Novel PCR-based detection methods for the lettuce bacterial leaf spot pathogen, Xanthomonas hortorum pv. vitians Morinière et al., 2020
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MDPI
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.
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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.
SUPPLEMENTARY MATERIAL 1 : FIGURE S1: BLASTx protein alignment for top hit (A) and BLASTn DNA alignments (B) to query B162 amplicon sequence. 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. SUPPLEMENTARY MATERIAL 2 : TABLE S1: The gene clusters identified using anvi’o and the in silico evaluation of their content and specificity. TABLE S2: Additional primers developed and tested for specificity to potential Xhv pathovar- or race-specific gene cluster targets.
SUPPLEMENTARY MATERIAL 1 : FIGURE S1: BLASTx protein alignment for top hit (A) and BLASTn DNA alignments (B) to query B162 amplicon sequence. 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. SUPPLEMENTARY MATERIAL 2 : TABLE S1: The gene clusters identified using anvi’o and the in silico evaluation of their content and specificity. TABLE S2: Additional primers developed and tested for specificity to potential Xhv pathovar- or race-specific gene cluster targets.
Keywords
Disease detection, Bacterial disease, Genetic diversity, Comparative genomics, PCR-based detection, Lactuca sativa, Xanthomonas hortorum pv. vitians (Xhv), Polymerase chain reaction (PCR)
Sustainable Development Goals
SDG-15: Life on land
SDG-02: Zero hunger
SDG-02: Zero hunger
Citation
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.