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Article

Novel PCR-Based Detection Methods for the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas hortorum pv. vitians Morinière et al., 2020

1
Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, PA 16802, USA
2
Embrapa Genetic Resources and Biotechnology, Federal District, Brasilia 70770-917, Brazil
3
Department of Plant and Soil Sciences, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0083, South Africa
*
Author to whom correspondence should be addressed.
Plants 2025, 14(6), 964; https://doi.org/10.3390/plants14060964
Submission received: 24 January 2025 / Revised: 11 March 2025 / Accepted: 13 March 2025 / Published: 19 March 2025
(This article belongs to the Special Issue Diagnosis and Control of Plant Bacterial Diseases)

Abstract

Bacterial leaf spot in lettuce is a sporadic but devastating disease that threatens lettuce production worldwide. Severe outbreaks have resulted in up to 100% crop loss, and even smaller outbreaks can cause a significant yield loss, as the affected tissue must be removed from lettuce heads prior to their sale. The pathogen, Xanthomonas hortorum pv. vitians (Xhv), has at least three races, with each defined by the disease or resistance phenotype it elicits in lettuce cultivars and accessions. Specific molecular detection of Xhv would facilitate the work of clinicians, growers, seed companies, and researchers in the lettuce industry. We present an Xhv-specific touchdown PCR method and progress toward race-specific methods. We used an alignment of 18 Xhv strains and 9 closely related, non-target strains to identify pathovar- and race-specific gene clusters as targets for PCR primers. We evaluated the specificity first using in silico methods and then empirically using a collection of Xanthomonas strains. Our protocol demonstrated Xhv-specific detection from two sample types, including genomic DNA extracts and bacterial suspensions. Additional research is required to refine the race-specific protocols.

1. Introduction

Bacterial leaf spot in lettuce poses a significant threat to the 4.1-billion-dollar lettuce industry in the United States [1]. Most of its large-scale production occurs in California, Arizona, and Florida, and this disease has been detected in these areas and worldwide where lettuce is grown [2,3]. Nearly all market lettuce is cultivated for its leaves, which are damaged in a bacterial leaf spot infection due to the disease’s symptoms: water-soaking, chlorosis, and small necrotic spots that later join to form large lesions [4]. Following an outbreak, crop loss and the removal of infected leaves from the surviving lettuce heads result in a sizable reduction in both the sellable yield and product value. Cool, wet weather favors disease development, and although sporadic, outbreaks have been shown to affect up to 100% of field-grown lettuce [5].
The successful management of any plant disease depends on the timely and accurate identification and detection of the pathogen. Bacterial leaf spot in lettuce is caused by a bacterium, Xanthomonas hortorum pv. vitians (Xhv, formerly X. campestris pv. vitians or Xcv) [6]. Previously, a conventional PCR was developed by Barak et al. [7]. This method using B162 primers resulted in target amplification from Xcv strains but not from the other X. campestris strains tested, suggesting that the protocol was Xcv-specific. However, Xcv and other members of X. campestris have since been transferred to X. hortorum as Xhv, X. hortorum pv. carotae, X. hortorum pv. cynarae, X. hortorum pv. gardneri, X. hortorum pv. hederae, X. hortorum pv. pelargonii, and X. hortorum pv. taraxaci [6,8]. Of the closely related pathovars, Barak et al. [7] only included X. hortorum pv. carotae and X. hortorum pv. pelargonii as outgroup strains during the development of their method. In this manuscript, we demonstrate that in addition to Xhv, the B162-based conventional PCR amplifies several other X. hortorum pathovars and closely related strains, though not X. hortorum pv. carotae and X. hortorum pv. pelargoni. This lack of specificity could lead to false positive results when seeking to identify or detect Xhv. We sought to develop a conventional PCR that would replace this method for the identification and detection of Xhv.
Similarly, Xhv can be detected using the loop-mediated isothermal amplification (LAMP) assay developed by Dia et al. [9], but its detection is not limited to Xhv strains. Instead, this method targets the clade containing Xhv, X. hortorum pv. gardneri, and X. hortorum pv. cynarae [9]. The continued lack of an Xhv-specific method among the previously published detection methods prompted our research to fill this technical gap.
The phenotypic variation among Xhv strains is an important consideration in the development of detection methods. Xhv consists of three distinct groups of strains capable of triggering a host hypersensitive response (HR), each in a different lettuce cultivar or accession [10,11]. The HR is a plant defense strategy; after the recognition of a pathogen, programmed cell death of the affected tissue is initiated to limit the spread of infection. It has been hypothesized that this variation in plant reactions to Xhv strains, used to define the three groups as “races”, is caused by genetic variation. This claim has been supported by both a multilocus sequence analysis [10] and a genome-wide SNP variant analysis [12]; in both cases, the Xhv strains were clustered by race. A set of race-specific PCR-based detection methods would need to target sequences common to strains within a single known race and absent from non-target, related strains.
Following this strategy, we developed touchdown PCR protocols based on the whole genome alignments of 18 Xhv strains and 9 closely related strains. The primers were designed to target gene clusters present in all of the Xhv strains but absent from all of the others. We originally designed a conventional PCR, but we found in testing subsets of strains that the touchdown method improved the specificity of the reactions. We then tested our protocol using a collection of 96 Xhv isolates from around the world representing the three known races, the 9 other closely related strains within X. hortorum and X. hydrangeae, and 25 plant pathogenic type strains within Xanthomonas. Once we observed the desired Xhv specificity with this collection, we evaluated our method using two Xhv isolates from a 2018 bacterial leaf spot outbreak in lettuce in Pennsylvania, which were correctly detected using our touchdown method. The same process was used to develop methods for race-specific detection; we observed mixed results due to the absence of target amplification, or the presence of non-target amplification, but here, we share our progress and insights for future development.

2. Results

2.1. The Specificity of the Previously Published Detection Methods

When we tested the B162 primer set developed by Barak et al. for Xhv-specific detection [7], we found that while it resulted in the amplification of the targeted 700-base-pair (bp) fragment from the Xhv strains (BS0347, race 1; ICMP 1408, race 2; BP5181, race 3), as expected, it also amplified the same fragment from four non-target yet closely related strains: X. hortorum pv. garderi (CFBP 8163PT), X. hortorum pv. cynarae (CFBP 4188PT), X. hortorum pv. taraxaci (CFBP 410PT), and an X. hortorum strain isolated from radicchio (BP5178) (Figure 1). The target fragment was not amplified from the other closely related strains, X. hortorum pv. hederae (CFBP 4925T), X. hortorum pv. pelargonii (CFBP 2533PT), and X. hortorum pv. carotae (CFBP 7900).
These results were corroborated by our in silico data. The B162 amplicon encoded a glycosyl hydrolase of family 3 that may play a role in starch hydrolysis, toxin degradation, and virulence promotion [13], and when we compared it to the NCBI whole genome database, it shared high sequence homology with a region in the whole genomes of the X. hortorum pvs. vitians, gardneri, cynarae, and taraxaci strains, as well as with a region in another close relative, X. campestris pv. nigromaculans, a pathogen of the Eurasian root vegetable and otherwise invasive weed known as greater burdock [14] (Supplemental Figure S1). Such high sequence homology could explain the ability of the B162 primers to bind to the off-target X. hortorum strains’ genomic DNA; further, these results suggested that X. campestris pv. nigromaculans could also be detected using this method. There were other X. campestris, X. arboricola, and Xyllela fastidiosa strains among the matches with the 700 bp B162 amplicon sequence, but they had significant variation that would likely prevent primer binding and subsequent target amplification from these strains.
Our laboratory and in silico experiments demonstrated that the conventional PCR procedure using the B162 primers does not have sufficient specificity to distinguish Xhv from several other agriculturally significant Xanthomonas plant pathogens, including many closely related X. hortorum pathovars. These results revealed the need for an improved, pathovar-specific protocol for Xhv detection that would remove the risk of false positive results in agricultural settings where multiple plant pathogens may be present.

