Development of a PCR-Based Assay for Identifying Members of the Pseudomonas syringae Species Complex from Environmental Samples

Progress report for GNE20-232

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:

There is a growing appreciation for the diversity of bacteria within the Pseudomonas syringae species complex (Pssc) that are capable of causing disease on tomato. Further, non-pathogenic lineages of the Pssc that are present in environmental reservoirs have been shown to be capable of adapting to agricultural settings and emerging as highly virulent pathogens. The goal of the proposed research is to develop an assay for screening various types of environmental samples that is both highly specific to the Pssc and offers a high degree of discriminatory ability within the Pssc. This assay involves the amplification of a genomic region partially encoding an R-type tailocin ubiquitous in the Pssc. Using primers that have been developed and tested both in-silico and in-planta, I will assess the ability of the assay to specifically amplify Pssc members from plant material, soil, and rain - all of which are important reservoirs for the Pssc. I will also determine the effectiveness and lower detection limit on commercial tomato seed, a common means of transmission for Pssc tomato pathogens. In addition to validating the molecular biology, I will also develop a software pipeline for accurately and reproducibly analyzing data obtained from the assay. The results of this study will provide extension staff and researchers with a valuable tool for studying and managing Pssc-mediated disease on tomato. Results will be communicated in publications and presentations geared toward extension staff, farmers, and agricultural researchers.

Project Objectives:

In this project, I propose to develop an assay for researchers to detect and monitor a broad range of Pssc lineages associated with disease in tomato.

 

The specific objectives of my proposal are:

  1. Assess the specificity and discriminatory ability of a Pssc-tailocin primer set compared to culture-based method of detection within natural microbial communities. Expected outcome: this objective will describe the ability of PCR assay to exclusively detect the Pssc while differentiating between different clades within Pssc communities.
  2. Determine the lower limits of detection in seed and water, both common sources of pathogen transmission. Expected outcome: This objective will determine how abundant different lineages need to be for the assay to detect them in common sources of pathogens. This has important implications for source tracking during epidemics and in the case of seed, screening contaminated seed that might have very low but still infectious numbers of pathogenic strains.
  3. Develop a software pipeline to predict identities of lineages detected through PCR-based assay. Expected outcome: This objective will provide extension faculty with a simple, accurate approach for analyzing the data obtained from PCR-amplification with the Pssc-tailocin primers. The output of the pipeline will include most probable identities of unique lineages detected as well as calculated measures of diversity.

Overall, the expected outcome for all three objectives together is to provide a fully-developed assay for extension faculty and plant epidemiologists to efficiently and accurately survey natural sources of Pssc members, identify known tomato pathogens throughout the complex, and better detect environmental lineages that might represent emerging tomato pathogens.

Introduction:

The purpose of this project is to provide extension faculty and growers with a tool that will allow for pathovar-level identification of the economically significant plant pathogen, Pseudomonas syringae

 

The Pseudomonas syringae species complex (Pssc) is composed of both environmental lineages of bacteria and plant-pathogenic lineages called pathovars. Phylogenetic evidence suggests that the pathogenic lineages currently infecting tomato, bean, and cucurbits emerged from environmental lineages (Morris et al. 2013), and there is still frequent intermixing between agricultural and non-agricultural Pssc strains (Monteil et al. 2013). The emergence of a highly virulent strain of P. syringae pathovar actinidiae (pv. actinidiae) in 2008, responsible for widespread destruction of kiwifruit that forced growers in many regions to switch to more resistant but less profitable cultivars, is currently hypothesized to be the result of such intermixing of pathogenic and environmental strains (Mccann et al. 2017).

