DETECTION, PHYLOGENY AND COMPARATIVE GENOMICS OF IMPORTANT BACTERIAL SPECIES OF TROPICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014144
• Project Start Date: Oct 11, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
Plant & Environmental Protection Sciences

Non Technical Summary
Plant pathogenic bacteria cause numerous diseases to crops (e.g. tomato, potato) and ornamentals (e.g. anthurium) of economic importance. Hawaii relies about 80% on imported foods, imported seeds and propagative stock of plants which have unintentionally introduced invasive pathogenic and new bacterial species and strain(s). New population(s) have emerged due to introduction of new strain(s) in the environment (results of import) and/or mutation in the previously existing populations which can change host specificity. Hence, the understanding of the current population structure is important; it provides the evidence about the sources and distribution of a pathogen. Also, effective and sensitive detection methods (lab and field-deployable) are required to detect latent infections with higher sensitivity and reliability; help to identify the disease(s) and the causal organism(s). Diverse pathogenic populations can vary widely with respect to virulence; interactions of pathogenic strains (diverse virulence) with their hosts have significance in disease management. Comparative genomics especially next generation sequencing-based would be significant tool for exploring the host specificity of bacterial pathogens and to identify generalist and specialist isolates/pathovars/species among important bacterial genera in tropics.Therefore, the objectives of this project are to use current and advanced techniques to develop effective diagnostic tools and map out the current population structure of important bacterial phytopathogens in the tropics with the ultimate goal to understand host-bacterial interactions and to facilitate the development of effective and integrated disease management strategies.

Animal Health Component

70%

Research Effort Categories

Basic

30%

Applied

70%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	4010	1100	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

phytobacteriology

genomics

phylogenetics

diagnostics

dickeya

clavibacter

xanthomonas

Goals / Objectives
The aim of this Hatch proposal is to understand population biology of important bacterial species of tropics and their interactions with the host to support effective disease management and mitigation procedures. The specific objectives are:Development of novel diagnostic methods for detection and quantification of important bacterial pathogens. Also, evaluation and improvement of existing diagnostic methods.Determination of population structure of important bacterial species of the tropics.Comparative genomics analyses of important bacterial species using next generation sequencing to better understand the biology of the pathogen, epidemiology and host-bacterial interactions.