2.2. A Pangenome Analysis Revealed Xhv Pathovar- and Race-Specific Gene Clusters

Pangenome alignment revealed gene clusters that were unique to the Xhv strains and not present in any of the genomes from the other Xanthomonas strains included in the alignment. Several other gene clusters appeared to be race-specific (encoded by all strains of a single Xhv race but not by the Xhv strains of the other races). A full list of the identified gene clusters is given in Supplemental Table S1, but our in silico evaluations of specificity using NCBI’s BLASTs led us to select gene clusters 3906, 4021, 4381, and 4980 to evaluate their specificity for Xhv, Xhv race 1, Xhv race 2, and Xhv race 3, respectively.
We chose gene cluster 3906 (GC3906) for Xhv specificity. For this gene cluster, we found no matches with NCBI’s database of conserved protein domains collected from most known forms of life. Comparing the gene cluster sequence to the NCBI BLAST database revealed that it had a 100% identity match with a hypothetical protein in Xhv but only a 90–98% identity match with the same protein in X. arboricola, X. nasturtii, X. campestris, X. phaseoli, and X. translucens (Supplemental Figure S2). Due to the observed sequence variation in all of the non-Xhv matches, it appeared to be a good candidate for the specific detection of Xhv strains. Comparisons of this cluster with the NCBI whole genome database revealed hits for the same Xhv strains that we included in our pangenome alignment, as expected, but also for several other X. hortorum strains that lack pathovar designations. X. hortorum pv. gardneri Xc69, which was re-classified from X. arboricola based on the average nucleotide identity (ANI) and genome-based DNA-DNA (gDDH) hybridization data [15], also appeared as a match. However, the ANI and gDDH are typically used for species determination and are not precise enough to distinguish between pathovars within the same species. While this strain may be a member of X. hortorum, its pathovar designation remains unclear. Two Xhv strains of unknown races and all of the known Xhv race 2 strains for which we had previously sequenced whole genomes [12] included the same SNP in this gene cluster, while all of the other matches described above were identical. All additional matches were not from Xhv strains and had significant sequence variation, including strains of X. nasturtii, X. campestris, and X. hortorum pv. cynarae. These results indicated that an Xhv-specific PCR-based detection method could be designed using the GC3906 gene cluster if the primers were designed to bind to segments that were conserved in all Xhv strains but showed variation in non-target strains.
Gene clusters that were unique among the Xhv strains to one of the three races were not necessarily absent from the related X. hortorum pathotype strains or other reference strains. Any race-specific detection method would need to be used in tandem with a pathovar-specific protocol for Xhv detection. For race 1, we investigated gene cluster 4021 (GC4021). We found a 100% identity match with an Xhv helix–turn–helix transcriptional regulator domain which is known to be activated in bacteria as a response to chemical stressors [16] (Supplemental Figure S3). This cluster matched with all Xhv race 1 strains for which we had previously obtained whole genome sequences [12] and no other Xhv strains. Many X. hortorum pv. pelargonii strains and one X. gardneri strain also matched but with sequence variations and truncations that could be exploited during the primer design to develop a Xhv race 1-specific reaction.
Neither GC4381 nor GC4980 had any matches with conserved domains, and each had a 100% identity match with different hypothetical proteins from Xhv (Supplemental Figures S4 and S5). GC4381 had identical sequence matches to all four Xhv race 2 strains that we had sequenced previously [12] and two Xhv strains of unknown races that we predict to also be Xhv race 2 strains. GC4980 had identical sequence matches to the two X. hortorum pv. vitians race 3 strains that were sequenced previously [12]. We therefore utilized GC4381 and GC4980 in an attempt to develop race 2- and race 3-specific primers, respectively.

2.3. The Development of an Xhv-Specific Detection Method

Many primer sets were designed to target the Xhv-specific gene clusters identified through the pangenome alignment, and while all of the others were eliminated during empirical testing due to the detection of off-targets, GC3906-152 (so called after the targeted gene cluster and amplicon size) proved to be an effective target for Xhv-specific detection (Table 1). The target fragment was amplified from 99% (95 out of 96) of the Xhv strains tested, including the pathotype strain CFBP 8686PT (Table 2; Supplemental Figure S6). However, the target fragment was not amplified from strain BS3126, a race 2 strain from France. The target fragment was not detected from any of the other X. hortorum pathotype strains, X. hydrangeae (LMG 31884T), X. campestris pv. coriandri (CFBP 8452PT), or X. hortorum from radicchio (BP5178) [17], or any of the 25 Xanthomonas species type strains included in this study, resulting in 100% exclusivity (Figure 2; Table 2). No amplification was observed for any of the sterile water controls.

2.4. Evaluating the Xhv-Specific Detection Method Using Environmental Samples

To evaluate whether the Xhv-specific PCR protocol designed could be used to identify Xhv from novel outbreaks, four isolates collected from a 2018 outbreak of BLS in lettuce in Pennsylvania were investigated. The Xhv-specific touchdown protocol using the GC3906-152 primer set amplified fragments from only two of the four strains. The expected 152 bp band was amplified for BP4476 and BP4477 but not for BP4478 and BP4479 (Figure 3). As expected, the positive control strain, Xhv race 1 BP5172, produced the expected 152 bp band, and the sterile water control did not. Strains BP4476 and BP4477 were identified as Xhv using the MLSA, which placed them within the clade containing the Xhv race 1 strains (Figure 4). Analysis of the 16S rRNA subunit sequence revealed that the two other isolates, BP4478 and BP4479, were members of Pseudomonas viridiflava (99.9% identity and 100% query cover) and Pseudomonas allivorans (100% identity and 100% query cover), respectively.

2.5. Progress Toward Xhv Race-Specific Detection

Many gene clusters were also evaluated as candidate targets for Xhv race-specific detection using our touchdown PCR method. The best-performing primer sets, those with the greatest number of target strains detected and the least number of non-targets, were selected and are shown in Table 1. These included GC4021-112, GC4381-138, and GC4980-178 for Xhv race 1-, 2-, and 3-specific detection, respectively. When used with our touchdown method, GC4021-112 resulted in strong amplification of a 112 bp fragment from 48 strains (59%) and its faint amplification from 2 strains (2%) out of the 82 strains that either demonstrated HR elicitation in the L. sativa cultivar Little Gem, leading to an Xhv race 1 designation, or clustered with the known Xhv race 1 strains in the MLSA, leading to the prediction that they belonged to Xhv race 1 (Table 2; Supplemental Figure S7). Among the thirty-four strains that were not detected using this method, two strains were known members of race 1 and were included in the pangenome alignments used to identify the race-specific gene clusters. This indicates that the whole genome sequence assemblies for these strains, BP5172 and BP5177, may not be representative of the true gene cluster sequence, possibly due to the limited ability of short reads to resolve certain types of genetic variations, such as long repetitive sequences. Such a consequence of the short-read assemblies may also have been true for the other twenty-eight known or predicted Xhv race 1 strains that did not show amplification of the 112 bp fragment. Long-read sequencing will be needed to evaluate these hypotheses and improve the positioning of the primers to improve the specificity or to identify stronger targets for race-specific detection. However, the protocol did successfully exclude all Xhv race 2 and 3 strains tested; none of these strains produced the 112 bp fragment, and this was expected, as the gene cluster GC4021 was absent from these strains’ alignments.
The same touchdown PCR protocol was used to evaluate the GC4381-178 primer set for Xhv race 2-specific detection. Among the race 2 strains tested, 8 out of 10 (80%) produced the expected amplicon, excluding BS3531 and BS3532 (Table 2; Supplemental Figure S8). The two strains that did not produce the amplicon were not included in the pangenome alignment used to identify the race-specific gene clusters because they lacked available whole genome sequence data. It is possible that these two strains, known to belong to race 2 due to their elicitation of an HR in L. serriola PI491114, do not encode GC4381 or that they encode a sufficient variation in this cluster to prevent primer binding. Other unexpected results included the detection of two known Xhv race 1 strains, one that was part of the pangenome alignment that showed GC4381 to be absent (BP5182) and one predicted to belong to Xhv race 1 based on the MLSA data (BS2996). It is possible that these strains retain GC4381 but that it is somehow inactive and unable to elicit an HR in L. serriola PI491114. These hypotheses will be investigated as we continue to improve the protocol; long-read sequencing will be needed to help resolve the conflicting results between the in silico sequence analyses and the empirical HR testing.
The touchdown PCR protocol used with the GC4980-138 primer set for Xhv race 3 detection showed amplification of the 138 bp target fragment in one known Xhv race 3 strain and three Xhv race 3 strains whose identities were predicted based on the MLSA data (Table 2; Supplemental Figure S9) [10]; these are all of the Xhv race 3 strains currently in curation. Non-target amplification of the 138 bp fragment occurred for X. hortorum pv. carotae CFBP 7900, though it was faint, which was part of our pangenome alignment, and for an Xhv strain predicted to belong to race 1 due to the MLSA results (BS3272). In combination with this protocol, the use of the described pathovar-specific protocol could eliminate any concern about the Xhv non-target amplification. HR screening using the BS3272 strain will be necessary to confirm its placement in Xhv race 1. However, no other Xhv race 1 strains, nor any Xhv race 2 strains, were amplified using this primer set. Again, long-read sequencing is expected to provide the resolution needed to confirm the appropriate targets, but these results represent major steps toward Xhv race-specific detection.

