Xanthomonas Strains Isolated from Hosts in the Family Araceae Reveal Diverse Phylogenetic Relationships and Origins.

The family Araceae, comprising ornamentals including Anthurium, Dieffenbachia, Philodendron, Colocasia, and Zantedeschia, is susceptible to Xanthomonas pathogens. Previous analyses have established heterogeneity in aroid strains, yet unresolved taxonomic positions and dynamics between Xanthomonas and frequently associated Stenotrophomonas in aroids necessitate in-depth genetic investigation to resolve these complex relationships. This study utilized multilocus sequence analysis of housekeeping genes atpD, dnaA, dnaK, gltA, and gyrB to investigate 59 aroid strains, selected based on hosts, time, and geographical origins. After adding sequences from additional strains from NCBI GenBank, analysis of 161 concatenated sequences indicated that all aroid strains fell within Xanthomonas and Stenotrophomonas. Thirty-six strains isolated from Anthurium grouped under X. phaseoli, with outliers including one strain each in X. arboricola and X. sacchari and two in Stenotrophomonas. Six strains from Caladium, Dieffenbachia, and Philodendron formed host-specific subgroups within X. euvesicatoria. One strain from Dieffenbachia aligned with X. campestris, whereas strains from Colocasia, Aglaonema, and Spathiphyllum clustered with X. sacchari. Apart from the zantedeschia strain described as X. arboricola pv. zantedeschiae, two colocasia, one epipremnum, and one anthurium strain joined the X. arboricola group. Overall, this study revealed significant heterogeneity among aroid strains, with anthurium strains clustering closely despite distant geographical origins. The analysis underscores the complexity of host-pathogen specificity within Xanthomonas and emphasizes the need for further taxonomic clarification through whole-genome analysis of representative strains. The findings of this research will facilitate strain selection for inclusivity and exclusivity panels in developing diagnostic assays for X. phaseoli and xanthomonads affecting aroids.

Elevation of Clavibacter michiganensis subsp. californiensis to species level as Clavibacter californiensis sp. nov., merging and re-classification of Clavibacter michiganensis subsp. chilensis and Clavibacter michiganensis subsp. phaseoli as Clavibacter phaseoli sp. nov. based on complete genome in silico analyses.

The Gram-positive genus Clavibacter is currently divided into seven species (Clavibacter michiganensis, Clavibacter nebraskensis, Clavibacter capsici, Clavibacter sepedonicus, Clavibacter tessellarius, Clavibacter insidiosus and Clavibacter zhangzhiyongii) and three subspecies (C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA-DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1 028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (= CECT 8144T= LMG 27667T) as the type strain.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri.

AIM: The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. METHODS AND RESULTS: A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10 fg genomic DNA and 1 CFU ml-1 bacterial culture. No change in the detection threshold (1 CFU ml-1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. CONCLUSION: The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

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).

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 Loop-Mediated Isothermal Amplification Assay for the Detection of Dickeya spp.

Dickeya and Pectobacterium spp. are responsible for soft-rotting diseases of several plant species, some with overlapping host range. On potato, symptoms caused by these pathogens cannot be clearly differentiated. Disease results in the downgrading and rejection of potato seed, thus requiring additional phytosanitary restrictions across Northern Europe and other parts of the world. In an effort to provide a more timely and accurate diagnostic to distinguish these two groups of pathogens, a method for detecting Dickeya spp. using loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay can be used to test crude extracts prepared directly from symptomatic lesions. The entire test can be completed in less than 30 min, making it faster than the current diagnostic standard, the pelADE conventional polymerase chain reaction. Additionally, the LAMP assay was able to detect Dickeya DNA in samples spiked with varying amounts of Pectobacterium DNA, thus demonstrating the highly specific and sensitive nature of the assay, which can be applied on survey samples with mixed soft-rotting bacterial populations.

Detection of Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis in Maize by Loop-Mediated Amplification.

The Goss's wilt pathogen, Clavibacter michiganensis subsp. nebraskensis, can cause considerable losses in maize (Zea mays) production. Diagnosis of Goss's wilt currently is based on symptomology and identification of C. michiganensis subsp. nebraskensis, following isolation on a semiselective medium and/or serological testing. In an effort to provide a more efficient identification method, a loop-mediated amplification (LAMP) assay was developed to detect the tripartite ATP-independent periplasmic (TRAP)-type C4-dicarboxylate transport system large permease component and tested using strains of C. michiganensis subsp. nebraskensis, all other C. michiganensis subspecies and several genera of nontarget bacteria. Only strains of C. michiganensis subsp. nebraskensis reacted positively with the LAMP assay. The LAMP assay was then used to identify bacterial isolates from diseased maize. 16S rDNA and dnaA sequence analyses were used to confirm the identity of the maize isolates and validate assay specificity. The Cmm ImmunoStrip assay was included as a presumptive identification test of C. michiganensis subsp. nebraskensis at the species level. The Cmn-LAMP assay was further tested using symptomatic leaf tissue. The Cmn-LAMP assay was run in a hand-held real-time monitoring device (SMART-DART) and performed equally to in-lab quantitative polymerase chain reaction equipment. The Cmn-LAMP assay accurately identified C. michiganensis subsp. nebraskensis and has potential as a field test. The targeted sequence also has potential application in other molecular detection platforms.

Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis.

The genus Clavibacter contains one recognized species, Clavibacter michiganensis. Clavibacter michiganensis is subdivided into subspecies based on host specificity and bacteriological characteristics, with Clavibacter michiganensis subsp. michiganensis causing bacterial canker of tomato. Clavibacter michiganensis subsp. michiganensis is often spread through contaminated seed leading to outbreaks of bacterial canker in tomato production areas worldwide. The frequent occurrence of non-pathogenic Clavibacter michiganensis subsp. michiganensis-like bacteria (CMB) is a concern for seed producers because Clavibacter michiganensis subsp. michiganensis is a quarantine organism and detection of a non-pathogenic variant may result in destruction of an otherwise healthy seed lot. A thorough biological and genetic characterization of these seed-associated CMB strains was performed using standard biochemical tests, cell wall analyses, metabolic profiling using Biolog, and single-gene and multilocus sequence analyses. Combined, these tests revealed two distinct populations of seed-associated members of the genus Clavibacter that differed from each other, as well as from all other described subspecies of Clavibacter michiganensis. DNA-DNA hybridization values are 70 % or higher, justifying placement into the single recognized species, C. michiganensis, but other analyses justify separate subspecies designations. Additionally, strains belonging to the genus Clavibacter isolated from pepper also represent a distinct population and warrant separate subspecies designation. On the basis of these data we propose subspecies designations for separate non-pathogenic subpopulations of Clavibacter michiganensis: Clavibacter michiganensis subsp. californiensis subsp. nov. and Clavibacter michiganensis subsp. chilensis subsp. nov. for seed-associated strains represented by C55(T) ( = ATCC BAA-2691(T) = CFBP 8216(T)) and ZUM3936(T) ( = ATCC BAA-2690(T) = CFBP 8217(T)), respectively. Recognition of separate subspecies is essential for improved international seed testing operations.

Three new species, Xanthomonas hawaiiensis sp. nov., Stenotrophomonas aracearum sp. nov., and Stenotrophomonas oahuensis sp. nov., isolated from the Araceae family.

Xanthomonas and Stenotrophomonas are closely related genera in the family Lysobacteraceae. In our previous study of aroid-associated bacterial strains, most strains isolated from anthurium and other aroids were reclassified as X. phaseoli and other Xanthomonas species. However, two strains isolated from Spathiphyllum and Colocasia were phylogenetically distant from other strains in the Xanthomonas clade and two strains isolated from Anthurium clustered within the Stenotrophomonas clade. Phylogenetic trees based on 16S rRNA and nine housekeeping genes placed the former strains with the type strain of X. sacchari from sugarcane and the latter strains with the type strain of S. bentonitica from bentonite. In pairwise comparisons with type strains, the overall genomic relatedness indices required delineation of new species; digital DNA-DNA hybridization and average nucleotide identity values were lower than 70 and 95%, respectively. Hence, three new species are proposed: S. aracearum sp. nov. and S. oahuensis sp. nov. for two strains from anthurium and X. hawaiiensis sp. nov. for the strains from spathiphyllum and colocasia, respectively. The genome size of X. hawaiiensis sp. nov. is ~4.88 Mbp and higher than S. aracearum sp. nov. (4.33 Mbp) and S. oahuensis sp. nov. (4.68 Mbp). Gene content analysis revealed 425 and 576 core genes present in 40 xanthomonads and 25 stenotrophomonads, respectively. The average number of unique genes in Stenotrophomonas spp. was higher than in Xanthomonas spp., implying higher genetic diversity in Stenotrophomonas.

Phylogeny and classification of Dickeya based on multilocus sequence analysis.

