Bacteriocin Repertoires in Pseudomonas syringae pv. Tomato

Progress report for GNE20-239

Project Type: Graduate Student
Funds awarded in 2020: $15,000.00
Projected End Date: 08/31/2022
Grant Recipient: The Pennsylvania State University
Region: Northeast
State: Pennsylvania
Graduate Student:
Faculty Advisor:
Kevin Hockett
The Pennsylvania State University
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Project Information

Summary:

Pseudomonas syringae pv. tomato (pto), the causal agent of bacterial speck of tomato, is an increasing problem in the Northeast and Mid-Atlantic region that can cause devastating crop losses. With the wide-spread emergence of chemical resistance in bacteria coupled with increasing severe weather events that favor the disease development, there is a need to find reliable long-term management strategies. One potential solution is by using bacteriocins. Bacteriocins are proteinaceous antimicrobial compounds bacteria use to kill closely related bacteria and are considered promising and safe potential agricultural bactericides and biological control agents. They could provide a sustainable alternative or complimentary strategy for managing problematic bacterial diseases in both conventional and organic systems. Current biological control strategies ignore complex community interactions, and therefore fail to produce reliable outcomes. In order to understand the role bacteriocins play in the development of bacterial speck of tomato, the competitive interactions that correlate to increased disease development due to bacteriocins in pto will be assessed. First, the targets of pto’s bacteriocin repertoire will be identified to determine susceptible community members. Then two ecological strategies (invasion & defending) will be evaluated with microbiome transplant experiments to understand when during the disease cycle (e.g. colonization & epiphytic survival) bacteriocins are most beneficial to the pathogen. This data can be applied to determine how and when to manipulate the foliar microbial community to effectively control the pathogen during critical points of the disease cycle for future microbial community-centered management strategies.

Project Objectives:

The specific objectives of this proposal are:

  1. Determine targets of individual bacteriocins in pto’s Expected outcome: This objective will identify the pathogen’s bacteriocin targets in order to understand how to limit these advantages in a community. I hypothesize that by identifying mutants resistant to knockout strains of pto that contain only 1 bacteriocin, I will be able to identify key components of the genome that contribute to mitigating competitive advantages within the context of a community.
  2. Assess level of foliar disease symptoms in tomato plants and determine the change in microbial community composition due to individual bacteriocins, after transplant communities are introduced to plants that have first been inoculated with pto. The purpose of this objective is to identify pto bacteriocins that contribute to disease severity of bacterial speck of tomato and identify microbial community changes due to ptobacteriocins after pto has penetrated the surface of the leaf. I hypothesize that individual bacteriocins will significantly contribute to different amounts of disease symptoms and to changes in community composition after pto has penetrated the leaf surface.
  3. Assess the level of foliar disease symptoms in tomato plants and determine the change in community composition due to individual bacteriocins after pto is introduced to a plant that has an established microbial transplant community. The purpose of this objective is to identify pto bacteriocins that contribute to disease severity of bacterial speck of tomato and identify microbial community changes due to pto bacteriocins before pto has penetrated the leaf surface. I hypothesize that individual bacteriocins will significantly contribute to different amounts of disease symptoms and to changes in community composition before pto has penetrated the leaf surface.
Introduction:

The purpose of this project is to determine targets of bacteriocin repertoires in complex microbial communities and assess when bacteriocins are beneficial for disease development to help create future biocontrol management strategies that reduce foliar disease symptoms in tomato.

Pseudomonas syringae pv. tomato (pto), the causal agent of bacterial speck of tomato, is an increasing problem in the Northeast and Mid-Atlantic region. Managing bacterial speck in the Northeast requires a season long proactive chemical and cultural management approach (Bogash, 2016) However, current chemical control measures (e.g. fixed copper) are becoming less reliable due to wide-spread resistance in pathogen populations (Obradovic, Jones, Momol, Balogh, & Olson, 2004; Stall & Thayer, 1962). The emergence of resistance coupled with increasing severe weather events that favor disease development, have made alternative control measures, like biological control agents (BCAs), a top priority for future tomato production.