3. Discussion

In previous studies, amplification using the B162 primer pair was used as the initial screening for Xhv isolates [52]. The touchdown PCR protocol using the GC3906-152 primer set can now be used for Xhv pathovar-specific detection from unknown samples. The specificity of our protocol represents an improvement over previous methods using the B162 primer set [7] designed to detect Xhv and LAMP assays [9] designed to detect all members of X. hortorum species. In addition to Xhv strains, the B162 primer set also amplifies the target 700 bp amplicon in X. hortorum pv. garderi, X. hortorum pv. cynarae, X. hortorum pv. taraxaci, and an X. hortorum strain isolated from radicchio. The two pathovars used as the outgroup strains to develop this protocol, X. hortorum pv. carotae and X. hortorum pv. carotae, were not amplified in our assays. The LAMP assay as designed by Dia et al. [28] detects an entire clade of X. hortorum species, including Xhv, X. hortorum pv. gardneri, and X. hortorum pv. cynarae.
The Xhv-specific primer set described here and the touchdown PCR protocol detected 99%, or 95 out of 96, of the known Xhv strains tested (including the pathotype strain CFBP 8686PT) and did not detect any of the 33 outgroup strains (demonstrating 100% exclusivity), including 9 closely related X. hortorum and X. hydrangeae strains and 23 additional Xanthomonas type strains. It also successfully detected two Xhv isolates from a 2018 bacterial leaf spot outbreak in lettuce in Pennsylvania and did not detect the two Pseudomonas isolates from symptomatic tissue from the same outbreak. It is unclear why the reactions with DNA from Xhv race 2 BS3126 did not produce the target amplicon using this protocol—this result requires further investigation to determine whether this strain encodes nucleotide variations in the target gene cluster that inhibit primer binding. However, based on its 99% success rate in detecting the collection of 96 known Xhv strains and its ability to correctly detect Xhv from environmental isolates, we can recommend the use of our touchdown PCR protocol for Xhv-specific detection from colony suspensions or DNA extracts. This could replace the less specific conventional PCR method that uses the B162 primers. Further research is necessary to evaluate the efficacy of our protocol for other sample types, such as tissue extracts and seed wash. Additionally, we designed the amplified targets to be within the size range for efficient Taqman RealTime PCR amplification. We are currently evaluating these genomic regions and incorporating additional Xhv genomes for the development of a Taqman RealTime PCR for this pathovar. Both its selectivity and sensitivity will be quantified in these assays.
Using the same touchdown PCR method, we evaluated primers designed for Xhv race-specific detection; our results appear to highlight the need for long-read sequencing for a variety of Xhv strains. The pangenome alignment of our short-read-based assemblies revealed race-specific gene clusters; however, these in silico analyses, along with applying BLAST to search these strains for DNA sequences homologous with these clusters, did not correspond to our laboratory results. GC4021, GC4981, and GC4890 were three gene clusters we identified from the pangenome alignment that were specific to Xhv races 1, 2, and 3, respectively. GC4021-112 did not detect any race 2 or 3 strains, but it also only detected 59% of the Xhv race 1 strains. GC4981-178 did not detect any race 3 strains, but it did detect 2% (2 out of 82) of the race 1 strains and failed to detect 20% (2 out of 10) of the race 2 strains. GC4980 did not detect any race 2 strains and detected all four race 3 strains, but it also detected 1% (1 out of 82) of the Xhv race 1 strains. Despite these drawbacks, we are now using these PCR protocols along with an MLSA to hypothesize about the race of isolates from symptomatic lettuce tissue from novel outbreaks. We hypothesize that our short-read-based sequence assemblies did not capture the full variation that exists in the Xhv strain genomes and thus expect that long-read sequences will help to improve our methods. These results also indicate strains for which HR screening will be valuable and may reveal whether any of the strains predicted to belong to a particular race based on MLSA data belong to a different race from that expected. Although the clonal nature of Xhv populations suggests that races may correspond to phylogenetic relationships [12,52], the race of a strain is defined by the lettuce cultivar or accession in which it elicits an HR. Differential germplasms for race determination have been described—L. sativa cv. Little Gem, L. serriola PI491114, and L. serriola ARM-09-161-10, for races 1, 2, and 3, respectively [11]—and are being used in subsequent studies.
We expect that our pathovar- and race-specific detection methods will be useful to clinicians, researchers, and seed companies. They will allow for the rapid detection of infection in suspect samples before they result in outbreaks, shown previously to be capable of causing 100% crop loss in severe cases. They may also become useful for seed companies to use to test isolates from seed lots prior to sale or for researchers to use to track the distribution and spread of the pathogen and its races. The race-specific methods, once optimized, could become useful for clinicians who want to provide recommendations to growers for cultivars resistant to the specific Xhv race detected from their prior plantings, as the pathogen has been shown to be harbored by weeds and crop debris and cause new outbreaks in subsequent seasons [7].

4. Materials and Methods

4.1. Isolation, Culturing, and Storage of the Bacterial Strains

Table 2 lists the strains used in this study. The bacterial strains were routinely grown at 28 °C on nutrient agar (NA) and in nutrient broth (NB). Long-term storage was achieved by adding bacteria from pure cultures to 50% nutrient broth and 50% glycerol, vortexing the combination to mix it, and storing it at −80 °C. All of the colony suspensions used in PCRs were created by isolating pure colonies from long-term storage on NA, incubating the plates for three to five days at room temperature, inoculating 30 µL of sterile water in a 1.5 mL Eppendorf tube with a single colony, and vortexing it to mix it.
Following a suspected bacterial leaf spot outbreak in Pennsylvania field-grown lettuce, green leaf and red leaf lettuce samples were submitted to the lab for pathogen isolation and identification. Symptoms appeared as chlorosis and necrotic spots on the leaves, and symptomatic leaves from each lettuce type were used to isolate bacteria using the following procedure. A small tissue fragment was cut out using a sterile scalpel at the margin of a necrotic spot and placed in a sterile 1.5 mL Eppendorf tube. To the tube, 1 mL of 10% sodium hypochlorite was added, and the tube was briefly vortexed. After 1 min of incubation at room temperature, the tube was again briefly vortexed, the sodium hypochlorite was discarded, and the leaf fragment was washed thrice with 1 mL of sterile water. After the final wash was discarded, 30 µL of sterile water was added to the tube, and the tissue was crushed with a sterile pestle. Aliquots (10 µL each) of the resulting plant extract were spread onto two NA plates and incubated for at least two days. Yellow, mucoid, round colonies (typical of Xanthomonas strains) that grew were sub-cultured until we achieved purified strains, which were then stored long term at −80 °C in 1.5 mL cryovials that contained 500 µL of sterile NB and 500 µL of sterile glycerol.