Bacterial heart rot of pineapple reported in Hawaii in 2003 and reoccurring in 2006 was caused by an undetermined species of Dickeya. Classification of the bacterial strains isolated from infected pineapple to one of the recognized Dickeya species and their phylogenetic relationships with Dickeya were determined by a multilocus sequence analysis (MLSA), based on the partial gene sequences of dnaA, dnaJ, dnaX, gyrB and recN. Individual and concatenated gene phylogenies revealed that the strains form a clade with reference Dickeya sp. isolated from pineapple in Malaysia and are closely related to D. zeae; however, previous DNA-DNA reassociation values suggest that these strains do not meet the genomic threshold for consideration in D. zeae, and require further taxonomic analysis. An analysis of the markers used in this MLSA determined that recN was the best overall marker for resolution of species within Dickeya. Differential intraspecies resolution was observed with the other markers, suggesting that marker selection is important for defining relationships within a clade. Phylogenies produced with gene sequences from the sequenced genomes of strains D. dadantii Ech586, D. dadantii Ech703 and D. zeae Ech1591 did not place the sequenced strains with members of other well-characterized members of their respective species. The average nucleotide identity (ANI) and tetranucleotide frequencies determined for the sequenced strains corroborated the results of the MLSA that D. dadantii Ech586 and D. dadantii Ech703 should be reclassified as Dickeya zeae Ech586 and Dickeya paradisiaca Ech703, respectively, whereas D. zeae Ech1591 should be reclassified as Dickeya chrysanthemi Ech1591.

Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific.

Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

Exploring taxonomic and functional microbiome of Hawaiian stream and spring irrigation water systems using Illumina and Oxford Nanopore sequencing platforms.

Irrigation water is a common source of contamination that carries plant and foodborne human pathogens and provides a niche for proliferation and survival of microbes in agricultural settings. Bacterial communities and their functions in irrigation water were investigated by analyzing samples from wetland taro farms on Oahu, Hawaii using different DNA sequencing platforms. Irrigation water samples (stream, spring, and storage tank water) were collected from North, East, and West sides of Oahu and subjected to high quality DNA isolation, library preparation and sequencing of the V3-V4 region, full length 16S rRNA, and shotgun metagenome sequencing using Illumina iSeq100, Oxford Nanopore MinION and Illumina NovaSeq, respectively. Illumina reads provided the most comprehensive taxonomic classification at the phylum level where Proteobacteria was identified as the most abundant phylum in the stream source and associated water samples from wetland taro fields. Cyanobacteria was also a dominant phylum in samples from tank and spring water, whereas Bacteroidetes were most abundant in wetland taro fields irrigated with spring water. However, over 50% of the valid short amplicon reads remained unclassified and inconclusive at the species level. In contrast, Oxford Nanopore MinION was a better choice for microbe classification at the genus and species levels as indicated by samples sequenced for full length 16S rRNA. No reliable taxonomic classification results were obtained while using shotgun metagenome data. In functional analyzes, only 12% of the genes were shared by two consortia and 95 antibiotic resistant genes (ARGs) were detected with variable relative abundance. Full descriptions of microbial communities and their functions are essential for the development of better water management strategies aimed to produce safer fresh produce and to protect plant, animal, human and environmental health. Quantitative comparisons illustrated the importance of selecting the appropriate analytical method depending on the level of taxonomic delineation sought in each microbiome.

Characterization of biofumigated Ralstonia solanacearum cells using micro-Raman spectroscopy and electron microscopy.

Essential oils of palmarosa, lemongrass, and eucalyptus have shown promise as biofumigants for control of the bacterial wilt disease of edible ginger (Zingiber officinale) caused by Ralstonia solanacearum race 4 in previous potting medium studies. Biochemical changes in R. solanacearum cells were evaluated with micro-Raman spectroscopy following treatment with essential oils at different concentrations (0.04, 0.07, and 0.14% [vol/vol] of culture medium) and changes in cell structure were observed using electron microscopy. All treatments except palmarosa oil at 0.04% caused significant reductions in levels of amino acids, purine and pyrimidine bases of nucleic acids, carbohydrates, and lipids, as indicated by significant reduction in Raman peak heights at 621, 1,003, and 1,031 inverse centimeters (cm(-1)) (phenylalanine); 643, 827, 852, 1,158, and 1,172 cm(-1) (tyrosine); 758 cm(-1) (tryptophan); 725, 782, 1,337, and 1,578 cm(-1) (adenine, cytosine plus uracil, adenine, and adenine plus guanine, respectively); 1,097 cm(-1) (carbohydrates); and 1,127, 1,450, and 2,932 cm(-1) (lipids) compared with untreated controls. Lemongrass oil treatments were the most effective in degrading cellular components. Scanning electron microscopy of palmarosa and lemongrass-oil-treated cells showed rupture of cell walls and cell debris but no degradation was noted for eucalyptus-oil-treated cells. Palmarosa- and lemongrass-oil-treated cells were positively stained with uranyl acetate when viewed by transmission electron microscopy whereas controls and eucalyptus-oil-treated cells were negatively stained, indicating that the cell membranes were intact. The viability of eucalyptus-oil-treated cells was confirmed by cell culture following treatment. Micro-Raman spectroscopy is a powerful tool which can be further employed to better understand effects of fumigants and other bactericides on bacterial cells.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis.

