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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.

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.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae), causing 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 10-fold 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.

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.

Bacterial wilt strains in the Ralstonia solanacearum species complex (RSSC) pose serious threats to economically important crops worldwide. In 2014, Safni and co-workers proposed the reclassification of RSSC into three genomospecies: R. solanacearum (Rsol), R. pseudosolanacearum (Rpseu), and R. syzygii (Rsyz). The revision requires the proper identification of strains for diagnostic and epidemiological studies. In response, we developed an inexpensive and user-friendly “RSSC-Lineage Multiplex PCR” that effectively detects plant pathogenic Ralstonia strains in general and also distinguishes between Rpseu, Rsol, Rsyz, and the high-security Select Agent “race 3 biovar 2” subgroup of Rsol, also known as the phylotype IIB-1 potato brown rot pandemic lineage. Genomes were retrieved from the NCBI GenBank database and screened for unique gene regions using OrthoMCL and other comparative genomic approaches. Specific primers were designed for each genomospecies, Ralstonia in general and for “race 3 biovar 2”. AT-rich flaps were added at 5’ position of each primer to optimize the reaction thermodynamics. The specificity was tested in silico using the NCBI GenBank genome database and an in-house database. In vitro specificity and accuracy of the tool was validated with 110 representative Ralstonia strains and 24 strains from other genera. The assay is highly specific, generating neither false positives nor false negatives. Primer set detection limits ranged from 10- to 100-pg. The assay also detected and differentiated strains from naturally and artificially inoculated plant hosts. This tool is highly specific, reliable, and economical for culture characterization, diagnostics, surveys, quarantine decisions, and epidemiological studies.

Pectobacterium, agenus comprising gram-negative, pectinolytic phytopathogens, is responsible for economic losses in a wide host range of plants. In this study, the bacterial strains PL152T and PL155 were isolated from taro corms in Hawai’i in 2018, and characterized using genomic and biochemical assays. The Next Generation Sequencing technologies, Oxford Nanopore MinION and Illumina NovaSeq, were used for whole genome sequencing of the PL152T strain. Short and long reads were assembled using the Unicycler tool accessible at the bioinformatic resource center, and PATRIC (PathoSystems Resource Integration Center) was used to generate a more accurate and reliable “hybrid” assembly. The 16S rRNA analysis of PL152T with type strains of other known Pectobacterium species showed a close relationship with P. fontis. Multi-locus sequence analysis using nine housekeeping genes (dnaA, gapA, gyrB, recA, dnaN, rpoS, mdh, rpoA and dnaK) differentiated strain PL152T from other species of Pectobacterium and formed a unique and well-defined clade. The concurrent results of average nucleotide identity (ANI) and digital DNA-DNA hybridization, with calculated values lower than 95 and 70%, respectively, supported the delineation of a novel bacterial species. Here, we proposed Pectobacterium colocasium, strain PL152T (=ICMP 24362T; LMG 32536 T) and PL155 as a novel species in the genus Pectobacterium.1.5RepositoriesCP091064; MZ542535 - MZ542540; OM457660

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.

The Gram-positive Clavibacter genus is currently divided into seven species (C. michiganensis, C. nebraskensis, C. capsici, C. sepedonicus, C. tessellarius, C. insidiosus and C. 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 taxonomy 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, 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 (dDDH), multi-locus sequence analysis (MLSA) of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), phylogenomic tree reconstructed from 1,028 core genes, and ANI-based phylogeny pinpointed conclusive evidence to raise C. michiganensis subsp. californiensis to the species status. These results led us to propose the establishment of C. 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 aforementioned bioinformatic strategies, revealed a high degree of homology between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these outcomes, we proposed to combine both subspecies into a single taxon and elevate its rank to the species level as C. chilensis sp. nov., with ZUM3936ᵀ (= ATCC BAA-2690ᵀ = CFBP 8217ᵀ) as the type strain.