4.2. Evaluation of the B162 Primer Set

Colony suspensions were prepared as described above for three Xhv strains representing the three known races, including Xhv BS0347 (race 1), ICMP 1408 (race 2), and BP5181 (race 3), as well as for Xanthomonas hortorum pv. gardneri (CFBP 8163PT), X. hortorum pv. cynarae (CFBP 4188PT), X. hortorum pv. hederae (CFBP 4925T), X. hortorum pv. taraxaci (CFBP 410PT), X. hortorum pv. pelargonii (CFBP 2533PT), X. hortorum from radicchio (BP5178), and X. hortorum pv. carotae (CFBP 7900). These suspensions were used as templates in conventional PCR using the primer set (B162) and the reaction conditions described in Barak et al. [7]. The reactions were run using an MJ Research PTC-100 thermocycler (Montarville, QC, Canada). The PCR products were analyzed using gel electrophoresis; a total of 5 µL of product mixed with 1 µL of loading dye (VWR Life Science EZ-Vision One Dye-as-Loading-Buffer; Radnor, PA, USA) was added to wells of 1% agarose gel, which was then subjected to 84 V for 45 min. Sterile water was loaded in place of the PCR product as the experimental control, and a 1 KB ladder was used as a size reference (New England Biolabs; Ipswich, MA, USA). The amplification bands were visualized using a Bio-Rad Gel Doc XR Imaging System (Hercules, CA, USA).

4.3. Genome Alignments and Target Selection

Whole genome sequences for thirteen Xhv race 1, four Xhv race 2, and two Xhv race 2 strains, along with strains from eight closely related Xanthomonas hortorum pathovars and one Xanthomonas hydrangea strain [12,28], were aligned with a Xanthomonas hortorum pv. carotae (M081) reference strain via the anvi’o pangenome pipeline [53]. Gene clusters unique to Xhv and each of the Xhv races were identified and extracted for further analysis. NCBI’s conserved domain tool, BLASTx (database = nr), and BLASTn (database = wgs; organism = ‘Xanthomonas’) were used to search for genes with known functions within these clusters and within the B162 amplicon sequence. Matches found in the non-redundant database were considered to be those with at least 90% query coverage, 90% identity, and an e-value threshold of 1 × 10−5. The NCBI BLAST versions used in our in silico analyses were 2.12.0 through 2.15.0.

4.4. DNA Isolation and Sequencing

Strains from our collection were streaked in quadrants to isolate single colonies, and then individual colonies were used to inoculate 50 µL of sterile NB into 100 mL sterile tubes. The liquid cultures were incubated overnight at 28 °C and shaken at 200 rpm. Genomic DNA was extracted from these cultures using the Qiagen DNeasy UltraClean Microbial Kit (Valencia, CA, USA) according to the manufacturer’s instructions. The DNA concentrations following the final elution were measured using the Thermo Fischer Qubit Fluorometer 3.0 and the Invitrogen dsDNA Broad Range Assay Kit (Waltham, MA, USA). The Genomics Core Facility at the Pennsylvania State University completed all of the Sanger sequencing.

4.5. Primer Development

NCBI’s Primer-BLAST was used to design the primers for each of the Xhv- or race-specific gene clusters (database = nr; organism = ‘Xanthomonas’). Primers were selected from the output that had low or no off-target amplification, low complementarity scores, similar melting temperatures near 60 °C, and a similar GC content near 50%. Custom DNA oligos were ordered from Thermo Fisher Scientific (Waltham, MA, USA). In total, 26 primer sets were evaluated using 19 Xhv strains, including strains of all three known races; selected from these primer sets were those that provided the desired pathovar or race specificity (Table 1). All of the other primer sets that were tested are shown in Supplemental Table S2. The detection protocol using the B162 primer set [7] was also evaluated.

4.6. Touchdown PCR Development

Initial testing using conventional PCR, involving cycling 34 times under the same conditions as those given in Table 3 but with a consistent annealing temperature of 58 °C during each cycle, did not provide the desired specificity for our detection reactions. Using the touchdown PCR method, using higher annealing temperatures in earlier PCR cycles to reduce non-specific primer binding [54], did provide the appropriate specificity for pathovar- and race-level detection. Each reaction was completed with the following reaction formulation: 9 µL of sterile water, 1.25 µL of each 10 µM primer, 12.5 µL of 2X Bioline Immomix containing IMMOLASE DNA polymerase, and 1 µL of the template, either DNA extract (30 ng/mL) or colony suspension. The colony suspensions were created by taking single colonies from the NA plates, inoculating them into 30 µL of sterile water, and vortexing the combination to mix it. As an experimental control, 1 µL of sterile water was substituted for the template. The reaction conditions are listed in Table 3, and the reactions were run in an MJ Research PTC-100 thermocycler (Montarville, QC, Canada).
Amplified products were separated using gel electrophoresis, as described above in Section 4.2, except that the gels were run with 84 V applied for 2 h given the larger size of these gels. The same 1 KB ladder described above was used as a reference for the amplicon size. The amplification bands were again visualized using the Bio-Rad Gel Doc XR Imaging System (Hercules, CA, USA). A collection of 96 Xhv strains, along with the pathotype strains for each of the other X. hortorum pathovars, 1 additional X. hortorum strain isolated from radicchio [15], and the type strain of X. hydrangeae (another close relative of X. hortorum), were used to evaluate the specificity of our selected primers. These experiments were conducted twice using colony suspensions from the same strain collection.

4.7. The Multilocus Sequence Analysis of the PA Isolates

Single colony suspensions of the strains isolated from the PA lettuce served as the templates for the PCR reactions to separately amplify four Xanthomonas housekeeping genes: rpoD (693 bp), fyuA (762 bp), gyrB (522 bp), and gap1 (774 bp) [10,55]. Bands of the correct sizes were confirmed using gel electrophoresis, the PCR products were cleaned using EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA), and the cleaned products were submitted to the Genetic Core Facility at Penn State University for Sanger sequencing. Using Qiagen’s CLC Genomic Workbench version 21 (Valencia, CA, USA), the forward and reverse sequences for each gene were assembled to the corresponding reference gene sequence of X. hortorum pv. hederae CFBP 4925T. The consensus sequences from each gene were merged into one file for each strain, in the order listed above, and each strain’s merged sequence was aligned with similarly made files but for a collection of Xhv strains of known races. The model testing tool, with the default options, was used to determine the appropriate nucleic acid substitution model. As recommended, the general time-reversible model with four categories of rate variation, estimated topology, and 1000 bootstraps was used to generate the maximum likelihood phylogeny.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14060964/s1. Figure S1: BLASTx protein alignment for top hit (A) and BLASTn DNA alignments (B) to query B162 amplicon sequence. Table S1: The gene clusters identified using anvi’o and the in silico evaluation of their content and specificity. Figure S2: BLASTx protein alignments for top hits (A) and BLASTn DNA alignments (B) to query GC3906 sequence. Figure S3: BLASTx protein alignments for top hits (A) and BLASTn DNA alignments (B) to query GC4021 sequence. Figure S4: BLASTx protein alignments for top hits (A) and BLASTn DNA alignments (B) to query GC4381 sequence. Figure S5: BLASTx protein alignments for top hits (A) and BLASTn DNA alignments (B) to query GC4980 sequence. Figure S6: Xhv-specific detection using the touchdown PCR method and the GC3906-152 primer set. Figure S7: Partial Xhv race 1 detection using the touchdown PCR method and the GC4021-112 primer set. Figure S8: Xhv race 2 detection using the touchdown PCR method and the GC4381-178 primer set. Figure S9: Xhv race 3 detection using the touchdown PCR method and the GC4980-138 primer set. Table S2: Additional primers developed and tested for specificity to potential Xhv pathovar- or race-specific gene cluster targets.