Project Methods
Objective 1: Development of novel diagnostic methods for detection and quantification of important bacterial pathogens. Also, evaluation and improvement in existing diagnostic methods.Accurate detection and identification of the causal agent is one of the most important part for an effective disease management. In this objective, we will be developing accurate, robust and sensitive detection methods for key bacterial pathogens of tropics. Genome-informed sequences will be used to design robust primers and probes. Endpoint-PCR and qPCR protocols will be developed for lab based identification; we will be using methodology described by Arif et al (2013a,b) and Ouyang et al (2013). Also, LAMP and RPA methods will be developed for on-filed detection (Arif et al, 2016a,b). Initially, we will be developing and evaluating tools for Clavibacter michiganensis and Xanthomonas. Detection tools for other bacterial species will also be developed based on their needs.Brief description: genome sequencing - pacbio sequencing method will be used for whole genome sequencing; target gene selection - genomes will be aligned using mauve/BRIGS and conserved/variable regions will be selected, selected target gene sequences will further be aligned against the NCBI GenBank microbial nucleotide database with closely related species/genus; primers and probes design - thermodynamically competent primers and probes specific for the target species will be designed and validated in silico using BLASTn tool of NCBI GenBank; in vitro validation for specificity - members of exclusivity and inclusivity panels will be selected from the near-neighbor species/genus and representative isolates of the target species; validation with infected plant samples - known infected samples will be tested with developed primers/probes; confirmation of detection limit by performing the sensitivity assays - ten-fold serial dilution will be used; confirmation of non- inhibitory effect by performing the spiked sensitivity assays - ten-fold serial dilution with host DNA will be used; and the final validation includes the multiple operators validation for robustness. The internal and artificial positive controls will be developed whenever required.Objective 2: Determination of population structure of important bacterial species of the tropics.The understanding of population structure is important for integrated diseases management strategies. The bacterial cultures (>5,000) of numerous bacterial species belong to different genus are available in our lab; these bacterial cultures represent strains from different geographical regions (national and international) and timespan (about four decades). To understand the population structure, representative isolates from the culture collection will be included in the study with new strains. The pure culture of new bacterial strains will be generated from infected plant samples; single colony will be picked for pure culture. The DNA will be isolated from pure cultured bacterial strains followed by assessment of genomic DNA quality and quantity using spectrophotometer and/or agarose gel electrophoresis. Different methods including MLST (Arif et al, 2016) and AFLP (Pruvost et al, 2014) will be used to determine the population structure of important bacterial species of tropics. Initially, we will study the species complex of X. axonopodis pv. dieffenbachiae. Other bacterial species will be included as needed.Brief descriptions:MLST- Primers design for conserve and/or genes involve in different cellular functions - gene regions will be selected based on the variable regions or SNPs; amplification of target gene regions from all isolates - the selected gene regions from all the isolates included in the study will be amplified using target gene specific primers; purification of amplified DNA fragments followed by sequencing - both sense and anti-sense strands will be sequenced; manual sequence formatting for accuracy followed by alignment - geneious will be used for manual editing and alignement; phylogenetic