Source: AUBURN UNIVERSITY submitted to NRP
ECOLOGY, EVOLUTION AND MOLECULAR BASIS OF PLANT-BACTERIAL INTERACTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1012760
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
May 15, 2017
Project End Date
May 14, 2022
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Plant Pathology
Non Technical Summary
Bacterial plant pathogens cause a significant amount of crop loss around the world. Plant pathogenic bacteria have evolved multiple strategies to colonize and infect host plants, in addition to their survival mechanism. The emergence of new strains poses problems for disease management and is particularly disruptive to breeding efforts that have focused on race-specific resistance. In this project, bacterial spot of tomato and pepper caused by four diverse Xanthomonas sp. will be used as a model system to understand the pathogen biology. It is not clear how and why new strains of bacterial spot xanthomonads emerged and, therefore, how it can be prevented or mitigated. Understanding the pathogen population dynamics and emergence of strains with novel pathogenicity factors is necessary for integrated and sustainable management of bacterial leaf spot. Other aspect for this project is aimed at identification and characterization of novel disease resistance genes and their efficacy upon combining with existing disease resistant genotypes. Identification of such combinatorial strategies to mitigate bacterial diseases is important for designing sustainable disease management approaches.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121469110030%
2124010104030%
2124010107020%
2124010108020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1469 - Solanaceous and related crops, general/other; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1070 - Ecology; 1100 - Bacteriology; 1080 - Genetics;
Goals / Objectives
A major goal of the proposed project is to understand plant-pathogen-environment interactions with the long-term goal of identifying sustainable disease management strategies. Bacterial spot of tomato and pepper is used as a model pathosystem in the proposed project. Following objectives have been designed towards the proposed goal.Understanding the bacterial spot pathogen ecology and population dynamics on tomato and pepper Understanding the pathogenicity/fitness factors of bacterial spot xanthomonads Identification and characterization of novel sources of disease resistance in tomato and pepper against bacterial spot xanthomonads
Project Methods
Understanding the bacterial spot pathogen ecology and population dynamics on tomato and pepper: The goal of this objective is to characterize the pathogen diversity in the southeastern U.S., more specifically, Alabama on tomato as well as pepper cultivars in comparison to the diversity existing in other parts of the tomato/pepper growing regions in the world. The PI has a strong collaboration with the University of Florida research group. The team at UF has obtained a collection of pathogen isolates from seed to the fruit stages on tomato from around the world. The PI's role in this collaborative project is to investigate the pathogen dynamics in Alabama on tomato and contribute to pathogen genome analyses. Sampling on pepper has been very limited. As a part of this objective, spatial and temporal sampling of pepper from seed to fruit stage (seed sources, transplant houses, greenhouses, high tunnel operations and commercial fields in southeastern states will be conducted). In addition to looking at pathogen diversity, ecological factors such as alternate hosts, pathogen survival mechanisms will be explored by such widespread sampling. Pepper pathogen isolates from other parts of the world will also be obtained. Representative strains from different parts of the U.S. and other parts of the world will be sequenced. A combination of Next-Generation Sequencing tools will be used in our studies. Comparative genome analyses and whole genome phylogenies will be performed to understand overall pathogen evolution on pepper, including host range shift and emergence of novel strains. Factors responsible for shaping the pathogen diversity will also be explored.Understanding the pathogenicity/fitness factors of bacterial spot xanthomonads: Based on whole genome comparisons of currently available bacterial spot xanthomonads, we have identified core and variable pathogenicity factors. Upon obtaining genome sequences of the additional strains from pepper proposed in objective 1, pathogenicity factors will be characterized for their role in different stages of disease development, pathogen fitness/adaptation including host range specificity. Deletion mutants will be constructed for single genes/in combinations and perform in planta assays to determine functional significance. Transcriptomics and functional genomic studies are expected to reveal candidates that would be ideal targets for disease control strategies.Identification and characterization of novel sources of disease resistance in tomato and pepper against bacterial spot xanthomonads : In collaboration with U.S.Vegetable laboratory, novel disease resistance genes from pepper Plant Introduction lines, that have been identified from the germplasm screen against Xanthomonas gardneri, will be characterized. USDA collaborators will be performing the crosses into the elite cultivars. The PI's research group will be involved in performing phenotypic assays for screening the lines. The overall goal is to identify the genes associated with the resistance and perform functional studies to verify contribution of the candidate genes in imparting resistance. These novel resistance gene sources will be tested and characterized along with the exisiting disease resistant genotypes including those imparting PAMP-triggered immunity as well as Effector-triggered immunity against bacterial spot xanthomonads.