PCR-based Assay for the Specific Detection of Pseudomonas syringae pv. tagetis using an AFLP-derived Marker
Article information
Abstract
A PCR method has been developed for the pathovar-specific detection of Pseudomonas syringae pv. tagetis, which is the causal agent of bacterial leaf spots and apical chlorosis of several species within the Compositae family. One primer set, PSTF and PSTR, was designed using a genomic locus derived from an amplified fragment length polymorphism (AFLP) fragment produced a 554-bp amplicon from 4 isolates of P. syringae pv. tagetis. In DNA dot-blot analysis with the PCR product as probe, a positive signal was identified in only 4 isolates of P. syringae pv. tagetis. These results suggest that this PCR-based assay will be a useful method for the detection and identification of P. syringae pv. tagetis.
Introduction
Pseudomonas syringae pv. tagetis was first described in Denmark as a pathogen that affects marigold production (Hellmers, 1955). It is now known as a phytopathogenic bacterium that is the causal agent of bacterial leaf spots and apical chlorosis in several species within the family Compositae: African marigold (Tagetes erecta L.), sunflower (Helianthus annuus L.), common ragweed (Ambrosia artemisiifolia L.), Jerusalem artichoke (Helianthus. tuberosus L.), dandelion (Taraxacum officinale Weber), compass plant (Silphium perfoliatum L.) and another sunflower species (Helianthus salicifolius A. Diter) (Gulya et al., 1981; Hellmers, 1955; Rhodehamel and Durbin, 1985; Rhodehamel and Durbin, 1989a; Shane and Baumer, 1984; Styer and Durbin, 1982).
P. syringae pv. tagetis produces a toxin (tagetitoxin) in host leaves that is then translocated to emerging leaves, where it inhibits RNA polymerase III, thereby preventing chloroplast biogenesis and resulting in apical chlorosis (Mathews and Durbin, 1990; Steinberg et al., 1990). The pathogens are divided into three classes based on their capability to produce tagetitoxin: class 1 and 2 strains produce tagetitoxin in plants; class 3 strains do not produce the toxin (Rhodehamel and Durbin, 1989b).
There are many reports that specific detection methods for phytotoxin-producing P. syringae pathovars have been developed based on genes required for their production (Bereswill et al., 1994; Lydon and Patterson, 2001; Schaad et al., 1995; Sorensen et al., 1998). Recently, a PCR protocol to distinguish P. syringae pv. tagetis from other P. syringae pathovars and closely related species was developed based on genes required for tagetitoxin production (Kong et al., 2004). However, this approach is unable to distinguish the bacterium from other Pseudomonas isolates at the pathovar level. Furthermore, Pseudomonas species other than P. syringae pv. tagetis have been reported to induce apical chlorosis in Canada thistle and pea (Suzuki et al., 2003; Zhang et al., 2002). Therefore, a PCR-based assay that is able to unambiguously distinguish P. syringae pv. tagetis from P. syringae pv. helianthi and other apical chlorosis-inducing Pseudomonas species is needed.
In this study, we report the development of a pathovar-specific marker derived from the AFLP technique for detecting and distinguishing P. syringae pv. tagetis from other pathovars and species of Pseudomonas and Xanthomonas. The specificity of the PCR-based assay using pathovar-specific primers was validated by testing 47 isolates collected from various geographical regions and host plants.
Material and Methods
Bacterial strains and DNA isolation
The bacterial strains that are listed in Table 1 were obtained from the Korean Agricultural Culture Collection (KACC) in Suwon, Korea, and the Belgian Co-ordinated Collections of Micro-organisms (BCCM) in Brussels, Belgium. The genomic DNA was isolated as described previously (Song et al., 2014).
AFLP PCR analysis
The AFLP assay was performed using a previously described method (Song et al., 2014), with minor modification. Genomic DNA (approximately 300 ng) was digested with EcoRI and MseI enzymes and was then ligated to the ends of the restricted DNA fragments with EcoRI adaptor and MseI adaptor (Table 2). A pre-selective PCR reaction was performed with the AccuPower PCR Premix (Bioneer, Daejeon, Korea) in a 25 ml reaction mixture containing 1 ml of DNA (50 ng/ml), 10 pmol of Eco0 (5’-GACTGCGTACCAATTC-3’), and 10 pmol of Ms0 (5’-GATGAGTCCTGAGTAA-3’). The pre-selective PCR and AFLP PCR amplification were conducted as described previously (Song et al., 2014). The amplified products were resolved in a 1.2% agarose gel with a 1-kb DNA ladder (TNT Research, Seoul, Korea) as a reference, stained with ethidium bromide, and visualized on a UV transilluminator.
Primer design and PCR amplification
The specific DNA fragment was eluted as described previously (Song et al., 2014). DNA was directly used in the ligation reaction with a pGEM-T Easy Vector (Promega, Madison, WI, USA) and was then transferred into competent DH5a (RBC Bioscience, Taipei, Taiwan) cells according to the supplier’s instructions. The sequencing reaction was performed with an ABI Prism 3730 DNA Sequencer (Life Technologies, Carlsbad, CA, USA). After trimming the vector sequence, one pair of primers was designed based on the obtained sequence.