analyses - both geneious and MEGA-7 software will be used for phylogenetic tree analyses.AFLP- Restriction digestion of genomic DNA using rare and frequent cutter restriction enzymes (EcoRI and MseI) - high quality genomic DNA will be isolated and combination of restriction enzymes will be used to restrict the genomic DNA; pre-amplification followed by selective amplification - fragmented DNA will be used as template for pre-amplification and diluted pre-amplified product will be used as template for selective amplification; preparation of samples for fragment analysis - includes dilution of selectively amplified product and addition of size standard, ROX; samples for fragment analysis - ABI capillary genetic analyzer will be used. Genemapper to convert the data into binary form data will be converted into the binary data for analysis and NTSys-PC software will be used to generate the phylogenetic tree.Objective 3: Comparative genomics analyses of important bacterial species using next generation sequencing to better understand the biology of the pathogen, epidemiology and host-bacterial interactions.The next generation sequencing-based methods provides a snapshot of constituent and/or combinations of genes and gene clusters to understand the biology and behavior of the isolate. In this objective, we will be using the PacBio (Pacific Biosciences of California, Inc.) and Illumina MiSeq (Illumina Inc.) based sequencing methods. PacBio is helpful to get the genome assemble faster and easier since its provide the longer reads (Arif et al, 2015b, 2016d). Illumina MiSeq provides the shorter reads which leads to get more contigs, but good for target selection or phylogenomics, if complete genome assembly is not required to fulfil the objectives of the study (Arif et al, 2015c). Initially, species in the genera Clavibacter, Xanthomonas and Dickeya will be included for comparative genomic analyses.Brief description: Isolate selection based on pathogenicity experiments - the isolate for whole genome sequencing will be selected based on pathogenicity test, phylogenetic groups and other characteristics; isolation of highly purified and intact genomic DNA from the targeted bacterial isolates - highly purified genomic DNA (about 2 µg) will be isolated and quantified; library preparation and sequencing run (Arif et al, 2016d) - DNA library for sequencing will be either prepared in the lab or direct DNA samples will be sent to the service provider. Analyses/bioinformatics part includes: Quality control, assembly, annotation, alignments and comparisons. We will be using different software and analyses pipelines including Geneious, BBDuk Trimmer, Cap3, Contig Sorter, Bowtie, Velvet, Glimmer Gene Prediction, Rapid Annotation using Subsystem Technology (RAST), NCBI Prokaryotic Genome Annotation Pipeline, Bacterial Annotation System (BASys), Integrated Microbial Genomes & Microbiomes (IMG/M) and MUMmer.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:1. Formal class and lab instructions 2. Stakeholders and seed certification agencies 3. Extension agents and farmers 4. Researchers and scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and researchers were trained for bacteriological and molecular techniques. They have also developed skills for bioinformatics analyses. Students and researcher attended and presented their research outcomes at APS annual meeting 2020. They interacted with scientists and other researchers. How have the results been disseminated to communities of interest?Through peer-reviewed publications, presentations in annual meeting, discussion with farmers and extension agents, dissemination through our lab website. What do you plan to do during the next reporting period to accomplish the goals?1. Genome sequencing of Pectobacterium and Diceya species strains to study evolutionary relationships 2. Transcriptomic analyses to understand the host adaptation 3. Diagnostic development for Xanthomonas oryzae 4. Preparing manuscripts for perr-reviewed journals 5. presentations in scientific meetings