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:Target audiences for this progress report include: Graduate student research in the lab (4 M.S. and 2 Ph.D. students); Undergraduate students through formal classroom teaching and research in the laboratory (Course: General Plant Pathology, Omics in Agriculture) as well as 4 students conducted undergraduate research Growers in Alabama and other neighboring states Research findings were presented by the students and postdoc at national and international meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project period, as a part of research mentoring, six graduate students and one postdoc were trained in molecular biology techniques such as constructing deletion mutants, complementation, shotgun metagenome analyses, and plant pathology techniques such as in planta population assays, disease severity ratings and pathogen isolations. Four undergraduate students were trained in basic microbiology and plant pathology techniques. Undergraduate students were also mentored for computational analyses. How have the results been disseminated to communities of interest?Results from our 2019 and 2020 surveys in Alabama were communicated with the growers during the Alabama fruit and vegetable grower association meeting. For our continued surveys now focusing on microbiome, we have been working with the tomato and pepper growers and extension agents across Alabama, Georgia, South Carolina, and North Carolina. This has provided us opportunities to learn about the current management practices growers have been implementing and see their effects on disease incidence. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I plan to continue investigating the proposed objectives. We are conducting spatio-temporal sampling across the southeastern US which will not only provide information on pathobiome but also co-occurring pathogens, and saprophytic organisms. We will also be able to implement modeling approaches that will allow us to predict risk factors for bacterial spot disease incidence as well as tap into its evolution. We will continue investigating the host specificity factors in pepper by constructing deletion mutants and conducting in planta assays. We expect to screen F2 crosses with newly identified resistance that can help in the identification of QTLs involved in resistance against Xanthomonas gardneri. New domestic and international collaborations are being developed to explore new areas of research related to plant-microbe interactions.

Impacts
What was accomplished under these goals? Obj. 1 Understanding the bacterial spot pathogen ecology and population dynamics on tomato and pepper:During this project period, we continued our extensive field survey of tomato and pepper fields in Alabama, Georgia, North Carolina, and South Carolina. We performed shotgun metagenomics to capture the pathogen diversity and associated phyllosphere microbiome in bacterial spot-infested tomato/pepper fields. Our results indicate that multiple pathogen genotypes can coexist in the fields. We have also obtained pure cultures of Xanthomonas perforans and X. euvesicatoria from these infected samples. X. perforans was dominant in tomato fields. While X. euvesicatoria was dominant in pepper fields, X. perforans was also recovered from some pepper genotypes. A mixed infection with Pseudomonas was also observed in many fields. Two field trials were conducted in Alabama for evaluating the efficacy of currently employed disease management methods, however, both trials were impacted by tropical storms that hit Alabama. Obj 2. Understanding the pathogenicity/fitness factors of bacterial spot xanthomonads: We completed writing of a manuscript summarizing findings from the experiments where we compared epiphytic fitness of DtssM-i3* mutant, WT Xanthomonas perforans and complement. This manuscript was accepted after revision in Phytopathology Journal. Briefly, our data revealed that functional TssM belonging to type VI secretion system cluster i3* confers epiphytic fitness to a hemibiotrophic pathogen, by providing higher osmotolerance, enhancing its dissemination, while at the same time, minimizing overall disease severity. To understand the contribution of T6SS towards ecology and evolution of Xanthomonas spp., we explored the distribution of the three distinguishable T6SS clusters, i3*, i3*** and i4, in ~1,500 Xanthomonas genomes, along with their conservation, genetic organization and their evolutionary patterns in this genus. Phylogenetic analysis demonstrated that T6SS clusters i3* and i3*** were likely acquired by the ancestor