Bacteriocins are one promising biological control option for foliar bacterial diseases (Riley & Wertz, 2002). Bacteriocins are toxic proteinaceous compounds that bacteria use to kill closely related bacteria. They are attractive options for BCAs because of their narrow target range, thus, avoiding off-target effects, such as killing beneficial community members, as well as not selecting for highly transmissible resistance genes as broad-spectrum biocides do. They are also deemed safe for human consumption, because they are easily degraded by human gut enzymes (Balciunas & Martinez, 2013). Currently, the use of bacteriocins as bactericides is widespread in the food preservation industry but hasn’t been duplicated in other industries like agriculture. Current BCAs in agriculture contain one or few distinct organisms (Johnson, 2010). Inconsistent efficacy of those BCAs can be attributed to the dynamic outcome of complex competition within their microbial community. For instance, many bacterial genomes are predicted to encode multiple bacteriocins, potentially allowing for the targeting of multiple competitors and/or multiple ways to target a single competitor by the producing strain. This suggests complex interactions are contributing to the outcomes of microbial competition due to bacteriocin content. Consequently, individual bacteriocin-producing BCAs may fail to thrive long term because of other dynamic interactions within the community or fail to outcompete the pathogen during critical points of the disease cycle in which the pathogen contains other advantages (Foster, 2017).

Understanding the role of bacteriocins in mediating foliar disease symptoms within a community context can lead to microbial community manipulation by either direct bacteriocin application or by the application of BCA cocktails at key time points during the disease cycle of the pathogen Therefore, I propose to use a metagenomic approach using different microbial communities to determine the target organisms of pto’s bacteriocin repertoire and assess when during the disease cycle the bacteriocins are providing substantial advantages to the pathogen. These data will help determine which organisms are critical competitors and detect key time points for intervention. The results can be incorporated into sustainable systems of both conventional and organic tomato production to support the long-term health of tomato agricultural systems.

Research

Materials and methods:

PRELIMINARY

The preliminary research supported by internal University funds will be conducted in the summer of 2020 will seek to outline and characterize the genomic bacteriocin content, and create triple and quadruple knockouts in order to quantify the effects of one active bacteriocin at a time has on the community in the community.

  • Knockouts: Preliminary data from mining 56 recently denovo assembled genomes from isolates given by Christine Smart’s lab at Cornell University, I found that they all contained the same 4 bacteriocins within its genome. These bacteriocins will be referred to as bacteriocins A, B, C, and D. Knockouts (KO) will include 4 triple knockouts that only contain one of the 4 bacteriocin (KO+A, KO+B, KO+C, and KO+D). Then one quadruple KO will be made not containing any of the 4 identified bacteriocins (KO-ABCD). To be used in objectives 2-3. KOs will be made by deleting the bacteriocin genes using overlap PCT.
  • Microbial Community Collections: Two plant communities plant selected microbial communities 1 month prior to this project, collected and kept at 4°C for the experiments to be performed for this project in 2021 and 2022. Leaf microbial community samples will be taken from tomato plants grown at the Pennsylvania State University research farm that are not undergoing experimental treatment in another study will be harvested for their and from communities on leaves from a non-agricultural native plant at least 500 feet away from managed land. Then they will be stored for no longer that 1 month at 4°C, before they will be dried in a drying oven at 25°C and fragmented by grinding the plant material finely with a mortar and pestle.

DURING

Objective One:

  • Pairing knockouts with sensitive target strains: Supernatants from knockouts, KO+A, KO+B, KO+C, and KO+D, containing one bacteriocin will be collected and soft agar inhibition test will be performed against the 56 strains of pto to pair the triple KO strains with their respective sensitive counterpart(Hockett & Baltrus, 2017).
  • Mutant Selection: Once the bacteriocin producing strain has been paired with the bacteriocin sensitive strain, then selection of bacteriocin mutants by mixing 50uL of the susceptible strain in log phase grown in KB 50uL of supernatant supernatant extracted from the 4 KO strains in a microcentrifuge tube (using a sterile KB as a control). After, vortexing the tube for 10 seconds I will let the supernatant incubate for 30 minutes at room temperature vortexing for an additional 10 seconds every 10 minutes. Following incubation, a 1/10 serial dilution series by spotting the mixture onto non-selective KB plate. Then the dilution will incubate on the plate at 26 degrees C for 24-48 hours. Finally, individual resistant colonies should emerge at one of the dilution points and will be picked and whole genome sequenced using Illumina to identify genetic mutations that indicate resistance by comparing the mutants to the wild type whole genome sequences that were assembled in April of 2020.

