Bacterial fruit blotch (BFB) was first discovered in Griffin, GA, USA, in 1965, from watermelon cotyledons that were introduced from Turkey (Webb and Goth, 1965). BFB is caused by the gram-negative seed-borne bacterium Paracidovorax citrulli (Du et al., 2023) (formerly known as Acidovorax citrulli), which infects cucurbits such as watermelon, melon, cucumber, and pumpkin (Azman Husni et al., 2021). The major inoculum source of BFB is contaminated seeds, which could be collected even from asymptomatic fruits (Burdman and Walcott, 2012). The symptoms of BFB on watermelon include water-soaking on the cotyledon at an early stage, followed by reddish-brown lesions emerging along the midveins of leaves with water-soaking spots, brown cracks, and effervescent ooze on the fruits that finally collapse into a watery rot (Burdman and Walcott, 2018). In Korea, BFB was first discovered in watermelons in Gochang, Jeonbuk province (Song et al., 1991), and then reported in other regions, including Gwangju and Naju of Jeonnam province (Seo et al., 2006), which resulted in a severe yield decrease.
Various detection methods have been developed to eliminate and manage the pathogen. In particular, polymerase chain reaction (PCR) detection of P. citrulli allows high levels of specificity and sensitivity of detection limits ranging from 10 to 104 cfu/PCR assay in seeds and diseased tissues with the combination of adequate sampling and DNA extraction methods (Burdman and Walcott, 2018). Several P. citrulli-specific molecular markers have been developed using 16S rDNA (Walcott and Gitaitis, 2000), 16S-23S internal transcribed spacer (Feng et al., 2013; Schaad et al., 2000; Song et al., 2003; Tian et al., 2013), rep-PCR fragments (Bahar et al., 2008; Ha et al., 2009), YD-repeat protein gene (Cho et al., 2015), and hrp (Öztürk and Basim, 2022). Nevertheless, it was difficult to distinguish P. citrulli from closely related species using these genes (Islam et al., 2019; Tian et al., 2016). In recent years, researchers have preferred selecting species-specific molecular markers from the information of genome archives and testing using the Basic Local Alignment Search Tool (BLAST) in silico (Cho et al., 2015; Islam et al., 2019; Tian et al., 2016). The development of next-generation sequencing techniques has enabled us to rapidly produce high-quality sequences from microorganisms, and their genome data in archives such as National Center for Biotechnology Information (NCBI) are updated in real-time. Therefore, it is necessary to verify and update the previously developed specific molecular markers because the results of BLAST can change over time.
In this study, we developed a new P. citrulli-specific gene and marker suitable for SYBR Green real-time PCR. Our specific gene matched only the sequences of P. citrulli when the nucleotides in NCBI GenBank were searched using BLAST modules. We evaluated the sensitivity of the primer set using cloned DNA, genomic DNA (gDNA), and cell suspensions. We also determined the detection limit of this primer set by direct real-time PCR using diseased plant tissues. Our results demonstrated the species-specific detection of P. citrulli from the plant and the applicability of exclusion from infested seeds by this marker.
Materials and Methods
Bacterial strains and DNA extraction
P. citrulli strains and related species listed in Table 1 were collected from the Korean Agricultural Culture Collection (KACC, Wanju, Korea). Each bacterium was cultured in tryptic soy agar (TSA; Difco, Detroit, MI, USA) at 27°C for 2 days. The bacterial cells grown in TSA were picked and pelleted for the extraction of gDNA using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The DNA concentration was measured and adjusted using a spectrophotometer (Hidex F1/Sense, Turku, Finland).