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.

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.

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.

Effect of Plant Essential Oils on Ralstonia solanacearum Race 4 and Bacterial Wilt of Edible Ginger.

Palmarosa (Cymbopogon martini), lemongrass (C. citratus), and eucalyptus (Eucalyptus globulus) oils were investigated for their effects on Ralstonia solanacearum race 4 and their potential use as biofumigants for reducing bacterial wilt disease of edible ginger (Zingiber officinale). Three concentrations of the oils (0.04, 0.07, and 0.14% vol/vol) were evaluated by culture amendment assays, epifluorescence microscopy, and studies in potting medium. In culture amendment assays with palmarosa and lemongrass oils at 0.04%, both oils significantly reduced the growth of the bacterium compared with the control, and at 0.07 and 0.14% they showed complete inhibition of bacterial growth. Epifluorescence microscopic observations showed cell deterioration in 95 to 100% of the cells at all concentrations of palmarosa and lemongrass oils, indicating its bactericidal properties. Eucalyptus oil treatments at 0.04 and 0.07% had bacteriostatic effects on the cells. The pathogen was not detected in R. solanacearum-infested potting medium after treatment with palmarosa and lemongrass oils at 0.07 and 0.14% in any of the experiments. Bacterial wilt incidence on edible ginger was significantly reduced when planted in essential oil-treated potting medium. None of the essential oil treatments reduced the growth or yield of edible ginger grown for 180 days in 5-liter pots.

Taxonomy and Phylogenetic Research on Ralstonia solanacearum Species Complex: A Complex Pathogen with Extraordinary Economic Consequences.

The bacterial wilt pathogen, first known as Bacillus solanacearum, has undergone numerous taxonomic changes since its first description in 1896. The history and significance of this pathogen is covered in this review with an emphasis on the advances in technology that were used to support each reclassification that finally led to the current separation of Ralstonia solanacearum into three genomic species. Frequent name changes occurred as methodology transitioned from phenotypic, biochemical, and molecular studies, to genomics and functional genomics. The diversity, wide host range, and geographical distribution of the bacterial wilt pathogen resulted in its division into three species as genomic analyses elucidated phylogenetic relationships among strains. Current advances in phylogenetics and functional genomics now open new avenues for research into epidemiology and control of the devastating bacterial wilt disease.

Phylogenetic Analyses of Xanthomonads Causing Bacterial Leaf Spot of Tomato and Pepper: Xanthomonas euvesicatoria Revealed Homologous Populations Despite Distant Geographical Distribution.

Bacterial leaf spot of tomato and pepper (BLS), an economically important bacterial disease caused by four species of Xanthomonas (X. euvesicatoria (Xe), X. vesicatoria (Xv), X. gardneri (Xg), and X. perforans (Xp)), is a global problem and can cause over 50% crop loss under unfavorable conditions. Among the four species, Xe and Xv are prevalent worldwide. Characterization of the pathogens is crucial for disease management and regulatory purposes. In this study, we performed a multilocus sequence analysis (MLSA) with six genes (hrcN, dnaA gyrB, gapA, pdg, and hmbs) on BLS strains. Other Xanthomonas species were included to determine phylogenetic relationships within and among the tested strains. Four BLS species comprising 76 strains from different serological groups and diverse geographical locations were resolved into three major clades. BLS xanthomonads formed distinct clusters in the phylogenetic analyses. Three other xanthomonads, including X. albilineans, X. sacchari, and X. translucens pv. undolusa revealed less than 85%, 88%, and 89% average nucleotide identity (ANI), respectively, with the other species of Xanthomonas included in this study. Both antibody and MLSA data showed that Xv was clearly separated from Xe and that the latter strains were remarkably clonal, even though they originated from distant geographical locations. The Xe strains formed two separate phylogenetic groups; Xe group A1 consisted only of tomato strains, whereas Xe group A2 included strains from pepper and tomato. In contrast, the Xv group showed greater heterogeneity. Some Xv strains from South America were closely related to strains from California, while others grouped closer to a strain from Indiana and more distantly to a strain from Hawaii. Using this information molecular tests can now be devised to track distribution of clonal populations that may be introduced into new geographic areas through seeds and other infected plant materials.