Bacterial pathogens identified as Dickeya sp. have recently been associated with a corm rot of wetland taro on Oahu, Hawaii, but the species designation of these strains was unclear. A Gram-negative, pectinolytic bacterial strain PL65T isolated from an infected taro corm was subjected to polyphasic analysis to determine its genomic and phenotypic characteristics. Multi-locus sequence analyses (MLSA) based on five housekeeping genes (dnaA, gapA, gyrB, atpD, and purA) revealed that Dickeya zeae and D. oryzae, were the closest relatives. Phylogenetic analysis based on 463 core gene sequences clearly showed two potentially new species within Dickeya oryzae. In silico DNA– DNA hybridization value of strain PL65T with 12 Type strains of Dickeya species was T was at the margin of the species delineation cut-off values with a 96% ANI value. The metabolic profile of strain PL65T using BIOLOG differentiated it from the type strains of all other known species of Dickeya. Based on the results of genome-to-genome comparisons and phenotypic data presented in this report, we propose establishment of a new species, Dickeya colocasiae sp. nov. with strain PL65T as the type strain (ICMP 24361T).

Ironwood (Casuarina equisetifoliasubsp.equisetifolia) trees on Guam have been in decline since 2002. This study applied proportional odds logistic multiple regression modeling to a set of biological variables in order to find significant decline predictors as a first step towards identifying pathogenic contributors. Based on the analysis of a set of 77 medium and large trees, the bacteriumRalstonia solanacearumand the fungusGanoderma australespecies complex were found to be significant predictors, withpvalues of R. solanacearumbyG. australeinteraction was not a strong effect, indicating additive rather than multiplicative behavior of these effects on decline severity (pvalue>0.087). When nine covariates were applied univariately to a data subset of 30 tree cross-sections, the significance ofR. solanacearumwas strongly upheld while that ofG. australewas moderately reduced. Also significant were percent cross-sectional area with bacterial wetwood and the formation of ooze within 24 h. Wetwood bacteria ofKlebsiellaspp. andK. oxytocawere found across all levels of decline and were not significant predictors. Other enteric bacteria identified includedKosakonia,Enterobacter, Pantoea, Erwinia,andCitrobacter.

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) (Helias 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).

AIMS Clavibacter michiganensis is an important bacterial plant pathogen that causes vast destruction to agriculturally important crops worldwide. Early detection is critical to evaluate disease progression and to implement efficient control measures to avoid serious epidemics. In this study, we developed a sensitive, specific and robust loop-mediated isothermal amplification (LAMP) assay for detection of all known subspecies of C. michiganensis. METHODS AND RESULTS Whole genome comparative genomics approach was taken to identify a unique and conserved region within all known subspecies of C. michiganensis. Primer specificity was evaluated in silico and with 64 bacterial strains included in inclusivity and exclusivity panels; no false positives or false negatives were detected. Both the sensitivity and spiked assay of the developed LAMP assay was 1 fg of the pathogen DNA per reaction. A 100% accuracy was observed when tested with infected plant samples. CONCLUSIONS The developed LAMP assay is simple, sensitive, robust and easy to perform using different detection platforms and chemistries. SIGNIFICANCE AND IMPACT OF THE STUDY The developed LAMP assay can detect all known subspecies of C. michiganensis. The LAMP process can be performed isothermally at 65°C and results can be visually assessed, which makes this technology a promising tool for monitoring the disease progression and for accurate pathogen detection at point-of-care.

Dickeya zeae, a bacterial plant pathogen in 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 soft rot of taro (Colocasia esculenta) and heart rot of pineapple (Ananas comosus). In this study, we used Pacific Biosciences to sequence two high quality complete genomes of novel strains of D. zeae: PL65 (size - 4.74997 MB; depth - 701; GC - 53.3%) and A5410 (size - 4.7792 MB; depth - 558; GC - 53.6%) isolated from economically important Hawaiian crops, taro and pineapple, respectively. Additional complete genomes of D. zeae representing two additional hosts (rice and banana), and other species for taxonomic comparison, were retrieved from the NCBI GenBank genome database. The genomic analyses indicated truncated type III and IV secretion systems (T3SS and T4SS) in the taro strain, which only harbors 1 and 2 genes of T3SS and T4SS, respectively, and showed high heterogeneity in the type VI secretion system. Unlike the EC1 strain, neither the PL65 nor the A5410 genome harbors the zeamine biosynthesis gene cluster, which plays a key role in bacterial virulence. The ANI and dDDH percentages between the two genomes were 94.47 and 57.00, respectively. In this study, we compared major virulence factors (plant cell wall-degrading extracellular enzymes and protease) produced by D. zeae strains and virulence ability on taro corms and pineapple. Both strains produced protease, pectate lyases and cellulases but no significant quantitative differences were observed (p>0.05) among the strains. All the strains produced symptoms on taro corms and pineapple leaves. Strain PL65 developed symptoms faster than the others. Our study highlights genetic constituents of pathogenicity determinants and genomic heterogeneity that will help understand the virulence mechanisms and aggressiveness of this plant pathogen.

