Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18-20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay's applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Genomic and Phenotypic Biology of Novel Strains of Dickeya zeae Isolated From Pineapple and Taro in Hawaii: Insights Into Genome Plasticity, Pathogenicity, and Virulence Determinants.

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.

First Report of Pectobacterium brasiliense causing soft rot on mizuna (Brassica rapa var. japonica) in the United States.

Mizuna (Brassica rapa var. japonica), a member of family Brassicaceae, is a leafy vegetable having phenolic and other compounds beneficial to human health, such as natural antioxidants (Khanam et al. 2012). In October 2020, a field of mizuna (variety: Early) on Oahu island was observed having 20-30% diseased plants. Four randomly selected infected mizuna plants, showing the symptoms of wilt and stem rot (Figure 1A-D), were collected and isolations were made to determine the pathogen. Small sections of infected stems were cut, surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The tissues were macerated in a sterile 1.5 ml centrifuge tube containing 100 µl sterile water-macerated tissues were streaked onto crystal violet pectate medium (CVP) (Hélias et al. 2011) and incubated at 26 ± 2°C for 48 h. Isolated bacterial colonies that formed pits on the CVP plates were re-streaked onto dextrose peptone agar: Peptone (10 g/L), Dextrose (5 g/L) and Agar (17 g/L) (DPA-without tetrazolium chloride; Norman and Alvarez 1989) to obtain purified colonies for DNA isolation using DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). The two housekeeping genes (dnaA and gapA) were amplified and sequenced following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), for identity confirmation and phylogenetic analysis. Cleaned PCR products were sent to the GENEWIZ facility (Genewiz, La Jolla, CA) for sequencing of sense and antisense strands. The obtained sequences were aligned, manually edited, and consensus sequences were analyzed with BLASTn using the NCBI GenBank nucleotide and genome databases for identity confirmation. The BLASTn results demonstrated 100% query coverage of all four strains (PL248-PL251); and showed 100% identity of PL248 and PL249, and 99% identity of PL250 and PL251 with Pectobacterium brasiliense. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: dnaA gene MW560271 - MW560274 (PL248 - PL251); and gapA gene MW560275 - MW560278 (PL248 - PL251). Pathogenicity was assessed by artificially inoculating 100 µl bacterial suspension of each strain (PL248 - 1.12x 108 CFU/ml; PL249 - 1.32x 108 CFU/ml; PL 250 - 1.2x 108 CFU/ml and PL251 - 1.15x 108 CFU/ml) onto four-week-old mizuna (variety: Leafy Asian Greens) plants in three replicates, using sterile pipette tips, which was stabbed into stem halfway and wrapped with parafilm. The inoculated plants were well maintained under controlled greenhouse conditions. As negative controls, three plants were inoculated with 100 µl distilled water. Soft rot and wilt symptoms (Figure 1E-H) were observed 24 hours post inoculation. No symptoms were observed on control plants (Figure 1F). All four strains were re-isolated from the inoculated plants and confirmed as P. brasiliense based on resequencing of the dnaA region and 100% homology with the sequences of original strain. In the phylogenetic tree (Figure 2), based on two housekeeping genes (dnaA and gapA), the bacterial strains from mizuna grouped with other P. brasiliense retrieved from the NCBI GenBank database. To our knowledge, this is the first report of P. brasiliense infecting mizuna plants in Hawaii or in the USA and is important because this species is one of the most aggressive pectolytic pathogens in the genus Pectobacterium. Understanding the diversity of different pectolytic phytopathogens is essential to formulating risk mitigation strategies as P. brasiliense could potentially pose a threat to additional vegetable crops, especially the crucifers vegetables (Arizala et al. 2019; Klair et al, 2021).

First Report of Bacterial Soft Rot Disease on Pak Choi (Brassica rapa subsp. chinensis) caused by Pectobacterium brasiliense in the United States.

Pak choi (Brassica rapa subsp. chinensis) is an important vegetable crop native to China, known for high water content and low caloric value, containing high quality of protein, carbohydrates, fiber, vitamins, minerals, and secondary plant metabolites (Acikgoz, 2016). A pak choi field (8,000 sq. ft.) on Oahu, Hawaii, was visited in May 2020. About 10% plants were infected and showed characteristic symptoms of soft rot, wet lesions, macerated infected stem and necrotic leaves (Figure1A-D); leading to the suspect of one of the most devastating bacterial pathogens within genus Pectobacterium (Boluk et al. 2020; Li et al. 2019; Arizala et al. 2020; Arizala and Arif, 2019). Four infected plants were collected from the field, and stems were surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The stems were aseptically macerated, streaked on Crystal violet pectate medium (CVP) (Hélias et al. 2011), and incubated for 48 h at 26 ± 2°C. The peculiar morphological characteristic of pectolytic bacterial pathogen, forming pits on CVP, were observed (Meng et al. 2016) (Figure 1E). Purification of bacterial colonies were done by re-streaking of a single colony on dextrose peptone agar (DPA-without tetrazolium chloride; Norman and Alvarez 1989). DNA was isolated from bacterial cultures using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA), respectively. Molecular identification of four strains (PL243-246) were performed by the sequencing region of the housekeeping gene dnaA (chromosomal replication initiation protein) using Pec. dnaA-F1/R1 primer set (Dobhal et al. 2020). The amplified PCR product was enzymatically cleaned using ExoSAP-ITTM (Affymetrix Inc, Santa Clara, CA), and sent for sequencing at the GENEWIZ facility (Genewiz, La Jolla, CA) using both forward and reverse primers. The dnaA gene sequences were aligned using Geneious, and manually edited to remove the errors. The consensus sequences were analyzed with the NCBI BLASTn tool and were deposited in the NCBI GenBank under the accession numbers MT899920-MT899923. The NCBI BLASTn report indicated that all the sequences shared 99-100% identity and query cover with Pectobacterium brasiliense accession numbers MN544627-29. A phylogenetic analysis, using Geneious, was performed with the dnaA sequences representing different Pectobacterium spp., all strains grouped within the clade of P. brasiliense (Figure 2; Arizala et al, 2020). A pathogenicity assay was carried out in three replications on pak choi grown in pots containing commercial pot mixture, and maintained in the controlled-greenhouse (temperature 26-30°C; relative humidity 50-58%). Three-weeks old plant stems were artificially inoculated with 100 µl bacterial suspensions of PL243 (1.3x 108 CFU/ml), PL244 (1.2x 108 CFU/ml), PL 245 (1.2x 108 CFU/ml) and PL246 (1.1x 108CFU/ml); control plants were inoculated with 100 µl of distilled water (Figure 1F). Two days after inoculation, the soft rot and wilting symptoms (Figure 1G-H), similar to the ones observed on the field, were developed for all four strains tested. Bacteria was successfully re-isolated from the inoculated plants; DNA was isolated, amplified, sequenced for dnaA region and analyzed for 100% homology with original strains, to fulfill Koch's postulates. Based on the molecular characteristics re-isolates were identical to the original strains. To the best of our knowledge, this is the first report of P. brasiliense on pak choi in the USA. Recent reports indicated that the pathogen could potentially pose a threat to cruciferous crops, therefore, highlighting a need to conduct a state-wide survey for pectinolytic bacteria, and implement better management strategies to combat the vegetable crop losses.

Genome-Informed Recombinase Polymerase Amplification Assay Coupled with a Lateral Flow Device for In-Field Detection of Dickeya Species.

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.

Development of a robust, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of potato pathogen Dickeya dianthicola targeting a unique genomic region.

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.

Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria.

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.