Author Contributions

Conceptualization: E.R.M., A.B.Z. and C.T.B. Data curation: E.R.M. and C.T.B. Methodology: E.R.M. Validation: E.R.M. and M.H. Formal analysis: E.R.M. and C.T.B. Investigation: E.R.M. and A.B.Z. Resources: C.T.B. Software: E.R.M. Writing—original draft preparation: E.R.M. Writing—review and editing: M.H., A.B.Z. and C.T.B. Visualization: E.R.M. Supervision: C.T.B. Project administration: C.T.B. Funding acquisition: E.R.M. and C.T.B. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the USDA National Institute of Food and Agriculture and Federal Appropriations under project PEN04926 (accession 7006350), Specialty Crop Multi State Program Agreement No. AGR00018712, and NSF GRFP Grant No. 2018265841.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

We thank the Pennsylvania State University Genomics Core Facility and the CIRM-CFBP French Collection for Plant-Associated Bacteria for their services. We thank Joel Pothier and his team for sharing the X. hydrangeae type strain from their collection with us.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
XhvXanthomonas hortorum pv. vitians
LAMPLoop-mediated isothermal amplification
HRHypersensitive response
SNPSingle-nucleotide polymorphism
PCRPolymerase chain reaction
DNADeoxyribonucleic acid
bpBase pair
KBKilobase
NCBINational Center for Biotechnology Information
BLASTBasic local alignment search tool
ANIAverage nucleotide identity
gDDHGenome-based DNA-DNA hybridization
NEBNew England Biolabs
MLSAMultilocus sequence analysis