Aim Clavibacter michiganensis (Cm) is a seed-borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples. Methods and results A specific region of Cm (clvG gene) was selected for primer design using comparative genomics approach. A fully synthetic universal internal control (UIC) was also designed to detect PCR inhibitors and false negative results in qPCR reactions. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter-specific primers. The assay was specific for Cm and detected upto 10 fg of Cm DNA in sensitivity and spiked assay. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. Conclusion The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. Significance and impact of the study This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titers in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified, and multiplex compatible with any other qPCR assay.

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 R. solanacearum has resulted in its inclusion in a “species complex” as genomic analyses of elucidated phylogenetic relationships among strains. Current advances in phylogenetics and functional genomics now open new avenues for research into the epidemiology and control of the devastating bacterial wilt disease.

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 and 100 fg was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1,000 fg when 1 µL of host sap was added into the RPA reaction containing 10-fold 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 for any plant pathogen.IMPORTANCEClavibacter species are prevalent worldwide as have the potential to result in systemic infection. In the past, detection attempts have relied on both molecular- and immunological-based assays; however, current detection methods are time consuming and laborious. Field-deployable tests are desirable to identify potential samples infected with Clavibacter species. This study demonstrates that the field-deployable isothermal multi-target recombinase polymerase amplification can be performed for the simultaneous detection of the genus Clavibacter in general (all species), and C. nebraskensis, in particular, without specialized equipment. Additionally, the multiplex RPA coupled with a LFD may confer the benefits of faster detection and discrimination of Clavibacter species that affect critical regions susceptible to infection. This user-friendly format offers a flexible assay to complement both nucleic acid amplification and novel diagnosis methods without the need for DNA purification; this assay may serve as a point-of-reference for developing multiplex RPA assay for other plant pathogens.

Potassium tetraborate tetrahydrate, K2B4O7⋅4H2O (PTB), was assessed as a preharvest spray in the greenhouse for reducing gray mold of tomato fruit caused by Botrytis cinerea. Efficacy of PTB was compared with a natural proprietary product formulated by Agrichem, which was previously shown to inhibit mycelial growth of B. cinerea in vitro and was used as a positive control for greenhouse studies. Mycelial growth and spore germination of B. cinerea were completely inhibited in vitro by PTB at 25 mM. Phytotoxicity was not observed on treated tomato plants with PTB at 25 mM. Preharvest sprays were applied at three ripening stages: turning, pink, and light red. After fruit reached the final ripening stage, all fruits were harvested and stored for 10 days at either 4 or 25°C. Disease severity was reduced compared with controls after storage at either temperature when PTB was applied at the turning stage. Preharvest applications of PTB improved the marketable quality of postharvest tomato fruit and significantly reduced the incidence of gray mold in comparison with inoculated control plants, which received no preharvest fungicidal treatment.