Primer design and polymerase chain reaction
Species-specific genes and primers were selected and designed according to the pipeline described by Lang et al. (2010). Briefly, genomes of 46 strains from 8 species (P. anthurii, P. avenae, P. cattleyae, P. citrulli, P. konjaci, P. oryzae, P. valerianellae, P. wautersii) of the genus Paracidovorax were collected from the NCBI GenBank FTP site (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/). The unique P. citrulli KACC 17001 loci were identified using a nucleotide BLAST (BLASTn) search (E = 1e-10) against Paracidovorax loci. The commonly present genes and strain-specific genes were removed to gather species-specific genes. Additionally, the genes previously reported as species-specific were excluded to get a novel specific gene. Finally, the species-specific gene (3-oxoacyl synthase III C-terminal domain-containing protein, WP_165842990.1) was selected. Primers with a product size of 90-350 bp were developed using the Primer 3 module from the NCBI by reverse ePCR (n = 2, g = 1). Among the five sets of primer candidates amplifying the specific gene, the primer set RS01560-164 (F: 5′-CGCTGGCCGCCGACCTTCTCC-3′, R: 5′-GCCCCGGCTGCTCCCCTTTTC-3′) were selected according to whether the melting peak converged at a single temperature and their shape was symmetric. The consensus nucleotide sequence of the gene, specific to the species, was compared by BLASTn from NCBI with options excluding “uncultured/environmental sample sequences” and optimized for “somewhat similar sequences.”
Specificity and sensitivity assays
The gDNA of bacterial strains listed in Table 1 was used as a template for the specificity test. The PCR mixture consisted of 1× reaction buffer, 0.2 mM dNTP, 4 mM MgCl2, 1.25 U GoTaq Flexi DNA polymerase (Promega), and 25 ng template DNA in a final volume of 25 μl. PCR was performed under the following conditions: predenaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 30 s, extension at 72°C at 30 s, and a final extension at 72°C for 10 min. Each amplicon was mixed with 6× LoadingSTAR (Dyn Bio, Seoul, Korea) and loaded onto a 1% agarose gel for electrophoresis, followed by visualization using a UV transilluminator.
The gDNA of P. citrulli KACC 17001 was used as a template for testing the sensitivity of the primer. For constructing the cloned DNA, 164 bp of the PCR product of the RS01560-164 primer set was purified using AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea). The amplicon was cloned into the pGEM-T Easy Vector (Promega) and transformed into Escherichia coli DH5α competent cells by heat shock, according to the manufacturer’s instructions. Then, the plasmid DNA was purified using GeneAll Hybrid-QTM Plasmid Rapidprep (GeneAll Biotechnology, Seoul, Korea) and used for the primer sensitivity test, wherein 5 ng of each gDNA and plasmid DNA was serially diluted 10-fold, and 1 μl of each DNA was used as a template. The number of copies of plasmid DNA was calculated using the following formula described by Yeates et al. (1998): [6.022 × 1023 (copies/mol) × DNA amount (g)]/[length (bp) × 660 (g/mol/bp)]. For preparing the cell suspension, P. citrulli KACC 17001 was cultured in TSA, and the cell concentration was adjusted at an optical density of 600 nm of 0.1 using a spectrophotometer (Hidex F1/Sense). The sample was serially diluted 10-fold, from which 1 μl was used as a template. The conditions for Real-time PCR were as follows: pre-denaturation at 95ºC for 3 min; 40 cycles of denaturation at 95ºC for 5 s and annealing at 63ºC for 30 s; followed by melting curve construction with increments from 65ºC to 95ºC at 0.5ºC. Real-time PCR was conducted in three replications for each sample to determine the mean of cycle threshold (Ct) values. The amplification efficiency (E) was calculated using the following formula: e = 10−1/slope, E (%) = (e − 1) × 100.
Inoculation and detection of P. citrulli using watermelon tissues
P. citrulli KACC 17001 was cultured at 27°C for 2 days. The cell concentration was adjusted at OD600 = 0.1 using a spectrophotometer and diluted 10-fold (final concentration: 9.6 × 105 cfu/ml). Then, 10 μl of cells was inoculated to the cotyledon of watermelon seedlings (cv. Dangdanghan) by syringe injection. As a negative control, 10 μl of sterilized water was inoculated into the watermelon seedling. The inoculated plants were incubated for 7 days at 28°C, maintaining a relative humidity of >95%. The marginal plant parts with disease symptoms were sterilized with 70% ethanol, and then five tissue pieces measuring 5 × 5 mm in size were ground and macerated with 500 μl of sterilized water for 30 min to extract the pathogens. P. citrulli colonies were counted from the bacterial extracts by spreading onto TSA media and diluting 10-fold from 1/10 to 1/10,000. Then, 1 μl of extracts from each dilution was used as a template for the real-time PCR. Moreover, 200 μl of bacterial extracts was used for the extraction of the total gDNA using the taco plant DNA/RNA extraction kit (GeneReach Biotechnology, Taichung, Taiwan) and then an automatic DNA extraction system (taco, GeneReach Biotechnology). The concentration of gDNA was adjusted to 3 ng/μl and serially diluted 10-fold until it reached 300 fg/μl. Each concentration of gDNA was used as a template for the real-time PCR, which was conducted in three replications to determine the mean of Ct values.