of the genus Xanthomonas, followed by conservation or loss of individual clusters upon diversification into subsequent clades and that individual T6SS clusters and associated effectors provided a selective advantage to the species during adaptation onto specific hosts. Genetic flux of T6 loci and associated effectors has been common among species and specific combinations of T6SS clusters and repertoires of effectors have been maintained within pathogenic and nonpathogenic species, that inform the role of T6SS in ecological adaptation. Genome-wide association study revealed the role of carbohydrate metabolism and amino acid transport-associated genes in pepper specificity. Earlier we conducted studies with allele exchange mutations swapping non-functional versions of TonB-dependent receptors in pepper pathogenic strains and functional versions of TonB dependent receptors in pepper non-pathogenic Xanthomonas perforans strains. The functional in planta assays using both infiltration and dip-inoculation method indicated the role of this TonB-dependent receptor during early colonization of the apoplast, possibly to evade recognition by the plant host, thus explaining its pseudogenization in pepper pathogenic strain. We are further exploring the role of cell-wall degrading enzymes and induced immunity in restricting the growth of X. perforans during early colonization on pepper. Obj 3. Identification and characterization of novel sources of disease resistance in tomato and pepper against bacterial spot xanthomonads: The two PI pepper lines that have been found to be resistant to X. gardneri have been used for generating crosses with susceptible parent. We will be screening the F1 and F2s against X. gardneri. We have also recently sequenced Xanthomonas population obtained from pepper fields. The epiphytic colonization of X. euvesicatoria was observed on resistant pepper cultivars.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Liyanapathiranage, P., Jones, J.B., Potnis N. 2021. A mutation of a single core gene, tssM, of type VI secretion system of Xanthomonas perforans influences virulence, epiphytic survival and transmission. Phytopathology. https://doi.org/10.1094/PHYTO-02-21-0069-R
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Osdaghi, E., Sharma, A., Goss, E.M., Abrahamian, P., Newberry, E., Potnis, N., Carvalho, R., Choudhary, M., Paret, M., Timilsina, S., Vallad, G., Jones, J.B. A Centenary for Bacterial Spot of Tomato and Pepper. Molecular Plant Pathology, Pathogen Profile. Early View. https://bsppjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/mpp.13125
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Potnis, N. 2021. Harnessing Eco-Evolutionary Dynamics of Xanthomonads on Tomato and Pepper to Tackle New Problems of an Old Disease. Annual Review of Phytopathology, 59 (1): null. https://doi.org/10.1146/annurev-phyto-020620-101612.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Stam, R., Gladieux, P., Vinatzer, B. A., Goss, E. M., Potnis, N., Candresse, T., Brewer, M. T. 2021. Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology. Phytopathology 111 (1): 811. https://doi.org/10.1094/PHYTO-11-20-0528-FI.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Shantharaj, D., Williams, M. A., Potnis, N., Liles, M. R. 2021. Burkholderia Gladioli C101 Metabolites Protect Tomato Plants against Xanthomonas Perforans Infection. J Plant Dis Prot, 128 (2): 379390. https://doi.org/10.1007/s41348-020-00416-9.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ge, Q., Liu, R., Cobine, P. A., Potnis, N., De La Fuente, L. 2021. Phenotypic and Phylogenetic Characterization of Cu Homeostasis among Xylella Fastidiosa Strains. Pathogens 10 (4): 495. https://doi.org/10.3390/pathogens10040495.
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Dia, N.C., Moriniere, L., Osdaghi, E., Bernal, E., Jacobs, J., Cottyn, B., Koebnik, R., Potnis, N., Pothier, J. Xanthomonas hortorum: a Species Complex or a Complex Species? Molecular Plant Pathology, Pathogen Profile. Accepted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Liyanapathiranage, P., and Potnis, N. Type VI secretion system of Xanthomonas perforans contributes to the incubation period and is involved in niche adaptation. APS Melhus Graduate Student Symposium 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Bhandari, R., and Potnis, N. 2021. Host genotype affects the microbial community structure on pepper. APS Melhus Graduate Student Symposium 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Liu, R., Castillo, A., Arias-Giraldo, L., Velasco-Amo, M., Almeida, R., Landa, B., Potnis, N., and De La Fuente, L. 2021. Natural competence and homologous recombination among Xylella fastidiosa strains. XF-ACTORS conference Europe 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Arias-Giraldo, L., Potnis, N., De La Fuente, L., Moralejo, E., Velasco-Amo, M.P., Roman-Ecija, M., Imperial, J., Landa, B.B. Detection of recombination events in Xylella fastidiosa genomes of different Spanish strains. XF-ACTORS conference Europe 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bhandari, R.*, Potnis, N. Shotgun metagenome sequencing provided intra-specific diversity in bacterial spot Xanthomonads. Tomato disease workshop 2020.