Objective 2:

  • Design: A randomized block design will be conducted blocking by the source of the starting microbial community (tomato vs. non-tomato). All objective 2 treatment groups will be considered “invading”. Invading treatment groups will have the bacteriocin producing strains applied as a foliar application after transplant communities have 72 hours to establish. Invading treatment groups are measuring the benefits that bacteriocins provide during colonization of an established community. This is important for disease spread within a field. Each treatment block will consist of 6 treatment groups with 5 plants each for a total of 60 plants for objective 2.
  • Metagenomic community identification: Next, I will compare the change in community compositions in all my treatments, 7 days after initial symptoms appear on the wt treatment groups, to the negative controls. Then, I will use a metagenomics approach for community profiling instead of the traditional 16SrDNA, since metagenomics provides a greater resolution of the community. This will likely be important based on knowledge of bacteriocin narrow spectrum targeting. Metagenomic comparisons will be addressed using the methods listed under Metagenomics.
  • Disease assessment: Additionally, the plant will be evaluated for disease severity using the methods listed under quantitative and qualitative disease assessments and evaluated for statistical significance using the methods under statistical analysis. If there is a statistical significance to a treatment group, the experiment will be performed again with a complimented version of the significantly different strain and with the wt and KO-ABCD strains as controls in order to provide evidence that the significant differences in the community are in fact due to a specific bacteriocin in the community and are not the result of some other interaction happening on the leaf surface.

Objective 3:

  • Design: A randomized block design will be conducted blocking by the source of the starting microbial community (tomato vs. non-tomato). All objective 3 treatment groups will be considered “defending”. Defending treatment groups will have the bacteriocin producing strains inoculated directly into the leaf. Then foliar application of transplant communities will be applied 72 hours after syringe inoculations. The purpose of the defending treatment groups is to measure the benefits a bacteriocin provides during establishment and persistence of the community. Each block will will consist of 6 treatment groups that contain 5 plants each for a total of 60 plants for objective 3.
  • Metagenomic community identification: Next, I will compare the change in community composition 7 days after initial symptoms appear on the wt treatment groups to the negative controls using a metagenomic approach for the same reasons stated in objective 2.
  • Disease assessment: Additionally, the plant will be evaluated for disease severity using the methods listed under quantitative and qualitative disease assessments and evaluated for statistical significance using the methods under statistical analysis. Finally, if there is a statistical significance within a block or between treatment groups, the experiment will be performed again with using complimented versions of the significant strains, combined with the wt and KO-ABCD strains as controls respective positive and negative controls. The purpose of this step is to provide evidence that the significant differences in the community are in fact due to a specific bacteriocin in the community.

Pathogen inoculations:

  • For plant inoculations, Pseudomonas syringae pv. tomato (pto) will be grown in liquid King’s B (KB) media at 28C for 24 hours. The cells are centrifuged at maximum speed, washed with 10 mM MgCl2 three times, then resuspended in 10 mM MgCl2. Bacterial suspensions are standardized to an optical density at 600 nm of 0.1 (Berg and Koskella 2018). Final inoculum will be quantified by plating serial dilutions of leaf wash onto selected media with antibiotics. At 3 weeks old, five tomato plants for all treatments will be sprayed with pathogen suspension until dripping. The plants will be lightly shaken to remove large droplets then covered with a plastic bag for 24 hours to increase relative humidity to allow for pathogen establishment (Williams and Marco 2014). The negative control group will be the treatment group that undergoes inoculation with pto that contains a quadrupole knockout (KO-ABCD), containing no active bacteriocins, and the positive control will be inoculation with a strain from our wild type (wt) representative from collection of Smart lab isolates that won’t have any of the bacteriocins knocked out. Then all treatments will be assessed for disease severity and pathogen load will be calculated using the methods outlined below.  

Metagenomic analysis:

  • Shotgun metagenomics will be performed on each of the treatment groups after the 72 hours after the initial transplant community or pathogen inoculation are established, then again 7 days after the first disease symptoms appear on the wild type treatment group. Reads will be assembled into contigs, then quality control will be performed to detect if sequences are too long or ambiguous. Next the contigs will be aligned to an established database and then classified by their estimated phylogenetic relationships. Last, OTUs will be defined and clustered using a 97% similarity index. Last diversity of the samples will be analyzed with Principle Coordinate Analysis using UniFrac as the similarity index in order to incorporate phylogenetic information and presence absence information into the coordinate analysis since relatedness of the bacterium plays a key role in targeting range of the bacteriocin.

Qualitative Disease Assessment:

  • Following inoculation of tomato plants with pathogen, all plants will be evaluated on a symptom index scale from 1 to 3 one week after symptoms develop on the plants that have been inoculated or sprayed with the pto strain without any knockouts (positive control). The disease severity scale that will be used is as followed: 1 = small spots and/or water-soaking, 0-10% leaf coverage, 2 = 20-30% leaf coverage, 3 = >30% leaf coverage. Disease severity and incidence will be calculated in comparison to the control groups. It is assumed that if disease doesn’t develop within a week of the control plants, it is unlikely to develop.  