Results
Species-specific gene selection and specificity test
The gene annotated as “3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III C-terminal domain-containing protein” (WP_165842990.1) in the NCBI was selected for constructing the P. citrulli-specific marker. Based on the BLASTn search of the gene nucleotide sequences consisting of 1,038 bp, only the sequences of P. citrulli matched with 100% query cover and >99.3% identity (Supplementary Table 1). Moreover, no significant similarity found in the BLASTn search from the amplicon sequences by RS01560-164 primer set when excluding the sequences of Acidovorax citrulli (taxid: 80869) and optimize for “somewhat similar sequences”. The species-specific gene and the primer sequences were aligned and compared between different P. citrulli strains in silico (Fig. 1). All the P. citrulli strains showed highly similar consensus sequences.
The primer set RS01560-164 identified the 164 bp of amplicons only from P. citrulli strains in conventional PCR. The amplicon was not produced from other Paracidovorax species, including P. avenae, P. valerianellae, and P. konjaci as well as Paenacidovorax monticola, Acidovorax defluvii, Burkholderia glumae, B. gladioli, B. plantarii, and Pseudomonas syringae (Table 1, Fig. 2). In the SYBR Green real-time PCR assay, the primer set RS01560-164 amplified only five P. citrulli strains with a melting temperature of 91°C and no other Paracidovorax spp. and related species. The melting curve converged into a single peak, indicating that there was no non-specific amplification (Fig. 3).
Sensitivity test
The sensitivity of the primer set RS01560-164 was determined by SYBR Green real-time PCR using the Ct values of P. citrulli KACC 17001. For constructing the standard curve, the concentration ranges of each cloned DNA, gDNA, and cell suspension for plotting the Ct values were as follows: 1.46 × 103-1.46 × 109 copies/μl, 5 × 10−15-5 × 10−9 g/μl, and 1.0 × 101-1.0 × 107 cfu/ml, respectively. The amplification and melting curves of each cloned DNA, gDNA, and cell suspension were consistent, showing a melting temperature of 91°C. The standard curves of Ct values and each cloned DNA, gDNA, and cell suspension concentration demonstrated linear correlations as follows: cloned DNA (R2 = 0.999, slope = −3.409, E = 96.5%), gDNA (R2 = 0.999, slope = −3.478, E = 93.9%), and cell suspension concentration (R2 = 0.999, slope = −3.458, E = 94.2%) (Fig. 4). The detection limits of the RS01560-164 primer set in P. citrulli for the cloned DNA, gDNA, and cell suspension were 1.46 × 103 copies/μl, 500 fg/μl, and 1.4 × 104 cfu/ml, with Ct values of 12.52-32.67, 20.71-34.33, and 26.56-37.00, respectively (Table 2).
Detection of P. citrulli from diseased watermelon tissues
P. citrulli KACC 17001 (GCA_030285825.1) belongs to a group II determined by the gltA gene of 439, 442, and 451 nucleotide positions, which is highly aggressive on watermelon compared with that on other cucurbit hosts (Walcott et al., 2004; Yan et al., 2013). The detection efficiency of the RS01560-164 primer set was determined by real-time PCR using watermelon tissues artificially infected with the P. citrulli KACC 17001 strain. The cell suspension of the three replicates of the watermelon cotyledon with BFB symptoms was clearly detected by the RS01560-164 primer set with a melting temperature of 91°C (Fig. 5). We also determined the detection limit of cell suspension and gDNA from the diseased sample using the Ct values obtained by real-time PCR. The tested range of the 10-fold serially diluted concentration was 2.4 × 102-2.4 × 106 cfu/ml for the cell suspension and 3 × 10−13-3 × 10−9 g/μl for gDNA. According to the SYBR Green real-time PCR, the detection limits for the cell suspension and gDNA were 2.4 × 104 cfu/ml and 3 pg/μl, with Ct values of 33.81 and 36.66, respectively (Table 3).