Gray mold and soft rot are the most important postharvest diseases of tomato worldwide. A survey of fresh-market tomato fruit was conducted in Oahu to determine which fungal and bacterial pathogens were most commonly associated with postharvest disease. Alternaria, Botrytis, Colletotrichum, Fusarium, Geotrichum, Mucor, Stemphyllium, Rhizopus and Penicillium were the most frequently isolated fungi and Acetobacter, Gluconobacter, Klebsiella, Leuconostoc and Pectobacterium were the prevalent bacteria. Fifty-one percent of the diseased tomatoes had been imported from California and Mexico and 49% had been grown locally at three sites in Oahu. Pathogenicity tests revealed that 33 of 99 fungal isolates and 10 of 17 bacterial isolates were pathogenic on tomato types known as common market, cherry and grape tomato. Based on fruit assays, Botrytis cinerea (B03) and Pectobacterium carotovorum (BA17) were the most virulent isolates. Tested leaf extracts of Capsicum annuum cv. Stocky Red, C. annuum cv. Criolla de cocina, Capsicum chinense cv. NuMexsuave, Tagetes tenuifolia, Aloe vera, Origanum vulgare and Azadirachta indica were ineffective as biopesticides and did not reduce spore germination or mycelial growth of B. cinerea (B03) nor P. carotovorum (BA17). In contrast, a proprietary product (PF) reduced mycelial growth of B. cinerea (B03) and was further evaluated at doubling concentrations ranging from 0.0625 to 1 ml/L. Mycelial growth of B. cinerea and other fungi was completely inhibited by exposure to PF at 1 ml/L. On the other hand, PF was not an effective biopesticides against P. carotovorum. PF shows promise for reducing gray mold and will be evaluated as a preharvest spray on tomato plants in the greenhouse. Key words: Survey, postharvest diseases, tomato, natural product, Botrytis cinerea, Pectobacterium carotovorum.

Susceptibility of anthurium cultivars to systemic infection by the bacterial blight pathogen, Xanthomonas campestris pv. dieffenbachiae, was examined using a bioengineered bioluminescent strain (V108LRUH1) and compared with susceptibility to foliar infection. Eight cultivars with different levels of susceptibility to foliar infection were evaluated for their susceptibility to systemic infection. Petioles of second youngest leaves cut near the main stem were inoculated with strain V108LRUH1, and subsequent movement of this bacterium into other petioles was monitored by observing bioluminescence from the plants. The actual extent of systemic movement was determined by reisolating V108LRUH1 from dissected segments of the remaining petioles. In susceptible cultivars, the pathogen advanced very rapidly and nearly reached the distal end of petioles. In resistant cultivars, the pathogen was detected in none (or very few) of the petiole segments. However, the susceptibility ranking among the tested cultivars for systemic infection did not always correspond to the ranking determined for foliar infection: i.e., one cultivar that was susceptible to foliar infection was highly resistant to systemic infection, and vice versa. This suggests that cultivar susceptibility of anthuriums to bacterial blight may differ depending on the phase of disease progression, and thus evaluation for both disease phases is essential for complete understanding of cultivar susceptibility.

Clavibacter is an agriculturally important genus comprising a single species, Clavibacter michiganensis, and multiple subspecies, including, C. michiganensis subsp. nebraskensis which causes Goss’s wilt/blight of corn and accounts for high yield losses - listed among the five most significant diseases of corn in the United States of America. Our research objective was to develop a robust and rapid multiplex TaqMan real-time PCR (qPCR) to detect C. michiganensis in general and C. michiganensis subsp. nebraskensis with enhanced reliability and accuracy by adding non-complementary AT sequences to the 5’ end of the forward and reverse primers. Comparative genomic analyses were performed to identify unique and conserved gene regions for primer and probe design. The unique genomic regions, ABC transporter ATP-binding protein CDS/ABC-transporter permease and MFS transporter were determined for specific detection of C. michiganensis and C. m. subsp. nebraskensis, respectively. The AT-rich sequences at the 5’ position of the primers enhanced the reaction efficiency and sensitivity of rapid qPCR cycling; the reliability, accuracy and high efficiency of the developed assay was confirmed after testing with 59 strains from inclusivity and exclusivity panels – no false positives or false negatives were detected. The assays were also validated through naturally and artificially infected corn plant samples; all samples were detected for C. michiganensis and C. m. subsp. nebraskensis with 100% accuracy. The assay with 5’ AT-rich sequences detected up to 10- and 100-fg of C. michiganensis and C. michiganensis subsp. nebraskensis genome targets, respectively. No adverse effect was observed when sensitivity assays were spiked with host genomic DNA. Addition of 5’ AT rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (with optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis subsp. nebraskensis, respectively; a increase of 10-fold sensitivity was also obtained with C. michiganensis primer set. The methodology proposed here can be used to optimize the reaction efficiency and to harmonize the diagnostic protocols which have prodigious applications in routine diagnostics, biosecurity and microbial forensics.