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Figure 1. Conventional PCR using B162 primers. The agarose gel shows the banding pattern upon the amplification of a 700 bp fragment using B162 primers [7] and X. hortorum genomic DNA as the templates. Xhv strains are included with overlays in blue (BS0347; race 1), red (ICMP 1408; race 2), and green (BP5181; race 3); the other strains included are Xanthomonas hortorum pv. gardneri (CFBP 8163PT), X. hortorum pv. cynarae (CFBP 4188PT), X. hortorum pv. hederae (CFBP 4925T), X. hortorum pv. taraxaci (CFBP 410PT), X. hortorum pv. pelargonii (CFBP 2533PT), X. hortorum from radicchio (BP5178), and X. hortorum pv. carotae (CFBP 7900). The first well includes a 1 KB ladder, and the final well was run with sterile water as a template.
Figure 1. Conventional PCR using B162 primers. The agarose gel shows the banding pattern upon the amplification of a 700 bp fragment using B162 primers [7] and X. hortorum genomic DNA as the templates. Xhv strains are included with overlays in blue (BS0347; race 1), red (ICMP 1408; race 2), and green (BP5181; race 3); the other strains included are Xanthomonas hortorum pv. gardneri (CFBP 8163PT), X. hortorum pv. cynarae (CFBP 4188PT), X. hortorum pv. hederae (CFBP 4925T), X. hortorum pv. taraxaci (CFBP 410PT), X. hortorum pv. pelargonii (CFBP 2533PT), X. hortorum from radicchio (BP5178), and X. hortorum pv. carotae (CFBP 7900). The first well includes a 1 KB ladder, and the final well was run with sterile water as a template.
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Figure 2. Evaluation of our Xhv-specific method using twenty-four Xanthomonas type strains. Genomic DNA from the following strains was used as templates for touchdown PCR using our GC3906-152 primer set designed for Xhv-specific detection: Xhv (CFBP 8686PT), X. axonopodis (CFBP 4924T), X. campestris (CFBP 5251T), X. translucens (CFBP 2054T), X. theicola (CFBP 4691T), X. arboricola (CFBP 2528T), X. cucurbitae (CFBP 2542T), X. alfalfae (CFBP 7686T), X. sacchari (CFBP 4641T), X. melonis (CFBP 4644T), X. hyacinthi (CFBP 1156T), X. phaseoli (CFBP 8462T), X. bromi (CFBP 1976T), X. euvesicatoria (CFBP 6864T), X. vasicola (CFBP 2543T), X. cassavae (CFBP 4642T), X. populi (CFBP 1817T), X. vesicatoria (CFBP 2537T), X. pisi (CFBP 4643T), X. dyei (CFBP 7245T), X. citri (CFBP 3369T), X. albilineans (CFBP 2523T), X. perforans (CFBP 7293T), and X. codiae (CFBP 4690T). The 1 KB DNA ladder from NEB was used as a size reference for the amplicon, and 1% agarose gels were run for 20 min at 120 V.
Figure 2. Evaluation of our Xhv-specific method using twenty-four Xanthomonas type strains. Genomic DNA from the following strains was used as templates for touchdown PCR using our GC3906-152 primer set designed for Xhv-specific detection: Xhv (CFBP 8686PT), X. axonopodis (CFBP 4924T), X. campestris (CFBP 5251T), X. translucens (CFBP 2054T), X. theicola (CFBP 4691T), X. arboricola (CFBP 2528T), X. cucurbitae (CFBP 2542T), X. alfalfae (CFBP 7686T), X. sacchari (CFBP 4641T), X. melonis (CFBP 4644T), X. hyacinthi (CFBP 1156T), X. phaseoli (CFBP 8462T), X. bromi (CFBP 1976T), X. euvesicatoria (CFBP 6864T), X. vasicola (CFBP 2543T), X. cassavae (CFBP 4642T), X. populi (CFBP 1817T), X. vesicatoria (CFBP 2537T), X. pisi (CFBP 4643T), X. dyei (CFBP 7245T), X. citri (CFBP 3369T), X. albilineans (CFBP 2523T), X. perforans (CFBP 7293T), and X. codiae (CFBP 4690T). The 1 KB DNA ladder from NEB was used as a size reference for the amplicon, and 1% agarose gels were run for 20 min at 120 V.
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Figure 3. Evaluation of our Xhv-specific detection method using environmental samples collected from a 2018 bacterial leaf spot outbreak in lettuce in Pennsylvania. Two Xhv isolates, BP4476 and BP4477, and two Pseudomonas isolates, BP4478 (Pseudomonas viridiflava) and BP4479 (Pseudomonas allivorans), isolated from the same symptomatic lettuce tissue were used as the templates in the Xhv-specific touchdown reaction with the GC3906-152 primers to test the primers’ specificity. Xhv race 1 strain BP5172 was included as a positive control, sterile water was included as an experimental control, and the size reference was a 1 KB DNA ladder from NEB. The 1% agarose gel was run for 20 min at 120 V.
Figure 3. Evaluation of our Xhv-specific detection method using environmental samples collected from a 2018 bacterial leaf spot outbreak in lettuce in Pennsylvania. Two Xhv isolates, BP4476 and BP4477, and two Pseudomonas isolates, BP4478 (Pseudomonas viridiflava) and BP4479 (Pseudomonas allivorans), isolated from the same symptomatic lettuce tissue were used as the templates in the Xhv-specific touchdown reaction with the GC3906-152 primers to test the primers’ specificity. Xhv race 1 strain BP5172 was included as a positive control, sterile water was included as an experimental control, and the size reference was a 1 KB DNA ladder from NEB. The 1% agarose gel was run for 20 min at 120 V.
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Figure 4. Multilocus sequence analysis (MLSA) of PA X. hortorum isolates. Maximum likelihood phylogeny generated from the alignment of concatenated fragments of the rpoD, fyuA, gyrB, and gap1 housekeeping genes using the neighbor-joining algorithm and the general time-reversible nucleotide substitution model (including rate variation and estimated topology). The bootstrap threshold was set to 70%, and the branch lengths represent the expected number of nucleotide substitutions per site. Branches shorter than 0.0002 are shown as having a length of 0.0002. Strains of a known Xhv MLSA type were included as a reference set in order to predict the races of the Pennsylvania strains that tested positive for Xhv detection using our touchdown PCR-based method. The type strain of X. hortorum pv. hederae was included as an outgroup.
Figure 4. Multilocus sequence analysis (MLSA) of PA X. hortorum isolates. Maximum likelihood phylogeny generated from the alignment of concatenated fragments of the rpoD, fyuA, gyrB, and gap1 housekeeping genes using the neighbor-joining algorithm and the general time-reversible nucleotide substitution model (including rate variation and estimated topology). The bootstrap threshold was set to 70%, and the branch lengths represent the expected number of nucleotide substitutions per site. Branches shorter than 0.0002 are shown as having a length of 0.0002. Strains of a known Xhv MLSA type were included as a reference set in order to predict the races of the Pennsylvania strains that tested positive for Xhv detection using our touchdown PCR-based method. The type strain of X. hortorum pv. hederae was included as an outgroup.
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Table 1. Primer sets developed for the specific detection of Xhv and its three known races.
Table 1. Primer sets developed for the specific detection of Xhv and its three known races.
Primer SetForward SequenceReverse SequenceAmplicon Size (bp)Target Strains
GC3906-152GTTCGGTCGCCATTTCGATGAGATAACCTCCCAGACCGCT152Xhv
GC4021-112GGTGGCCTACTTTCATGCGAGAGCAAGCCCTTCACAAGGT112Xhv race 1
GC4381-178TATGATGCGGCACACAACCTCGTATTGCGGTGCGAACTTT178Xhv race 2
GC4980-138TCACTCAAAAGCCCACCCTCACATTCCTCGGCTATCCCCT138Xhv race 3
Table 2. A list of the bacterial strains that were included in this study.
Table 2. A list of the bacterial strains that were included in this study.
OrganismStrain *Other Strain IDs Host of IsolationKnown or Hypothesized RaceGC3906-152 DetectionGC4021-112 DetectionGC4381-178 DetectionGC4980-138 DetectionOriginCollector or Citation
X. hortorum pv. vitiansBP5172 Xav 98-37 2/01Lactuca sativaXhv race 1YesNoNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0339 ‡Salinas 2/01Lactuca sativaXhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0340 ‡Xav 98-23 2/01Lactuca sativaXhv race 1YesNoNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0347 ‡Xcv 5/01Lactuca sativaXhv race 1YesFaintNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBP5176 ‡Xcv 5/01Lactuca sativaXhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBP5177 ‡“Edge A”Lactuca sativaXhv race 1YesNoNoNoCO, USAS. Koike