Impacts
What was accomplished under these goals? Objective 1. Many diagnostics protocols have been developed including (A) RPA for Ralstonia solanaceaarum r3bv2, (B) RPA for Dickeya sp. (3) LAMP and qPCR multiplex assay for Pectobacterium parmentieri. These protocols were developed for federal quarantine labs and routine diagnostics. Three peer-reviewed publications have been published and 4 presentations at APS annual meeting (please see the list of publications/presentations). Objective 2. During this period, the population genetics work was done on Xanthomonas axonopodis. Different pipelines and software were used to perform evolutionary and phylogenetic relationships. Publications are in preparations for peer-reviewed journals. Objective 3. Many genomes of Pectobacterium, Dickaya, Ralstonia and Clavibacter were sequenced and analyzed for evolutionary and phylogenomic analyses. Pathogenicity determinants were analyzed--many bioinformatic pipelines were installed and setup for big dataset analyses. We have setup our own server for bioinformatics analysis. Many peer-reviewed articles were published during this period and a few are in preparation. Many presentations at APS annual meeting.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Larrea-Sarmiento, A., Stack, J.P., Alvarez, A.M., and Arif, M. 2020. Multiplex recombinase polymerase amplification (RPA) developed using unique genomic region and coupled with a lateral flow device for rapid on-site detection of genus Clavibacter and C. nebraskensis. bioRxiv. doi.org/10.1101/2020.08.22.262824v1.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, J., Arif, M., Shen, H., Hu, J., Sun, D., Pu, X., and Yang, Q. 2020. Genomic divergence between Dickeya zeae strain EC2 isolated from rice and previously identified strains, suggests a different rice foot rot strain. PLoS ONE, 15(10), e0240908. doi.org/10.1371/journal.pone.0240908
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Arif, M., Busot, G.Y., Mann, R., Rodoni, B., and Stack, J.P. 2020. Multi-internal controls enhance reliability and accuracy for PCR and qPCR detection and population-level discrimination of the Select Agent Rathayibacter toxicus. Scientific Reports (submitted)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Andreason, S.A., Arif, M., Brown, J.K., Ochoa-Corona, F., Fletcher, J., and Wayadande, A. 2020. Exploring the use of high resolution melting analysis and helicase dependent amplification for discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species and Trialeurodes vaporariorum, J Econ Entomol. doi.org/10.1093/jee/toaa180
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Boluk, G., Arizala, D., Ocenar, J., Mokwele, J., Silva, J., Dobhal, S., Uyeda, J., Alvarez, A.M., and Arif, M. 2020. First report of Pectobacterium brasiliense causing soft rot on Brassica oleracea var. sabellica L. in Hawaii, United States. Plant Dis. doi.org/10.1094/PDIS-04-20-0701-PDN
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Arizala, D., Dobhal, S., Paudel, S., Boluk, G., Silva, J., Ahmad, A.A., Uyeda, J., Sugano, J., Alvarez, A.M., and Arif, M. 2020. First report of Pectobacterium brasiliense causing bacterial soft rot and blackleg diseases of potato in Hawaii. Plant Dis. doi.org/10.1094/PDIS-02-20-0395-PDN
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yasuhara-Bell, J., Arif, M., Busot, G., Mann, R., Rodoni, B., and Stack, J. 2020. Comparative genomic analysis confirms five genetic populations of the Select Agent, Rathayibacter toxicus. Microorganisms, 8, 366; doi:10.3390/microorganisms8030366
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Boluk, G., Dobhal, S., Crockford, A.B., Melzer, M.J., Alvarez, A.M., and Arif, M. 2020. Genome-informed recombinase polymerase amplification assay coupled with a lateral flow device for in-field detection of Dickeya species. Plant Dis. doi.org/10.1094/PDIS-09-19-1988-RE
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Arizala, D., Dobhal, S., Paudel, S., Seo, H.N., Alvarez, A.M., and Arif, M. 2020. Evolutionary genomics reveals recombination events involved in speciation, host specificity and pathogenicity in the genus Clavibacter (Abstract; APS Annual Meeting, Virtual).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Boluk, G., Arizala, D., Dobhal, S., Alvarez, A.M., and Arif, M. 2020. Comparative Genomics analyses revealed distinct pathogenicity determinants and distinct features between Dickeya zeae strains from taro and pineapple (Abstract; APS Annual Meeting, Virtual).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Dobhal, S., Paudel, S., Stulberg, M.J., Rascoe, J., Nakhla, M.K., Alvarez, A.M., and Arif, M. 2020. A unique region revealed through genome-wide analyses was used to develop an RPA assay for detection of the Select Agent Ralstonia solanacearum R3bv2 (Abstract; APS Annual Meeting, Virtual).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Boluk, G., Dobhal, S., Alvarez, A.M., and Arif, M. 2020. Complete genomic analysis of plant-pathogenic Pectobacterium species found associated with soft rot disease of kale (Abstract; APS Annual Meeting, Virtual).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Dobhal, S., Boluk, G., Arizala, D., Alvarez, A.M., and Arif, M. 2020. Multitrophic interactions of chromosomally labelled Pectobacterium and Dickeya species with their host and analysis of pathogenicity determinants (Abstract; APS Annual Meeting, Virtual).
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Dobhal, S., Arizala, D., Chuang, S.C., Pal, K., Amore, T.D., Alvarez, A.M., and Arif, M. 2020. Comparative genomics analyses of the bacterial blight pathogen of anthurium, Xanthomonas phaseoli pv. dieffenbachiae (Abstract; APS Annual Meeting, Virtual).
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Boluk, G., Dobhal, S., Alvarez, A.M., and Arif, M. 2020. Evolutionary relationships and phylogeny of Dickeya zeae strains based on phenotypic, biochemical and genomic characteristics (Abstract; APS Annual Meeting, Virtual).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Dobhal, S., Santillana, G., Stulberg, M.J., Boluk, G., Rascoe, J., Nakhla, M.K., Alvarez, A.M., and Arif, M. 2020. Multigene based TaqMan qPCR multiplex assay for sensitive and reliable detection of Dickeya solani (Abstract; APS Annual Meeting, Virtual).