Edible ginger is a popular spice crop that is grown in Hawaii primarily for the fresh market, and as such, rhizome quality is of paramount importance. In our studies, a Gram-negative, facultative anaerobic, rod-shaped bacterium was consistently isolated from decayed as well as symptomless ginger rhizomes. The bacterium was identified as Enterobacter cloacae by biochemical assays and 16S rDNA sequence analysis. Rot symptoms, which usually occurred in the central cylinder of the rhizome, were characterized by yellowish-brown to brown discolored tissue and firm to spongy texture. In inoculation experiments, ginger strains of E. cloacae produced basal stem and root rot, with foliar chlorosis and necrosis in tissue-cultured ginger plantlets, and discolored and spongy tissue in mature ginger rhizome slices and whole segments. In other hosts, ginger strains of E. cloacae caused internal yellowing of ripe papaya fruit and internal rot of onion bulbs. All strains that caused symptoms in inoculated plants were reisolated and identified as E. cloacae. Our studies suggest that E. cloacae can exist as an endophyte of ginger rhizomes, and under conditions that are favorable for bacterial growth, or host susceptibility, including maturity of tissues, rhizome rot may occur. Rhizome quality may be impacted by the presence of E. cloacae under conditions such as high temperature, high relative humidity, and low oxygen atmosphere that may affect the development of decay, and such conditions should be avoided during post-harvest handling and storage. The association of E. cloacae with a rhizome rot of ginger is a new finding.

The first reported outbreak of bacterial heart rot of pineapple (Ananas comosus var. comosus) in Hawaii occurred in December 2003. Of immediate concern was the differentiation of heart rot caused by Erwinia chrysanthemi from a soft rot caused by E. carotovora subsp. carotovora because of regulatory issues. Presumptive identifications of the isolated bacteria were made using bacteriological tests (including reactivity with an Erwinia-specific monoclonal antibody, E2) and compared with identifications obtained by two general methods: carbon source utilization profiling (Biolog) and 16S rDNA sequence analysis. The panel of bacteriological tests consistently differentiated E. chrysanthemi from E. carotovora subsp. carotovora and other nonquarantine organisms. BOX-polymerase chain reaction fingerprint patterns further differentiated the pineapple-isolated E. chrysanthemi strains from those obtained from other plants and irrigation water. Pineapple leaf inoculations revealed that only E. chrysanthemi from pineapple produced watersoaking and rot similar to that observed on the original symptomatic plants, thus identifying these strains as the causal agents of the outbreak. In this situation, where rapid identification of an unknown pathogen was necessary, standard bacteriological tests then available in the laboratory provided reliable differentiation of E. chrysanthemi from E. carotovora subsp. carotovora. Additional strain characterization is needed before the pineapple-isolated E. chrysanthemi strains can be classified into a species of the new genus Dickeya.

Bacterial heart rot caused by a yet undetermined species of Dickeya was first observed in December 2003 in Oahu, HI, on a pineapple cultivar (Ananas comosus ‘PRI 73-114’) recently imported from Central America. Identical symptoms were later seen in the same plantation in fields that had been planted with propagules from the Philippines. Dickeya strains isolated from symptomatic plants and irrigation water collected over subsequent years were identified using bacteriological tests and partial 16S ribosomal DNA sequencing and characterized using repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer (BOX-PCR), pathogenicity on pineapple leaves, and reactivity with two monoclonal antibodies (MAbs). Strains exhibited variability in pathogenicity and in reactivity with MAbs. BOX-PCR separated the plant-isolated Dickeya strains into seven haplotypes that were placed into four fingerprint groups (A to D). Strains from the A and B groups were isolated from the Central American stocks, whereas strains in the D group were identified from Philippine material. Strains from the C group were isolated from both planting materials. Most strains from water sources were placed into three haplotypes that loosely formed group E. BOX-PCR polymorphisms between the Dickeya strains isolated from foreign pineapple plants, a local collection of Dickeya strains, and strains from the plantation's water sources support the possibility that at least two separate introductions of genetically distinct strains occurred via imported planting stocks.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3′T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States. References: (1) F.-H. Chu et al. Phytopathology 91:361, 2001. (2) M. J. Melzer et al. Virus Genes 40:111, 2010. (3) M. Okuda and K. Hanada. J. Virol. Methods 96:149, 2001.

Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.