X. hortorum pv. vitiansBP5179 ‡“Daniel Rom”Lactuca sativaXhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBP5182 ‡“Moreno Let”Lactuca sativaXhv race 1YesYesYesNoSanta Maria, CA, USAJ. Barak
X. hortorum pv. vitiansNCPPB 4058 ‡N/ALactuca sativaXhv race 1YesYesNoNoUKH. Stanford
X. hortorum pv. vitiansCFBP 8686PTLMG 938PT, NCPPB 2248PT, MR20213PTLactuca sativaXhv race 1YesYesNoNoZimbabwe[6,8]
X. hortorum pv. vitiansBP5191 ‡VT111Lactuca sativaXhv race 1YesNoNoNoCanadaV. Toussaint
X. hortorum pv. vitiansBP5192 ‡Xcv-2Lactuca sativaXhv race 1YesYesNoNoCA, USAC. T. Bull
X. hortorum pv. vitiansICMP 1408 ‡PDDCC 1408, Burkholder XL5Lactuca sativaXhv race 2YesNoYesNoIthaca, NY, USAW. H. Burkholder
X. hortorum pv. vitiansICMP 4165 ‡LMG 7508, PDDCC 4165Lactuca sativaXhv race 2YesNoYesNoNew ZealandH. J. Boesewinkel
X. hortorum pv. vitiansBS3127 ‡VT106Lactuca sativaXhv race 2YesNoYesNoCanadaV. Toussaint
X. hortorum pv. vitiansBP5194 ‡917Lactuca sativaXhv race 2YesNoYesNoOH, USA[18]
X. hortorum pv. vitiansBS2861 ‡“Christy BuLet 2”Lactuca sativaXhv race 3YesNoNoYesKing City, CA, USAS. Koike and Rianda
X. hortorum pv. vitiansBP5181 ‡“Christy BuLet 3”Lactuca sativaXhv race 3YesNoNoYesKing City, CA, USAS. Koike and Rianda
X. hortorum pv. vitiansBS0313A674-2B (e1)Lactuca sativaHypothesized Xhv race 1YesYesNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0341C 5/20/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0342Xcv 5/20/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0343Xcv 5/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0345Xcv 5/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0346Xcv 5/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS2849“Mike Lombard Reeves”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2850“Mike Lombard Harden”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2852“Daniel Rom”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2855“Frank Let”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2857“Frank Let”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2858John DeCarli Rom1-1Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2859John DeCarli Rom1-2Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2870“Moreno Let”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSanta maria, CA, USAS. Koike
X. hortorum pv. vitiansBS2871“Keller Rom”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2872“Keller Rom”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2873“Greg greenleaf”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2874“Greg greenleaf”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2875“Greg redleaf”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2876“Greg let”Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2908“Matt romaine”Lactuca sativaHypothesized Xhv race 1YesYesNoNoWatsonville, CA, USAS. Koike
X. hortorum pv. vitiansBS2994ICMP 6656, DAR 30547Lactuca sativaHypothesized Xhv race 1YesYesNoNoNew South Wales, AustraliaR. Fitzell
X. hortorum pv. vitiansBS2996ICMP 6735, Watson B2578Lactuca sativaHypothesized Xhv race 1YesYesYesNoPalmerston North, WI, New ZealandD. R. W. Watson
X. hortorum pv. vitiansBS2997ICMP 7423, Watson J2928.Lactuca sativaHypothesized Xhv race 1YesYesNoNoPatumahoe, AK, New ZealandD. R. W. Watson
X. hortorum pv. vitiansBS3050LMG 8688; ICMP 6461; Watson D2538Lactuca sativaHypothesized Xhv race 1YesNoNoNoNew ZealandD. R. W. Watson
X. hortorum pv. vitiansBS3051LMG 8690; Hill AS3997; ICMP 6857Lactuca sativaHypothesized Xhv race 1YesYesNoNoNew ZealandD. R. W. Watson
X. hortorum pv. vitiansBS3054CFBP 3980, Audusseau 11.72Lactuca sativaHypothesized Xhv race 1YesNoNoNoVaucluse, FranceC. Audusseau
X. hortorum pv. vitiansBS3056CFBP 3996 Audusseau 17.09Lactuca sativaHypothesized Xhv race 1YesYesNoNoIsère, FranceC. Audusseau
X. hortorum pv. vitiansBS3128105 VT107Lactuca sativaHypothesized Xhv race 1YesYesNoNoCanadaV. Toussaint
X. hortorum pv. vitiansBS3131119 VT41Lactuca sativaHypothesized Xhv race 1YesYesNoNoCanadaV. Toussaint
X. hortorum pv. vitiansBS3132‡B07-007, ID200707ALactuca sativaXhv race 1YesYesNoNoCanadaV. Toussaint
X. hortorum pv. vitiansBS3272L11Lactuca sativaHypothesized Xhv race 1YesYesNoYesFL, USA[2]
X. hortorum pv. vitiansBS3300Xcv-4Lactuca sativaHypothesized Xhv race 1YesYesNoNoCA, USAC. T. Bull
X. hortorum pv. vitiansBS3301Xcv-5Lactuca sativaHypothesized Xhv race 1YesYesNoNoCA, USAC. T. Bull
X. hortorum pv. vitiansBS3303Xcv-7Lactuca sativaHypothesized Xhv race 1YesYesNoNoCA, USAC. T. Bull
X. hortorum pv. vitiansBS3304Xcv-8Lactuca sativaHypothesized Xhv race 1YesYesNoNoCA, USAC. T. Bull
X. hortorum pv. vitiansBS3306Xcv-10Lactuca sativaHypothesized Xhv race 1YesYesNoNoCA, USAC. T. Bull
X. hortorum pv. vitiansBS030110S7-2 (a2)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS030210TB7 (a4)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS030410TB9 (a6)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS030510TB12-1 (a7)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0309S4-1(K) (c5)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0310S5-2 (c6)Lactuca sativaHypothesized Xhv race 1YesFaintNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0311S5-2(K) (c7)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0314A674-4B (e2)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS031610TB9 (e4)Lactuca sativaHypothesized Xhv race 1YesNoNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0318QR71B (e8)Lactuca sativaHypothesized Xhv race 1YesYesNoNoHI, USAA. Alvarez
X. hortorum pv. vitiansBS0335Xav 98-05 2/01Lactuca sativaHypothesized Xhv race 1YesNoNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0337Xav 98-67 2/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0338Xav 98-76 2/01Lactuca sativaHypothesized Xhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS054210S7-2 (a2)Lactuca sativaHypothesized Xhv race 1YesNoNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS0543“Spot A”Lactuca sativaHypothesized Xhv race 1YesNoNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS2946Julio Rodrigues Neto IBSBF 1553 Embrapa K 532Lactuca sativaHypothesized Xhv race 1YesNoNoNoBrazilI. M. G. Almeida
X. hortorum pv. vitiansBS2998ICMP 7465, IBSBF 325, C.F. Robbs: ENA2008Lactuca sativaHypothesized Xhv race 1YesNoNoNoBrazilC. F. Robbs
X. hortorum pv. vitiansBS3035NCPPB 970, ICPB XL 102, Thornberry 1-49Lactuca sativaHypothesized Xhv race 1YesNoNoNoUSAH. H. Thornberry
X. hortorum pv. vitiansBS3036NCPPB 1839, ICPB XV169, Robbs ENA-250Lactuca sativaHypothesized Xhv race 1YesNoNoNoBrazilA. P. Viegas
X. hortorum pv. vitiansBS3041NCPPB 3663, Neto ISBF 473Lactuca sativaHypothesized Xhv race 1YesNoNoNo J. R. Neto
X. hortorum pv. vitiansBS3042NCPPB3931, Sellwood/Wilson A6520/1Lactuca sativaHypothesized Xhv race 1YesNoNoNoThe UKJ.E. Sellwood and J.K. Wilson
X. hortorum pv. vitiansBS3047LMG 7453; ATCC 11525; Burkholder XL3; CNBP 500; Dye YA3; ICMP 337; ICPB XL3; NCPPB 992; PDDCC 337Lactuca sativaHypothesized Xhv race 1YesNoNoNoUSAH. Anderson
X. hortorum pv. vitiansBS3049LMG 7510; Fahy DAR30526; ICMP 6655; PDDCC 6655Lactuca sativaHypothesized Xhv race 1YesNoNoNoAustraliaP. Fahy
X. hortorum pv. vitiansBS3053CFBP 3973, Audusseau 11.08Lactuca sativaHypothesized Xhv race 1YesNoNoNo C. Audusseau
X. hortorum pv. vitiansBS3055CFBP 3983, Audusseau 14.28Lactuca sativaHypothesized Xhv race 1YesNoNoNoIsère, FranceC. Audusseau
X. hortorum pv. vitiansBS3130118 VT25Lactuca sativaHypothesized Xhv race 1YesNoNoNo V. Toussaint
X. hortorum pv. vitiansBS3271L43Lactuca sativaHypothesized Xhv race 1YesNoNoNoFL, USAC. T. Bull
X. hortorum pv. vitiansBS3526B55Lactuca sativaHypothesized Xhv race 1YesNoNoNoCA, USA[18]
X. hortorum pv. vitiansBS3527B57Lactuca sativaHypothesized Xhv race 1YesNoNoNoCA, USA[18]
X. hortorum pv. vitiansBS0344 ‡Xcv 5/01Lactuca sativaXhv race 1YesYesNoNoSalinas, CA, USAJ. Barak
X. hortorum pv. vitiansBS2851 ‡“Mike Lombard Harden”Lactuca sativaXhv race 1YesYesNoNoSalinas, CA, USAS. Koike