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

Outputs
Target Audience:Target audiences for this progress report include: (1) Graduate student research in the lab (2 M.S. and 2 Ph.D. students); (2) undergraduate students through formal classroom teaching and research in the laboratory (Course: General Plant Pathology) as well as 3 students conducted undergraduate research; (3) growers in Alabama and other neighboring states (4) Research findings were presented by the students and postdoc at national and international meetings. Changes/Problems:There were three issues related to pandemic. The lab was shut down from March 2020 to May 2020. This slowed our progress on all objectives. What opportunities for training and professional development has the project provided?During this project period, as a part of research mentoring, four graduate students and one postdoc were trained in molecular biology techniques such as constructing deletion mutants, complementation, shotgun metagenome analyses and plant pathology techniques such as in planta population assays, disease severity ratings and pathogen isolations. Undergraduate students were trained in basic microbiology and plant pathology techniques. Undergraduate students conducted computational project during Summer 2020 analyzing sequences of pathogenicity factors from published bacterial spot pathogen genomes. How have the results been disseminated to communities of interest?Results from our 2017/2018 and 2019 surveys in Alabama were communicated with the growers during Alabama fruit and vegetable grower association meeting. For our continued surveys now focusing on microbiome, we have been working with the tomato and pepper growers and extension agents across Alabama, Georgia, South Carolina and North Carolina. This has provided us opportunities to learn about the current management practices growers have been implementing and seeing their effects on disease incidence. For example, spread of copper resistance is not uniform across all the fields. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I plan to continue investigating the proposed objectives. We are conducting spatio-temporal sampling across southeastern US which will not only provide information on pathobiome but also co-occurring pathogens, and saprophytic organisms. We will also be able to implement modeling approaches that will allow us to predict risk factors for bacterial spot disease incidence as well as tap into its evolution. We will continue investigating the host specificity factors in pepper by constructing deletion mutants and conducting in planta assays. Role of type VI secretion system and its regulation during pathogenesis will be investigated. We expect to generate F2 crosses with newly identified resistance that can help in identification of QTLs involved in resistance aginst Xanthomonas gardneri.New domestic and international collaborations are being developed to explore new areas of research related to plant microbe interactions.

Impacts
What was accomplished under these goals? Obj. 1 Understanding the bacterial spot pathogen ecology and population dynamics on tomato and pepper During this project period, we have optimized a shotgun metagenome method to capture the pathogen diversity and associated phyllosphere microbiome in bacterial spot infested tomato/pepper fields. One of the problems with the culture-dependent isolate genome sequencing approach is inherent bias in selecting dominant strain. We sampled 12 fields and sequenced tomato, pepper and weed microbiome to resolve pathogen diversity at intra-subspecific level and identify co-occurrence patterns for different bacterial species. Our results indicated that multiple pathogen genotypes can coexist in the fields. Pseudomonas cichorii was also common co-occurring pathogen on tomato/pepper. Other opportunistic pathogens such as Xanthomonas arboricola were also identified in these samples along with common phyllosphere residents. This method also provided a snapshot into the pathobiome of tomato/pepper fields. We have published this work in Environmental Microbiology. Such approach when applied at spatial and temporal scale is important to understand eco-evolutionary dynamics of the pathogen population. Obj 2. Understanding the pathogenicity/fitness factors of bacterial spot xanthomonads Genome-wide association study has been conducted on bacterial spot xanthomonads to identify pepper specificity factors. The candidate genes pointed towards the role of metabolism, more specifically carbohydrate and amino acid transport, important in host specificity. Interestingly, allele exchange mutations swapping non-functional version of TonB-dependent receptor in pepper pathogenic strains and functional version of TonB dependent receptor in pepper non-pathogenic Xanthomonas perforans strains indicated role of this TonB-dependent receptor during early colonization of the apoplast, possibly to evade recognition by the plant host, thus explaining its pseudogenization