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:1. Formal class and lab instructions 2. Stakeholders and seed certification agency 3. Extension agentsand farmers 4. Researchers and scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate students and researchers were trained for bacteriological and molecular methods. They have developed skills for bioinformatics analyses. Students attended the APS annual meeting and presented their research work--theyinteracted with the leading scientists and researchers. How have the results been disseminated to communities of interest?The research findings were disseminated through peer-reviewed publications and presentations. What do you plan to do during the next reporting period to accomplish the goals?1. We will develop diagnostics for X. oryzae, R. solanacearum, Pectobacterium sp. and Dickeya sp. 2. Population genetics studies of Xanthomonasaxonopodis pv. dieffenbachiae andR.solanacearuminfecting aroids and ironwood, respectively. 3. Pathogenicity determinants of Clavibacter michiganensis subsp. michiganensis and their effects on disease expression; Molecular evolutionof all know Clavibacter species; comparative genomics of Xanthomonasaxonopodispv.dieffenbachiae isolated from anthurium; genome-wide evolutionary relationships of D. zeae strains involved in pineapple heart rot outbreaks in Hawaii.

Impacts
What was accomplished under these goals? Objective 1: Many diagnostic protocols were developed and published; these protocols are being used by diagnostic laboratories and federal quarantine labs. The diagnostic protocols developed during this period: LAMP assay for Dickeya dianthicola, and Dickeya solani;RPA assay for genus Dickeya,genus Clavibacter, C. michiganensissubsp. michiganensis; and qPCR assays for genus Dickeya, genus Pectobacterium, P. parmentieri, D. dianthicola, D. solani andD. zeae. Many other protocols are being validated.A new approach "One lab-One Protocol" has been developed to synchronize the qPCR protocols--by using this approach all qPCR protocols work at the same PCR condition, and users have the opportunity to mix and match the target primers. Dr. Arif has presented this work in APS Annual Meeting atCleveland, OH, in August 2019. In this objective, 4 peer-reviewed manuscripts were published, and 6 presentations were presented in the APS annual meeting. Objective 2: During this period, we worked on two pathogens, Dickeya zeae and Xanthomonas euvesicatoria. Both pathogens are economically important for Hawaii, particularly, D. zeae which has caused a tremendous loss to the pineapple industry. In our studies, we are investigating the source of inoculation as well as their evolution. Peer-reviewed publication on X. euvesicatoria has already been published in Microorganisms, however, D. zeae paper is under preparation and will be communicated soon. Two presentations were presented in APS annual meeting at Cleveland, OH. Objective 3. Multiple genomes of Pectobacterium sp., Dickeya sp., Clavibacter sp., and Xanthomonas sp. were sequenced to understand the biology and evolution of these strains. These bacterial pathogens are economically important for agriculture crops in the United States. We have developed our own bioinformatics pipelines for evolutionary and comparative genomics studies. Weinvestigated the host-bacterial interactions with EPS producing and non-producing strains; their genomes were also analyzed. Genome-wide analyses of Pectobacterium species has already been published in Pathogens.Two presentations were presented in APS annual meeting at Cleveland, OH.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dobhal, S., Boluk, G., Babler, B., Stulberg, M.J., Rascoe, J., Nakhla, M., Chapman, T., Crockford, A.B., Melzer, M., Alvarez, A.M., and Arif, M. 2019. Comparative genomics approach for identifying signature regions to develop a robust and highly reliable multiplex TaqMan real-time qPCR assay for sensitive detection of the genus Dickeya and Dickeya dianthicola. Journal of Applied Microbiology, https://doi.org/10.1111/jam.14579
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Arizala, D., and Arif, M. 2019. Comprehensive comparative genomics analyses revealed remarkable heterogeneity in pathogenicity determinants, antimicrobial compounds, and CRISPR-Cas systems of complex phytopathogenic genus Pectobacterium. Pathogens, 8, 247, doi:10.3390/pathogens8040247
