Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D . dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D . dianthicola , which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase , of D . dianthicola . Assay specificity was assessed using strains present in inclusivity (16 D . dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D . dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection.
Dickeya zeae, a bacterial plant pathogen of the family Pectobacteriaceae, is responsible for a wide range of diseases on potato, maize, rice, banana, pineapple, taro, and ornamentals and significantly reduces crop production. D. zeae causes the soft rot of taro (Colocasia esculenta) and the heart rot of pineapple (Ananas comosus). In this study, we used Pacific Biosciences single-molecule real-time (SMRT) sequencing to sequence two high-quality complete genomes of novel strains of D. zeae: PL65 (size: 4.74997 MB; depth: 701x; GC: 53.6%) and A5410 (size: 4.7792 MB; depth: 558x; GC: 53.5%) isolated from economically important Hawaiian crops, taro, and pineapple, respectively. Additional complete genomes of D. zeae representing three additional hosts (philodendron, rice, and banana) and other species used for a taxonomic comparison were retrieved from the NCBI GenBank genome database. Genomic analyses indicated the truncated type III and IV secretion systems (T3SS and T4SS) in the taro strain, which only harbored one and two genes of T3SS and T4SS, respectively, and showed high heterogeneity in the type VI secretion system (T6SS). Unlike strain EC1, which was isolated from rice and recently reclassified as D. oryzae, neither the genome PL65 nor A5410 harbors the zeamine biosynthesis gene cluster, which plays a key role in virulence of other Dickeya species. The percentages of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) between the two genomes were 94.47 and 57.00, respectively. In this study, we compared the major virulence factors [plant cell wall-degrading extracellular enzymes and protease (Prt)] produced by D. zeae strains and evaluated the virulence on taro corms and pineapple leaves. Both strains produced Prts, pectate lyases (Pels), and cellulases but no significant quantitative differences were observed (p > 0.05) between the strains. All the strains produced symptoms on taro corms and pineapple leaves, but the strain PL65 produced symptoms more rapidly than others. Our study highlights the genetic constituents of pathogenicity determinants and genomic heterogeneity that will help to understand the virulence mechanisms and aggressiveness of this plant pathogen.
Bacterial spot (BS), caused by Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, is an economically important bacterial disease of tomato and pepper. Symptoms produced by all four species are nearly indistinguishable. At present, no point-of-care diagnostics exist for BS. In this research, we examined genomes of X. euvesicatoria, X. vesicatoria, X. gardneri, X. perforans and other species of Xanthomonas; the unique gene recG was chosen to design primers to develop a loop-mediated isothermal amplification (LAMP) assay to rapidly and accurately identify and differentiate X. euvesicatoria from other BS causing Xanthomonas sp. using a field-deployable portable BioRangerTM instrument. Specificity of the developed assay was tested against 39 strains of X. euvesicatoria and 41 strains of other species in inclusivity and exclusivity panels, respectively. The assay detection limit was 100 fg (~18 genome copies) of genomic DNA and 1,000 fg in samples spiked with tomato DNA. The assay unambiguously detected X. euvesicatoria in infected tomato plant samples. Concordant results were obtained when multiple operators performed the test independently. No false positives and false negatives were detected. The developed LAMP assay has numerous applications in diagnostics, biosecurity and disease management.
Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.
Aims Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola. Methods and Results A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome‐based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed a detection limit of 10 fg DNA. Conclusion The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. Significance and Impact of the Study The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.
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