in pepper pathogenic strain. We have also characterized TssM deletion mutant, a mutant of core Type VI secretion system in Xanthomonas perforans. In planta experiments at different stages of plant development, such as seed to seedling or 4-5-week old plants or transplants indicated that type VI secretion system plays role in epiphytic fitness of the pathogen and prolongs its colonization with the host by delaying symptom development. Obj 3. Identification and characterization of novel sources of disease resistance in tomato and pepper against bacterial spot xanthomonads The two PI pepper lines that have been found to be resistant to X. gardneri have been selfed and checked for homozygosity in each generation by phenotyping (dip-inoculation followed by assessing resistance and susceptibility in the plants), and selfing the resistant lines until 6th selfing. We have S6 lines now that seem to have no segregation for disease resistance. These are currently being crossed with susceptible parent.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Newberry, E. A.; Bhandari, R.; Minsavage, G. V.; Timilsina, S.; Jibrin, M. O.; Kemble, J.; Sikora, E. J.; Jones, J. B.; Potnis, N. Independent Evolution with the Gene Flux Originating from Multiple Xanthomonas Species Explains Genomic Heterogeneity in Xanthomonas Perforans. Appl. Environ. Microbiol. 2019, 85 (20). https://doi.org/10.1128/AEM.00885-19.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Newberry, E.; Bhandari, R.; Kemble, J.; Sikora, E.; Potnis, N. Genome-Resolved Metagenomics to Study Co-Occurrence Patterns and Intraspecific Heterogeneity among Plant Pathogen Metapopulations. Environmental Microbiology 2020, 22 (7), 2693⿿2708. https://doi.org/10.1111/1462-2920.14989.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Timilsina, S.; Potnis, N.; Newberry, E. A.; Liyanapathiranage, P.; Iruegas-Bocardo, F.; White, F. F.; Goss, E. M.; Jones, J. B. Xanthomonas Diversity, Virulence and Plant⿿Pathogen Interactions. Nature Reviews Microbiology 2020, 18 (8), 415⿿427. https://doi.org/10.1038/s41579-020-0361-8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Potnis, N.; Timilsina, S.; Jones, J. B. Whole Genome Sequencing of Plant Pathogenic Bacteria; APSNET, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Rosenthal, E. R.; Sebastian, A.; Potnis, N.; Albert, I.; Bull, C. Comparative Genomic Analysis of the Lettuce Bacterial Leaf Spot Pathogen; APSNET, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bibi, S.; Bhandari, R.; Minsavage, G. V.; Timilsina, S.; Jones, J. B.; Potnis, N. Chromosomally Encoded Copper Resistance Genes from Xanthomonas Perforans Reside on the Genomic Island; APSNET, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Nampijja, M.; Boyd, L. N.; Crane, S.; Dundore-Arias, J. P.; Gaulke, E.; Herschlag, R.; Huerta, A. I.; Kulesza, E.; Kan, Y.; Newberry, E. A.; Potnis, N. Integrated Management of Emerging Seedborne Pseudomonas Syringae Pathogens of Cucurbitaceae and Chenopodiaceae; APSNET, 2020.


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

Outputs
Target Audience:Target audiences for this progress report include: (1) Graduate student research in the lab (1 M.S. and 1 Ph.D. student); (2) undergraduate students through formal classroom teaching and research in the laboratory (Course: General Plant Pathology); (3) Graduate student classroom teaching (A new graduate course was taught during this project period; PLPA 7880 Plant Microbial Ecology and Omics, that included lecture class with concepts in microbial ecology, phytobiome and a lab class for hands-on training in analysis of Omics data).; (3) growers in Alabama and the US. Participated as an invited speaker at Alabama Fruit and Vegetable Growers Association meeting and communicated my research findings with the growers.; (4) Research findings were presented by the students and postdoc at national and international meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project period, as a part of research mentoring, two graduate students and one postdoc were trained in molecular biology techniques such as constructing deletion mutants, complementation, developing computational pipelines for genomic data analyses. How have the results been disseminated to communities of interest?We have been working with the tomato and pepper growers across Alabama. Growers have been contacting us to investigate the sources of infection in their fields. We have been assisting them by sampling various reservoirs, sampling throughout the season, assessing bacterial populations for bactericides. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I plan to continue investigating the proposed objectives. We will continue investigating the host specificity factors in pepper. We are in the process of phenotyping crosses of resistant and susceptible pepper genotypesfor disease resistance. New domestic and international collaborations are being developed to explore new areas of research related to plant microbe interactions.

