiester bonds in DNA (endonucleases with a role in DNA repair) or phosphorylated sugars (isomerases) (Ref: NCBI Conserved Domain Database). It is possible that both the C. necator strain and Rsol lineage horizontally acquired a similar DUF692-containin gene.
Of the 55 Rpseu strain tested, 53 yielded the expected bands with the RpseuWF5/R5 primer set. Two Rpseu strains, A5713 (Reunion Island) and A5714 (Zimbabwe), did not amplify with the primer set RpseuWF5/R5. When the new primers were designed to amplify a larger fragment of the same region, A5713 and A5714 DNA still did not yield a band, suggesting the that these strains lack genes encoding the Flp family type IV b pilin, the TPM domain containing protein, or both. When a new primer set was designed for a different Rpseu specific target, the product amplified from A5713 and A5714 DNA but failed to amplify a band in A5711 and A5715 from Burkina Faso and Cameroon, respectively (data not shown). Nevertheless, all four African strains (A5711, A5713, A5714, A5715) yielded the expected band with the RSSC primer set. The results may be due to the high diversity among the phylotype III African strains of the RSSC. Only a few whole genome sequences are available for the African strains, but preliminary analysis indicates that phylotype III is significantly more diverse than the phylotype I Rpseu strains (Paudel et al. 2020; Sharma et al. 2022). In future, it would be interesting to analyze the complete genomes of more phylotype III African strains.
An important goal of the RSSC-Lineage Multiplex PCR is to facilitate routine diagnostic work and colony identification. Diagnostic assays are more likely to be adopted by research and diagnostic laboratories if they are cost-effective, use accessible reagents and techniques, and work effectively on a diversity of sample types (Arif et al, 2014). Well-designed routine endpoint PCR assays meet these requirements. The RSSC-Lineage Multiplex PCR assay identified strains in infected plant samples from a farmer’s field and correctly identified RSSC strains in artificially inoculated plants, including seedlings of woody host Ironwood (Casuarina equisetifolia) grown in the greenhouse. We tested the RSSC-Lineage Multiplex PCR against multiple PCR reaction mixes and showed that although it works better in the economical GoTaq Green PCR Mix, the assay also worked in the Qiagen Multiplex Kit. Unlike the GoTaq Green-based PCR, the Qiagen Multiplex Kit did not amplify all five targets simultaneously in the same reaction. This should not present a problem for detection of RSSC from the infected plant parts, as to our knowledge, no two genomospecies of Ralstonia have ever been isolated from the same host plant.
The limit of detection (lowest concentration of the template that can be accurately detected) is an important characteristics of a diagnostic tool (Armbruster and Pry, 2008; Arif et al, 2021). In this assay, no differences in detection limits were observed when either pure genomic DNA or DNA spiked with host was used. No noticeable differences were observed in detection limits when comparing the GoTaq Green-based single and multiplex PCR. Likewise, we found no change in limit of detection when either a single DNA template or all five DNA targets were used in the same multiplex reaction with GoTaq green-based mix (100 pg each for Rsol, Rsyz, R3bv2, 10 pg for Rpseu). However, using Qiagen Multiplex PCR kit increased the detection limit by 10–100 fold when a single DNA target was tested against all primers, which may be due to the use of a more effective polymerase, PCR buffers, or the Q-solution in the kit. This kit is specifically designed for multiplex assay but it will increase the cost of detection per sample.
The developed assay has potential uses in characterizing bacterial cultures and in routine diagnostics and screening of plant materials. Our data demonstrate that it can accurately identify members of the RSCC in a timely and cost effective way.