X. hortorum pv. vitiansBS2909“Matt romaine”Lactuca sativaHypothesized Xhv race 1YesYesNoNoWatsonville, CA, USAS. Koike
X. hortorum pv. vitiansBS3528 ‡B59Lactuca sativaXhv race 1YesYesNoNoCA, USA[18]
X. hortorum pv. vitiansBS3034 ‡NCPPB 2969, ICPB XL6Lactuca sativaXhv race 2YesNoYesNoUSAW. H. Burkholder
X. hortorum pv. vitiansBS3043 ‡NCPPB 4033, Sahin/Miller 700aLactuca sativaXhv race 2YesNoYesNoUSAF. Sahin
X. hortorum pv. vitiansBS3126 ‡99 VT101Lactuca sativaXhv race 2NoNoYesNoIsère, FranceC. Audusseau
X. hortorum pv. vitiansBS3529 ‡906Lactuca sativaXhv race 2YesNoYesNoOH, USAS. Miller
X. hortorum pv. vitiansBS3531 ‡923Lactuca sativaXhv race 2YesNoNoNoOH, USA[18]
X. hortorum pv. vitiansBS3532 ‡924Lactuca sativaXhv race 2YesNoNoNoOH, USA[18]
X. hortorum pv. vitiansBS2860Christy BuLet 1Lactuca sativaHypothesized Xhv race 3YesNoNoYesKing City, CA, USAS. Koike
X. hortorum pv. vitiansBS2863Christy BuLet 4Lactuca sativaHypothesized Xhv race 3YesNoNoYesKing City, CA, USAS. Koike
X. hortorum pv. vitiansBP4476N/ALactuca sativaHypothesized Xhv race 1YesNot testedNot testedNot testedPA, USAThis study
X. hortorum pv. vitiansBP4477N/ALactuca sativaHypothesized Xhv race 1YesNot testedNot testedNot testedPA, USAThis study
Pseudomonas viridiflavaBP4478N/ALactuca sativaN/ANoNot testedNot testedNot testedPA, USAThis study
Pseudomonas allivoransBP4479N/ALactuca sativaN/ANoNot testedNot testedNot testedPA, USAThis study
X. hortorumBP5178N/ACichorium intybus (radicchio)N/ANoNoNoNoSalinas, CA, USA[17]
X. hortorum pv. hederaeCFBP 4925TICMP 453T, NCPPB 939T, LMG 733THedera helix (English ivy)N/ANoNoNoNoUSA[8,19,20]
X. hortorum pv. taraxaciCFBP 410PTATCC 19318PT, NCPPB 940PT, LMG 870PTTaraxacum kok-sahgyz (Russian dandelion)N/ANoNoNoNoIthaca, NY, USA[8,19,20,21]
X. hortorum pv. pelargoniiCFBP 2533PTICMP 4321PT, LMG 7314PT, NCPPB 2985PTPelargonium peltatum (pelargonium)N/ANoNoNoNoAuckland, New Zealand[20,22]
X. hortorum pv. gardneriCFBP 8163PTLMG 962PT, ATCC19865PT, NCPPB 881PT, PDCC 1620PTSolanum lycopersicum (tomato)N/ANoNoNoNoYugoslavia[23,24]
X. hortorum pv. cynaraeCFBP 4188PTICMP 16775PTCynara scolymus (artichoke)N/ANoNoNoNoFrance[25]
X. hortorum pv. carotaeCFBP 7900M081Daucus carota (carrot)N/ANoNoNoFaintHungary[8,20,26]
X. campestris pv. coriandriCFBP 8452PTLMG 687PT, ATCC 17996PT, ICMP 5725PT, NCPPB 1758PT, PDDCC 5725PTCoriandrum sativum (coriander)N/ANoNoNoNoIndia[8,20,27]
X. hydrangeaeLMG 31884TVan Vaerenbergh gbbc963T; GBBC 2123T, CCOS 1956THydrangea arborescens (smooth hydrangea)N/ANoNot testedNot testedNot testedFlanders, Belgium[28]
X. axonopodisCFBP 4924TATCC 19312T, CFBP 2156T, ICMP 50T, ICPB Xa103T, LMG 538T, LMG 982T, NCPPB 457TAxonopus scoparius (carpet grass)N/ANoNot testedNot testedNot testedColombia[29]
X. campestrisCFBP 5251TATCC 33913T, CFBP 2350T, CFBP 5241T, DSM 3586T, ICMP 13T, LMG 568T, Labo 11405T, NCPPB 528TBrassica oleracea var. gemmifera (Brussels sprout)N/ANoNot testedNot testedNot testedThe UK[30,31]
X. translucensCFBP 2054TATCC 19319T, ICMP 5752T, ICPB XT2T, LMG 876T, NCPPB 973THordeum vulgare (barley)N/ANoNot testedNot testedNot testedUSA[8,32]
X. theicolaCFBP 4691TICMP 6774T, LMG 8684TCamellia sinensis (tea tree)N/ANoNot testedNot testedNot testedJapan[8]
X. arboricolaCFBP 2528TATCC 49083T, ICMP 35T, LMG 747T, NCPPB 411TJuglans regia (walnut)N/ANoNot testedNot testedNot testedNew Zealand[8,33]
X. cucurbitaeCFBP 2542TICMP 2299T, LMG 690T, NCPPB 2597TCucurbita maxima (squash)N/ANoNot testedNot testedNot testedNew Zealand[8,34]
X. alfalfaeCFBP 7686TATCC 11765T, ICPB 10701T, ICPB XA 121T, LMG 495TMedicago sativa (alfalfa)N/ANoNot testedNot testedNot testedIndia[35,36]
X. sacchariCFBP 4641TLMG 471TSaccharum officinarum (sugarcane)N/ANoNot testedNot testedNot testedGuadeloupe, France[8]
X. melonisCFBP 4644TIBSBF 68T, ICMP 8682T, LMG 8670T, NCPPB 3434TCucumis melo (melon)N/ANoNot testedNot testedNot testedBrazil[8]
X. hyacinthiCFBP 1156TATCC 19314T, ICMP 189T, LMG 739T, NCPPB 599THyacinthus orientalis (hyacinth)N/ANoNot testedNot testedNot testedNetherlands[8,37]
X. phaseoliCFBP 8462TATCC 49119T, LMG 29033TPhaseolus vulgaris (common bean)N/ANoNot testedNot testedNot testedNE, USA[38,39]
X. bromiCFBP 1976TICMP 12545T, LMG 947TBromus carinatus (bromegrass)N/ANoNot testedNot testedNot testedFrance[8]
X. euvesicatoriaCFBP 6864TATCC 11633T, DSM 19128T, ICMP 109T, ICMP 98T, NCPPB 2968TCapsicum frutescens (wild chili pepper)N/ANoNot testedNot testedNot testedUSA[23]
X. vasicolaCFBP 2543TICMP 3103T, LMG 736T, NCPPB 2417TSorghum vulgare (sorghum)N/ANoNot testedNot testedNot testedNew Zealand[8,40]
X. cassavaeCFBP 4642TICMP 204T, LMG 673T, NCPPB 101TManihot esculenta (cassava)N/ANoNot testedNot testedNot testedMalawi[8,41]
X. populiCFBP 1817TATCC 51165T, ICMP 5816T, ICPB XP 240T, LMG 5743TPopulus x canadensis cv. Regenerata (Canadian poplar)N/ANoNot testedNot testedNot testedNoyon, Oise, France[42,43]
X. vesicatoriaCFBP 2537TATCC 35937T, CFBP 4645T, ICMP 63T, LMG 911T, NCPPB 422TLycopersicon esculentum (tomato)N/ANoNot testedNot testedNot testedNew Zealand[8,44]
X. pisiCFBP 4643TATCC 35936T, ICMP 570T, ICMP 570T, LMG 847T, Labo 13356T, NCPPB 762TPisum sativum (pea)N/ANoNot testedNot testedNot testedJapan[8,45]
X. dyeiCFBP 7245TDSM 110537T, ICMP 12167T, NCPPB 4446TMetrosideros excelsa (New Zealand Christmas tree)N/ANoNot testedNot testedNot testedOmahanui, Bay of Plenty, New Zealand[46]
X. citriCFBP 3369TATCC 49118T, LMG 9322TCitrus aurantifolia (key lime)N/ANoNot testedNot testedNot testedFL, USA[38,47,48]
X. albilineansCFBP 2523TATCC 33915T, ICMP 196T, LMG 494T, NCPPB 2969TSaccharum officinarum (sugarcane)N/ANoNot testedNot testedNot testedFiji[49,50,51]
X. perforansCFBP 7293TDSM 18975T, NCPPB 4321TSolanum lycopersicum L. (tomato)N/ANoNot testedNot testedNot testedFL, USA[23]
X. codiaeCFBP 4690TICMP 9513T, LMG 8678TCodiacum variegatum (croton)N/ANoNot testedNot testedNot testedFL, USA[8]
* Strains in bold were used in the whole genome sequence alignments to identify Xhv-specific and race-specific gene cluster targets for PCR-based detection. Xhv strains marked with ‡ have had their race confirmed via HR screening [10,11], while those lacking the ‡ symbol have been hypothesized to belong to their race based on a multilocus sequence analysis (MLSA).
Table 3. Touchdown PCR cycling conditions.
Table 3. Touchdown PCR cycling conditions.
StepInstructionPurpose
195 °C for 1 minTaq polymerase activation
2 *95 °C for 30 sDenaturation
3 *68 °C for 30 s, −1 °C every cycleAnnealing
4 *72 °C for 30 sExtension
5 *GOTO Step 2, 10 timesCycling
695 °C for 30 sDenaturation
758 °C for 30 sAnnealing
872 °C for 30 sExtension
9GOTO Step 6, 23 timesCycling
1072 °C for 5 minFinal extension
* The touchdown phase of our protocol was when the annealing temperature was kept higher than that in typical PCR for 11 cycles to reduce non-specific primer binding. The remaining cycling temperatures were typical of general PCR.
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Martinez, E.R.; Hamidizade, M.; Zacaroni, A.B.; Bull, C.T. Novel PCR-Based Detection Methods for the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas hortorum pv. vitians Morinière et al., 2020. Plants 2025, 14, 964. https://doi.org/10.3390/plants14060964

AMA Style

Martinez ER, Hamidizade M, Zacaroni AB, Bull CT. Novel PCR-Based Detection Methods for the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas hortorum pv. vitians Morinière et al., 2020. Plants. 2025; 14(6):964. https://doi.org/10.3390/plants14060964

Chicago/Turabian Style

Martinez, Emma R., Mozhde Hamidizade, Ana B. Zacaroni, and Carolee T. Bull. 2025. "Novel PCR-Based Detection Methods for the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas hortorum pv. vitians Morinière et al., 2020" Plants 14, no. 6: 964. https://doi.org/10.3390/plants14060964

APA Style

Martinez, E. R., Hamidizade, M., Zacaroni, A. B., & Bull, C. T. (2025). Novel PCR-Based Detection Methods for the Lettuce Bacterial Leaf Spot Pathogen, Xanthomonas hortorum pv. vitians Morinière et al., 2020. Plants, 14(6), 964. https://doi.org/10.3390/plants14060964

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