The specificity of the designed primers was evaluated against P. syringae pv. tagetis and other Pseudomonas and Xanthomonas species. The PCR reaction was performed with premixed polymerase (Taq PreMix; TNT Research, Seoul, Korea) in a 20 ml reaction mixture containing 1 ml of DNA (50 ng/ml), 10 pmol of PSTF (5’-AATGAGCTGAAATTCAACGG-3’), and 10 pmol of PSTR (5’-CGACCTGGATATAAGTTGCC-3’). The PCR amplification was performed with a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation (5 min at 96°C), 25 cycles (15 s at 96°C; 15 s at 62°C; and 30 s at 72°C), and a final extension (5 min at 72°C). Subsequently, 5 ml of each reaction mixture was resolved in a 1.2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.
DNA dot-blot analysis
A DNA dot-blot analysis was performed using a previously described method (Kang et al., 2007), with some modifications. A total volume of 5 ml of genomic DNA (approximately 250 ng) was spotted onto an Amersham Hybond-N+ nylon membrane (GE Healthcare, Little Chalfont, UK), which was then air-dried and baked at 80°C for 2 h. The PCR product from P. syringae pv. tagetis LMG 5090 was labeled with [α-32P] dCTP using a random primer method according to the manufacturer’s instructions (Ladderman Labeling Kit, Takara Bio, Otsu, Japan). The pre-hybridization and hybridization were conducted as described by Sambrook and Russell (2001). The membrane was exposed to an imaging screen (Fuji, Tokyo, Japan) for 3 h, and captured radiation was visualized using a Personal Molecular Imager system (Bio-Rad).
Results
Specificity of an AFLP-derived marker
In order to develop the pathovar-specific marker, 100 AFLP primer combinations (EcoRI + 3 / MseI + 3) were tested with 4 isolates of P. syringae pv. tagetis and 12 isolates of P. syringae pathovars, including the closely related P. syringae pv. helianthi (data not shown). From this, a specific 594-bp amplicon for P. syringae pv. tagetis was cloned and sequenced. The sequence was analyzed for similarity with sequences in the National Center for Biotechnology Information (NCBI) GenBank database. BLASTN results showed that no significant similarity was found. Based on this sequence, the PSTF/PSTR primer set was designed to amplify a 554-bp amplicon.
The specificity of the designed primers was evaluated by testing all isolates shown in Table 1. The PCR product was amplified from only 4 isolates of P. syringae pv. tagetis from among 47 isolates of other pathovars and species of Pseudomonas and Xanthomonas (Fig. 1). These results indicate that the pathovar-specific primers are highly specific for detecting this pathogen.
DNA dot-blot analysis
To confirm whether the entire 554-bp amplicon was unique to P. syringae pv. tagetis, the amplicon was used as a probe against genomic DNA extracted from P. syringae pv. tagetis and other Pseudomonas and Xanthomonas isolates shown in Table 1. Positive signals were found in only 4 isolates of P. syringae pv. tagetis from among 47 isolates of other pathovars and species of Pseudomonas and Xanthomonas, including the closely related P. syringae pv. helianthi and other apical chlorosis-inducing Pseudomonas species (Fig. 2). This result revealed that this amplicon is highly conserved in P. syringae pv. tagetis and does not share considerable homology with other bacteria.
Discussion
The plant pathogen P. syringae pv. tagetis causes apical chlorosis and bacterial leaf spots in various Asteraceae, including the weeds common ragweed and dandelion (Gulya et al., 1981; Hellmers, 1955; Rhodehamel and Durbin, 1985; Rhodehamel and Durbin, 1989a; Shane and Baumer, 1984; Styer and Durbin, 1982). Since the isolation of this pathogen from weeds displaying apical chlorosis, it has been evaluated as a biological agent to control Canada thistle in soybean and woollyleaf bursage in cotton (Gronwald et al., 2002; Sheikh et al., 2001). Apical chlorosis-inducing Pseudomonas species other than this pathogen have also been reported in Canada thistle and pea (Suzuki et al., 2003; Zhang et al., 2002). However, pathovar-specific primers, which could be used for identifying a particular P. syringae pv. tagetis, are still lacking. Therefore, we utilized the AFLP technique to identify a specific polymorphic amplicons for P. syringae pv. tagetis. Polymorphic band produced only from this pathogen was cloned and sequenced. The sequence was used to design pathovar-specific primers that precisely distinguished P. syringae pv. tagetis from other pathovars and species of Pseudomonas and Xanthomonas (Fig. 1).
Previously, Kong et al. (2004) described a PCR method for the identification of P. syringae pv. tagetis based on genes required for tagetitoxin production, but was unable to differentiate between P. syringae pv. tagetis and P. syringae pv. helianthi or P. syringae pv. atrofaciens. In contrast, the PCR technique described in this study was able to unambiguously differentiated 4 isolates of P. syringae pv. tagetis from among other Pseudomonas and Xanthomonas isolates, including both P. syringae pathovars (Fig. 1). Furthermore, a DNA dot-blot analysis using the PCR product as a probe showed a positive signal for all the P. syringae pv. tagetis (Fig. 2), confirming that the entire 554-bp amplicon was highly conserved in this pathogen. This fragment was analyzed by a BLASTN search and showed no significant matches with known nucleotide sequences. BLASTX results revealed that the sequences showed relatively low similarity (32%) to the hypothetical protein from Paenibacillus sp. WLY78. These results suggest that the specificity of the primers for P. syringae pv. tagetis described in the present study is due to the uniqueness of the DNA sequence within the amplified region.
In conclusion, the results presented herein indicate that this PCR-based assay could be a reliable and useful method for the specific detection of P. syringae pv. tagetis strains.
Acknowledgements
This study was supported by the 2014 Post-doctoral Fellowship Program (Project No. PJ0100852014) of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.