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Arizala, D., Dobhal, S., Paudel, S., Gunarathne, S., Boluk, G., Arif, M. 2019. First report of bacterial soft rot and blackleg on potato caused by Pectobacterium parmentieri in Hawaii. Plant Dis. doi.org/10.1094/PDIS-09-19-1894-PDN
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dhakal, U., Dobhal, S., Alvarez, A.M., and Arif, M. 2019. Phylogenetic analyses of xanthomonads causing bacterial leaf spot of tomato and pepper: Xanthomonas euvesicatoria revealed homologous populations despite distant geographical distribution. Microorganisms, 7, 462, doi:10.3390/microorganisms7100462
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Larrea-Sarmiento, A., Alvarez, A.M., Stack, J.P., and Arif, M. 2019. Synergetic effect of non-complementary 5 AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis. PLoS ONE 14(7):e0218530, doi.org/10.1371/journal.pone.0218530
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ocenar, J., Arizala, D., Boluk, G., Dhakal, U., Gunarathne, S., Paudel, S., Dobhal, S., and Arif, M. 2019. Development of a robust, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of potato pathogen Dickeya dianthicola targeting a unique genomic region. PLoS ONE, 14 (6): e0218868, doi.org/10.1371/journal.pone.0218868
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Boluk, G., and Arif, M. 2019. First report of Dickeya dianthicola as a causal agent of bacterial soft rot of potato in Hawaii. Plant Dis, doi.org/10.1094/PDIS-11-18-2094-PDN
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Arif, M. 2019. One Lab - One Protocol: synergetic effect of 5AT-rich flap to harmonize qPCR protocols for easy, sensitive and cost-effective diagnostics (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Larrea, A., Dobhal, S., Alvarez, A., and Arif, M. 2019. Insights into pathogenicity determinants of Clavibacter michiganensis subsp. michiganensis and their effects on disease expression (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Arizala, D., and Arif, M. 2019. Comparative genomics of Pectobacterium species revealed remarkable heterogeneity in pathogenicity determinants, antimicrobial compounds and CRISPR Cas (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dobhal, S., Boluk, G., Babler, B., Stulberg, M.J., Rascoe, J., Nakhla, M., Chapman, T., Crockford, A.B., Melzer, M., Alvarez, A.M., and Arif, M. 2019. Comparative genomics approach to develop a highly reliable duplex TaqMan qPCR assay for sensitive detection of genus Dickeya and Dickeya dianthicola (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Boluk, G., Dobhal, S., Alvarez, A., and Arif, M. 2019. Phylogeny of Dickeya zeae isolated from different hosts and irrigation water using multi-locus sequence analysis (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dhakal, U., Alvarez, A.M., and Arif, M. 2019. Xanthomonas euvesicatoria populations are notably clonal irrespective of distant geographical distribution (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dobhal, S., Boluk, G., Stulberg, M.J., Rascoe, J., Nakhla, M., and Arif, M. 2019. Robust and highly reliable loop-mediated isothermal amplification (LAMP) assay for specific and sensitive detection of Dickeya solani (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Arizala, D., Dobhal, S., Crockford, A.B., Ochoa-Corona, F., Alvarez, A.M., and Arif, M. 2019. Multiplex TaqMan qPCR targeting unique genomic regions for specific, sensitive and robust detection of Pectobacterium species and P. parmentieri (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Boluk, G., Dobhal, S., Crockford, A.B., Melzer, M., Alvarez, A.M., and Arif, M. 2019. Genome-informed recombinase polymerase amplification assay for specific and sensitive detection of Dickeya species at point-of-care (Abstract; APS Annual Meeting, Cleveland, OH).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Larrea, A., Alvarez, A., and Arif, M. 2019. Detection of Clavibacter michiganensis and C. michiganensis ssp. nebraskensis using multiplex recombinant polymerase amplification coupled with LFD (Abstract; APS Annual Meeting, Cleveland, OH).