Discussion
Climate change induces unpredictable weather anomalies worldwide. The climate of the Korean Peninsula is shifting from temperate to subtropical (Lee and Bae, 2012). P. citrulli can survive in hot and humid environments where it generally emerges not only in regions with temperate climates such as Korea and the US but also in regions with tropical climates such as Southeast Asia and South America (Burdman and Walcott, 2018). Therefore, considering that BFB in cucurbits could very soon become more problematic in Korea, it is necessary to implement preventive measures. Hence, accurate and rapid detection of P. citrulli in cucurbit hosts is essential.
Using the 16S rRNA gene has been a standard method for the identification of bacterial species, genera, and families (Gürtler and Stanisich, 1996). Nevertheless, because of the indistinguishable limitations of some of the closely related species, other molecular markers such as 16S-23S rRNA have been used. The 16S-23S rRNA region exhibits more variable sequences among different species (Liu et al., 2012). Furthermore, these universal regions are used for detecting bacteria in combination with real-time PCR for sensitive and rapid detection and quantification. However, because of the limitations of discriminating the pathogen from closely related species, TaqMan-based PCR methods have been developed using an additional probe (Ha et al., 2009; Schaad et al., 2000). TaqMan-based real-time PCR techniques improve detection specificity and enable robust identification of the target organisms (Maurin, 2012). In recent years, exploring novel species-specific as well as strain-specific markers from abundant genome data has emerged as a trend. The combination of specific markers and real-time PCR techniques enables high specificity and sensitivity, making it a powerful method for accurately detecting trace amounts of pathogens (Cho et al., 2015; Öztürk and Basim, 2022; Tian et al., 2016). Nonetheless, when we searched the amplification regions of the seven different markers developed for detecting P. citrulli using the BLASTn module, most of them showed sequence similarity with other species such as P. avenae. Only the RS01560-164F/R and A. citrulli-F/R (Tian et al., 2016) primers exhibited no significant similarity with other bacteria (Supplementary Tables 2 and 3). A. citrulli-F/R primers were constructed from the hypothetical protein (Aave_1909, Gene ID: 4669443) for TaqMan real-time PCR with propidium monoazide to detect viable cells. The detection limit of this method was 103 cfu/ml in pure cell suspensions; however, the efficacy of direct real-time PCR was not determined. Consequently, the SYBR direct real-time PCR using RS01560-164F/R primers allow simple, fast, and cost-effective detection of the pathogen directly from plant samples whereas TaqMan real-time PCR using A. citrulli-F/R primers allow detection of viable cells from contaminated seeds. Additionally, the genes used in these studies would provide valuable information for further development of P. citrulli detection techniques with other purposes.
In the present study, we developed a new P. citrulli species-specific gene termed 3-oxoacyl-ACP synthase III (fabH) C-terminal domain-containing protein through a comparative genome analysis. FabH is an essential enzyme for the survival of bacteria by initiating the reaction chain of the fatty acid synthase in E. coli (Davies et al., 2000). This gene provides the advantage of quantification in real-time PCR analysis because it is present as a single copy in P. citrulli. Moreover, the constructed primer set RS01560-164 demonstrated stable and robust detection results from gDNA, cell suspensions, and plant tissues by the SYBR Green real-time PCR method.
Our study demonstrated the usability of the molecular marker for the specific detection of P. citrulli and its potential use in distinguishing contaminated seeds through the optimization of sample preparation from cucurbit seeds. This method could be applicable for the specific detection of P. citrulli in symptomless plants or seeds, providing the potential to prevent BFB in cucurbits.
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