Impacts
What was accomplished under these goals? During this project period, we analyzed genome sequences of strains obtained from disease outbreaks in Alabama. Our results indicated presence of novel lineages of X. perforans that contained novel pathogenicity factors. We also identified signatures of host specificity factors from these genome comparisons. Genesinvolved in carbohydrate metabolism and TonB-dependent receptors were identified to be associated with host specificity. We have functionally characterized these candidate genes for their contribution towards pathogenicity on pepper. We have also characterized type VI secretion system cluster for its role in disease progression. We are continuing to assess the resistance germplasm for the levels of disease resistance against different lineages of pathogen.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Potnis, Neha, Sandra E. Branham, Jeffery B. Jones, and W. Patrick Wechter. Genome-Wide Association Study of Resistance to Xanthomonas Gardneri in the USDA Pepper (Capsicum) Collection. Phytopathology" 109, no. 7 (February 18, 2019): 121725. https://doi.org/10.1094/PHYTO-06-18-0211-R.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Newberry, Eric A., Mohamed Ebrahim, Sujan Timilsina, Nevena Zlatkovi?, Aleksa Obradovi?, Carolee T. Bull, Erica M. Goss, et al. Inference of Convergent Gene Acquisition Among Pseudomonas Syringae Strains Isolated From Watermelon, Cantaloupe, and Squash. Frontiers in Microbiology 10 (2019). https://doi.org/10.3389/fmicb.2019.00270.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: An, Shi-Qi, Neha Potnis, Max Dow, Frank-J�rg Vorh�lter, Yong-Qiang He, Anke Becker, Doron Teper, et al. Mechanistic Insights into Host Adaptation, Virulence and Epidemiology of the Phytopathogen Xanthomonas. FEMS Microbiology Reviews. https://doi.org/10.1093/femsre/fuz024.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Abrahamian, Peter, Sujan Timilsina, Gerald V. Minsavage, Neha Potnis, Jeffrey B. Jones, Erica M. Goss, and Gary E. Vallad. Molecular Epidemiology of Xanthomonas Perforans Outbreaks in Tomato Plants from Transplant to Field as Determined by Single-Nucleotide Polymorphism Analysis. Applied and Environmental Microbiology 85, no. 18 (September 15, 2019). https://doi.org/10.1128/AEM.01220-19.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Iruegas-Bocardo, Fernanda, Sujan Timilsina, Mustafa O. Jibrin, Gerald V. Minsavage, Peter Abrahamian, Daniel S. Egel, Tom Creswell, et al. Effector Profile Analysis of the Tomato and Pepper Pathogens Xanthomonas Perforans and X. Euvesicatoria. ISMPMI, 2019. https://ismpmi.confex.com/ismpmi/2019/meetingapp.cgi/Paper/3724.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Iruegas-Bocardo, Fernanda, Sujan Timilsina, Mustafa Jibrin, Gerald V. Minsavage, Peter Abrahamian, Daniel S. Egel, Tom Creswell, et al. Effector Profiles of Xanthomonas Perforans and X. Euvesicatoria Provide Insights into the Evolution of Host Range and Virulence in BLS Pathogens. APSNET, 2019. https://apsnet.confex.com/apsnet/2019/meetingapp.cgi/Paper/14405.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bhandari, Rishi, and Neha Potnis. Investigation of in Vitro and in Planta Transfer of Chromosomally-Encoded Copper Resistance in Xanthomonas Perforans. APSNET, 2019. https://apsnet.confex.com/apsnet/2019/meetingapp.cgi/Paper/14293.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Timilsina, Sujan, Juliana A. Pereira-Martin, Gerald V. Minsavage, Fernanda Iruegas-Bocardo, Peter Abrahamian, Neha Potnis, Bryan Kolaczkowski, Gary E. Vallad, Erica M. Goss, and Jeffrey B. Jones. Multiple Recombination Events Drive the Current Genetic Structure of Xanthomonas Perforans in Florida. Frontiers in Microbiology 10 (2019). https://doi.org/10.3389/fmicb.2019.00448.