Progress 10/11/17 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Teaching a Special Topic (PEPS 690; one lecture and 2 labs per week): In this course, I am giving the opportunities to the students to develop new skill setsfornew job opportunity in industry, academia and government.They workin aproject - I help them to design the experiments, they execute the experiments, use the generateddata to write the manuscript and finally they publish it. They have already published one article (Larreaet al, 2018, Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria),in Scientific Reports. Students from this year are currently preparing the manuscript and will submit to journalPloS One soon. This course is opening new job opportunities for the students and provided them excellent molecular skills. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?1. Developing newfield-deployable sensitive detection methods 2. Population genetics on Dickeya species 3. Comparative genomics of Clavibacter michiganensis and Dickeya species.

Impacts
What was accomplished under these goals? A. Major activities completed: 1. We have developed field-deployable Loop-mediated isothermal amplification (LAMP) methods to detect all known subspecies of C. michiganensis (Dobhal et al, 2018) and X. euvesicatoria (Larrea et al, 2018). We have also developed LAMP and multiplex TaqMan qPCR methods for D. dianthicola, an aggressive pathogen of potato (both publications are under preparation). Moreover, we have developed recombinase polymerase amplification (RPA) methods to detect genus Dickeya, and a RPA multiplex to detect C. michiganensis and C. michiganensis subsp. nebraskensis (publication is under preparation). We also investigated the effect of non-complementary sequences at 5' position of the primers for optimizing the reaction thermodynamics to lead the development of sensitive methods (manuscript communicated). 2. In population genetics/phylogenetics, we have worked on X. euvesicatoria and X. vesicatoria population genetics; both cause the bacterial spot on pepper and tomato. ELISA and MLSA of partial sequences of five housekeeping gene (dnaA, gapA, gyrB, hHmbs and pdg) and one gene in type 3 secretion system (hrcN) were used to characterize the diversity within and among X. euvesicatoria and X. vesicatoria from wide range of geographic locations (publication is under preparation). 3. We have completed research on comparative genomics of Pectobacterium species; we have included all know species within the genus Pectobacterium; this genus comprises devastating pathogens of soft rot/blackleg with broad host range. The different clusters involve in different functions (Type Secretion Systems, motility, cell attachment and agglutination, phytotoxins, polysaccharide synthesis, iron uptake, antimicrobial and antibiotics, arsenic resistance etc.) were compared and evaluated; high plasticity among the different species was observed (publication is in preparation). 4. We aim to determine the role of EPS in tomato bacterial canker disease development. The disease is caused by C. michiganensis subsp. michiganensis (Cmm); the genomes of virulent EPS producing and non-EPS producing strains as well as a virulent EPS producing strain have been sequenced. The four clusters involved in EPS productions have been identified and compared; pathogenicity islands and other important regions have also been identified and evaluated. Electron microscopic studies to understand the colonization pattern inside the host tissues were also investigates. A few greenhouse experiments are still underway. B. Specific objectives met: We havedeveloped and published thediagnostic results from objective 1 and we more protocols under development and validation stage. In objective 2 and 3, we have completed two studies on X. euvesicatoria/X. vesicatoria and genus Pectobacterium but publications are under preparation. C. Significant results achieved: We have developed 1st multiplex RPA for any plant pathogen that will provide the reference for the researcher/scientist to develop for other plant pathogens. We have demonstrated that RPA doesn't need DNA isolation; it can be performed directly from macerated plant tissue- no or leasteffect of plant inhibitors. We also demonstrated comprehensive genome analyses of Pectobacterium species. D. Key Outcomes 1. Publications: three has already been published from this project and we have five more to be communicated soon. 2. Five graduate students are working in this project. They learn new methods and developed new skill sets. 3.Collaborating with scientists/researcher/students of other institutions. 4. Student presentation award: 1st rank in MS poster presentation in CTAHR Research Symposium 2018. 5. Teaching a Special Topic (PEPS 690; one lecture and 2 labs per week): In this course, I am giving the opportunities to the students to develop new skill setsfornew job opportunity in industry, academia and government.They workin aproject - I help them to design the experiments, they execute the experiments, use the generateddata to write the manuscript and finally they publish it. They have already published one article, Larreaet al (2018),in Scientific Reports. Students from this year are currently preparing the manuscript and will submit to journalPloS One soon.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ahmed, F.A., Larrea-Sarmiento, A., Alvarez, A.M., and Arif, M. 2018. Genome-informed diagnostics for specific and rapid detection of Pectobacterium species using recombinase polymerase amplification coupled with a lateral flow device. Scientific Reports, 8:15972. doi:10.1038/s41598-018-34275-0
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Dobhal, S., Larrea-Sarmiento, A., Alvarez, A.M., and Arif, M. 2018. Development of a loop-mediated isothermal amplification assay for specific detection of all known subspecies of Clavibacter michiganensis. J Appl Microbiol, doi: 10.1111/jam.14128
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Larrea-Sarmiento, A., Dhakal, U., Boluk, G., Fatdal, L., Alvarez, A.M., Strayer-Scherer, A., Paret, M., Jones, J., Jenkins, D., and Arif, M. 2018. Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria. Scientific Reports. 2018 Sep 24;8(1):14298. doi: 10.1038/s41598-018-32295-4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ahmed, F.A., Larrea, A., Arif, M., and Alvarez, A.M. 2018. Development of genome-informed diagnostics for detection of Pectobacterium species using recombinase polymerase amplification coupled with LFD. Phytopathology, 108: 52