 

Understanding the diversity of Pssc lineages co-existing in and near agricultural settings is crucial for sustainable management of current and emerging pathogens. An important example of why studying the full diversity of Pssc pathogens is crucial to disease management can be seen by examining an economically important pathovar found in the northeast United States: Pseudomonas syringae pv. tomato (Pto), the causative agent of bacterial speck in tomato. Although Pto is the most common Pssc pathovar to cause disease in tomato, many other members of the group have been identified as tomato pathogens, including pv. maculicola, pv. appi, pv. antirrhini, pv. syringae and Pseudomonas viridiflava, all causing symptoms very similar to bacterial speck (Morris et al. 2019, Goumas et al. 1999). In a recent preliminary survey of bacterial isolates taken from tomato plants throughout New York exhibiting speck-like symptoms, only 44 out of 57 were confirmed with whole genome sequencing to be Pto. Two of the disease-associated isolates were identified as P. syringae pv. syringae, whereas the majority of the others were various non-syringae pseudomonads, including one instance of P. viridiflava. The extent to which these and other Pssc lineages might be intermixing has consequences for pathogen evolution and emergence that affects short- and long-term management strategies.

 

I propose to develop a PCR-based assay for the detection of bacterial speck and speck-like pathogens in common bacterial reservoirs of the pathogen. Amplification and sequencing of an antibacterial protein (known as an R-type tailocin) encoding region in P. syringae’s genome would form the basis of an assay that is both highly specific for the Pssc and discriminatory at the pathovar-level within the complex. This proposal would enable the positive identification of Pto, as well as closely-related pseudomonads known to cause bacterial speck-like symptoms in tomato throughout the Northeast. 

Research

Materials and methods:

Primer design and preliminary testing of primer specificity conducted in the summer of 2019 found that roughly 80% of sequences on tomato leaves obtained from a tomato research field at The University of Florida and amplified by the Pssc-tailocin primers belonged to P. syringae. Analysis of in-silico PCR results from 191 published Pssc genomes showed that phylogenetic relationships inferred by amplification products broadly recapitulated known phylogeny based on multilocus sequence analysis (MLSA). Further, the amplicon-derived phylogeny was able to accurately discriminate between the known biovars within pv. actinidiae, including being able to identify strains belonging to the recently emerged and highly virulent lineage responsible for the 2008 kiwifruit pandemic. Further validation of the technique is necessary to determine usefulness under field conditions and will be accomplished through the following approaches:

Objective 1

SAMPLE COLLECTION

Sampling will take place in the summer of 2020 at a tomato research plot at Rock Springs Research Farm maintained by Penn State. To assess the ability of the PCR assay to detect Pssc members in a variety of likely reservoirs, I will be sampling host plant material, non-host plant material, soil, and rainwater. Specifically, a diverse collection of weeds surrounding the perimeter of the field, six leaflets randomly selected from tomato plants within the field, and six 1 ml soil samples taken from the top 1 cm will be collected. Rainwater will be collected throughout the growing season automatically via a sterile collection apparatus in the field. The number of replicates will be in part dependent on the number of rain events that occur at the research plot in the summer of 2020.

All plant material and soil samples will be kept on ice during transport, and subsequently stored at 4C for no more than 48 hours before being processed.

SAMPLE PROCESSING

Plant material: Mechanical removal of microbial communities from plant material will be done by bath sonication of 100 grams of plant material for 5 minutes in 30ml of buffer solution containing 0.02% surfactant.

Soil: soil will be suspended in 30ml buffer solution.

Water: 500 ml of water will be passed through a 0.2 micron filter to concentrate bacteria on filter membrane.

Each processed sample will be subsequently assayed for Pssc members using two methods to compare the sensitivity and accuracy of the proposed PCR-based assay with a traditional culture-based method of detection. 

PCR-based detection of Pssc members: Each microbial community will undergo PCR amplification using currently designed primers. The following conditions were previously found to be ideal for selectively amplifying the desired genomic region of Pssc members using Phusion High-Fidelity DNA Polymerase: 98C for 30 seconds, 30 cycles of: 98C for 5 seconds, 62C for 30 seconds, and 72C for 120 seconds, followed by a final extension at 72C for 10 minutes. These conditions produce bands of 3-5.5kb depending on the strains being amplified. 

Before undergoing PCR, soil samples will have their DNA extracted using the Qiagen DNeasy PowerSoil kit to remove PCR inhibitors. Long-read sequencing via PacBio will be performed to ensure that accurate, full-length sequences of the amplicons are obtained.