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Potnis, Neha, Prem P. Kandel, Marcus V. Merfa, Adam C. Retchless, Jennifer K. Parker, Drake C. Stenger, Rodrigo P. P. Almeida, et al. Patterns of Inter- and Intrasubspecific Homologous Recombination Inform Eco-Evolutionary Dynamics of Xylella Fastidiosa. The ISME Journal 13, no. 9 (September 2019): 231933. https://doi.org/10.1038/s41396-019-0423-y.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Newberry, Eric A., Rishi Bhandari, and Neha Potnis. Identification of a New Class of Transcription Activation-Like Effector, pthXp1 Among Xanthomonas Perforans Strains Collected in Alabama. APSNET, 2019. https://apsnet.confex.com/apsnet/2019/meetingapp.cgi/Paper/14291.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Timilsina, Sujan, Gilcianny P. Cavalcante, Roxanita Ramrez, Maria Do Socorro Bezerra de Araujo, Jichen Tian, Gerald V. Minsavage, Erica M. Goss, Gary E. Vallad, Jeffrey B. Jones, and Neha Potnis. The Effect of XopQ and XopJ4 on Host Specificity Varies with the Xanthomonas Perforans Genetic Background. APSNET, 2019. https://apsnet.confex.com/apsnet/2019/meetingapp.cgi/Paper/14494.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Liyanapathiranage, Prabha and Neha Potnis. Probing infectious strategies of Xanthomonas perforans during latent infection period. APSNET, 2019. https://apsnet.confex.com/apsnet/2019/meetingapp.cgi/Paper/14293.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Target audiences for this progress report include: (1) Graduate student research in the lab (1 M.S. and 1 Ph.D. student); (2) undergraduate students through formal classroom teaching and research in the laboratory (Course: General Plant Pathology); (3) Graduate student classroom teaching (A new graduate course was taught during this project period; PLPA 7880 Plant Microbial Ecology and Omics, that included lecture class with concepts in microbial ecology, phytobiome and a lab class for hands-on training in analysis of Omics data).; (3) growers in Alabama and the US. Participated as an invited speaker at Alabama Fruit and Vegetable Growers Association meeting and communicated my research findings with the growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project period, one undergraduate student was trained in the bacteriology techniques used toisolatebacterial pathogens from infected plant samples, and in identification and characterization of isolates using molecular approaches. As a part of research mentoring, two graduate students and one postdoc were trained in molecular biology techniques such as constructing deletion mutants, complementation, developing computational pipelines for genomic data analyses. How have the results been disseminated to communities of interest?We have been working with the tomato and pepper growers across Alabama. Growers have been contacting us to investigate the sources of infection in their fields.We have been assisting them by sampling various reservoirs, sampling throughout the season, assessing bacterial populations for bactericides. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I plan to continue investigating the proposed objectives. We will continue investigating the host specificity factors in pepper. We will also explore the novel features acquired by diverse strains of X. perforans in Alabama. New domestic and international collaborations are being developed to explore new areas of research related to plant microbe interactions.