Quantitative Disease Assessment:

  • After the qualitative assessment of disease, pto leaf exterior populations will be dislodged from the surface of three tomato leaves per plant by submerging the leaves in tubes with 10 mM MgCl2 buffer and sonicating for 10 minutes. The leaves will then be removed from the buffer and the bacterial cells will be centrifuged and resuspended in 10 mM MgCl2 buffer. Following removal from buffer, the leaves will be surface sterilized by placing individual leaves in a beaker containing 150 mL of 10% (vol/vol) bleach solution for 5 minutes with gentle shaking. For the enumeration of the leaf interior pto population, the leaves will be rinsed in sterile distilled water and dried inside a laminar flow hood for 1 hour.
  • The leaf interior populations will be determined by homogenization of individual leaves in 10 mM MgCl2 by mechanical disruption. Both the exterior and interior leaf pto populations will be calculated by serial diluting onto KB media amended with selective antibiotics. The plates will then be incubated at 28°C for 24 to 48 hours. The colonies that formed after incubation will be counted and transformed into the number of colony-forming units per leaf. This approach will add a quantitative measurement to support the qualitative disease assessment.

Statistical analysis of disease:

  •  Qualitative disease analysis: Objectives 2 and 3 will each be a randomized block design that is blocked by source of transplant community communities (tomato and non-tomato). Each block will undergo 6 treatments each. A positive and negative control along with 4 KOs containing only 1 bacteriocin each. Analysis of variance (ANOVA) and Tukey pairwise comparisons will be used to infer statistically significant relationships overall between the treatment groups and between the blocks. Blocking by community helps determine if the starting community contributes significantly to the effectiveness of an individual bacteriocin. Last, final data from objectives 2 & 3 will be compared using ANOVA and Tukey pairwise comparisons to compare disease severity between invading vs. defending treatments.

  • Quantitative disease analysis: For pathogen enumeration, the pathogen populations will be analyzed for normality and heteroscedasticity, and log transformed as needed. The means of the pathogen populations for both the exterior and interior part of the leaf will also be compared using ANOVA multiple comparison with a Tukey post-hoc test. All analyses will be performed by using open source software in R.

Research results and discussion:

Currently due to the pandemic, and the strict regulations on lab space and time. I am still in the process of creating the knockouts with overlap PCT. 

Research conclusions:

NA

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary

Education/outreach description:

Throughout the entire duration of this project, the progress and preliminary results will be presented to multiple audiences from local to international level events. Currently no work has been presented from this project, but I am still planning to attend the online versions of events outlined below.

February of 2021& February 2022 preliminary results of objectives 1 & 2 respectively will be presented at the Mid-Atlantic Fruit and Vegetable Convention. This convention attracts a diverse annual audience of 2,200 people focused on vegetable production. It has become one of the top grower meetings in the Northeast, providing an avenue where growers, industry personnel, and researchers to learn and progress towards their common goals together. In June of 2021, preliminary data from objective 1 will be presented at the international Pseudomonas syringae conference, that is taking place in Iceland, where researchers from all over the world that work with P. syringae come together to share their expertise ranging from new disease management strategies to specialized novel biological techniques with each other every 5 years. I will also present results at the Northeastern American Phytopathological Society meeting (APS) in 2021 & 2022, to share my work with local researchers and extension agents focused on local plant disease related questions. The final outcomes produced from this project will also be shared at the 2022 National APS meeting, where researchers from the United States and abroad come to share work from a range of plant disease focused research topics. This will allow the results to reach and receive feedback from a broader audience than regional APS meetings. The results and concepts from this project will be published in a peer-reviewed journal such as Phytopathology, or one with similar scope. If this project is successful, community-based microbial disease resistance experiments will be designed to provide proof-of-concept. Finally, once-proof-of concept has been satisfied, further outreach to tomato growers and extension specialist will be required to create the best plan for growers to successfully integrate bacteriocin and community-based microbial disease resistance strategies into conventional and organic tomato production.

Project Outcomes

Project outcomes:

Not applicable at this point in the project. 

Knowledge Gained:

Not applicable at this point in the project. 

Assessment of Project Approach and Areas of Further Study:

Not applicable at this point in the project. 

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.