Pectobacterium species cause serious bacterial soft rot diseases worldwide on economically important fruit and vegetable crops including tomato and potato. Accurate and simple methods are essential for rapid pathogen identification and timely management of the diseases. Recombinase polymerase amplification (RPA) combined with a lateral flow device (LFD) was developed for specific detection of Pectobacterium sp. directly from infected plant materials with no need for DNA isolation. The specificity of RPA-LFD was tested with 26 Pectobacterium sp. strains and 12 non-Pectobacterium species and no false positive or false negative outcomes were observed. RPA primers and probe for host control were also developed to detect the host genome for enhanced reliability and accuracy of the developed assay. The detection limit of 10 fg was obtained with both sensitivity and spiked sensitivity assays. No inhibitory effects were observed on the RPA assay when targets (pathogen and host) were directly detected from infected potato and tomato sap. The developed RPA assay has numerous applications from routine diagnostics at point-of-care, biosecurity, surveillance and disease management to epidemiological studies. In addition, this tool can also be used to discover reservoir hosts for Pectobacterium species.

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.

A loop-mediated amplification (LAMP) assay developed previously to specifically identify Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato, was compared with a commercial lateral flow immunoassay, three PCR assays, and culturing on agar medium for efficacy in detecting the pathogen in pepper and tomato tissues and tomato seeds. The LAMP assay was as effective in identifying C. m. michiganensis-positive tomato seed and tissue samples as the Cmm ImmunoStrip®. The LAMP assay was performed using a handheld real-time fluorescence-monitoring device (Smart-DARTTM) and the iQTM5 Multicolor Real-Time PCR Detection System to compare both systems. Both devices produced equivalent results, thus demonstrating the potential of this LAMP detection system for field detection. A side-by-side comparison of the C. m. michiganensis-LAMP with the Cmm ImmunoStrip® assay, two conventional PCR assays, using Cmm5/6 and PSA8/R primer sets, and one quantitative PCR assay (primers...

Ironwood (Casuarina equisetifolia subsp. equisetifolia) is a nitrogen-fixing tree of considerable social, economic and environmental importance that commonly occurs in tropical/subtropical zones of Asia, the Pacific, Africa, and Central America. Ironwood decline was first noticed on Guam in 2002 and is now affecting thousands of trees and impacting the ecosystem. In 2012, a survey showed that Ralstonia solanacearum and Klebsiella spp. were associated with wetwood symptoms of declining trees. R. solanacearum strains isolated from diseased ironwood in Guam were similar to R. solanacearum strain GMI1000, having similar BOX-PCR profiles and belonging to phylotype I and biovar 3. Two Klebsiella species (K. variicola and K. oxytoca) were recovered, with K. variicola being the more prevalent species. Pathogenicity tests revealed that R. solanacearum caused wilt in tomato and ironwood seedlings, whereas neither Klebsiella spp. produced symptoms. There were no differences in virulence between Guam R. solanacearum and control strains following inoculation into tomato and ironwood from Hawaii. Additionally, no observable differences in ironwood susceptibility to Ralstonia strains from Guam or Hawaii, were observed, suggesting that the association of Guam R. solanacearum with Guam ironwood is not specific. Co-inoculation studies with both R. solanacearum and Klebsiella variicola and K. oxytoca revealed that Klebsiella sp. did not affect symptoms produced by R. solanacearum alone. In planta studies were feasible only on seedlings and young trees in Hawaii; thus, possible interactions between R. solanacearum and Klebsiealla sp. in adult trees remain to be investigated. A new in-field survey of declining ironwood is needed to better understand the role of Klebsiella and Ralstonia in ironwood tree decline in Guam.

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.

Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), occurs worldwide. This study focuses on three genes, clvA (clavicidin) and two associated genes (clvF and clvG, encoding a putative ABC-type multidrug transport system ATPase and permease component, respectively). Loop-mediated amplification of clvA and PCR of clvA, clvF and clvG showed that these genes were present only in Cmm and not in other Clavibacter species/subspecies, as well as other genera of plant-associated bacteria. Sequences of clvA, clvF and clvG from 48 geographically diverse strains of Cmm were analyzed phylogenetically to determine sequence variation. Maximum parsimony (MP), neighbour-joining (NJ) and maximum likelihood (ML) analyses placed strains into subgroups irrespective of their geographical origins. Multi-locus sequence analysis (MLSA) of these three genes produced the same results. In sum, all three clv genes are unique and conserved in Cmm, further supporting the use of the clvA gene for identification of Cmm, using either a previously designed LAMP assay or PCR with additional primers developed in this study. The genes investigated here are novel targets for specific detection of Cmm and have numerous molecular diagnostic applications.