BIOCHEMICAL DETECTION OF PSSC MEMBERS

Each microbial community will initially be plated on King’s B (KB) medium agar plates. When grown on KB, Pssc members typically produce the fluorescent compound pyoverdin, which is visible under UV illumination. Fluorescent colonies will be streaked onto a second plate to obtain pure isolates, which will undergo further biochemical tests to determine the strains most likely to be Pssc members, as described in (Vicente 2003). 

IDENTIFICATION OF PCR-DETECTED PSSC MEMBERS

To assign an identity to each unique sequence obtained, Mothur (Schloss et al. 2009) will be used to perform a pairwise local alignment to a custom reference database of 251 amplicons generated via in-silico-PCR amplification of currently available Pssc genomes. This reference database will be expanded as sequenced genomes are made available and will represent the currently known diversity of the Pssc-tailocin region.

IDENTIFICATION OF CULTURE-BASED ISOLATES

MLSA will be performed based on the genes 16S, aroE, glnS, gyrB, ileS and rpoD, in accordance with previously published methods (Vasquez-Ponce et al. 2018, Andreani et al. 2014). To compare the discriminatory ability of my proposed PCR assay to MLSA when performed on the same set of unknown organisms, all isolates undergoing MLSA analysis will also undergo the PCR assay as described above.

STATISTICAL ANALYSIS

The specificity of each assay will be assessed by calculating the proportion of unique organisms identified as Pssc members by each assay, from each sample type. For the PCR assay, this will be calculated as (reads identified as Pssc members/total reads). For the culture-based assay, this will be calculated as (isolates identified as Pssc members with MLSA/isolates selected for MLSA). The discriminatory ability of each assay will be determined by building maximum-likelihood trees of all isolates based on MLSA sequences and Pssc-tailocin sequences. The generalized Robinson-Foulds distance between the two trees will be calculated using the DendroPy python library.

Objective 2

INOCULATION OF SEED

commercial tomato seed will be sterilized with bleach as per (Benson et al. 2017) and separated into 21 treatment groups consisting of 100 seeds, each with six technical replicates. Each treatment group will be dip-inoculated in water containing a representative of one of the following pathovars: pv. tomato, pv. syringae, or pv. maculicola, at a concentration ranging from 102-108 cfu/ml. Seeds will be allowed to air dry before the assay is performed. Colony forming units per seed (cfu/seed) will be calculated through removal of seedborne bacteria from a subset of the inoculated seed via sonication, plating on KB agar and counting fluorescent colonies after 24 hours. 

PCR-BASED ASSAY OF PSSC MEMBERS

will be performed as described in Objective 1

STATISTICAL ANALYSIS

The sensitivity for each treatment will be calculated as true positives / (true positives + false negatives). A simple linear regression of sensitivity per CFU/seed will be performed to quantify sensitivity as a function of inoculum concentration.

Objective 3

A pipeline for sequence analysis will be compiled based on Mothur (Schloss et al. 2009) and a curated reference database of amplicons that will allow extension staff and researchers to quickly and consistently analyze sequence data generated by the PCR assay. Web-based and command-line executable versions of the pipeline will be developed and released. This will allow for wider adoption of the tool and consistent results among researchers.

 

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

Project results and progress will be presented through multiple venues that target researchers, extension faculty, and farmers. I will present preliminary results at the 2021 American Phytopathological Society (APS) Northeastern division meeting, which will be attended by researchers and extension staff throughout the Northeastern United States. This will provide an opportunity to introduce the project and communicate early findings, and to receive feedback from stakeholders. Results will again be communicated at the 2022 Pennsylvania Association for Sustainable Agriculture (PASA) conference.

Final results will be submitted to a peer-reviewed plant pathology journal, such as Plant Disease or Phytopathology, and any publication will be accompanied by articles in Penn State’s Extension Report and Journal of Integrated Pest Management, summarizing findings and discussing implications for disease management. 

The software pipeline developed for analysis of the assay results will be published on GitHub with full documentation on installation, usage and interpretation of software outputs. The native ‘pull request’ and ‘issues’ functionality of GitHub will allow timely troubleshooting and communication with software users and will allow for active maintenance and expansion of the software based on the needs of the user base. The software will be open source, which along with thorough documentation will allow users to implement and alter the pipeline based on their specific needs. 

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.