Impacts
What was accomplished under these goals? During this project period, we continued sampling efforts from infected tomatoes and peppers around the state of Alabama. As mentioned in the previous report, we obtained whole genome sequences of the select isolates (10). Based on core genome phylogenies, the isolates collected in Alabama presented novel diversity compared to what has been observed in neighboring states. We are currently in the process of writing a manuscript to communicate our findings. Xanthomonas perforans was identified as a dominant species on peppers, with random occurrence of X. euvesicatoria,indicating that similar to tomato, X. perforans has also dominated the pepper fields in Alabama. Hierarchical clustering method applied to define sequence clusters within X. perforans strains identified two novel clusters in Alabama, interestingly, one being highly aggressive on both tomato and pepper. Genome sequences are being deposited to NCBI genome database. To investigate host specificity of X. perforans on pepper, we have employed a genome-wide association analysis approach to identify SNPs/genes that are significantly associated with pepper pathogenicity. We have identified two highly significant associations, both genes associated with metabolic pathway. We have constructed deletion mutants and complements in the pepper pathogenic and non-pathogenic strains and are conducting in planta assays to assess the contribution of these GWAS identified genes in pepper pathogenicity and, in turn, in host range expansion. We are continuing to investigate the chromosomal integration of copper resistance genes in the novel strains identified in both Florida and Alabama. We have conducted plasmid profiles on select strains and have confirmed the presence of both plasmid-borne copper resistance and chromosomal copper resistance co-existing in the fields.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gochez, A.M., Huguet-Tapia, J.C., Minsavage, G.V., Shantaraj, D., Jalan, N., Strau-f^-A, Lahaye, T., Wang, N., Canteros, B.I., Jones, J.B., Potnis, N., 2018. Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains. BMC Genomics 19, 16. https://doi.org/10.1186/s12864-017-4408-9 Jibrin, M.O., Potnis, N., Timilsina, S., Minsavage, G.V., Vallad, G.E., Roberts, P.D., Jones, J.B., Goss, E.M., 2018. Genomic Inference of Recombination-Mediated Evolution in Xanthomonas euvesicatoria and X. perforans. Appl. Environ. Microbiol. 84, e00136-18. https://doi.org/10.1128/AEM.00136-18


Progress 05/15/17 to 09/30/17

Outputs
Target Audience:The target audience for this progress report include: i) graduate student research in the laboratory; ii) undergraduate and graduate students through formal classroom teaching and research in the laboratory. These efforts during this progress period included undergraduate level plant pathology course, undergraduate research mentoring for hands-on molecular techniques in bacteriology and graduate students; iii) growers in Alabama and the US Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project period, two undergraduate students have been trained in techniques used in isolation of bacterial pathogens from infected samples, routine bacteriology methods and identification and characterization of isolates using molecular based techqniues including PCR on housekeeping genes. As a part of research mentoring, undergarduate student was also trained for molecular biology techniques including gene deletion/insertion/complementation. Additionally, bioinformatic pipelines have been developed to analyze bacterial genomes in the laboratory. How have the results been disseminated to communities of interest?We have been working with growers over the past years by characterizing pathogen isolates collected from the fields for resistance to various bactericides that growers are using in the fields over the years. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I plan to continue investigating the proposed objectives. We will continue sampling extensively in tomato and pepper fields in addition to conducting field trials in various locations in Albama.Graduate students and a post-doc will work on these objectives. We are also developing new domestic and international collaborations to develop new areas of research related to plant microbe interactions.

Impacts
What was accomplished under these goals? The changing landscapes of agriculture, climate and global trade have been responsible for changes in pathogen population structures that have influenced vegetable production across the world. Xanthomonas sp. responsible for bacterial spot of tomato and pepper are no exception. Recent studies have pointed out several new lineages appearing to cause outbreaks in US challenging the diseae resistant lines employed under field conditions. We surveyed pathogen population in Alabama in 2017 season and identified two novel lineages that have not yet been observed in other parts of the southeastern US. Interestingly, we observed presence of at least two species in Alabama affecting tomato and pepper production. While X. perforans has been major pathogen of tomato and X. euvesicatoria on pepper in other neighboring states in the southeast US, we observed X. perforans being increasingly isolated in Alabama on both tomato and pepper during 2017 season. The chromosomal profiles of these isolates were analyzed by repetitive PCR method revealing a high degree of variability in the isolates collected from a single field. The representative isolates belonging to each unique profile are being sequenced. The pathogenicity tests of these isolates on tomato and pepper have been carried out. In addition, plasmid profiles of representative isolates have revealed possible chromsomal integration of copper resistance. Strains collected in 1980s and 1990s werecharacterized as copper resistant withplasmid-borne copper resistance genes. The finding of chromosomal integration of the plasmid with heavy metal resistance genes as carrier genes is further being investigated in terms of its implication in integrated management strategies.Once genomes of representative strains are obtained, we will conduct extensive genome analyses to correlate phenotype including host range to the genotype. Further studies will include generation of mutants targeting candidate genes to confirm contribution of the candidate genes towards the phenotype.

Publications