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.

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.

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.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

Grey mold is the one of most important postharvest disease of tomato fruit worldwide. Treatments with edible, natural products are needed to reduce losses and contribute to food sustainability. Based on the hypothesis that inhibition of Botrytis spore germination will significantly reduce postharvest losses, botanicals were tested for their effects on conidia. Ten Botrytis isolates from rotting tomato fruit collected at seventeen different sites in Hawaii were identified morphologically and confirmed by DNA sequence analysis. Effects of plant extracts on spore germination were assessed at several dilutions. Leaves of candidate biocontrol plants were frozen at -20°C and plant fluids were sterilized by passing through a 0.22 μm millipore. The effect of plant extracts on germination of Botrytis spores (106/ml) in sterile malt extract broth was evaluated in multi-well microplates using preparations ranging from 10 to 40%. Changes in absorbance measured at 6, 12, and 24 hours after inoculation were analyzed with Gen5 software. Capsicum chinense cultivars Datil and C. annuum Carnival completely inhibited fungal germination at all evaluation times. Extracts from Capsicum species were superior to all other extracts tested. Treatments with 40% extracts increased the generation of intercellular reactive oxygen species in the fungal conidia. Plasma membrane damage was shown with fluorescence microscopy when extract-treated conidia were stained with propidium iodide. Loss of integrity in the spore plasma membrane appears to account for the inhibition of Botrytis spore germination. Extracts from the two pepper cultivars, Datil and Carnival, showed promise as pre-harvest sprays in the greenhouse and as edible coatings on tomato fruit postharvest to reduce grey mold.

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.

New rapid diagnostic methods are urgently needed to discriminate the quarantine pathogen Ralstonia solanacearum (Rs) race 3 biovar 2 (R3B2) from other populations of Rs that lack the adaptation to cause bacterial wilt disease in temperate regions. We used an in silico bioinformatic approach to identify several genome sequences potentially specific to R3B2 strains. Primer sets were designed to PCR-amplify sequences in these regions, and four sets were ultimately shown to be >99% accurate for detection of R3B2 strains. On the basis of these results, several primers were designed to enable development of a loop-mediated isothermal amplification assay that was rapid, technologically simple, and essentially 100% accurate for identification of R3B2 when applied to a comprehensive collection of geographically diverse Rs strains. We fortuitously found that a sequence in one of the “R3B2-specific” regions has ~90% identity to a sequence present in strains of the blood disease bacterium (BDB), a member of the Rs species complex that infects banana. Alignments of these sequences allowed design of a second PCR primer set that proved 100% accurate for identification of BDB strains when tested on the 22 BDB strains available to us. These results demonstrate the power of in silico genomic subtraction for rapid identification of population-specific DNA sequences and for the development of simple, reliable detection methods for Rs subpopulations.

Isothermal nucleic acid amplification offers compelling opportunities for simple gene-based diagnostics, although the technologies for monitoring these reactions in real-time are still relatively undeveloped compared to PCR-specific probes. In this article, we describe the adaptation of an innovative FRET-based probe architecture for monitoring the isothermal LAMP reaction in real-time, allowing single-step, direct identification of different isolates of the agricultural pathogen Ralstonia solanacearum. The probe design is analogous to a two-component molecular zipper used for monitoring rolling circle amplification (RCA), in which a quenching oligonucleotide strand is displaced from a fluorescent strand by a strand-displacing polymerase during the reaction. Because the fluorescent strand is stably assimilated into the LAMP amplicon in our configuration, we use the nomenclature assimilating probe to describe the probe. Using these probes, we have demonstrated the ability to perform real-time, quantitative LAMP analysis of DNA, detecting fewer than ten gene copies. Probe compositions were optimized to prevent noticeable inhibition of the LAMP reaction, allowing observation of kinetics for different LAMP reactions and inference of primer design rules for accelerated detection. The sequence-specific nature of the assimilating probes offers compelling advantages over non-specific methods for real-time detection of LAMP amplification, including improved selectivity and the potential for multiplexed detection.

We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 µL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics.

Ralstonia solanacearum (Rs) race 4 strains cause bacterial wilt of edible ginger (Zingiber officinale). The survival of the pathogen was studied in plant-free soil and potting medium in the presence of plants inoculated by different methods (non-wounded, rhizome-wounded, and stem-wounded) and irrigated on different schedules (alternate and daily). Detection thresholds for Rs were determined for an enzyme-linked immunoabsorbent assay (ELISA), immunostrip assay, and polymerase chain reaction (PCR) using drainage water from soil and potting medium containing known concentrations of Rs. In the absence of a plant or in the presence of non-wounded plants, Rs populations declined rapidly in drainage water from potting medium during the first 9 days and were undetectable after 81 days. When plants were stem- or rhizome-wounded, Rs populations increased by two to three orders of magnitude from the initial population levels for the first 9 to 19 days and then gradually declined and became undetectable after 89 days. Results were similar in experiments with soil except for non-wounded ginger plants, where the initial decline in Rs populations was followed by an abrupt increase after day 11, reaching 7 log cfu/mL on day 21, then declining gradually to non-detectable levels after 137 days. The increase was attributed to natural infection of the plants followed by release of high populations of Rs into the irrigation water when plants wilted. When rhizome-inoculated plants were watered on alternate days, Rs was recovered from 97 to 129 days in soil and potting medium, but when the plants were watered daily, Rs was recovered in soil and potting medium up to 153 days after plant inoculation. ELISA using Ps1a monoclonal antibody detected the pathogen from >95% of the samples from soil and potting medium when viable populations were >5 log cfu/mL. The immunostrip assay (using the same antibody) detected the pathogen from 100% of the samples when viable populations were >3 log cfu/mL. PCR based on the flagellin gene fliC detected the pathogen from >95% of the samples from soil and from >74% of the samples from potting medium when viable populations were >4 log cfu/mL.

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.

Raman spectra of Gram-positive and Gram-negative plant bacteria have been measured with micro-Raman spectrometers equipped with 785 and 514.5 nm lasers. The Gram-positive bacteria Microbacterium testaceum, Paenibacillus validus, and Clavibacter michiganensis subsp. michiganensis have strong carotenoid bands in the regions 1155–1157 cm−1 and 1516–1522 cm−1 that differentiate them from other tested Gram-negative bacteria. In the Raman spectrum of Gram-positive bacteria Bacillus megaterium excited with 785 nm laser, the Raman bands at 1157 and 1521 cm−1 are weak in intensity compared to other Gram-positive bacteria, and these bands did not show significant resonance Raman enhancement in the spectrum recorded with 514.5 nm laser excitation. The Gram-positive bacteria could be separated from each other based on the bands associated with the in-phase C=C (ν1) vibrations of the polyene chain of carotenoids. None of the Gram-negative bacteria tested had carotenoid bands. The bacteria in the genus Xanthomonas have a carotenoid-like pigment, xanthomonadin, identified in Xanthomonas axonopodis pv. dieffenbachiae, and it is a unique Raman marker for the bacteria. The representative bands for xanthomonadin were the C–C stretching (ν2) vibrations of the polyene chain at 1135–1136 cm−1 and the in-phase C=C (ν1) vibrations of the polyene chain at 1529–1531 cm−1, which were distinct from the carotenoid bands of other tested bacteria. The tyrosine peak in the region 1170–1175 cm−1 was the only other marker present in Gram-negative bacteria that was absent in all tested Gram-positives. A strong-intensity exopolysaccharide-associated marker at 1551 cm−1 is a distinguishable feature of Enterobacter cloacae. The Gram-negative Agrobacterium rhizogenes and Ralstonia solanacearum were differentiated from each other and other tested bacteria on the basis of presence or absence and relative intensities of peaks. The principal components analysis (PCA) of the spectra excited with 785 nm laser differentiated the various strains of bacteria based on the unique pigments these bacteria do or do not possess. Raman spectroscopy of diverse plant bacteria that are pathogenic and non-pathogenic to plants, and isolated from plants and soil, indicates the possibilities of using the method in understanding plant–bacterial interactions at the cellular level.